WO2023196881A2

WO2023196881A2 - Bcl6 inhibitory peptide compositions and methods

Info

Publication number: WO2023196881A2
Application number: PCT/US2023/065408
Authority: WO
Inventors: Brant E. ISAKSON; Abigail Grace WOLPE
Original assignee: University of Virginia Patent Foundation
Current assignee: UVA Licensing and Ventures Group
Priority date: 2022-04-05
Filing date: 2023-04-05
Publication date: 2023-10-12
Anticipated expiration: 2024-10-05
Also published as: WO2023196881A3

Abstract

Provided are compositions and methods for treating cancer and mimicking oxidative stress in a subject. In some embodiments, the methods include modulating vascular oxidative stress, inflammation, and/or hypertension in a subject. Also provided are methods for evaluating therapeutic compositions in a subject with modulated vascular oxidative stress, inflammation, and/or hypertension.

Description

BCL6 INHIBITORY PEPTIDE COMPOSITIONS AND METHODS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 63/327,658 filed April 5, 2022; the disclosure of which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING XML

The Sequence Listing XML associated with the instant disclosure has been electronically submitted to the United States Patent and Trademark Office via the Patent Center as a 10,469 byte UTF-8-encoded XML file created on April 5, 2023 and entitled “3062_184_PCT.xml”. The content of the Sequence Listing XML submitted via Patent Center is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. HL137112 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

The presently disclosed subject matter relates generally to methods for treating cancer, methods for modulating hypertension, and compositions therefor.

BACKGROUND

B-Cell lymphoma 6 (BCL6) is an oncogenic nuclear sequence-specific transcriptional repressor (Chang et al., 1996; Deweindt et al., 1995; Seyfert et al., 1996). BCL6 has a broad range of physiological functions including (1) regulating immune cell differentiation and proliferation (Alinikula et al., 2011; Choi & Crotty, 2021; Duy et al., 2010; Misawa et al., 2020; Nurieva et al., 2009), (2) repressing pro-inflammatory NFKB activity (Barish et al., 2010; Perez-Rosado et al., 2008), and (3) facilitating pathological immune responses in autoimmune encephalitis (Li et al., 2020) and systemic lupus erythematosus (Venkatadri et al., 2022). Dysregulation of BCL6 expression and activity has also been implicated in an expanding scope of soft tissue and solid tumors (e.g. diffuse large B Cell Lymphoma, DLBCL (Cardenas et al., 2016; Krull et al., 2020; Polo et al., 2007); B-acute lymphoblastic leukemia (Geng et al., 2012; Hurtz et al., 2019); chronic myeloid leukemia (Hurtz et al., 2011; Madapura et al., 2017); breast cancer (Sultan et al., 2021; Walker et al., 2015); non-small cell lung cancer (Deb et al., 2017; Marullo Rossella et al., 2016); and glioma (Xu et al., 2017)). In all of these diseases, BCL6 protein is upregulated (Cardenas et al., 2016; Deb et al., 2017; Geng et al., 2012; Hurtz et al., 2011, 2019; Krull et al., 2020; Madapura et al., 2017; Marullo Rossella et al., 2016; Polo et al., 2007; Sultan et al., 2021; Venkatadri et al., 2022; Walker et al., 2015; Xu et al., 2017).

Reactive oxidative species (ROS) are produced as a consequence of oxygen metabolism and include highly reactive free radicals, such as superoxide (O²‘), as well as more stable, nonradical peroxide species, such as hydrogen peroxide (H2O2). While ROS participate in many physiological signaling mechanisms, their overproduction can overwhelm antioxidant capacity, causing deleterious effects on the cardiovascular system. Thus, the expression of ROS generating enzymes and endogenous antioxidant systems must be tightly regulated. Transcriptional control of oxidative stress related genes is paramount to achieving homeostatic redox balance.

B Cell Lymphoma 6 (BCL6) is a transcription factor that has been shown to differentially regulate expression of oxidant stressors as well as redox proteins. As part of its well-documented role in the humoral response, BCL6 transcriptional activity promotes B cell survival during genotoxic and oxidative challenge of germinal center formation (Dent et al., 1997; Fukuda et al., 1997; Nakagawa et al., 2021; Ye et al., 1997). In non-Hodgkin’s lymphoma, overexpression of BCL6 in lymphoma has been shown to promote resistance to chemotherapeutics such as etoposide by preventing apoptotic ROS generation (Kurosu et al., 2003). Similarly, a subset of Diffuse Large B Cell Lymphoma (DLBCL) cells will undergo a metabolic shift to oxidative phosphorylation and require BCL6-dependent expression of thioredoxin to tolerate the oxidative challenge (Sewastianik et al., 2016). Oxidative protection by BCL6 is not limited to its ability to directly alter expression of oxidants and reductants. BCL6 exhibits mutual antagonism with pro-oxidant, pro-inflammatory transcription factor NFKB, repressing many of the transcriptional targets that NFKB activates (Barish et al., 2010; Perez-Rosado et al., 2008). BCL6 engages in a diverse repertoire of protein-protein interactions to fulfill these roles. In the cytosol, MAPK can phosphorylate BCL6 PEST motifs, resulting in rapid recruitment of BCL6 to SCF ubiquitin ligase through interactions with FBXO11 resulting in proteasomal degradation(Duan et al., 2012; Niu et al., 1998). However, functional roles have not been identified for all BCL6 interactions. For example, an unbiased screen for binding partners of BCL6 identified an interaction with pannexin 3 (Panx3) (Miles et al., 2005), though the products of this interaction are not known.

Panx3 belongs to the family of pannexin channels of which there are three isoforms: Panxl, Panx2 and Panx3. Panxl has well-documented roles at the plasma membrane contributing to purinergic, adrenergic and calcium signaling in the vasculature (Billaud et al., 2011, 2015; Chekeni et al., 2010; DeLalio et al., 2018, 2019, 2020; Good et al., 2018; Lohman et al., 2015; Yang et al., 2020). While low levels of Panx3 expression has been noted in RNA- Seq datasets from endothelium throughout the systemic microvasculature (Khan et al., 2019), its functional role has yet to be described. Published reports on the localization and functional implications of Panx3 demonstrate a high degree of plasticity across cell types and expression systems. When localized to the endoplasmic reticulum, Panx3 has been suggested to contribute to calcium store release (Ishikawa et al., 2011). However, Panx3 has also been reported to localize to the plasma membrane where it has been implicated in dye uptake (Celetti et al., 2010; Penuela et al., 2007, 2009) and ATP release (Ishikawa et al., 2011; Iwamoto et al., 2010). The data described herein suggest that Panx3 and BCL6 interact in endothelium, which shields BCL6 from degradation and protects against vascular oxidative stress. Targeting the BCL6- Panx3 interaction may prove to be a valuable mechanism by which to modulate the abundance and transcriptional activity of BCL6 in clinically relevant settings such as cancer or autoimmune disorders.

Therefore, the BCL6-Panx3 interaction remains a site of interest for therapeutic development and as a model of vascular oxidative stress and hypertension.

SUMMARY

This Summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments of the presently disclosed subject matter. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.

The presently disclosed subject matter relates in some embodiments to inhibitor of an oncogenic nuclear sequence-specific transcriptional repressor, optionally B-Cell lymphoma 6 (BCL6), the inhibitor comprising a peptide that promotes degradation of the oncogenic nuclear sequence-specific transcriptional repressor and/or BCL6 in vitro and/or in vivo. In some embodiments, the inhibitor comprises a BCL6 inhibitory peptide (BCLiP), wherein the BCLiP comprises a peptide comprising an amino acid sequence that mimics amino acids L328-P344, (SEQ ID NO. 10), of native human BCL6 (SEQ ID NO. 1), optionally wherein the BCLiP comprises SEQ ID NO. 2. In some embodiments, the inhibitor comprises a conserved region containing degradation-targeting phosphorylation sites S333 and S343 of native human BCL6 (SEQ ID NO. 1). In some embodiments, the inhibitor comprises an about 17 amino acid sequence, optionally SEQ ID NO. 2 or 10, optionally a peptide sequence having about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identity to SEQ ID NO. 2 or 10, optionally a biologically active fragment and/or homolog of any of the foregoing sequences including SEQ ID NO. 2 or 10. In some embodiments, the inhibitor comprises a cell entry tag. In some embodiments, the cell entry tag is selected from the group consisting of a lipid tag, peptide sequences and any combination thereof. In some embodiments, the cell entry tag comprises an N-terminal Stearyl tag. In some embodiments, the inhibitor comprises one or more phosphorylation sites to target BCL6 protein for proteasomal degradation. In some embodiments, the inhibitor further comprises a pharmaceutically acceptable carrier.

In some embodiments the inhibitor for use in inducing degradation of an oncogenic nuclear sequence-specific transcriptional repressor and/or B-Cell lymphoma 6 (BCL6) in vitro and/or in vivo. In some embodiments the inhibitor for use in treating a cancer and/or a tumor in a subject in need thereof. In some embodiments the inhibitor for use, the cancer and/or tumor comprises a cancer and/or tumor driven by oncogenic BCL6 transcriptional activity, optionally diffuse large B Cell Lymphoma, B-acute lymphoblastic leukemia, chronic myeloid leukemia, breast cancer, non-small cell lung cancer, and/or glioma. In some embodiments the inhibitor for use, in modeling pathological autoimmunity, modulating Pannexin (Panx) channels in vitro and/or in vivo, modulating inflammation in a subject, modulating vascular oxidative stress in a subject, and/or modulating hypertension in a subject in which said modeling and/or modulating is desired.

The presently disclosed subject matter also relates in some embodiments to an inhibitor of a transcription factor, optionally B-Cell Lymphoma 6 (BCL6), the inhibitor comprising a peptide that blocks the interaction of the transcription factor with a pannexin channel in vitro and/or in vivo. In some embodiments, the inhibitor comprises a BCL6 inhibitory peptide (BCLiP), wherein the BCLiP comprises a peptide comprising an amino acid sequence that mimics amino acids L328-P344, (SEQ ID NO. 10) of native human BCL6 (SEQ ID NO. 1), optionally wherein the BCLiP comprises SEQ ID NO. 2. In some embodiments, the inhibitor comprises a conserved region containing degradation-targeting phosphorylation sites S333 and S343 of native human BCL6 (SEQ ID NO. 1). In some embodiments, the inhibitor comprise an about 17 amino acid sequence, optionally SEQ ID NO. 2 or 10, optionally a peptide sequence having about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identity to SEQ ID NO. 2 or 10, optionally a biologically active fragment and/or homolog of any of the foregoing sequences including SEQ ID NO. 2 or 10. In some embodiments, the inhibitor comprises a cell entry tag. In some embodiments, cell entry tag is selected from the group consisting of a lipid tag, a peptide sequence, and any combination thereof. In some embodiments, the cell entry tag comprises an N-terminal tag, optionally an N-terminal stearyl tag. In some embodiments, the inhibitor comprises one or more phosphorylation sites to target BCL6 protein for proteasomal degradation. In some embodiments, wherein the pannexin channel is optionally one of three isoforms, optionally Panxl, Panx2 and/or Panx3. In some embodiments, the pannexin channel is Panx3. In some embodiments, the inhibitor further comprises a pharmaceutically acceptable carrier.

The presently disclosed subject matter also relates in some embodiments to a method of inducing degradation of an oncogenic nuclear sequence-specific transcriptional repressor and/or B-Cell lymphoma 6 (BCL6) in vitro and/or in vivo, the method comprising contacting a cell in vivo or in vitro with an inhibitor, as described herein and above, and/or administering to a subject the inhibitor as described herein and above.

The presently disclosed subject matter also relates in some embodiments to a method treating a cancer and/or a tumor in a subject, the method comprising administering to a subject in need of treatment a composition comprising an inhibitor, described herein and above, optionally wherein the inhibitor is BCLiP, further optionally wherein the BCLiP comprises SEQ ID NO. 2 or 10. In some embodiments, the cancer and/or tumor comprises a cancer and/or tumor driven by oncogenic BCL6 transcriptional activity, optionally diffuse large B Cell Lymphoma, B-acute lymphoblastic leukemia, chronic myeloid leukemia, breast cancer, nonsmall cell lung cancer, and/or glioma.

The presently disclosed subject matter also relates in some embodiments to a method of modeling pathological autoimmunity in a subject, the method comprising administering to a subject a composition comprising an inhibitor as described above and herein, optionally wherein the inhibitor is BCLiP, further optionally wherein the BCLiP comprises SEQ ID NO. 2 or 10.

The presently disclosed subject matter also relates in some embodiments to a method of modulating Pannexin (Panx) channels in vitro and/or in vivo, the method comprising contacting a cell in vivo or in vitro with an inhibitor of any of the above claims, and/or administering to a subject an inhibitor as described above and herein, wherein the modulating comprises disruption of BCL6 binding with Panx, wherein the Panx is optionally one of three isoforms, optionally Panxl, Panx2 and/or Panx3.

The presently disclosed subject matter also relates in some embodiments to a method of modulating inflammation, hypertension, and/or vascular oxidative stress in a subject, the method comprising administering to a subject a composition comprising an inhibitor as described above and herein, optionally wherein the inhibitor is BCLiP, further optionally wherein the BCLiP comprises SEQ ID NO. 2 or 10.

The presently disclosed subject matter also relates in some embodiments to use of an inhibitor of a transcription factor, optionally B-Cell Lymphoma 6 (BCL6), for the treatment of cancer and/or a tumor in a subject in need thereof, optionally wherein the inhibitor is an inhibitor as described above and herein, optionally wherein the inhibitor is BCLiP, further optionally wherein the BCLiP comprises SEQ ID NO. 2 or 10. In some embodiments, the cancer and/or tumor comprises a cancer and/or tumor driven by oncogenic BCL6 transcriptional activity, optionally diffuse large B Cell Lymphoma, B-acute lymphoblastic leukemia, chronic myeloid leukemia, breast cancer, non-small cell lung cancer, and/or glioma.

The presently disclosed subject matter also relates in some embodiments to a method of evaluating a candidate therapeutic composition, the method comprising providing a subject modulated with an inhibitor of a transcription factor, optionally B-Cell Lymphoma 6 (BCL6); administering a candidate therapeutic composition to the subject; and assessing an effect of the candidate therapeutic composition, optionally wherein the inhibitor is an inhibitor as described above and herein, optionally wherein the inhibitor is BCLiP, further optionally wherein the BCLiP comprises SEQ ID NO. 2 or 10.

Therefore, it is an object of the presently disclosed subject matter to provide methods for treating cancer, methods for modulating hypertension, and compositions therefor. This and other obj ects are achieved in whole or in part by the presently disclosed subj ect matter. Further, objects of the presently disclosed subject matter having been stated above, other objects and advantages of the presently disclosed subject matter will become apparent to those skilled in the art after a study of the following description, Figures, and Examples. Additionally, various aspects and embodiments of the presently disclosed subject matter are described in further detail below.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A-1D are graphs showing that hypertension is associated with attenuated vascular Panx3 expression in humans and mice. Figures 1 A and IB show that Treatmentresistant (HT) human participants exhibit attenuated expression of Panx3 mRNA in adipose arteries compared to normotensive (NT) participants. Figures 1C and ID show genetically inbred, spontaneously hypertensive BPH/2 mice show increased expression of Panxl and reduced expression of Panx3. Each dot represents one human (Fig. 1A, Fig. IB) or one mouse (Fig. 1C, Fig. ID). * is p<0.05; ** is p<0.01; **** is p<0.0001 via unpaired t-test.

Figures 2A-2D are immunostaining, graphs and western blots showing endothelial Panx3 localizes to the Golgi Apparatus. Figure 2A shows immunostaining of murine third- order mesenteric arteries prepared en face. Cellular compartment markers (shown in color, yellow; Top: CD31/PECAM-1, interendothelial junctions; Middle: Calnexin, endoplasmic reticulum; Bottom: GM- 130, Golgi Apparatus), Panx3 (shown in color, shown in color, magenta), nuclei (shown in color, cyan). Figure 2B shows colocalization between Panx3 and each cellular compartment was quantified via unpaired t-tests of Manders’ Overlap Coefficients (** is p<0.01, *** is p<0.001). Each dot represents one mouse (averaged from 3 fields of view). Figure 2C shows polarization of endothelial Panx3 is dependent on vessel type , in a manner consistent with localization to the Golgi Apparatus. Panx3 is polarized upstream (white arrowheads) of the nucleus in arteries, but polarization is lost in veins. Black arrowheads indicate downstream polarization. Blood flow arrow indicates direction of flow. Quantification at right. *** indicates p<0.001 via two-way ANOVA (p=0.0003). Artery N = 7 mice, Vein N = 3 mice. Figure 2D is a Western blotting for Panx3 (mesenteric vascular lysate) and Panxl (lung lysate) following deglycosylation by PNGase F. Scale bars are 20pm throughout.

Figures 3A-3G are an exemplary gene targeting strategy model, graphs, blots and immunohistochemistry of the generation of an inducible, endothelial cell-specific Panx3 knockout mouse (EC Panx3 ^/A). Figure 3A show the genetic targeting strategy for the generation of EC Panx3^{A A} mice using the inducible Cdh5-CreER^T2 system. Figure 3B shows gDNA excision of the second exon of Panx3. Figure 3C shows Panx3 mRNA (left column) abundance is reduced -85% in endothelial-rich lung tissue. * is p<0.05 via unpaired t-test with Welch’s correction. Figures 3D and 3E show loss of Panx3 protein in EC Panx3^{A A} mice is shown by western blot from mesenteric vascular tissue and immunohistochemistry of the endothelium of third-order mesenteric arteries viewed en face. Figure 3F shows Fluorescence intensity quantification of Panx3^n/ri (left column) vs Panx3^A/A (right column). * * * * is p<0.0001 via unpaired t-test with Welch’s correction. Figure 3G shows Panxl mRNA abundance is unchanged by loss of EC Panx3^AfA (right column) in endothelial-rich lung tissue. Throughout the figure, each dot represents one mouse.

Figures 4A-4L are graphs and blots showing that genetic deletion of endothelial Panx3 causes spontaneous hypertension. Figures 4A and 4B show EC Panx3^A/A mice (right column) exhibit significantly increased mean arterial and systolic blood pressure at each time period assessed as measure by implanted radiotelemetry. * is p<0.05; ** is pO.Ol via unpaired t-test. Figures 4C and 4D show that diastolic pressure and heart rate remain unchanged in Panx3^AfA mice (right column). Figure 4E shows cardiac output as measured by cardiac MRI in Panx3^AfA mice (right column). Figures 4F and 4G show blood levels of sodium and potassium in Panx3^{A A} mice (right column). Figure 4H shows renin concentrations in blood plasma collected during the active period Panx3^AfA mice (right column). Figure 41 shows acetylcholine (Ach)-induced dilation of third-order mesenteric arteries from EC Panx3^A/A (shown in color as magenta) mice (bottom line) is severely impaired as compared to Panx3fl^/fl littermates (top line) via pressure myography. N = 5 mice/group with 1-3 arteries averaged per condition per mouse. * is p<0.05; ** is p<0.005; *** is p<0.0005 between genotypes via two-way ANOVA with Sidak’s multiple comparison test. Figure 4J shows constriction to KC1. Figure 4K is a western blot analysis of eNOS expression and phosphorylation at Thr⁴⁹⁵. Figure 4L shows dilation of third-order mesenteric arteries to IpM NS309. * is p<0.05 via unpaired t-test. Each dot represents one mouse.

Figures 5A-5I are graphs, immunohistochemistry and blots showing that channelindependent association of Panx3 and transcriptional repressor BCL6 in intact endothelium. Figures 5 A and 5B show loss of endothelial Panx3 (right column) does not alter intracellular ATP or ATP released following Ach stimulation (10pM, 5 min) of third-order mesenteric arteries. Each dot represents one mouse. Figure 5C shows the number of transient calcium events per field of view under basal conditions or following 10pM carbachol (CCh) stimulation in third-order mesenteric arteries. Each dot represents one artery. N = 4 mice per group with 1-2 arteries per mouse. * indicates p<0.05 via paired t-test. Unpaired t-tests were used to compare genotypes. Figure 5D shows Golgi neutralization via treatment with 2.5mM NH4Q displaces B4GALT1 (showin in color, red) from the Golgi (shown in color with eNOS, yellow) in HCAEC. Nuclei are depicted in cyan. Figure 5E shows that intact mesenteric endothelium exhibits no change in the subcellular distribution of B4GALT1 (shown in color, red) following loss of EC Panx3. Golgi is depicted in color, yellow (eNOS), nuclei are shown in color, cyan. Figure 5F shows colocalization analysis of B4GALT1 and eNOS in resistance endothelium. Figure 5G blots show Top: Crosslinking of endogenous Panx3 from mesenteric vascular tissue with 0.5mM (+) or 2.5mM (++) BS³ results in a doublet at ~90 kDa but larger heptameric species are not observed. Bottom: Crosslinking of Panxl from lung tissue reveals heptameric state. Figure 5H shows immunostaining for Panx3 (show in color, magenta) and BCL6 (show in color, yellow) in intact resistance endothelium. Nuclei are depicted in color, cyan. Figure 51 shows proximity ligation puncta formation which suggests Panx3 and BCL6 are within close proximity in intact resistance endothelium. Negative (No Primary, Panx3 Primary alone) and positive controls (eNOS/Cavl) shown at left. Proximity ligation puncta generation quantified at right. Each dot represents one mouse, except where otherwise noted. Scale bars are 20pm.

Figures 6A-6N are graphs, blots and JASPAR sequence showing that genetic deletion of endothelial Panx3 is associated with decreased BCL6 protein, increased NFKB activity and the specific upregulation of Nox4, which can be predicted by evidence of BCL6 chromatin binding activity. Figure 6A shows that BCL6 mRNA is unchanged following deletion of EC Panx3^A'^A (right column). Figure 6B is Western blotting for BCL6 in endothelial-rich lung tissue. Figures 6C and 6D show the JASPAR sequence logo for murine BCL6 (MA0463.1; SEQ ID NO. 5) was used for in silico assessments of the lOOObp sequences preceding the transcriptional start side (TSS) of genes of interest. Figures 6E-6I show BCL6 ChlP-Seq (Barish et al., 2010; X. Liu et al., 2016; Sommars et al., 2019) binding profiles in the promoter region of genes of interest. Figures 6J-6N show transcript abundance for the same genes of interest assessed in lung tissue from PanxS ¹ (left column) and EC Panx3^^A (right column) mice. Each data point represents one mouse. * indicates p <0.05; ** indicates p < 0.001 via unpaired t-test.

Figures 7A-7E are graphs, blots and immunohistochemistry showing that endothelial Panx3 protects against HiOi-induced oxidative stress and prevents endothelial-mediated vascular dysfunction in resistance arteries. Figure 7A shows western blot and quantification of Nox4 and p22^phox in lung lysates. Figure 7B shows relative abundance of 3 -Nitrotyrosine protein adducts assessed from plasma. * indicates p < 0.05 via unpaired t-test with Welch’s correction. Figure 7C shows quantification of H2O2 in deproteinized plasma collected via AmplexRed fluorescence. *** indicates p < 0.001 via unpaired t-test. Figure 7D shows immunodetection and quantification of hyperoxidized peroxiredoxin (PrxSCh, yellow) in transverse sections of third-order mesenteric arteries. Nuclei are shown in color, cyan. In images, * indicates vessel lumen. ‘+ H2O2’ positive controls were exposed to ImM H2O2 and ImM NaNCh prior to antigen retrieval. Mean fluorescent signal was normalized to positive controls. Scale bars are 50pm. ** indicated p < 0.005 via unpaired t-test. Figure 7E shows acetylcholine (ACh)-induced dilation of third-order mesenteric arteries. Vehicle conditions replicated from Figure 41. Pretreatment with PEG- catalase (lOOOU/mL) results in partial rescue of the dilatory response. Repeated measures oneway ANOVA with Geisser-Greenhouse correction and Sidak's multiple comparisons test was used to compare the dose response curves between each condition. *** indicates p < 0.001, **** indicates p < 0.0001. Each dot represents one mouse.

Figures 8A-8M are sequence listings, an exemplary amino acid structure, blots and graphs showing the inhibition of the Panx3/BCL6 interaction via BCLiP peptide phenocopies genetic deletion of endothelial Panx3. Figure 8 A shows that the BCLiP amino acid sequence (SEQ ID NO. 2) mimics a highly evolutionarily conserved region of BCL6 (L328-P344, SEQ ID NO. 10) with an N-terminal stearyl tag. SEQ ID NO. 3 shows a scramble peptide with an N-terminal stearyl tag. Sequence ID NO. 4 shows the Homo sapiens BCL6. Sequence ID NO. 5 shows the Mus musculus BCL6. Sequence ID NO. 6 shows the Rattus norvegicus BCL6. SEQ ID NO. 7 shows the Macaca mulatta BCL6. S333 and S343 are highlighted (shown in box) to denote MAPK phosphorylation sites which drive BCL6 degradation (see also Table 3). Figure 8B provides a side view (top) and top-down view (bottom) of dimeric Panx3 (monomers in shades of grey) interaction with BCLiP (SEQ ID NO. 2) (shown in color). Panx3 residues involved with peptide interactions are depicted in color, maroon. Figure 8C shows immunoprecipitation of BCL6 using Panx3-Flag as bait from HEK cells transfected with PANX3-Flag and BCL6. Figures 8D and 8E show mRNA of BCL6 is unchanged following five days of BCLiP (SEQ ID NO. 2; right column) or scramble peptide (SEQ ID NO. 3, left column) administration (12.5mg/kg via I.P.), while BCL6 protein is significantly reduced. Figures 8F and 8G show that mRNA expression of autoregulated NFKB family members Nfkbia a \ Nfkb2 is increased following BCLiP (SEQ ID NO. 2, right column) administration. Nfkbia was compared via unpaired t-test with Welch’s correction. Figures 8H- 8J show that BCLiP (SEQ ID NO. 2; right column) treatment significantly increased Nox4 expression but not Noxl or Cybb. Figures 8K-8M show that radiotelemetry reveals elevation of mean arterial (Fig. 8K), systolic (Fig. 8L) and diastolic (Fig. 8M) blood pressure throughout the period of BCLiP (SEQ ID NO. 2) administration. * is p<0.05; ** is p<0.01 via unpaired t- test. Each dot represents one mouse.

Figures 9A-9B are immunohistochemistry showing the abundance of vascular Panx3 is attenuated by hypertension in humans and mice. Figure 9A shows immunohistological staining of Panx3 (show in color, magenta) expression in cross sections of human omental arteries from normotensive and hypertensive human participants. Figure 9B shows that genetically inbred hypertensive BPH/2 mice exhibit reduced endothelial expression of Panx3 in murine thoracodorsal arteries. Panx3 is shown in color, magenta. CD31/PECAM- 1 highlights endothelium shown in color, yellow. Autofluorescence from internal elastic laminae (IEL) is shown in color, green. Nuclei are represented in color, cyan. Asterisks denote vessel lumen. Scale bars are 20pm.

Figures 10A-10D are immunohistochemistry, blots and graphs showing Endogenous Panx3 expression in vascular endothelium. Figure 10A shows Panx3 (shown in color, magenta) expression in third-order mesenteric vasculature prepared en face from control and Panx3 global knockout mice (Panx3'^/_). Interendothelial junctions are visualized by staining for CD31 (shown in color, yellow) and nuclei are represented in color, cyan. Scale bar is 20pm. Figure 10B is a Western blot of Panx3 protein from control and Panx3'^/_ generated from lung tissue using the same Panx3 antibody (Thermofisher #433270) depicted in Figure 10 A. Figure 10C shows murine thoracodorsal artery cross sections reveal Panx3 (shown in color, red) visualized in endothelium via an alternative Panx3 antibody (Panx3 CT-379⁷'⁹). Secondary only control shown at right. Autofluorescence from internal elastic laminae (IEL) is shown in color, green. Nuclei are represented in color, blue. Asterisks denote vessel lumen. Figure 10D shows immunogold labeling of Panx3 in third-order mesenteric resistance arteries demonstrates a preference to localize to intracellular membranes. Scale bar in lower magnification view is 10pm and 5nm in the inset. Abundance and distribution of gold beads conjugated to Panx3 antibody quantified at right.

Figures 11A-11L are graphs showing that EC Panx3^AA exhibit unremarkable gross anatomy, blood lipids, heart and kidney functions, and splenic B-cell development. Figures 11 A, 1 IB and 11C show body length (nose to anus), body weight, and gonadal fat pad weight are unchanged by loss of endothelial Panx3^A/A (right column) . Figures 1 ID-11G show blood lipids were unchanged by genotype. Figure 11H shows that cardiac MRI revealed no significant differences in functional parameters including ejection fraction. Figures 11I-11K show blood testing for serum creatinine, creatine kinase, and Blood Urea Nitrogen (BUN) reveal no difference in kidney function between EC Pctnx3^{A A} (right column) mice and controls (left column). Figure 1 IL flow cytometry reveals the abundance of B220⁺GL7⁺FasL⁺ splenic germinal centers are unchanged by endothelial Panx3 expression.

Figures 12A-12B are immunohistochemistry showing that genetic deletion of endothelial Panx3 does not alter Golgi luminal pH regulation but does ablate interaction between Panx3/BCL6 in intact endothelium. Figure 12A provides visualization of MGAT1 (shown in color, red) in mesenteric resistance endothelium with Golgi apparatus depicted in color, yellow (eNOS), and nuclei are shown in color, cyan. Figure 12B shows proximity ligation assay for Panx3 and BCL6 in PanxS⁰⁷® and EC Pctnx3^A resistance endothelium. Scale bars are 20pm. Figures 13A-13C are immunohistochemistry and graphs showing Evidence of oxidative stress in the aortic vascular wall in EC Panx3^AA mice. Figure 13 A shows immunodetection and quantification of hyperoxidized peroxiredoxin (PrxSCh, shown in color, yellow) in transverse sections of descending thoracic aorta. * indicates p<0.05 via unpaired t- test between genotypes. Scale bars are 50pm. Figure 13B shows that 3 -Nitrotyrosine protein adducts are significantly elevated in the vascular wall of EC Panx3^A/A (right column) mice as compared to controls (left column). *** indicates p<0.005 via unpaired t-test between genotypes. Figure 13C shows that lipid peroxidation as observed via 4-hydroxynonenal staining exhibits a similar trend (p = 0.0846 via unpaired t-test). In all graphs, ‘+H2O2’ indicates that samples were briefly exposure to ImM H2O2 and ImM NaNCh prior to antigen retrieval to be used as positive controls. Mean fluorescent signal was normalized to positive controls. Each dot represents the average from 2-3 cross sections per mouse.

Figures 14A-14F are graphs and a blot assessing other potential sources of oxidative stress following genetic deletion of endothelial Panx3. Figure 14 A shows transcript levels of Nos3 (eNOS) are unchanged in endothelial-rich lung tissue by endothelial Panx3 expression. Figure 14B shows structural eNOS coupling is not altered by loss of Panx3^A'^A (right column) in thoracic aorta and lung tissue. Dimer to monomer ratio is normalized to controls (left column). Figures 14C-14F show mRNA abundance for Xdh (xanthine oxidase), Eroll (endoplasmic reticulum oxidoreductase la), Prx4 (peroxiredoxin 4), Cyb5r3 (cytochrome B5 reductase 3) are consistent in lung tissue between EC Panx3^A/A (right column) mice and controls (left column).

Figure 15 is a plot showing structural analysis of BCLiP and other candidate mimetic peptides. Figure 15 shows Phi -psi plots of all peptides bound to Panx3 dimers. The residues of the BCLIP peptide (SEQ ID NO. 2) are highlighted in color, green (large dot), while the other peptides are shown in black (small dot). The plot shows that the monomer-monomer interface is capable of binding a wide array of secondary structures. Plots were generated using the MolProbity program.

Figure 16 is an exemplary schematic of endothelial Panx3/BCL6 interaction which can be perturbed by BCLiP administration. Under normal conditions, BCL6 and Panx3 interact at the Golgi Apparatus, preventing the cytoplasmic degradation of BCL6. In this state, BCL6 repressive activity is preserved, and the vascular oxidative state is normal. However, following BCLiP administration, the BCL6/Panx3 interaction is inhibited and BCL6 is readily degraded. In the absence of BCL6 repressive activity, gene expression is dysregulated, likely due, in part, increased in NFKB activity. The expression of Nox4 is selectively increased and this constitutively-active enzyme generates an overabundance of hydrogen peroxide which is associated with spontaneous hypertension.

DETAILED DESCRIPTION

Administration of a newly developed peptide, BCLiP (e. g., SEQ ID NO. 2), to inhibit the Panx3-BCL6 interaction recapitulates the knockout of Panx3 from endothelium, with a specific increase in Nox4 and blood pressure. Channel-independent function of Panx3, wherein its interactions with a perinuclear pool of BCL6, can dictate transcriptional repression. While not being bound by theory, as the BCL6 protein is upregulated in cancers, BCLiP (e. g., SEQ ID NO. 2) can prevent BCL6 transcriptional activity, acting as a therapeutic treatment for cancers.

The presently disclosed subject matter will now be described more fully. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein below and in the accompanying Examples. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art.

All references listed herein, including but not limited to all patents, patent applications and publications thereof, and scientific journal articles, are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or compositions employed herein.

I. DEFINITIONS

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the presently disclosed subject matter.

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

In describing the presently disclosed subject matter, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques.

Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the presently disclosed and claimed subject matter.

Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including in the claims. For example, the phrase “an antibody” refers to one or more antibodies, including a plurality of the same antibody. Similarly, the phrase “at least one,” when employed herein to refer to an entity, refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more of that entity, including but not limited to whole number values between 1 and 100 and greater than 100.

Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” The term “about”, as used herein when referring to a measurable value such as an amount of mass, weight, time, volume, concentration, or percentage, is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1 % from the specified amount, as such variations are appropriate to perform the disclosed methods and/or employ the disclosed compositions. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

A disease or disorder is “alleviated” if the severity of a symptom of the disease, condition, or disorder, or the frequency at which such a symptom is experienced by a subject, or both, are reduced.

As used herein, the term “and/or” when used in the context of a list of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.

The terms “additional therapeutically active compound” and “additional therapeutic agent,” as used in the context of the presently disclosed subject matter, refers to the use or administration of a compound for an additional therapeutic use for a particular injury, disease, or disorder being treated. Such a compound, for example, could include one being used to treat an unrelated disease or disorder, or a disease or disorder which may not be responsive to the primary treatment for the injury, disease, or disorder being treated.

As used herein, the term “adjuvant” refers to a substance that elicits an enhanced immune response when used in combination with a specific antigen.

As used herein, the terms “administration of’ and/or “administering” a compound should be understood to refer to providing a compound of the presently disclosed subject matter to a subject in need of treatment.

The term “comprising,” which is synonymous with “including” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements and/or method steps. “Comprising” is a term of art that means that the named elements and/or steps are present, but that other elements and/or steps can be added and still fall within the scope of the relevant subject matter.

As used herein, the phrase “consisting essentially of’ limits the scope of the related disclosure or claim to the specified materials and/or steps, plus those that do not materially affect the basic and novel characteristic(s) of the disclosed and/or claimed subject matter. For example, a pharmaceutical composition can “consist essentially of’ a pharmaceutically active agent or a plurality of pharmaceutically active agents, which means that the recited pharmaceutically active agent(s) is/are the only pharmaceutically active agent(s) present in the pharmaceutical composition. It is noted, however, that carriers, excipients, and/or other inactive agents can and likely would be present in such a pharmaceutical composition, and are encompassed within the nature of the phrase “consisting essentially of.”

As used herein, the phrase “consisting of’ excludes any element, step, or ingredient not specifically recited. It is noted that, when the phrase “consists of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

With respect to the terms “comprising,” “consisting of,” and “consisting essentially of,” where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms. For example, a composition that in some embodiments comprises a given active agent also in some embodiments can consist essentially of that same active agent, and indeed can in some embodiments consist of that same active agent.

As use herein, the terms “administration of’ and or “administering” a compound should be understood to mean providing a compound of the presently disclosed subject matter or a prodrug of a compound of the presently disclosed subject matter to a subject in need of treatment.

The term “adult” as used herein, is meant to refer to any non-embryonic or non-juvenile subject. For example, the term “adult adipose tissue stem cell,” refers to an adipose stem cell, other than that obtained from an embryo or juvenile subject.

As used herein, an “agent” is meant to include something being contacted with a cell population to elicit an effect, such as a drug, a protein, a peptide. An “additional therapeutic agent” refers to a drug or other compound used to treat an illness and can include, for example, an antibiotic or a chemotherapeutic agent.

As used herein, an “agonist” is a composition of matter which, when administered to a mammal such as a human, enhances or extends a biological activity attributable to the level or presence of a target compound or molecule of interest in the mammal.

An “antagonist” is a composition of matter which when administered to a mammal such as a human, inhibits a biological activity attributable to the level or presence of a compound or molecule of interest in the mammal.

As used herein, “alleviating a disease or disorder symptom,” means reducing the severity of the symptom or the frequency with which such a symptom is experienced by a patient, or both.

As used herein, an “analog” of a chemical compound is a compound that, by way of example, resembles another in structure but is not necessarily an isomer (e.g., 5 -fluorouracil is an analog of thymine).

As used herein, amino acids are represented by the full name thereof, by the three letter code corresponding thereto, and/or by the one-letter code corresponding thereto, as summarized in the following Table 1 : Table 1

Amino Acid Codes and Functionally Equivalent Codons

The expression “amino acid” as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids. “Standard amino acid” means any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid residue” means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source. As used herein, “synthetic amino acid” also encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the peptides of the presently disclosed subject matter, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the peptide’s circulating half-life without adversely affecting their activity. Additionally, a disulfide linkage may be present or absent in the peptides of the presently disclosed subject matter.

The term “amino acid” is used interchangeably with “amino acid residue,” and may refer to a free amino acid and to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

Amino acids have the following general structure:

Amino acids may be classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.

The nomenclature used to describe the peptide compounds of the presently disclosed subject matter follows the conventional practice wherein the amino group is presented to the left and the carboxy group to the right of each amino acid residue. In the formulae representing selected specific embodiments of the presently disclosed subject matter, the amino-and carboxy -terminal groups, although not specifically shown, will be understood to be in the form they would assume at physiologic pH values, unless otherwise specified.

The term “basic” or “positively charged” amino acid as used herein, refers to amino acids in which the R groups have a net positive charge at pH 7.0, and include, but are not limited to, the standard amino acids lysine, arginine, and histidine.

The term “antibody,” as used herein, refers to an immunoglobulin molecule which is able to specifically or selectively bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the presently disclosed subject matter may exist in a variety of forms. The term “antibody” refers to polyclonal and monoclonal antibodies and derivatives thereof (including chimeric, synthesized, humanized and human antibodies), including an entire immunoglobulin or antibody or any functional fragment of an immunoglobulin molecule which binds to the target antigen and or combinations thereof. Examples of such functional entities include complete antibody molecules, antibody fragments, such as F_v, single chain F_v (scFv), complementarity determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), Fab, F(ab’)2 and any combination of those or any other functional portion of an immunoglobulin peptide capable of binding to target antigen.

Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab’)2 a dimer of Fab which itself is a light chain joined to VH -CHI by a disulfide bond. The F(ab’)2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab’)2 dimer into an Fabi monomer. The Fabi monomer is essentially a Fab with part of the hinge region (see Paul, 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies.

An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules.

An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules.

The term “single chain antibody” refers to an antibody wherein the genetic information encoding the functional fragments of the antibody are located in a single contiguous length of DNA. For a thorough description of single chain antibodies, see Bird et al., 1988; Huston et al., 1988).

The term “humanized” refers to an antibody wherein the constant regions have at least about 80% or greater homology to human immunoglobulin. Additionally, some of the nonhuman, such as murine, variable region amino acid residues can be modified to contain amino acid residues of human origin. Humanized antibodies have been referred to as “reshaped” antibodies. Manipulation of the complementarity-determining regions (CDR) is a way of achieving humanized antibodies. See for example, Jones et al., 1986; Riechmann et al., 1988, both of which are incorporated by reference herein. For a review article concerning humanized antibodies, see Winter & Milstein, 1991, incorporated by reference herein. See also U.S. Patent Nos. 4,816,567; 5,482,856; 6,479,284; 6,677,436; 7,060,808; 7,906,625; 8,398,980; 8,436,150; 8,796,439; and 10,253,111; and U.S. Patent Application Publication Nos. 2003/0017534, 2018/0298087, 2018/0312588, 2018/0346564, and 2019/0151448, each of which is incorporated by reference in its entirety.

By the term “synthetic antibody” as used herein, means an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

The term “antigen” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. An antigen can be derived from organisms, subunits of proteins/antigens, killed or inactivated whole cells or lysates.

As used herein, the term “antisense oligonucleotide” or antisense nucleic acid means a nucleic acid polymer, at least a portion of which is complementary to a nucleic acid which is present in a normal cell or in an affected cell. “Antisense” refers particularly to the nucleic acid sequence of the non-coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences. The antisense oligonucleotides of the presently disclosed subject matter include, but are not limited to, phosphorothioate oligonucleotides and other modifications of oligonucleotides.

An “aptamer” is a compound that is selected in vitro to bind preferentially to another compound (for example, the identified proteins herein). Often, aptamers are nucleic acids or peptides because random sequences can be readily generated from nucleotides or amino acids (both naturally occurring or synthetically made) in large numbers but of course they need not be limited to these. The term “aqueous solution” as used herein can include other ingredients commonly used, such as sodium bicarbonate described herein, and further includes any acid or base solution used to adjust the pH of the aqueous solution while solubilizing a peptide.

The term “binding” refers to the adherence of molecules to one another, such as, but not limited to, enzymes to substrates, ligands to receptors, antibodies to antigens, DNA binding domains of proteins to DNA, and DNA or RNA strands to complementary strands.

“Binding partner,” as used herein, refers to a molecule capable of binding to another molecule.

The term “biocompatible,” as used herein, refers to a material that does not elicit a substantial detrimental response in the host.

As used herein, the terms “biologically active fragment” and “bioactive fragment” of a peptide encompass natural and synthetic portions of a longer peptide or protein that are capable of specific binding to their natural ligand and/or of performing a desired function of a protein, for example, a fragment of a protein of larger peptide which still contains the epitope of interest and is immunogenic.

The term “biological sample,” as used herein, refers to samples obtained from a subject, including but not limited to skin, hair, tissue, blood, plasma, cells, sweat, and urine.

As used herein, the term “chemically conjugated,” or “conjugating chemically” refers to linking the antigen to the carrier molecule. This linking can occur on the genetic level using recombinant technology, wherein a hybrid protein may be produced containing the amino acid sequences, or portions thereof, of both the antigen and the carrier molecule. This hybrid protein is produced by an oligonucleotide sequence encoding both the antigen and the carrier molecule, or portions thereof. This linking also includes covalent bonds created between the antigen and the carrier protein using other chemical reactions, such as, but not limited to reactions as described herein. Covalent bonds may also be created using a third molecule bridging the antigen to the carrier molecule. These cross-linkers are able to react with groups, such as but not limited to, primary amines, sulfhydryls, carbonyls, carbohydrates, or carboxylic acids, on the antigen and the carrier molecule. Chemical conjugation also includes non-covalent linkage between the antigen and the carrier molecule.

A “coding region” of a gene comprises the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene. “Complementary” as used herein refers to the broad concept of subunit sequence complementarity between two nucleic acids (e.g., two DNA molecules). When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other at a given position, the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other when a substantial number (in some embodiments at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides that can base pair with each other (e.g., A:T and G:C nucleotide pairs). Thus, it is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. By way of example and not limitation, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, in some embodiments at least about 50%, in some embodiments at least about 75%, in some embodiments at least about 90%, and in some embodiments at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In some embodiments, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

A “compound,” as used herein, refers to a polypeptide, an isolated nucleic acid, or other agent used in the method of the presently disclosed subject matter.

A “control” cell, tissue, sample, or subject is a cell, tissue, sample, or subject of the same type as a test cell, tissue, sample, or subject. The control may, for example, be examined at precisely or nearly the same time the test cell, tissue, sample, or subject is examined. The control may also, for example, be examined at a time distant from the time at which the test cell, tissue, sample, or subject is examined, and the results of the examination of the control may be recorded so that the recorded results may be compared with results obtained by examination of a test cell, tissue, sample, or subject. The control may also be obtained from another source or similar source other than the test group or a test subject, where the test sample is obtained from a subject suspected of having a condition, disease, or disorder for which the test is being performed.

A “test” cell is a cell being examined.

As used herein, the term “conservative amino acid substitution” is defined herein as an amino acid exchange within one of the five groups summarized in the following Table 2: Table 2: Conservative Amino Acid Substitutions

Group Characteristics Amino Acids

A. Small aliphatic, nonpolar or slightly polar residues Ala, Ser, Thr, Pro, Gly

B. Polar, negatively charged residues and their amides Asp, Asn, Glu, Gin

C. Polar, positively charged residues His, Arg, Lys

D. Large, aliphatic, nonpolar residues Met Leu, He, Vai, Cys

E. Large, aromatic residues Phe, Tyr, Trp

A “pathoindicative” cell is a cell that, when present in a tissue, is an indication that the animal in which the tissue is located (or from which the tissue was obtained) is afflicted with a condition, disease, or disorder.

A “pathogenic” cell is a cell that, when present in a tissue, causes or contributes to a condition, disease, or disorder in the animal in which the tissue is located (or from which the tissue was obtained).

A tissue “normally comprises” a cell if one or more of the cells are present in the tissue in an animal not afflicted with a condition, disease, or disorder.

As used herein, the terms “condition,” “disease condition,” “disease,” “disease state,” and “disorder” refer to physiological states in which diseased cells or cells of interest can be targeted with the compositions of the presently disclosed subject matter. In some embodiments, a disease is cancer, which in some embodiments comprises a solid tumor.

As used herein, the term “diagnosis” refers to detecting a risk or propensity to a condition, disease, or disorder. In any method of diagnosis exist false positives and false negatives. Any one method of diagnosis does not provide 100% accuracy.

A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

As used herein, an “effective amount” or “therapeutically effective amount” refers to an amount of a compound or composition sufficient to produce a selected effect, such as but not limited to alleviating symptoms of a condition, disease, or disorder. In the context of administering compounds in the form of a combination, such as multiple compounds, the amount of each compound, when administered in combination with one or more other compounds, may be different from when that compound is administered alone. Thus, an effective amount of a combination of compounds refers collectively to the combination as a whole, although the actual amounts of each compound may vary. The term “more effective” means that the selected effect occurs to a greater extent by one treatment relative to the second treatment to which it is being compared.

“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of an mRNA corresponding to or derived from that gene produces the protein in a cell or other biological system and/or an in vitro or ex vivo system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence (with the exception of uracil bases presented in the latter) and is usually provided in Sequence Listing, and the noncoding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

The term “epitope” as used herein is defined as small chemical groups on the antigen molecule that can elicit and react with an antibody. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly five amino acids or sugars in size. One skilled in the art understands that generally the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity.

As used herein, an “essentially pure” preparation of a particular protein or peptide is a preparation wherein in some embodiments at least about 95% and in some embodiments at least about 99%, by weight, of the protein or peptide in the preparation is the particular protein or peptide. A “fragment,” “segment,” or “subsequence” is a portion of an amino acid sequence, comprising at least one amino acid, or a portion of a nucleic acid sequence comprising at least one nucleotide. The terms “fragment,” “segment,” and “subsequence” are used interchangeably herein.

As used herein, the term “fragment,” as applied to a protein or peptide, can ordinarily be at least about 3-15 amino acids in length, at least about 15-25 amino acids, at least about 25- 50 amino acids in length, at least about 50-75 amino acids in length, at least about 75-100 amino acids in length, and greater than 100 amino acids in length.

As used herein, the term “fragment” as applied to a nucleic acid, may ordinarily be at least about 20 nucleotides in length, typically, at least about 50 nucleotides, more typically, from about 50 to about 100 nucleotides, in some embodiments, at least about 100 to about 200 nucleotides, in some embodiments, at least about 200 nucleotides to about 300 nucleotides, yet in some embodiments, at least about 300 to about 350, in some embodiments, at least about 350 nucleotides to about 500 nucleotides, yet in some embodiments, at least about 500 to about 600, in some embodiments, at least about 600 nucleotides to about 620 nucleotides, yet in some embodiments, at least about 620 to about 650, and most in some embodiments, the nucleic acid fragment will be greater than about 650 nucleotides in length. In the case of a shorter sequence, fragments are shorter.

As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property by which it can be characterized. A functional enzyme, for example, is one that exhibits the characteristic catalytic activity by which the enzyme can be characterized.

“Homologous” as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 3’-ATTGCC-5’ and 3’-TATGGC-5’ share 50% homology.

As used herein, “homology” is used synonymously with “identity.” The determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin & Altschul, 1990, modified as in Karlin & Altschul, 1993). This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, and can be accessed, for example at the National Center for Biotechnology Information (NCBI) world wide web site. BLAST nucleotide searches can be performed with the NBLAST program (designated “blastn” at the NCBI web site), using the following parameters: gap penalty = 5; gap extension penalty = 2; mismatch penalty = 3; match reward = 1; expectation value 10.0; and word size = 11 to obtain nucleotide sequences homologous to a nucleic acid described herein. BLAST protein searches can be performed with the XBLAST program (designated “blastn” at the NCBI web site) or the NCBI “blastp” program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997. Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.) and relationships between molecules which share a common pattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the length of the formed hybrid, and the G:C ratio within the nucleic acids.

The term “ingredient” refers to any compound, whether of chemical or biological origin, that can be used in cell culture media to maintain or promote the proliferation, survival, or differentiation of cells. The terms “component,” “nutrient,” “supplement,” and ingredient” can be used interchangeably and are all meant to refer to such compounds. Typical nonlimiting ingredients that are used in cell culture media include amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins and the like. Other ingredients that promote or maintain cultivation of cells ex vivo can be selected by those of skill in the art, in accordance with the particular need.

As used herein “injecting”, “applying”, and administering” include administration of a compound of the presently disclosed subject matter by any number of routes and modes including, but not limited to, topical, oral, buccal, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, ophthalmic, pulmonary, vaginal, and rectal approaches.

Used interchangeably herein are the terms: 1) “isolate” and “select”; and 2) “detect” and “identify”.

The term “isolated,” when used in reference to compositions and cells, refers to a particular composition or cell of interest, or population of cells of interest, at least partially isolated from other cell types or other cellular material with which it naturally occurs in the tissue of origin. A composition or cell sample is “substantially pure” when it is at least 60%, or at least 75%, or at least 90%, and, in certain cases, at least 99% free of materials, compositions, cells other than composition or cells of interest. Purity can be measured by any appropriate method, for example, by fluorescence-activated cell sorting (FACS), or other assays which distinguish cell types. Representative isolation techniques are disclosed herein for antibodies and fragments thereof.

An “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

As used herein, a “ligand” is a compound that specifically or selectively binds to a target compound. A ligand (e.g., an antibody) “specifically binds to,” “is specifically immunoreactive with,” “having a selective binding activity”, “selectively binds to” or “is selectively immunoreactive with” a compound when the ligand functions in a binding reaction which is determinative of the presence of the compound in a sample of heterogeneous compounds. Thus, under designated assay (e.g., immunoassay) conditions, the ligand binds preferentially to a particular compound and does not bind to a significant extent to other compounds present in the sample. For example, an antibody specifically or selectively binds under immunoassay conditions to an antigen bearing an epitope against which the antibody was raised. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with an antigen. See Harlow & Lane, 1988, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

A “receptor” is a compound that specifically or selectively binds to a ligand.

A ligand or a receptor (e.g., an antibody) “specifically binds to”, “is specifically immunoreactive with,” “having a selective binding activity,” “selectively binds to” or “is selectively immunoreactive with” a compound when the ligand or receptor functions in a binding reaction which is determinative of the presence of the compound in a sample of heterogeneous compounds. Thus, under designated assay (e.g., immunoassay) conditions, the ligand or receptor binds preferentially to a particular compound and does not bind in a significant amount to other compounds present in the sample. For example, a polynucleotide specifically or selectively binds under hybridization conditions to a compound polynucleotide comprising a complementary sequence; an antibody specifically or selectively binds under immunoassay conditions to an antigen bearing an epitope against which the antibody was raised. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane 1988 for a description of immunoassay formats and conditions that can be used to determine specific or selective immunoreactivity. See also the EXAMPLES set forth herein below for additional formats and conditions that can be used to determine specific or selective immunoreactivity. As used herein, the term “linkage” refers to a connection between two groups. The connection can be either covalent or non-covalent, including but not limited to ionic bonds, hydrogen bonding, and hydrophobic/hydrophilic interactions.

As used herein, the term “linker” refers to a molecule that joins two other molecules either covalently or noncovalently, such as but not limited to through ionic or hydrogen bonds or van der Waals interactions.

The terms “measuring the level of expression” and “determining the level of expression” as used herein refer to any measure or assay which can be used to correlate the results of the assay with the level of expression of a gene or protein of interest. Such assays include measuring the level of mRNA, protein levels, etc. and can be performed by assays such as northern and western blot analyses, binding assays, immunoblots, etc. The level of expression can include rates of expression and can be measured in terms of the actual amount of an mRNA or protein present. Such assays are coupled with processes or systems to store and process information and to help quantify levels, signals, etc. and to digitize the information for use in comparing levels.

The term “modulate,” as used herein, refers to changing the level of an activity, function, or process. The term “modulate” encompasses both inhibiting and stimulating an activity, function, or process. The term “modulate” is used interchangeably with the term “regulate” herein.

The term “nucleic acid” typically refers to large polynucleotides. By “nucleic acid” is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil).

As used herein, the term “nucleic acid” encompasses RNA as well as single and double-stranded DNA and cDNA. Furthermore, the terms, “nucleic acid,” “DNA,” “RNA” and similar terms also include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, the so-called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the presently disclosed subject matter. By “nucleic acid” is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil). Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5 ’-end; the left-hand direction of a doublestranded polynucleotide sequence is referred to as the 5 ’-direction. The direction of 5’ to 3’ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the “coding strand;” sequences on the DNA strand which are located 5’ to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3’ to a reference point on the DNA are referred to as “downstream sequences”.

The term “nucleic acid construct,” as used herein, encompasses DNA and RNA sequences encoding the particular gene or gene fragment desired, whether obtained by genomic or synthetic methods.

The term “oligonucleotide” typically refers to short polynucleotides, generally, no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.”

The term “otherwise identical sample,” as used herein, refers to a sample similar to a first sample, that is, it is obtained in the same manner from the same subject from the same tissue or fluid, or it refers a similar sample obtained from a different subject. The term “otherwise identical sample from an unaffected subject” refers to a sample obtained from a subject not known to have the disease or disorder being examined. The sample may of course be a standard sample. By analogy, the term “otherwise identical” can also be used regarding regions or tissues in a subject or in an unaffected subject. As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.

The term “peptide” typically refers to short polypeptides.

The term “pharmaceutical composition” refers to a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, without limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.

“Pharmaceutically acceptable” means physiologically tolerable, for either human or veterinary application. Similarly, “pharmaceutical compositions” include formulations for human and veterinary use.

As used herein, the term “pharmaceutically acceptable carrier” means a chemical composition with which an appropriate compound or derivative can be combined and which, following the combination, can be used to administer the appropriate compound to a subject.

As used herein, the term “physiologically acceptable” ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.

“Plurality” means at least two.

A “polynucleotide” means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double-stranded nucleic acid.

“Polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. “Synthetic peptides or polypeptides” refers to non-naturally occurring peptides or polypeptides. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid phase peptide synthesis methods are known to those of skill in the art.

The term “prevent,” as used herein, means to stop something from happening, or taking advance measures against something possible or probable from happening. In the context of medicine, “prevention” generally refers to action taken to decrease the chance of getting a disease or condition. It is noted that “prevention” need not be absolute, and thus can occur as a matter of degree.

A “preventive” or “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs, or exhibits only early signs, of a condition, disease, or disorder. A prophylactic or preventative treatment is administered for the purpose of decreasing the risk of developing pathology associated with developing the condition, disease, or disorder, such as, but not limited to, in a subject that has been exposed to a pathogen, who is at risk for exposure to a pathogen, and/or who would be particularly susceptible to suffering from severe disease if exposed to a pathogen or after exposure to a pathogen.

“Primer” refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase. A primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.

As used herein, the term “promoter/regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulator sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

A “constitutive” promoter is a promoter which drives expression of a gene to which it is operably linked, in a constant manner in a cell. By way of example, promoters which drive expression of cellular housekeeping genes are considered to be constitutive promoters.

An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living cell substantially only when an inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

As used herein, “protecting group” with respect to a terminal amino group refers to a terminal amino group of a peptide, which terminal amino group is coupled with any of various amino-terminal protecting groups traditionally employed in peptide synthesis. Such protecting groups include, for example, acyl protecting groups such as formyl, acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethane protecting groups such as benzyloxy carbonyl; and aliphatic urethane protecting groups, for example, tert-butoxy carbonyl or adamantyloxycarbonyl. See Gross & Mienhofer, 1981 for suitable protecting groups.

As used herein, “protecting group” with respect to a terminal carboxy group refers to a terminal carboxyl group of a peptide, which terminal carboxyl group is coupled with any of various carboxyl-terminal protecting groups. Such protecting groups include, for example, tert-butyl, benzyl, or other acceptable groups linked to the terminal carboxyl group through an ester or ether bond.

The term “protein” typically refers to large polypeptides. Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.

As used herein, the term “purified” and like terms relate to an enrichment of a molecule or compound relative to other components normally associated with the molecule or compound in a native environment. The term “purified” does not necessarily indicate that complete purity of the particular molecule has been achieved during the process. A “highly purified” compound as used herein refers to a compound that is in some embodiments greater than 90% pure, that is in some embodiments greater than 95% pure, and that is in some embodiments greater than 98% pure.

“Recombinant polynucleotide” refers to a polynucleotide having sequences that are not naturally joined together. An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell.

A recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.

A host cell that comprises a recombinant polynucleotide is referred to as a “recombinant host cell.” A gene which is expressed in a recombinant host cell wherein the gene comprises a recombinant polynucleotide, produces a “recombinant polypeptide.”

A “recombinant polypeptide” is one which is produced upon expression of a recombinant polynucleotide.

The term “regulate” refers to either stimulating or inhibiting a function or activity of interest.

As used herein, the term “regulatory elements” is used interchangeably with “regulatory sequences” and refers to promoters, enhancers, and other expression control elements, or any combination of such elements.

As used herein, the term “secondary antibody” refers to an antibody that binds to the constant region of another antibody (the primary antibody).

As used herein, the term “single chain variable fragment” (scFv) refers to a single chain antibody fragment comprised of a heavy and light chain linked by a peptide linker. In some cases scFv are expressed on the surface of an engineered cell, for the purpose of selecting particular scFv that bind to an antigen of interest.

As used herein, the term “mammal” refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.

The term “subject” as used herein refers to a member of species for which treatment and/or prevention of a disease or disorder using the compositions and methods of the presently disclosed subject matter might be desirable. Accordingly, the term “subject” is intended to encompass in some embodiments any member of the Kingdom Animalia including, but not limited to the phylum Chordata (e.g., members of Classes Osteichythyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Aves (birds), and Mammalia (mammals), and all Orders and Families encompassed therein.

The compositions and methods of the presently disclosed subject matter are particularly useful for warm-blooded vertebrates. Thus, in some embodiments the presently disclosed subject matter concerns mammals and birds. More particularly provided are compositions and methods derived from and/or for use in mammals such as humans and other primates, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economic importance (animals raised on farms for consumption by humans) and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), rodents (such as mice, rats, and rabbits), marsupials, and horses. Also provided is the use of the disclosed methods and compositions on birds, including those kinds of birds that are endangered, kept in zoos, as well as fowl, and more particularly domesticated fowl, e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans. Thus, also provided is the use of the disclosed methods and compositions on livestock, including but not limited to domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.

As used herein, “substantially homologous amino acid sequences” or “homolog” includes those amino acid sequences which have at least about 75% homology, in some embodiments at least about 80% homology, in some embodiments at least about 85% homology, in some embodiments at least about 90% homology, in some embodiments at least about 95% homology, in some embodiments at least about 96% homology, more in some embodiments at least about 97% homology, in some embodiments at least about 98% homology, and most in some embodiments at least about 99% or more homology to an amino acid sequence of a reference antibody chain. Amino acid sequence similarity or identity can be computed by using the BLASTP and TBLASTN programs which employ the BLAST (basic local alignment search tool) 2.0.14 algorithm. The default settings used for these programs are suitable for identifying substantially similar amino acid sequences for purposes of the presently disclosed subject matter.

“Substantially homologous nucleic acid sequence” or “homolog” means a nucleic acid sequence corresponding to a reference nucleic acid sequence wherein the corresponding sequence encodes a peptide having substantially the same structure and function as the peptide encoded by the reference nucleic acid sequence; e.g., where only changes in amino acids not significantly affecting the peptide function occur. In some embodiments, the substantially identical nucleic acid sequence encodes the peptide encoded by the reference nucleic acid sequence. The percentage of identity between the substantially similar nucleic acid sequence and the reference nucleic acid sequence is at least about 50%, 65%, 75%, 85%, 95%, 99% or more. The substantial identity of nucleic acid sequences can be determined by comparing the sequence identity of two sequences, for example by physical/chemical methods (i.e., hybridization) or by sequence alignment via computer algorithm. Suitable nucleic acid hybridization conditions to determine if a nucleotide sequence is substantially similar to a reference nucleotide sequence are: 7% sodium dodecyl sulfate SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 2X standard saline citrate (SSC), 0.1% SDS at 50°C; in some embodiments in 7% (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C; in some embodiments 7% SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C; and more in some embodiments in 7% SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C. Suitable computer algorithms to determine substantial similarity between two nucleic acid sequences include, GCS program package (Devereux et al., 1984), and the BLASTN or FASTA programs (Altschul et al., 1990a; Altschul et al., 1990b; Altschul et al., 1997). The default settings provided with these programs are suitable for determining substantial similarity of nucleic acid sequences for purposes of the presently disclosed subject matter.

A “sample,” as used herein, refers in some embodiments to a biological sample from a subject, including, but not limited to, normal tissue samples, diseased tissue samples, biopsies, blood, saliva, feces, semen, tears, and urine. A sample can also be any other source of material obtained from a subject which contains cells, tissues, or fluid of interest. A sample can also be obtained from cell or tissue culture.

The term “standard,” as used herein, refers to something used for comparison. For example, it can be a known standard agent or compound which is administered and used for comparing results when administering a test compound, or it can be a standard parameter or function which is measured to obtain a control value when measuring an effect of an agent or compound on a parameter or function. Standard can also refer to an “internal standard,” such as an agent or compound which is added at known amounts to a sample and is useful in determining such things as purification or recovery rates when a sample is processed or subjected to purification or extraction procedures before a marker of interest is measured. Internal standards are often a purified marker of interest which has been labeled, such as with a radioactive isotope, allowing it to be distinguished from an endogenous marker.

A “subject” of analysis, diagnosis, or treatment is an animal. Such animals include mammals, in some embodiments, humans.

As used herein, a “subject in need thereof’ is a patient, animal, mammal, or human, who will benefit from the method of this presently disclosed subject matter. In some embodiments, the subject in need of treatment is a mammal. In some embodiments, the subject is a human.

The term “substantially pure” describes a compound, e.g., a protein or polypeptide, which has been separated from components which naturally accompany it. Typically, a compound is substantially pure when in some embodiments at least 10%, in some embodiments at least 20%, in some embodiments at least 50%, in some embodiments at least 60%, in some embodiments at least 75%, in some embodiments at least 90%, and in some embodiments at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, gel electrophoresis, or HPLC analysis. A compound, e.g., a protein, is also substantially purified when it is essentially free of naturally associated components or when it is separated from the native contaminants which accompany it in its natural state.

The term “symptom,” as used herein, refers to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease. In contrast, a “sign” is objective evidence of disease. For example, a bloody nose is a sign. It is evident to the patient, doctor, nurse, and other observers.

A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.

As used herein, the phrase “therapeutic agent” refers to an agent that is used to, for example, treat, inhibit, prevent, mitigate the effects of, reduce the severity of, reduce the likelihood of developing, slow the progression of, and/or cure, a disease or disorder.

The terms “treatment” and “treating” as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition, prevent the pathologic condition, pursue or obtain beneficial results, and/or lower the chances of the individual developing a condition, disease, or disorder, even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have or predisposed to having a condition, disease, or disorder, or those in whom the condition is to be prevented.

As used herein, the terms “vector,” “cloning vector,” and “expression vector” refer to a vehicle by which a polynucleotide sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transduce and/or transform the host cell in order to promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc.

All genes, gene names, and gene products disclosed herein are intended to correspond to homologs and/or orthologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates.

One of ordinary skill in the art will appreciate that based on the sequences of the components of the peptides disclosed herein they can be modified independently of one another with conservative amino acid changes, including, insertions, deletions, and substitutions, and that the valency could be altered as well. Amino acid changes (fragments and homologs) can be made independently in an antibody as well when they are being used in a therapy.

The presently disclosed subject matter provides peptides and biologically active fragments and homologs thereof as well as methods for preparing and testing new peptides for the properties disclosed herein.

In some embodiments, the presently disclosed subject matter uses a biologically active peptide, or biologically active fragment or homolog thereof. In some embodiments, the isolated peptide comprises a mammalian molecule at least about 30% homologous to a peptide having the amino acid sequence of at least one of the sequences disclosed herein. In some embodiments, the isolated peptide is at least about 35% homologous, more in some embodiments, about 40% homologous, more in some embodiments, about 45% homologous, in some embodiments, about 50% homologous, more in some embodiments, about 55% homologous, in some embodiments, about 60% homologous, more in some embodiments, about 65% homologous, in some embodiments, more in some embodiments, about 70% homologous, more in some embodiments, about 75% homologous, in some embodiments, about 80% homologous, more in some embodiments, about 85% homologous, more in some embodiments, about 90% homologous, in some embodiments, about 95% homologous, more in some embodiments, about 96% homologous, more in some embodiments, about 97% homologous, more in some embodiments, about 98% homologous, and most in some embodiments, about 99% homologous to at least one of the peptide sequences disclosed herein.

The presently disclosed subject matter further encompasses modification of the peptide, and/or fragments thereof disclosed herein, including amino acid deletions, additions, and substitutions, particularly conservative substitutions. The presently disclosed subject matter also encompasses modifications to increase in vivo half-life and decrease degradation in vivo. Substitutions, additions, and deletions can include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 changes as long as the activity disclosed herein remains substantially the same.

The presently disclosed subject matter includes an isolated nucleic acid comprising a nucleic acid sequence encoding a polypeptide, of the presently disclosed subject matter, or a fragment or homolog thereof. In some embodiments, the nucleic acid sequence encodes a peptide comprising a sequence of the presently disclosed subject matter, or a biologically active fragment or homolog thereof.

In some embodiments, a homolog of a peptide (including a fragment of a peptide) of the presently disclosed subject matter is one with one or more amino acid substitutions, deletions, or additions, and with the sequence identities described herein. In some embodiments, the substitution, deletion, or addition is conservative.

In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

The presently disclosed subject matter encompasses the use of purified isolated, recombinant, and synthetic peptides.

Peptide Modification and Preparation

It will be appreciated, of course, that the proteins or peptides of the presently disclosed subject matter may incorporate amino acid residues which are modified without affecting activity. For example, the termini may be derivatized to include blocking groups, i.e., chemical substituents suitable to protect and/or stabilize the N- and C-termini from “undesirable degradation”, a term meant to encompass any type of enzymatic, chemical or biochemical breakdown of the compound at its termini which is likely to affect the function of the compound, i.e. sequential degradation of the compound at a terminal end thereof.

Blocking groups include protecting groups conventionally used in the art of peptide chemistry which will not adversely affect the in vivo activities of the peptide. For example, suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N- terminus. Examples of suitable N-terminal blocking groups include C1-C5 branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, as well as substituted forms thereof, such as the acetamidomethyl (Acm) group. Desamino analogs of amino acids are also useful N-terminal blocking groups, and can either be coupled to the N-terminus of the peptide or used in place of the N-terminal reside. Suitable C-terminal blocking groups, in which the carboxyl group of the C-terminus is either incorporated or not, include esters, ketones or amides. Ester or ketone-forming alkyl groups, particularly lower alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups such as primary amines (-NH2), and mono- and di-alkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino and the like are examples of C-terminal blocking groups. Descarboxylated amino acid analogues such as agmatine are also useful C-terminal blocking groups and can be either coupled to the peptide’s C-terminal residue or used in place of it. Further, it will be appreciated that the free amino and carboxyl groups at the termini can be removed altogether from the peptide to yield desamino and descarboxylated forms thereof without effect on peptide activity.

Acid addition salts of the presently disclosed subject matter are also contemplated as functional equivalents. Thus, a peptide in accordance with the presently disclosed subject matter treated with an inorganic acid such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like, or an organic acid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tataric, citric, benzoic, cinnamie, mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicyclic and the like, to provide a water soluble salt of the peptide is suitable for use in the presently disclosed subject matter.

The presently disclosed subject matter also provides for analogs of proteins, e.g., analogs of peptides. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function. To that end, 10 or more conservative amino acid changes typically have no effect on peptide function.

Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

Also included are polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or non-standard synthetic amino acids. The peptides of the presently disclosed subject matter are not limited to products of any of the specific exemplary processes listed herein.

It will be appreciated, of course, that the polypeptides or peptides, derivatives, or fragments thereof may incorporate amino acid residues which are modified without affecting activity. For example, the termini may be derivatized to include blocking groups, i.e., chemical substituents suitable to protect and/or stabilize the N- and C-termini from “undesirable degradation”, a term meant to encompass any type of enzymatic, chemical or biochemical breakdown of the compound at its termini which is likely to affect the function of the compound, i.e. sequential degradation of the compound at a terminal end thereof.

Other modifications can also be incorporated without adversely affecting the activity and these include, but are not limited to, substitution of one or more of the amino acids in the natural L-isomeric form with amino acids in the D-isomeric form. Thus, the peptide may include one or more D-amino acid resides, or may comprise amino acids which are all in the D-form. Retro-inverso forms of peptides in accordance with the presently disclosed subject matter are also contemplated, for example, inverted peptides in which all amino acids are substituted with D-amino acid forms.

Substantially pure protein obtained as described herein may be purified by following known procedures for protein purification, wherein an immunological, enzymatic or other assay is used to monitor purification at each stage in the procedure. Protein purification methods are well known in the art, and are described, for example in Deutscher et al., 1990.

As discussed, modifications or optimizations of peptide ligands of the presently disclosed subject matter are within the scope of the application. Modified or optimized peptides are included within the definition of peptide binding ligand. Specifically, a peptide sequence identified can be modified to optimize its potency, pharmacokinetic behavior, stability and/or other biological, physical and chemical properties.

Amino Acid Substitutions

In certain embodiments, the disclosed methods and compositions may involve preparing peptides with one or more substituted amino acid residues.

In various embodiments, the structural, physical and/or therapeutic characteristics of peptide sequences may be optimized by replacing one or more amino acid residues.

The skilled artisan will be aware that, in general, amino acid substitutions in a peptide typically involve the replacement of an amino acid with another amino acid of relatively similar properties (i.e., conservative amino acid substitutions). The properties of the various amino acids and effect of amino acid substitution on protein structure and function have been the subject of extensive study and knowledge in the art.

For example, one can make the following isosteric and/or conservative amino acid changes in the parent polypeptide sequence with the expectation that the resulting polypeptides would have a similar or improved profile of the properties described above:

Substitution of alkyl-substituted hydrophobic amino acids: including alanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid, S-cyclohexylalanine or other simple alpha-amino acids substituted by an aliphatic side chain from Cl-10 carbons including branched, cyclic and straight chain alkyl, alkenyl or alkynyl substitutions.

Substitution of aromatic-substituted hydrophobic amino acids: including phenylalanine, tryptophan, tyrosine, biphenylalanine, 1 -naphthylalanine, 2-naphthylalanine, 2- benzothienylalanine, 3 -benzothienylalanine, histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro, bromo, or iodo) or alkoxy-substituted forms of the previous listed aromatic amino acids, illustrative examples of which are: 2-, 3- or 4-aminophenylalanine, 2-,3- or 4-chlorophenylalanine, 2-, 3- or 4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or 5-methoxytryptophan, 2’-, 3’-, or 4’-amino-, 2’-, 3’-, or 4’- chloro-, 2,3, or 4-biphenylalanine, 2’, -3’,- or 4’-methyl-2, 3 or 4-biphenylalanine, and 2- or 3- pyridylalanine.

Substitution of amino acids containing basic functions: including arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, alkyl, alkenyl, or arylsubstituted (from Cl -CIO branched, linear, or cyclic) derivatives of the previous amino acids, whether the substituent is on the heteroatoms (such as the alpha nitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon, in the pro-R position for example. Compounds that serve as illustrative examples include: N-epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine, N,N-gamma, gamma’ -di ethyl-homoarginine. Included also are compounds such as alpha methyl arginine, alpha methyl 2,3-diaminopropionic acid, alpha methyl histidine, alpha methyl ornithine where alkyl group occupies the pro-R position of the alpha carbon. Also included are the amides formed from alkyl, aromatic, heteroaromatic (where the heteroaromatic group has one or more nitrogens, oxygens, or sulfur atoms singly or in combination) carboxylic acids or any of the many well-known activated derivatives such as acid chlorides, active esters, active azolides and related derivatives) and lysine, ornithine, or 2,3-diaminopropionic acid.

Substitution of acidic amino acids: including aspartic acid, glutamic acid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, and heteroaryl sulfonamides of 2,4- diaminopriopionic acid, ornithine or lysine and tetrazole-substituted alkyl amino acids.

Substitution of side chain amide residues: including asparagine, glutamine, and alkyl or aromatic substituted derivatives of asparagine or glutamine.

Substitution of hydroxyl containing amino acids: including serine, threonine, homoserine, 2,3 -diaminopropionic acid, and alkyl or aromatic substituted derivatives of serine or threonine. It is also understood that the amino acids within each of the categories listed above can be substituted for another of the same group.

For example, the hydropathic index of amino acids may be considered (Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (- 0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In making conservative substitutions, the use of amino acids whose hydropathic indices are within +/-2 is preferred, within +/-1 are more preferred, and within +/- 0.5 are even more preferred.

Amino acid substitution may also consider the hydrophilicity of the amino acid residue (e.g., U.S. Patent No. 4,554,101). Hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). Replacement of amino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. For example, it would generally not be preferred to replace an amino acid with a compact side chain, such as glycine or serine, with an amino acid with a bulky side chain, e.g., tryptophan or tyrosine. The effect of various amino acid residues on protein secondary structure is also a consideration. Through empirical study, the effect of different amino acid residues on the tendency of protein domains to adopt an alpha-helical, beta-sheet or reverse turn secondary structure has been determined and is known in the art (see e.g., Chou & Fasman, 1974, Biochemistry, 13:222- 245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979, Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables of conservative amino acid substitutions have been constructed and are known in the art. For example: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R) gin, asn, lys; Asn (N) his, asp, lys, arg, gin; Asp (D) asn, glu; Cys (C) ala, ser; Gin (Q) glu, asn; Glu (E) gin, asp; Gly (G) ala; His (H) asn, gin, lys, arg; He (I) val, met, ala, phe, leu; Leu (L) val, met, ala, phe, ile; Lys (K) gin, asn, arg; Met (M) phe, ile, leu; Phe (F) leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W) phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or not the residue is located in the interior of a protein or is solvent exposed. For interior residues, conservative substitutions would include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; lie and Val; Val and Leu; Leu and lie; Leu and Met; Phe and Tyr; Tyr and Trp. (See e.g., PROWL Rockefeller University website). For solvent exposed residues, conservative substitutions would include: Asp and Asn; Asp and Glu; Glu and Gin; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and lie; lie and Val; Phe and Tyr. Various matrices have been constructed to assist in selection of amino acid substitutions, such as the PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlan matrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix, Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider the existence of intermolecular or intramolecular bonds, such as formation of ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) or disulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in an encoded peptide sequence are well known and a matter of routine experimentation for the skilled artisan, for example by the technique of site-directed mutagenesis or by synthesis and assembly of oligonucleotides encoding an amino acid substitution and splicing into an expression vector construct.

Pharmaceutical Compositions and Administration

The presently disclosed subject matter is also directed to methods of administering the compounds of the presently disclosed subject matter to a subject.

Pharmaceutical compositions comprising the present compounds are administered to a subject in need thereof by any number of routes including, but not limited to, topical, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

In accordance with one embodiment, a method of treating a subject in need of such treatment is provided. The method comprises administering a pharmaceutical composition comprising at least one compound of the presently disclosed subject matter to a subject in need thereof. Compounds identified by the methods of the presently disclosed subject matter can be administered with known compounds or other medications as well.

The pharmaceutical compositions useful for practicing the presently disclosed subject matter may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day.

The presently disclosed subject matter encompasses the preparation and use of pharmaceutical compositions comprising a compound useful for treatment of the diseases and disorders disclosed herein as an active ingredient. Such a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

The compositions of the presently disclosed subject matter may comprise at least one active peptide, one or more acceptable carriers, and optionally other peptides or therapeutic agents.

For in vivo applications, the peptides of the presently disclosed subject matter may comprise a pharmaceutically acceptable salt. Suitable acids which are capable of forming such salts with the compounds of the presently disclosed subject matter include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like.

Pharmaceutically acceptable carriers include physiologically tolerable or acceptable diluents, excipients, solvents, or adjuvants. The compositions are in some embodiments sterile and nonpyrogenic. Examples of suitable carriers include, but are not limited to, water, normal saline, dextrose, mannitol, lactose or other sugars, lecithin, albumin, sodium glutamate, cysteine hydrochloride, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, ethoxylated isosteraryl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methahydroxide, bentonite, kaolin, agar-agar and tragacanth, or mixtures of these substances, and the like.

The pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary pharmaceutical substances or excipients and/or additives, such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like). Suitable additives include, but are not limited to, physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions (e.g., 0.01 to 10 mole percent) of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA or CaNaDTPA-bisamide), or, optionally, additions (e.g., 1 to 50 mole percent) of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). If desired, absorption enhancing or delaying agents (such as liposomes, aluminum monostearate, or gelatin) may be used. The compositions can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Pharmaceutical compositions according to the presently disclosed subject matter can be prepared in a manner fully within the skill of the art.

The peptides of the presently disclosed subject matter, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising these compounds may be administered so that the compounds may have a physiological effect. Administration may occur enterally or parenterally; for example, orally, rectally, intraci sternally, intravaginally, intraperitoneally, locally (e.g., with powders, ointments or drops), or as a buccal or nasal spray or aerosol. Parenteral administration is preferred. Particularly preferred parenteral administration methods include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature), peri- and intra-target tissue injection (e.g., peri -tumoral and intra-tumoral injection), subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps), intramuscular injection, and direct application to the target area, for example by a catheter or other placement device.

Where the administration of the peptide is by injection or direct application, the injection or direct application may be in a single dose or in multiple doses. Where the administration of the compound is by infusion, the infusion may be a single sustained dose over a prolonged period of time or multiple infusions.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

It will be understood by the skilled artisan that such pharmaceutical compositions are generally suitable for administration to animals of all sorts. Subjects to which administration of the pharmaceutical compositions of the presently disclosed subject matter is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs, birds including commercially relevant birds such as chickens, ducks, geese, and turkeys.

A pharmaceutical composition of the presently disclosed subject matter may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the presently disclosed subject matter will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

In addition to the active ingredient, a pharmaceutical composition of the presently disclosed subject matter may further comprise one or more additional pharmaceutically active agents. Particularly contemplated additional agents include anti-emetics and scavengers such as cyanide and cyanate scavengers.

Controlled- or sustained-release formulations of a pharmaceutical composition of the presently disclosed subject matter may be made using conventional technology.

As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the presently disclosed subject matter are known in the art and described, for example in Genaro, 1985, which is incorporated herein by reference.

Typically, dosages of the compound of the presently disclosed subject matter which may be administered to an animal, in some embodiments a human, range in amount from 1 pg to about 100 g per kilogram of body weight of the animal. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration. In some embodiments, the dosage of the compound will vary from about 1 mg to about 10 g per kilogram of body weight of the animal. In another aspect, the dosage will vary from about 10 mg to about 1 g per kilogram of body weight of the animal.

The compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type of cancer being diagnosed, the type and severity of the condition or disease being treated, the type and age of the animal, etc.

Suitable preparations include injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, suspension in, liquid prior to injection, may also be prepared. The preparation may also be emulsified, or polypeptides encapsulated in liposomes. The active ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine preparation may also include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants.

The presently disclosed subject matter also includes a kit comprising the composition of the presently disclosed subject matter and an instructional material which describes administering the composition to a subject. In some embodiments, this kit comprises a (in some embodiments sterile) solvent suitable for dissolving or suspending the composition of the presently disclosed subject matter prior to administering the compound to the subject.

As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of a composition of the presently disclosed subject matter in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material may describe one or more methods of using the compositions for diagnostic or identification purposes or of alleviation the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the presently disclosed subject matter may, for example, be affixed to a container which contains a composition of the presently disclosed subject matter or be shipped together with a container which contains the composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

As used herein in some embodiments, the terms “model,” and “mimic” refer to simulated in vivo or in vitro conditions used to study some aspect of mammalian, such as human, physiology or disease. Additional characterization of modeling and mimicking can be found in the Examples herein and below.

The term “pathological autoimmunity” refers in some embodiments, to diseases, disorders and/or conditions in the presence of antibodies directed against normal components, autoantigens and/or self-antigens of a subject. Additional characterization of pathological autoimmunity can be found in the Examples herein and below.

As used herein, the term “hypertension” refers in some embodiments to when blood pressure or the force of blood flowing through a subject’s blood vessels is consistently too high relative to pressure generally viewed as a normal physiological level. By way of example, in humans, hypertension has a defined blood pressure greater than 130/80 mmHg or higher. Additional characterization of hypertension can be found in the Examples herein and below.

As used herein, the term “inflammation” refers in some embodiments to when a factor, such as a physical factor, triggers an immune reaction. This physical factor may include, but is not limited to, pathogens, bacteria, viruses, autoimmune diseases and/or chronic diseases. Additional characterization of inflammation can be found in the Examples herein and below.

As used herein, the term “vascular oxidative stress” and “oxidative stress” refers in some embodiments to an imbalance between oxidants and antioxidants and more recently as a disruption of redox signaling and control leading to cell and tissue injury in a subject. Additional characterization of oxidative stress can be found in the Examples herein and below.

II. GENERAL CONSIDERATIONS In order to promote degradation of BCL6 using the cells nascent machinery, we have designed BCLiP , a BCL6 inhibitory peptide, which in some embodiments comprises, consists essentially of, or consists of SEQ ID NO. 2 or 10, or modified or substantially homologous versions thereof, including biologically active fragments and homologs thereof. In some embodiments, the BCLiP peptide is a 17- amino acid sequence which mimics L328-P344 of BCL6, (SEQ ID NO. 10), a highly evolutionarily conserved region containing degradationtargeting phosphorylation sites S333 and S343 (Figure 8A), which in some embodiments, comprises, consists essentially of, or consists of SEQ ID NO. 2 or 10, or modified or substantially homologous versions thereof, including biologically active fragments and homologs thereof. In a representative embodiment, an N-terminal stearyl group was added to facilitate cell entry.

Human BCL6 sequence (SEQ ID NO. 1) information:

LOCUS NP 001697 706 aa linear PRI 15 -MAR-2022

DEFINITION B-cell lymphoma 6 protein isoform 1 [Homo sapiens], ACCESSION NP 001697 VERSION NP 001697.2 DBSOURCE REFSEQ: accession NM 001706.5 KEYWORDS RefSeq; MANE Select. SOURCE Homo sapiens (human)

SEQ ID NO. 1 (native or wild-type BCL6)

NP 001697.2 B-cell lymphoma 6 protein isoform 1 [Homo sapiens]

MASPADSCIQFTRHASDVLLNLNRLRSRDILTDWIWSREQFRAHKTVLMACSG LFYSIFTDQLKCNLSVINLDPEINPEGFCILLDFMYTSRLNLREGNIMAVMATAMY LQMEHWDTCRKFIKASEAEMVSAIKPPREEFLNSRMLMPQDIMAYRGREVVEN NLPLRSAPGCESRAFAPSLYSGLSTPPASYSMYSHLPVSSLLFSDEEFRDVRMPVA NPFPKERALPCDSARPVPGEYSRPTLEVSPNVCHSNIYSPKETIPEEARSDMHYSV AEGLKPAAPSARNAPYFPCDKASKEEERPSSEDEIALHFEPPNAPLNRKGLVSPQS PQKSDCQPNSPTESCSSKNACILQASGSPPAKSPTDPKACNWKKYKFIVLNSLNQ NAKPEGPEQAELGRLSPRAYTAPPACQPPMEPENLDLQSPTKLSASGEDSTIPQAS RLNNIVNRSMTGSPRSSSESHSPLYMHPPKCTSCGSQSPQHAEMCLHTAGPTFPE EMGETQSEYSDSSCENGAFFCNECDCRFSEEASLKRHTLQTHSDKPYKCDRCQA SFRYKGNLASHKTVHTGEKPYRCNICGAQFNRPANLKTHTRIHSGEKPYKCETC GARFVQVAHLRAHVLIHTGEKPYPCEICGTRFRHLQTLKSHLRIHTGEKPYHCEK CNLHFRHKSQLRLHLRQKHGAITNTKVQYRVSATDLPPELPKAC

BCL6 is uniquely suited as a promising target for the therapeutic strategy of inducing protein degradation, as its activity in vivo is largely regulated by its targeted degradation. BCL6 was initially discovered in lymphoma, where upregulation of BCL6 promotes lymphangiogenesis by forming germinal centers during humoral immune response (Dent et al., 1997). Regulated degradation of BCL6 is important for transcriptional control, which is accomplished by multiple MAPK phosphorylation sites within PEST motifs driving ubiquitin- proteasome-mediated degradation (Moriyama et al., 1997; Niu et al., 1998).

In B cell development, BCL6 is a master regulator of germinal center development. Upon immune activation, germinal centers develop in secondary lymphoid organs, generating massive clonal expansion of B cells and affinity maturation of the antibodies they produce. During this process, BCL6 transcriptional activity represses DNA damage response and inhibits transcription of cell cycle genes, for the ultimate goal of promoting B Cell proliferation and survival²⁸. While this process is required to mount an appropriate immune response to foreign antigens, overriding the cellular checkpoints that regulate proliferation and cell death are fundamental mechanisms of oncogenesis. In fact, mutations and chromosomal rearrangements of BCL6 are the most common genetic abnormalities associated with DLBCL (Ye et al., 1993), the most common type of Non-Hodgkin’s Lymphoma. Minor alternations in the precise balance of BCL6 expression and degradation can significantly alter the transcriptome. In fact, Duan et al found that the intracellular half-life of BCL6 was extended in diffuse large b cell lymphoma cells (DBLCL) in culture following the mutagenesis of FBX011, which forms a SCF ubiquitin ligase complex that targets BCL6 for degradation (Duan et al., 2012). Interestingly, researchers also observed that the gene encoding FBXO11 was commonly mutated or deleted in human primary DBLCLs (Duan et al., 2012), further supporting that dysregulation of BCL6 degradation can have deleterious effects on the tissue and the system.

In addition to its role in B cells, BCL6 is also expressed in macrophages as well as in several subtypes of T cells. In macrophages, BCL6 inhibits expression of the cytokine IL-6 (Li et al., 2020), which promotes macrophage crosstalk with T cells and pathological Thl7 differentiation while inhibiting differentiation of immunosuppressive Treg cells (Heink et al., 2017). Furthermore, CD4+ T follicular helper cells (Tfh), which are characterized in part by their expression of BCL6, have been implicated in autoimmunity (Bocharnikov et al., 2019; Gensous et al., 2017; Venkatadri et al., 2022). Inhibition of BCL6 has been shown to rescue experimental models of autoimmunity, effectively reducing the generation of autoantibodies and preventing immune complex deposition in the kidney (Venkatadri et al., 2022). Altogether, this suggests that BCL6 upregulation is associated with immune overactivation observed and demonstrates that BCL6 is a promising therapeutic target in autoimmune diseases.

In preliminary studies of BCL6 inhibition via BCLiP peptide (which in some embodiments comprises, consists essentially of, or consists of SEQ ID NO. 2 or 10, or modified or substantially homologous version thereof, including biologically active fragments and homologs thereof) administration, mice were injected intraperitoneally with BCLiP (which in some embodiments comprises, consists essentially of, or consists of SEQ ID NO. 2 or 10, or modified or substantially homologous version thereof) or scrambled control (SEQ ID NO. 3) peptide once daily for five days. BCL6 protein abundance is significantly reduced in lung tissue following treatment with BCLiP (which in some embodiments comprises, consists essentially of, or consists of SEQ ID NO. 2 or 10, or modified or substantially homologous version thereof, including biologically active fragments and homologs thereof). In fact, the two doses assayed (2.5 mg/kg and 12.5m/kg) showed a dose-dependent loss of BCL6 protein.

BCLiP Peptide (which in some embodiments comprises, consists essentially of, or consists of SEQ ID NO. 2 or 10, or modified or substantially homologous version thereof, including biologically active fragments and homologs thereof) can used as a therapeutic to promote degradation of the protein in cancers driven by oncogenic BCL6 transcriptional activity (e.g. DLBCL (Cardenas et al., 2016; Krull et al., 2020; Polo et al., 2007); B-acute lymphoblastic leukemia (Geng et al., 2012; Hurtz et al., 2019); chronic myeloid leukemia (Hurtz et al., 2011; Madapura et al., 2017); breast cancer (Sultan et al., 2021; Walker et al., 2015); non-small cell lung cancer (Deb et al., 2017; Marullo Rossella et al., 2016); glioma (Xu et al., 2017)). As a research reagent, BCLiP (which in some embodiments comprises, consists essentially of, or consists of SEQ ID NO. 2 or 10, or modified or substantially homologous version thereof, including biologically active fragments and homologs thereof) could be applied in vitro or administered in vivo to model pathological autoimmunity.

Further applications and uses of BCLiP (which in some embodiments comprises, consists essentially of, or consists of SEQ ID NO. 2 or 10, or modified or substantially homologous version thereof, including biologically active fragments and homologs thereof) are described in the Figures herewith and forming part of the instant disclosure, particularly with respect to modulating blood pressure by interfering with the interaction between Bcl6 and Pannexin 3 (Panx3) channels in vitro and/or in vivo.

Because pannexins have been implicated in hypertension (Billaud et al., 2011, 2015; DeLalio et al., 2018, 2020; Good et al., 2018), we examined mRNA transcripts from hypertensive humans and mice. In treatment resistant hypertensive humans (mean systolic pressure 158.5+13.35mmHg; Figure 1A), Panx3 mRNA (Figure IB) was significantly reduced compared to controls (mean systolic pressure 118.8+14.19mmHg), which corresponded with expression patterns observed via immunofluorescence (Figure 9A). Panxl mRNA trended upwards with variation, and Panx2 transcripts were unchanged (Figure IB). A significant decrease in Panx3 was also observed in spontaneous hypertensive mice (Figure 1C-1D; Figure 9B)

Because Panx3 has not previously been reported in resistance arteries, we examined its localization. Parix3~ ~ mice were used to demonstrate Panx3 antibody specificity (Figure 10A- B). Transverse sections of thoracodorsal arteries indicated Panx3 was only in endothelium and not smooth muscle (Figure IOC). Next, mesenteric arteries were labeled with gold beads conjugated to Panx3 (Figure 10D). Qualitative assessment of the distribution of gold beads across four subcellular membranous domains (apical, intracellular, nuclear, and basal membranes) suggested Panx3 preferentially localizes to intracellular membranes (Figure 10D) Subcellular distribution was further assessed by immunohistochemical staining of Panx3 with markers of interendothelial junctions (CD31), endoplasmic reticulum (calnexin), and the Golgi Apparatus (GM130) in mesenteric arteries prepared en face (Figure 2A). Panx3 was restricted to the Golgi Apparatus, which was validated with Manders’ colocalization analysis (Figure 2B). Because Golgi is polarized in arterial endothelium due to flow, but not in low- flow veins, we corroborated Panx3 localization to Golgi by examining mesenteric arteries and veins. Distribution of Panx3 in both vessel types identified a polarization pattern similar to Golgi (Figure 2C). Last, glycosylation states of Panxl and Panx3 help to traffic the channels to the cell surface (Penuela et al., 2009). Using PNGase F to remove glycosylation on the channels, we could not identify glycosylation of endogenous Panx3 in mesenteric arteries, unlike Panxl (Figure 2D). Thus, Panx3 is endogenously expressed in endothelium at the Golgi Apparatus.

III. REPRESENTATIVE METHODS AND COMPOSITIONS

In some embodiments, the presently disclosed subject matter provides an inhibitor of an oncogenic nuclear sequence-specific transcriptional repressor, optionally B-Cell lymphoma 6 (BCL6). In some embodiments, the inhibitor comprises a peptide that promotes degradation of the oncogenic nuclear sequence-specific transcriptional repressor and/or BCL6 in vitro and/or in vivo. In some embodiments, targeting oncogenic transcription factors, such as BCL6, for the development of therapeutic inhibitors is a desirable approach due to their significant influence on the cellular phenotype. Overabundance of BCL6 is associated with several types of soft and solid tumors. In some embodiments, soft and solid tumors comprise a cancer. In some embodiments, the cancer is selected from a group consisting of diffuse large B Cell Lymphoma, DLBCL; B-acute lymphoblastic leukemia, chronic myeloid leukemia; breast cancer; non-small cell lung cancer; glioma; angioimmunoblastic T-cell lymphoma; Follicular Lymphoma; Burkitt Lymphoma; Acute Lymphoblastic Leukemia, systemic lupus erythematosus and any combination thereof.

In some embodiments, the inhibitor comprises a BCL6 inhibitory peptide (BCLiP), wherein BCLiP comprises a peptide comprising an amino acid sequence that mimics amino acids L328-P344, (SEQ ID NO. 10), of native human BCL6 (SEQ ID NO. 1).

In some embodiments, cell penetrating peptide sequences are be attached to the 17- amino acid mimetic BCL6 sequence to facilitate membrane permeation, including TAT peptides (Dent et al., 1997; Marullo Rossella et al., 2016; Xu et al., 2017), penetratin (Moriyama et al., 1997; Niu et al., 1998), or the amino acid sequence RRRRWWW (SEQ ID NO. 8) (Hatzi & Melnick, 2014). In some embodiments, the BCLiP peptide amino acid sequence (SEQ ID NO. 2 or 10) is linked to one or more polyethylene glycol (PEG) chains to stabilize the drug in the body and reduce peptide immunogenicity. For more focused delivery of the BCLiP peptide, there are peptide sequences which enable subcellular targeting. In some embodiments, addition of the peptide sequence FLWRIFCFRK (SEQ ID NO. 9) to the C- terminus of the BCLiP peptide (SEQ ID NO. 2 or 10) promotes localization to the Golgi Apparatus (Ye et al., 1993). In some embodiments, the addition of any of the aforementioned tags include the use of a chemical linker to inhibit steric hindrance.

In some embodiments, the inhibitor comprises a conserved region containing degradation-targeting phosphorylation sites S333 and S343 of native human BCL6 (SEQ ID NO. 1). In some embodiments, the inhibitor comprise an about 17 amino acid sequence, optionally SEQ ID NO. 2, optionally a peptide sequence comprising LVSPQSPQKSDCQPNSP (SEQ ID 10), optionally a peptide sequence having about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identity to SEQ ID NO. 2 or 10, optionally a biologically active fragment and/or homolog of any of the foregoing sequences including SEQ ID NO. 2 or 10. In some embodiments, the BCLiP inhibitor comprises, consists essentially of, or consists of SEQ ID NO. 2 or 10, or modified or substantially homologous version thereof, including biologically active fragments and homologs thereof.

In some embodiments, the inhibitor, e.g., BCLiP inhibitor as described herein comprises a cell entry tag, such as but not limited to an N-terminal cell entry tag. A C-terminal entry tag could also be employed, in accordance with the techniques that would be apparent to one of ordinary skill in the art upon a review of the instant disclosure. In some embodiments, the cell entry tag (e.g., N-terminal cell entry tag) comprises a lipid tag (e.g., farnesyl, formyl, myristoyl, or palmitoyl). In some embodiments, the cell entry tag comprises cell penetrating peptide sequences (e.g., SEQ ID NO. 9). In some embodiments, the cell entry tag (e.g., N- terminal cell entry tag) cell entry tag is selected from the group consisting of a lipid tag, a cell penetrating peptide sequence and any combination thereof. In some embodiments, the cell entry tag (e.g., N-terminal cell entry tag) is configured to optimize drug dosing, peptide localization and stability. In some embodiments, the cell entry tag (e.g., N-terminal cell entry tag) comprises a stearyl tag, e.g., an N-terminal stearyl tag. In some embodiments, the cell entry (e.g., N-terminal entry tag) stearyl tag facilitates cell entry due to its hydrophobicity. In some embodiments, the inhibitor has an N-terminal stearyl group attached to SEQ ID NO. 10 with the layout of: CH₃(CH2)i7-LVSPQSPQKSDCQPNSP (SEQ ID NO. 2). In some embodiments, the inhibitor comprises one or more phosphorylation sites to target BCL6 protein for proteasomal degradation. In some embodiments, the inhibitor is configured to inhibit binding of BCL6 to Panx3.

In some embodiments, the inhibitor further comprises a pharmaceutical acceptable carrier. In some embodiments, the inhibitor comprises and/or is provided in a composition, such as a pharmaceutical composition, comprising an effective amount or a therapeutically effect amount of inhibitor sufficient to produce a selected effect of degradation of an oncogenic nuclear sequence-specific transcriptional repressor and/or B-Cell lymphoma 6 (BCL6) in vitro and/or in vivo, of modeling pathological autoimmunity in a subject, of modulating hypertension in a subject, of modulating inflammation in a subject and/or of modulating oxidative stress in a subject. In some embodiments, the inhibitor comprises an effective amount of the inhibitor to treat cancer and/or a tumor and/or is provided in a pharmaceutical composition comprising an effective amount of the inhibitor to treat cancer and/or a tumor.

In some embodiments, there is provided the inhibitor, as described above and herein, for use in inducing degradation of an oncogenic nuclear sequence-specific transcriptional repressor and/or B-Cell lymphoma 6 (BCL6) in vitro and/or in vivo. In some embodiments, there is provided the inhibitor, as described above and herein, for use in treating a cancer and/or a tumor in a subject in need thereof. In some embodiments for use, the cancer and/or tumor comprises a cancer and/or tumor driven by oncogenic BCL6 transcriptional activity, optionally diffuse large B Cell Lymphoma, B-acute lymphoblastic leukemia, chronic myeloid leukemia, breast cancer, non-small cell lung cancer, and/or glioma.

In some embodiments, there is provided the inhibitor, as described above and herein, for use in in modeling pathological autoimmunity, modulating Pannexin (Panx) channels in vitro and/or in vivo, modulating inflammation in a subject, modulating vascular oxidative stress in a subject, and/or modulating hypertension in a subject in which said modeling and/or modulating is desired.

In some embodiments for use, the inhibitor further comprises a pharmaceutical acceptable carrier. In some embodiments for use, the inhibitor comprises and/or is provided in a composition, such as a pharmaceutical composition, comprising an effective amount or a therapeutically effect amount of inhibitor sufficient to produce a selected effect of degradation of an oncogenic nuclear sequence-specific transcriptional repressor and/or B-Cell lymphoma 6 (BCL6) in vitro and/or in vivo, of modeling pathological autoimmunity in a subject, of modulating hypertension in a subject, of modulating inflammation in a subject and/or of modulating oxidative stress in a subject. In some embodiments for use, the inhibitor comprises an effective amount of the inhibitor to treat cancer and/or a tumor and/or is provided in a pharmaceutical composition comprising an effective amount of the inhibitor to treat cancer and/or a tumor.

In some embodiments, the presently disclosed subject matter provides a method of inducing degradation of an oncogenic nuclear sequence-specific transcriptional repressor and/or B-Cell lymphoma 6 (BCL6) in vitro and/or in vivo. In some embodiments, the method comprises contacting a cell in vivo or in vitro with an inhibitor, as described herein and above, and/or administering to a subject the inhibitor as described herein and above.

In some embodiments, the presently disclosed subject matter provides a method of treating a cancer and/or a tumor in a subject. In some embodiments, the method comprises administering to a subject in need of treatment a composition comprising an inhibitor, described herein and above. In some embodiments, the inhibitor is BCLiP. In some embodiments, the BCLiP inhibitor comprises, consists essentially of, or consists of SEQ ID NO. 2 or 10, or modified or substantially homologous version thereof, including biologically active fragments and homologs thereof. In some embodiments, the cancer and/or tumor comprises a cancer and/or tumor driven by oncogenic BCL6 transcriptional activity, optionally diffuse large B Cell Lymphoma, B-acute lymphoblastic leukemia, chronic myeloid leukemia, breast cancer, non-small cell lung cancer, and/or glioma.

In some embodiments, the presently disclosed subject matter provides a method of modeling pathological autoimmunity in a subject. In some embodiments, the method comprises administering to a subject a composition comprising an inhibitor as described above and herein. In some embodiments, the inhibitor is BCLiP, further optionally wherein the BCLiP inhibitor comprises, consists essentially of, or consists of SEQ ID NO. 2 or 10, or modified or substantially homologous version thereof, including biologically active fragments and homologs thereof.

In some embodiments, the presently disclosed subject matter provides a method of modulating Pannexin (Panx) channels in vitro and/or in vivo. In some embodiments, the method comprises contacting a cell in vivo or in vitro with an inhibitor as described above and herein, and/or administering to a subject an inhibitor as described above and herein, wherein the modulating comprises disruption of BCL6 binding with Panx. In some embodiments, the Panx is optionally one of three isoforms, optionally Panxl, Panx2 and/or Panx3.

In some embodiments, the presently disclosed subject matter provides a method of modulating inflammation, hypertension, and/or vascular oxidative stress in a subject. In some embodiments, the method comprises administering to a subject a composition comprising an inhibitor as described above and herein. In some embodiments, the inhibitor is BCLiP. In some embodiments, the BCLiP inhibitor comprises, consists essentially of, or consists of SEQ ID NO. 2 or 10, or modified or substantially homologous version thereof, including biologically active fragments and homologs thereof.

In some embodiments of the presently disclosed methods, the inhibitor further comprises a pharmaceutical acceptable carrier. In some embodiments of the method, the inhibitor comprises and/or is provided in a composition, such as a pharmaceutical composition, comprising an effective amount or a therapeutically effect amount of inhibitor sufficient to produce a selected effect of degradation of an oncogenic nuclear sequence-specific transcriptional repressor and/or B-Cell lymphoma 6 (BCL6) in vitro and/or in vivo, of modeling pathological autoimmunity in a subject, of modulating hypertension in a subject, of modulating inflammation in a subject and/or of modulating oxidative stress in a subject . In some embodiments of the methods, the inhibitor comprises an effective amount of the inhibitor to treat cancer and/or a tumor and/or is provided in a pharmaceutical composition comprising an effective amount of the inhibitor to treat cancer and/or tumor.

In some embodiments, the presently disclosed subject matter provides use of an inhibitor of a transcription factor, optionally B-Cell Lymphoma 6 (BCL6), for the treatment of cancer and/or a tumor in a subject in need thereof. In some embodiments, the inhibitor is an inhibitor as described above and herein. In some embodiments, the inhibitor is BCLiP, further optionally wherein the BCLiP inhibitor comprises, consists essentially of, or consists of SEQ ID NO. 2 or 10, or modified or substantially homologous version thereof, including biologically active fragments and homologs thereof. In some embodiments, the cancer and/or tumor comprises a cancer and/or tumor driven by oncogenic BCL6 transcriptional activity, optionally diffuse large B Cell Lymphoma, B-acute lymphoblastic leukemia, chronic myeloid leukemia, breast cancer, non-small cell lung cancer, and/or glioma.

In some embodiments, the presently disclosed subject matter provides a method of evaluating a candidate therapeutic composition. In some embodiments, the method comprises providing a subject modulated with respect to a transcription factor, optionally B-Cell Lymphoma 6 (BCL6). In some embodiments, the subject is a model of inhibited BCL6, such as a model as described herein, such as in the Examples. In some embodiments, the modulation is due to administering an inhibitor of a transcription factor, optionally B-Cell Lymphoma 6 (BCL6) to the subject. In some embodiments, the method comprises administering a candidate therapeutic composition to the subject; and assessing an effect of the candidate therapeutic composition. In some embodiments, the inhibitor is an inhibitor as described above and herein, optionally wherein the inhibitor is BCLiP, further optionally wherein the BCLiP inhibitor comprises, consists essentially of, or consists of SEQ ID NO. 2 or 10, or modified or substantially homologous version thereof, including biologically active fragments and homologs thereof. In some embodiments, the subject comprises an in vitro culture and/or cell suspension. In some embodiments, the subject comprises an in vivo subject. In some embodiments, the in vivo subject comprises a non-human animal model. In some embodiments, the animal model comprises a rat model. In some embodiments, the animal model comprises a mouse model.

In some embodiments, the effect comprises reducing hypertension, such as reducing hypertension to a homeostatic level. In some embodiments, the effect comprises reducing inflammation, such as reducing to a homeostatic level. In some embodiments, the effect comprises reducing vascular oxidative stress, such as reducing vascular oxidative stress to a homeostatic level. Approaches to investigate these effects are presented in the Examples herein below.

In some embodiments, the inhibitor as described above and herein comprises sequence modifications using the approaches described under the peptide modification and preparation discussion above. In some embodiments, the inhibitor as described above and herein comprises amino acid modifications (e.g., substitutions and/or deletions) using the approaches described under the amino acid section discussion above. In some embodiments, the inhibitor as described above and herein comprises a pharmaceutical composition as described in the pharmaceutical compositions and administration above. In some embodiments, the inhibitor as described above and herein is administered to a subject in need thereof by any number of routes including, but not limited to, topical, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal approaches. In some embodiments, the effect comprises treating cancers related to BCL6 transcriptional activity. In some embodiments, the effect comprises preventing cancers related to BCL6 transcriptional activity. In some embodiments, the effect comprises reducing BCL6 transcriptional activity. In some embodiments, a pharmaceutical composition comprises an inhibitor which comprises, consists essentially of, or consists of SEQ ID NO. 2 or 10, or modified or substantially homologous version thereof, including biologically active fragments and homologs thereof.

In some embodiments, the presently disclosed subject matter provides a kit comprising an inhibitor as disclosed herein and above and instructional materials for carrying out any of the methods disclosed herein.

Additional techniques as would be apparent to one skilled in the art to implement the methods disclosed herein or otherwise use the presently disclosed subject matter are provided in this disclosure.

Table 3

EXAMPLES

The following Example have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. Because pannexins have been implicated in hypertension (Billaud et al., 2011, 2015; DeLalio et al., 2018, 2020; Good et al., 2018), we examined mRNA transcripts from hypertensive humans and mice. In treatment resistant hypertensive humans (mean systolic pressure 158.5+13.35mmHg; Figure 1A), Panx3 mRNA (Figure IB) was significantly reduced compared to controls (mean systolic pressure 118.8+14.19mmHg), which corresponded with expression patterns observed via immunofluorescence (Figure 9A). Panxl mRNA trended upwards with variation, and Panx2 transcripts were unchanged (Figure IB). A significant decrease in Panx3 was also observed in spontaneous hypertensive mice (Figure 1C-1D; Figure 9B)

Because Panx3 has not previously been reported in resistance arteries, we examined its localization. Panx3'^/_ mice were used to demonstrate Panx3 antibody specificity (Figure 10A- 10B) Transverse sections of thoracodorsal arteries indicated Panx3 was only in endothelium and not smooth muscle (Figure 10C). Next, mesenteric arteries were labeled with gold beads conjugated to Panx3 (Figure 10D). Qualitative assessment of the distribution of gold beads across four subcellular membranous domains (apical, intracellular, nuclear, and basal membranes) suggested Panx3 preferentially localizes to intracellular membranes (Figure 10D) Subcellular distribution was further assessed by immunohistochemical staining of Panx3 with markers of interendothelial junctions (CD31), endoplasmic reticulum (calnexin), and the Golgi Apparatus (GM130) in mesenteric arteries prepared en face (Figure 2A). Panx3 was restricted to the Golgi Apparatus, which was validated with Manders’ colocalization analysis (Figure 2B). Because Golgi is polarized in arterial endothelium due to flow, but not in low- flow veins, we corroborated Panx3 localization to Golgi by examining mesenteric arteries and veins. Distribution of Panx3 in both vessel types identified a polarization pattern similar to Golgi (Figure 2C). Last, glycosylation states of Panxl and Panx3 help to traffic the channels to the cell surface (Penuela et al., 2009). Using PNGase F to remove glycosylation on the channels, we could not identify glycosylation of endogenous Panx3 in mesenteric arteries, unlike Panxl (Figure 2D). Thus, Panx3 is endogenously expressed in endothelium at the Golgi Apparatus.

Materials and Methods

Human Samples

The collection and use of human adipose tissue biopsies was approved by the University of Virginia’s Institutional Review Board for Health Sciences Research (Study 17194). Human volunteers with resistant hypertension (elevated blood pressure above 140 mmHg systolic) and healthy controls had an approximate 40 mm><20 mm biopsy of adipose tissue removed from the abdomen (no additional procedures were performed), which was immediately placed in ice- cold KREBS buffer and arterioles dissected manually within 30 minutes of removal. Samples collected for immunostaining were placed in 4% paraformaldehyde, and paraffin embedded. Samples collected for mRNA quantification were collected in Trizol and frozen at -80C. All subjects were males (N = 4). Hypertensive participants had an average age of 47.75+/-4.23 years, with a body mass index of 36.75+/-5.24.

Animals

All mice were mixed sex, 10-20 weeks of age, on a C57B1/6 genetic background, and were cared for under the provisions of the University of Virginia Animal Care and Use Committee and followed the National Institutes of Health guidelines for the care and use of laboratory animals. C57Bl/6n mice were purchased from Taconic. The inducible, EC-specific Panx3 knockout mice (VECadER^T2+/Panx3f^l/fl') were generated by crossing VECadER^r2 Panx3^wt/wt mice (a kind gift from Dr Ralf Adams, Max Plank Institute, Germany) with VEXEtdEld²~ Panx3^{il il} _mjce (Moon et al., 2015). To conditionally induce Panx3 deletion in the vascular endothelium, VECadElX ² /Panxl^fl (EC Panx3^^A) and VECadEPX²', Paiix 1 ¹ (PanxlEfl') littermates received intraperitoneal (I.P.) injections of Tamoxifen (1 mg in 0.1 ml peanut oil) at six weeks of age for 10 consecutive days. All animal experiments were performed at least 14 days from the final injection with tamoxifen. The global Panx3 knockout mice were generated by crossing B6.Cg-Edil3^Tg(Sox2'^cre)1Amc/J⁺/Panx3^wt/wt mice (Jackson, #008454) with B6.Cg-Edil3^Tg(Sox2’^cre)1Amc/J’/Panx3^fl/fl mice (Moon et al., 2015). All experiments were performed on a minimum of three mice. For all assessments of blood, blood was collected via terminal cardiac puncture using a syringe fitted with 25G needle, coated with EGTA to prevent clotting. To assess plasma renin levels, ~100uL of whole blood was collected via tail vein into gold microtainers two hours into the dark/active time period. Following centrifugation, renin concentration was assessed in isolated plasma using a Mouse Renin 1 ELISA (Ray BioTech, ELM-Reninl-1). To assess circulating hydrogen peroxide concentration, plasma was isolated from whole blood and passed through deproteinization columns within 30 minutes of collection (Abeam, ab93349). Deproteinized plasma was processed using the Hydrogen Peroxide Assay Kit (Abeam, ab 102500). Cardiac MRI data was collected from anesthetized mice using a black blood sequence following imaging in the 7T ClinScan MRI. Western blotting

Cell and tissue lysates were generated in RIPA (50mmol/L Tris-HCL, 150mmol/L NaCl, 5mmol/L EDTA, 1% deoxycholate, 1% Triton-XlOO) in PBS and pH adjusted to 7.4) supplemented with protease inhibitor cocktail (Sigma). Lysates were rocked at 4°C for 30-60 min to solubilize proteins, sonicated briefly, and centrifuged for 15 min at 12,000 rpm to pellet cell debris. Protein concentration was determined using the BCA method (Pierce). 30 pg of total protein was loaded into each sample well. For crosslinking studies, mesenteric vascular and aortic lysates were made in non-amine lysis buffer (137mM NaCl, 5.4mM KC1, 0.34mM Na₂HPO₄, 0.35mM KH2PO4, 0.8mM MgSCU, 2.7mM CaCh, ImM NaF, 250mM sucrose, 20mM Hepes, 10% glycerol). lOOug of mesenteric vascular and aortic protein was incubated with 0.5 or 2.5 mM bis-sulfosuccinimidyl-suberate (BS³) prior to inactivation with Tris HC1 pH 7.5. Samples were subjected to SDS gel electrophoresis using 8% Bis-Tris gels (Invitrogen) and transferred to PVDF (when blotting for Panx3) or nitrocellulose membranes for immunoblotting. Membranes were blocked for 1 hour at room temperature in a solution containing 3% milk (Panx3) in phosphate buffered saline or 3% BSA in Tris buffered saline, then incubated overnight at 4°C with primary antibodies against Panx3 (Thermo Fisher, 433270; 1 : 500), eNOS (BD Biosciences, 610297; Cell Signaling Tech, 9572S), peNOS Thr495 (BD Biosciences, 612706), BCL6 (BD Bioscience 561520, Cell Signaling Tech, 5650S), Panxl (Cell Signaling Tech, 91127S), GAPDH (Santa Cruz, sc-51907), Nox4 (Abeam, abl09225), p22^phox (Santa Cruz, sc-271968), 3 -nitrotyrosine (Millipore, 06-284), Flag (Sigma, F3165). Membranes were washed and incubated in LiCOR IR Dye secondary antibodies (1 : 10,000) for 1 hour and viewed/quantified using the LiCOR Odyssey CLx with Image Studio software. Licor Total Protein stain or GAPDH was used for loading normalization. Representative western blot images have been cropped for presentation.

Real-time quantitative PCR

Total RNA was extracted from mouse tissues using the Aurum Total RNA Fatty and Fibrous Tissue Extraction kit (Biorad, #732-6870). RNA concentration was measured using the Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific). RNA was stored at -80°C before reverse transcription with SuperScript III First-Strand Synthesis system (Thermo Fisher Scientific, #18080051) using random hexamer primers on Ipg of template RNA. Real-time quantitative PCR was performed using Taqman Gene Expression Master Mix (Thermo Fisher, 4369016) and Taqman Real-Time PCR assays in MGB-FAM for Pannexin 3!Panx3 (Hs00364808_ml; Mm00552586_ml), Pannexin UPanxl (Hs00209790_ml;

Mm00450900_ml), B-Cell Lymphoma-6//A7.6 (HsOO153368_ml; Mm00477633_ml), v-rel reticuloendotheliosis viral oncogene homolog HIRelB (Mm00485664_ml), Nuclear factor KB pl 00 subunit/N/##2 (Mm00479807), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (lKBa)/Nfkbia (Mm00477798_ml), NADPH Oxidase-2/ AA (Mm01287743_ml), NADPH Oxidase-4/Mw-/ (Mm00479246_ml), and were normalized to P-2-microglobin/7GA7 in VIC -PL (Hs00364808_ml; Mm00437762_ml). Reactions were run in a CFX Real-Time Detection System (Applied BioSystems), and threshold cycle number (CT) was used as part of the 2'^AACT method to calculate fold change from control.

Immunohistochemistry, Histology & Microscopy

Vascular tissues (aortae, third-order mesenteric resistance arteries and veins) were collected and fixed in 4% paraformaldehyde or ice-cold acetone-m ethanol. For en face preparation, arteries were cut longitudinally with microdissection scissors and pinned open on polymerized Sylgard 184 (Electron Microscopy Sciences) using tungsten wire (0.0005”, ElectronTubeStore). Vessels prepared en face were then permeabilized in 0.2% NP-40 in PBS for 30 minutes at room temperature, blocked in 1% bovine serum albumin, Fraction V (BSA, Sigma) in 0.2%NP40/PBS and stained with primary antibodies overnight at 4°C in 0.1% BSA in 0.2%NP40/PBS. For paraffin sections, 5pm tissue sections were deparaffinized with heat (1 hour at 65°C) and histoclear (National Diagnostics, 5989-27-5). Following rehydration, tissues were blocked in 5%BSA, Fraction V, 0.05% fish skin gelatin (Sigma, G7765), and 0.2% Triton-XlOO for 1 hour at room temperature in a humified chamber. Primary antibodies include Panx3 (Thermo Fisher, 433270; 1 :50), Panx3 CT-379 (Penuela et al., 2007, 2009), BCL6 (Invitrogen #14-9887-82), PECAM (Santa Cruz, sc-376764, 1 :50), calnexin (Abeam, #ab219644, 1 : 100), eNOS (BD Transduction, 610297, 1 : 100), GM130 (R&D Systems, AF8199), Claudin 5 (Invitrogen, 35-2500), B4GALT1 (Invitrogen, PA5-106617, 1 :50), MGAT1 (Invitrogen, PA5-121001), Panxl (Cell Signaling Tech, 91137S), PrxSo3 (Abeam, abl6830), 3 -nitrotyrosine (Millipore Sigma, 06-28), 4-hydroxynonenal (Invitrogen, MAS- 27570). Samples were incubated with secondary antibodies at 1:500 for 1-2 hours at room temperature. Images of endothelium were collected on LSM 880 with Airyscan or Olympus FV1000 and post processing was completed using Fiji. Nuclear staining was achieved with a brief incubation with DAPI (Invitrogen, D1306, final concentration O. lpg/mL) in addition to mounting with Prolong Gold Antifade Mountant (Invitrogen, P36930). For colocalization analyses, images were assessed in Fiji using the Coloc 2 plugin following rolling ball background subtraction.

Blood Pressure Assessments via Radiotelemetry

Blood pressure was measured using telemetry equipment (Data Sciences International, DSI) as previously described (Billaud et al., 2015). Mice were surgically implanted with radiotelemetry units (PA-C10 or HD-X10). Briefly, while under isoflurane anesthesia, the catheter of a radiotelemetry unit was placed in the left carotid artery and positioned such that the probe reached the aortic arch. The radiotransmitter was placed in a subcutaneous pouch at the right flank. Buprenorphine was used as an analgesic. Mice were allowed to recover for seven days prior to the initiation of recordings. Mice that were injected with tamoxifen underwent implantation at least 10 days after the last injection. Baseline blood pressure measurements, including systolic pressure, diastolic pressure, mean arterial pressure (MAP) and heart rate, were recorded every minute for a continuous period of 5 days using Dataquest A.R.T. 20 software (DSI). Change in MAP (AMAP) was calculated by subtracting the average baseline MAP to the MAP after treatment administration. Diurnal (inactive period) MAP was measured during animal’s light cycle: 6:00 a.m. to 5:59 p.m., and nocturnal (active period) MAP was measured during the animal’s dark cycle: 6:00 p.m. to 5:59 a.m.

Pressure Myography

Freshly isolated third-order mesenteric arteries were placed into ice-cold Krebs-HEPES buffer (containing (in mM) NaCl 118.4, KC14.7, MgSO4 1.2, NaHCO3 4, KH2PO4 1.2, CaC12 2, Hepes 10, glucose 6; pH 7.40-7.42). The vessels were then mounted in a pressure arteriograph (Danish MyoTechnology, DMT) with the lumen filled with Krebs-HEPES buffer as previously described (Billaud et al., 2011, 2015; DeLalio et al., 2018, 2019; Good et al., 2018). The vessels were equilibrated for 30 min at 80 mmHg and 37°C. Increasing concentrations of ACh were added into the bath to examine the endothelial-dependent vasodilation of these vessels. Full ACh curves were obtained for each vessel under a given treatment condition. Smooth muscle cell health was verified by constriction to KC1. Next, arteries were exposed to NS309 (small- and intermediate-conductance calcium activated potassium (SKca/IKca) channel agonist). The vessels were then washed with a Ca2+-free Krebs-HEPES solution supplemented with 1 mM Ethyleneglycol-O,O'-bis(2-aminoethyl)- N,N,N',N'- tetraacetic acid and 10 pM sodium nitroprusside to obtain maximal passive diameter of the vessels. Internal diameter was measured at each step using the DMT MyoVIEW software. Vasodilation to ACh or NS309 was calculated as a % relaxation: % relaxation = ((DACH - D one) * 100)/(Dmax - Dione), where Dione was the diameter of the artery after the establishment of stable basal tone, DACH was the diameter after application of a given dose of ACh, and Dmax was the maximal diameter measured at the end of experiment.

Immunogold Labeling and Electron Microscopy

Third-order mesenteric resistance arteries were collected in cellulose capillary tubes (Leica Microsystems, Vienna, Austria) with an inner diameter of 200 pm. The tissues were transferred within the capillary tube to membrane carriers and cryo-immobilized using an EM ICE high-pressure freezer (Leica Microsystems, Vienna, Austria) (Redemann et al., 2018). Freeze substitution was performed over 2-3 days at -90C using an automatic freeze substitution machine (EM AFS Leica Microsystems). Samples were embedded in LR White (Electron Microscopy Sciences, 14380) and polymerized at 65°C. Semithick sections (200nm) were collected on pioloform-coated nickel grids (Electron microscopy sciences, EMS300-NI). Immunostaining was completed using the gird-on-drop technique in humidified chambers. All staining solutions were passed through a 0.2pm syringe filter prior to use. Briefly, grids were washed with PBS followed by 0.1% glycine/PBS. Blocking solutions contained 0.1% cold- water fish skin gelatin (Sigma, G7765), 5% BSA, 5% goat serum and were incubated with samples for 1 hour are room temperature. Primary antibodies were diluted to 1 :20 in blocking solution diluted 1 : 1 in PBS and incubated with sample for one hour at room temperature. Samples were briefly washed in diluted blocking solution prior to secondary antibody incubated for 2 hours at room temperature (Electron Microscopy Sciences, 15-25nm, 1 :20, #25116 and #25133). Samples underwent 10 washed in filtered diEEO to remove salts. Contrast staining was achieved by brief incubation in 4% uranyl acetate followed by incubations in 0.4% lead citrate. Electron micrographs were collected on a TECNAI F20 transmission electron microscope (Thermo-Fisher, formerly FEI) operated at 200kV and recorded on a Gatan US4000 (4000 px x 4000 px) CCD or a Teitz TVIPS XF416 camera.

Cell Culture

Human coronary artery endothelial cells (HCAEC, Cell Applications, 300-05a, Lot#3159) were cultured in Endothelial Cell Growth Medium 2 with the manufacturer’s recommended supplements (Promocell, C-39211). For Golgi neutralization, HCAEC were plated on fibronectin-coated coverslips (Sigma, FC0105) and exposed to 2.5mM ammonium chloride (Sigma, A9434) or vehicle (deionized water) for 48 hours prior to fixation in 4% paraformaldehyde.

Proximity ligation assay

Third-order mesenteric arteries were prepared en face as described above. Following fixation, arteries were permeabilized in 0.2% NP40 in PBS at room temperature for 30 minutes with gentle shaking. Proximity ligation staining was achieved by following the Duolink In Situ PLA fluorescent protocol (Millipore Sigma). Briefly, samples were blocked in Duolink blocking for 1 hour at room temperature. Next, samples were incubated with primary antibodies diluted 1 :25 in Duolink antibody buffer at 4°C overnight. Primary antibodies were used against Panx3 (Invitrogen; 433270), BCL6 (Invitrogen #14-9887-82), endothelial nitric oxide synthase (BD; 610297), and caveolin-1 (Abeam, ab32577). Despite staining on whole tissue, small reaction volumes (25pL) were achieved by placing sylgard squares holding arteries prepared en face within a 24-well plate and balancing the drop on the center of the sylgard. Damp filter paper was placed over the wells of the plate and the lid to generate a humified chamber. Reactions occurred under static conditions to avoid breaking surface tension and reduce the risk of sample drying. Anti-rabbit MINUS and anti-mouse PLUS probes (Millipore Sigma; DU092005-30RXN; DU092001-30RXN) in antibody diluent and incubated with samples for one hour at 37°C as per the manufacturer’s instructions. Ligation reactions were then allowed to proceed for 30 minutes at 37°C, followed by amplification reactions which occurred for 100 minutes at 37°C. Samples were then mounted with Prolong Gold Antifade Mountant (Invitrogen, P36930). Images were collected LSM 880 with Airyscan and post processing was completed using Fiji. Briefly, the PLA puncta channel was first isolated and converted to greyscale. From this, a binary image was generated with a threshold of 0.55-0.65 in preparation to analyze particles (0.003-Inifinity). The number of PLA puncta was normalized to the number of endothelial nuclei in view. Data from cells with nuclei that are not entirely visualized in the image were excluded from analysis. A minimum of four fields of view were analyzed and averaged per mouse.

Immunoprecipitation

HEK293T were cultured in DMEM, high glucose (Gibco, 11965-092) supplemented with ImM sodium pyruvate, 1% Pen-Strep (Gibco, 15140122), 10% FBS (Avantor, 97068- 085). Cells were transfected with pcDNA3.1-3xFlag-hPANX3 (NovoPro, 74275501, accession #: NM_052959) and pCMV6-AC-GFP-hBCL6 (Origene, RG226102, accession#: NM OOl 130845) using Lipofectamine 3000 (Thermo Fisher, L3000001). For BCLiP (SEQ ID NO. 2) treated samples, 50 pM BCLiP (SEQ ID NO. 2) was introduced to the cells 6 hours after transfection. Lysates were collected in IP RIPA (125mM NaCl, 5mM EDTA, 1% sodium deoxy cholate, 0.5% Triton-x 100 in phosphate buffered saline supplemented with lOpM AEBSF, lOmM NaF, lOmM NEM, 500pM Na3VO4 and protease inhibitor cocktail (Sigma, P8340). M-280 Sheep anti-mouse IgG Dynabeads (Invitrogen, 11201D) were washed in a blocking solution containing 0.5% BSA, 0.2% fish skin gelatin in PBS prior to incubation with Flag antibody (Sigma, F3165). Beads were mixed with 800pg HEK lysate in IP RIPA for three hours at 4C with gentle agitation prior to elution in 5x Lamelli buffer (0.5M TrisHCl pH6.8, 5% SDS, 0.5% Bromophenol blue, 12.5% P-mercaptoethanol).

Calcium imaging

Ca²⁺ imaging studies were performed as described previously (Ottolini et al., 2020). Third-order mesenteric arteries (~ 100 pm) were cut open and pinned down en face on Sylgard blocks. Mesenteric arteries were then incubated with fluo-4 AM (10 pM) and pluronic acid (0.04%) at 30°C for 45 minutes. Ca²⁺ images were acquired at 30 frames per second using Andor Revolution WD (with Borealis) spinning-disk confocal imaging system (Andor Technology, Belfast, UK) comprising of an upright Nikon microscope with a 60X water dipping objective (numerical aperture 1.0) and an electron multiplying charge coupled device camera. Mesenteric arteries were super fused with physiological salt solution (PSS; 119 mM NaCl, 4.7 mM KC1, 1.2 mM KH2PO4, 1.2 mM MgCh 160 hexahydrate, 2.5 mM CaCh dihydrate, 7 mM dextrose, and 24 mMNaHCCh) bubbled with 21% O2 and 5% CO2 to maintain the pH at 7.4. All the experiments were performed at 37°C. Fluo-4 was excited using a 488 nm solid-state laser and emitted fluorescence was captured using a 525/36 nm band-pass filter. Ca²⁺ images were analyzed using a custom-designed SparkAn software (developed by Dr. Adrian Bonev, University of Vermont) as described previously (https://github.com/vesselman/SparkAn) (Y. L. Chen et al., 2021). Calcium transients were automatically detected using an ROI of 5 x 5 pixels, and a threshold of 1.25 F/Fo. The number of calcium transients per field before or after 10 pM carbachol (CCh) stimulation was determined, and the numbers were averaged for each artery.

Predictions of Panx3 Structure and docking with BCLiP Candidate peptide sequences were identified based on literature review. Initial peptide and Panx3 dimer structures were generated using AlphaFold2 on the ColabFold (Mirdita et al., 2022) webserver. The highest-scored structures were used for docking studies. The structure of Panx3 dimer was generated by generating a structure of Panx3 heptamer, then removing all but two monomers. This was done to simulate the smallest protomer of Panx3, avoiding biasing the simulation with a higher-order oligomer. Docking between peptides and protein dimer was simulated with LightDock (Roel-Touris et al., 2020) using 400 swarms of 200 glowworms. Each docking run was comprised of 100 steps, Protein and peptide were treated as flexible structures through the course of the docking run and positional restraints were applied to bias peptide docking towards the intraluminal side of Panx3. Specifically, docking only considered peptide orientations in close proximity of Glu¹⁷⁷, Gin¹⁵⁸, Glu¹⁸³, Asp ³⁵⁴, Ala³⁷², Thr³⁶³, Gly³⁷³, or His⁵³⁸. Docking results were scored using the pyDock algorithm (Cheng et al., 2007), which generates a score by considering binding energetics due to electrostatic interactions and desolvation effects. Ramachandran plots were generated using the MolProbity program (Williams et al., 2018).

Generation and administration of BCL6 mimetic peptide

A region of BCL6 (residues 328-344 of BCL6; LVSPQSPQKSDCQPNSP; SEQ ID NO. 10) was selected due to its important role in regulating targeted degradation of BCL6 (Niu et al., 1998). This region of BCL6 was found to have a high degree of sequence conservation across mammalian species (as determined by multiple sequence alignment on NCBI blastp for Homo sapiens (SEQ ID NO. 4), Mus musculus (SEQ ID NO. 5), Rattus norvegicus (SEQ ID NO. 6) and Macaca mulatta (SEQ ID NO. 7), see Figure 8A). Peptides mimicking this region of BCL6 (BCLiP; SEQ ID NO. 2) or a scrambled peptide (CH3(CH2)i7- SPSDPPVLNSQCQSQPK; SEQ ID NO 3.) were synthesized with a N-terminal stearyl group to facilitate membrane permeability. For in vivo studies, BCLiP (SEQ ID NO. 2) or the scrambled peptide (SEQ ID NO. 3) were administered to C57Bl/6n mice via intraperitoneal (I.P.) injection at 2.5mg/kg and 12.5 mg/kg in sterile saline daily for a period of five days. All tissues were collected 6-8 hours following the last dose of BCLiP (SEQ ID NO. 2) or scramble peptide (SEQ ID NO. 3) . Protein and RNA were isolated from flash frozen lung tissue to be used for quantification. Third-order mesenteric resistance arteries were collected, fixed in 4%PFA and prepared en face for proximity ligation assays (PLA). BCL6 DNA binding assessments

For in silico assessments: Using the mm39 reference genome, the cis sequences Ikb upstream of the indicated genes were retrieved. The mouse Bcl-6 binding motif PSWM was retrieved from JASPAR (ID: MA0463.1). The Find Instances of Motif Occurrence (FIMO) algorithm (Bailey et al., 2015) was then applied to these sequences to find a motif occurrence of the Bcl-6 binding motif, using a threshold of 0.0005. The number of intervals above this threshold were then plotted for each gene. For assessments of publicly available BCL6 ChlP- Seq datasets: A comprehensive set of BCL-6 ChlP-Seq reads were assembled and retrieved from the gene expression omnibus and aligned to the mm39 reference genome. Signal was then plotted, in aggregate, for Ikb upstream of genes of interest. The GSM accession numbers used for this analysis are: GSM1857225, GSM1857226, GSM3347610, GSM3347618, GSM3347625, GSM3347623, GSM3347624, GSM3347613, GSM3347612, GSM3347611, GSM419049, GSM419050, GSM611114, GSM611115, GSM851557.

Example 1: Genetic deletion of endothelial Panx3 results in spontaneous hypertension.

We posited the hypertension associated with attenuated Panx3 observed in Figure 1 may be recapitulated if we removed Panx3 from the endothelium. Thus, EC PanxS^ mice were generated by crossing mice carrying loxP sites flanking exon 2 of the murine Panx3 gene (Moon et al., 2015) with mice carrying the tamoxifen-inducible driver of Cre recombinase under the vascular endothelial cadherin promoter (Cdh5-CreER^T2+; Figure 3A). Following tamoxifen administration, Panx3 gDNA is excised (Figure 3B), and Panx3 mRNA and protein is significantly reduced (Figure 3C-3F), with no corresponding change in Panxl (Figure 3G). The EC Panx3^A/A mice (right column) do not recapitulate phenotypes reported in global Panx3‘ ^/_ mice; there is no change in body length (Ishikawa et al., 2016), body fat (Wakefield et al., 2022), gonadal fat (Wakefield et al., 2022), or blood lipids (Halliwill et al., 2016) (Figure HAUG) However, similar to the observation in humans and genetically inbred hypertensive mice in Figure 1, EC PanxS^ mice (right column) present with spontaneous hypertension with significantly increased mean arterial and systolic pressure (Figure 4A-4B). There was no significant change in diastolic pressure, heart rate or cardiac output (Figure 4C-4E). To determine if salt and water retention functions of the kidney were part of the etiology of the spontaneous hypertension, we examined plasma sodium, potassium and renin, and found no significant differences (Figure 4F-4H). Ejection fraction, creatinine, creatine kinase, and blood urea nitrogen levels in the blood were unchanged (Figure 11H-11K). However, acetylcholine (ACh) cumulative dose-response curves from third-order mesenteric arteries demonstrated inhibition of dilatory capacity (Figure 41). The dilation impairment was not due to loss of smooth muscle function, as the arteries constricted to KC1 (Figure 4J), or changes in eNOS protein expression or inhibitory eNOS phosphorylation at Thr495 (Figure 4K); however, dilation to NS309 was significantly impaired (Figure 4L). Based on this data, we find mice with genetic deletion of Panx3 in endothelium develop spontaneous hypertension that is likely due to endothelial dysfunction impairing peripheral resistance, possibly through endothelial SK/IK inhibition.

Example 2: Panx3 exerts channel-independent functions through interactions with BCL6.

Because genetic deletion of endothelial Panx3 causes severe vascular impairments at the tissue and systemic level, we wanted to determine the functional role Panx3 in endothelium and first tested properties associated with Panxl. Mesenteric arteries exhibited no change in intracellular or stimulated release of ATP (Figure 5A-5B) suggesting Panx3 may not contribute to endothelial purinergic signaling. Panx3 has previously been reported to facilitate IP3-induced calcium store release (Ishikawa et al., 2011; Iwamoto et al., 2010). However, assessments of calcium signaling in mesenteric arteries revealed no impairments between genotypes at baseline or following stimulation with muscarinic agonist carbachol (Figure 5C), suggesting endogenous Panx3 has no role in IP3-mediated calcium mobilization. Next, because Panx3 is localized to Golgi, we hypothesized it may regulate luminal pH. Subcellular distribution of glycosyltransferases was used as an indirect measurement of Golgi homeostasis as neutralization of the Golgi luminal pH causes redistribution of Golgi-resident protein into the endosomal system (Figure 5D) (Axelsson et al., 2001; Bachert et al., 2001). However, the colocalization of B4GALT1 and MGAT1 with Golgi marker eNOS are unchanged by Panx3 expression in intact endothelium (Figure 5E-5F; Figure 12A), indicating no impairments in Golgi homeostasis or luminal pH regulation. The sum of this data precipitated the question of whether Panx3 monomers oligomerize into a channel endogenously. To test this, the oligomeric state of endogenous Panx3 was assessed in mesenteric vascular lysates following exposure to chemical crosslinker bis-sulfosuccinimidyl-suberate (BS³). Crosslinking of vascular lysates was insufficient to identify an oligomeric species of Panx3, unlike Panxl, instead revealing a Panx3 doublet at ~80 kDa, which suggests the presence of a stable dimeric Panx3 protomer (Figure 5G). Without evidence for Panx3 channel function or confirmation of the oligomeric species, we next interrogated the possibility that Panx3 may exert its cellular function through protein-protein interactions. In an unbiased protein-protein interaction screen, Panx3 was reported to bind transcriptional repressor BCL6 (Miles et al., 2005). Immunostaining indicates both proteins can be detected in the Golgi Apparatus of intact endothelium (Figure 5H). Proximity ligation assays on mesenteric endothelium corroborated Panx3 and BCL6 interaction, with eNOS and Cavl used as a positive control (Figure 51; Figure 12B).

Example 3: Panx3 interactions with BCL6 protect against H O -induced oxidative stress via repression of Nox4

In efforts to investigate how Panx3/BCL6 interactions would affect BCL6 transcriptional activity, BCL6 expression was first assessed in EC Panx3^^A tissue. BCL6 mRNA is unaffected by loss of Panx3 (right column), though BCL6 protein is significantly decreased, suggesting BCL6 may be destabilized in the absence of Panx3 (right column) (Figure 6A-6B). Notably, this reduction in vascular BCL6 abundance was not associated with alterations in the development of B220⁺GL7⁺FasL⁺ splenic germinal centers via flow cytometry (Figure 11L). To understand how this might alter oxidative-related genes, we first predicted BCL6 transcriptional activity via in silico sequence assessments of the BCL6 DNA binding motif (Figure 6C). The BCL6 DNA binding motif was detected in the promoter region of BCL6-sensitive genes in endothelium (Ccna2, Ccnbl, Hesl, and DU4) (Buchberger et al., 2017), as well as Nfkbia, Nfkb2, and Nox-l, but not Noxl or Cybb (Figure 6C-6D). This was further corroborated through evaluation of all publicly available murine BCL6 ChlP-Seq datasets (Barish et al., 2010; X. Liu et al., 2016; Sommars et al., 2019), which revealed numerous instances of BCL6 binding of autoregulated NFKB family members Nfkbia and Nfkb2 (Figure 6E-6F). Intriguingly, demonstration of BCL6 repression predicts upregulation of transcript abundance in vascular tissues of EC Panx3^AfA mice for multiple genes, suggesting loss of BCL6 transcriptional activity in the absence of endothelial Panx3. Nfkbia and Nfkb2 are significantly upregulated (Figure 6J-6K), suggesting a BCL6-dependent increase in NFKB activity following loss of endothelial Panx3. Because NFKB activity is associated with increased expression of vascular NADPH Oxidase enzymes (Kim et al., 2019; Manea et al., 2008), we next interrogated their expression following deletion of endothelial Panx3. Nox4 transcripts are uniquely upregulated following loss of endothelial Panx3 (Figure 6L-6N), which can be explained by differential regulation of Nox expression by BCL6 (Figure 6G-6I). In the absence of endothelial Panx3, BCL6 is destabilized resulting in de-repression of NFKB family members and Nox4. To examine the vascular oxidative state, the abundance of Nox4 was assessed via western blot. Following loss of Panx3, Nox4, but not cofactor p22^phox, was upregulated (Figure 7A). Because Nox4 is understood to be a constitutively active oxidase (Nisimoto et al., 2010, 2014) largely generating H2O2 (Nisimoto et al., 2014), we next assessed oxidative damage in the circulation and vascular wall. EC Panx3^^A mice (right column) exhibited increased 3 -nitrotyrosine (3NT) protein adducts (Figure 7B) and increased H2O2 levels in deproteinized blood plasma (Figure 7C). Small arteries from EC Panx3^^A mice (right column) exhibit increased levels of hyperoxidized peroxiredoxin, a specific measure of chronic H2O2 generation (Cox et al., 2010) (Figure 7D). In large arteries, hyperoxidized peroxiredoxin and 3NT protein adducts are significantly increased, while 4-hydroxynonenal, marker of lipid peroxidation, exhibited a similar trend (Figure 13A-13C). No changes were observed in eNOS mRNA or coupling status (Figure 14A-14B). Similarly, other redox related genes and H2O2- generating enzymes Xdh, Eroll, Prx4 and Cyb5r3 were not altered by Panx3 expression (Figure 14C-14F). Finally, ACh dose response curves were repeated on mesenteric arteries in the presence of HzCh-scavenger catalase (lOOOU/mL Pegylated-Catalase). Catalase treatment abolished the significant difference in dilation between Panx3fl^/fl and EC Panx3^A'^A arteries (Figure 7E). These data suggest Panx3-BCL6 interactions stabilize BCL6, which represses endothelial Nox4 expression under normal physiological conditions. In the absence of Panx3, Nox4 de-repression results in increased abundance and resultant chronic H2O2 generation in the vascular wall, impairing endothelial-mediated dilation of small arteries.

Example 4: Blocking Panx3-BCL6 interactions phenocopies genetic deletion of endothelial Panx3

To further demonstrate the role for Panx3-BCL6 interactions in maintaining oxidative balance, a mimetic peptide was designed to disrupt the Panx3-BCL6 complex. BCLiP (SEQ ID NO. 2), which mimics L328-P344 of BCL6 (SEQ ID NO. 10), was selected from a pool of 10 candidate mimetic sequences for its potential to competitively inhibit the Panx3-BCL6 interaction (Figure 8A-B, Table 3, Figure 15). To assess the ability of BCLiP (SEQ ID NO. 2) to disrupt the Panx3-BCL6 interaction, PANX3 and BCL6 were expressed in HEK293T cells and exposed to BCLiP (SEQ ID NO. 2). Following immunoprecipitation of PANX3- Flag, BCL6 pull down was reduced by BCLiP (SEQ ID NO. 2) treatment (Figure 8C). Next, stearylated BCLiP (SEQ ID NO. 2) peptide or scramble control (SEQ ID NO. 3) was administered to C57Bl/6n mice (12.5mg/kg via i.p. for five days) to assess its ability to disrupt the Panx3-BCL6 interaction in vivo. Similar to the observation in EC Panx3^A/A mice, BCL6 transcripts were unaffected by BCLiP (SEQ ID NO. 2, right column) exposure (Figure 8D), but BCL6 protein abundance was significantly reduced by BCLiP (SEQ ID NO. 2) treatment at 2.5 mg/kg and 12.5 mg/kg (Figure 8E), supporting the hypothesis that BCLiP (SEQ ID NO. 2) promotes BCL6 degradation, likely due to inhibition of Panx3-BCL6 interactions. NFKB activity was increased following BCLiP (SEQ ID NO. 2, right column) exposure (Figure 8F- 8G), further mimicking the EC Panx3^A/A phenotype. Specific upregulation of Nox4, but not Noxl or Cybb, was also induced following BCLiP (SEQ ID NO. 2) administration (Figure 8H- 8J). Finally, BCLiP (SEQ ID NO. 2) administration induced a sustained increase in mean arterial, systolic and diastolic blood pressure (Figure 8K-8M), recapitulating hypertension observed following genetic deletion of endothelial Panx3.

Discussion of Examples:

Using both genetic and pharmacological systems, we present the first demonstration of the Panx3-BCL6 interaction as a regulator of vascular oxidative stress and systemic blood pressure. In our current model (Figure 16), endothelial Panx3 distributes to the Golgi membrane where it can interact with the extranuclear pool of BCL6. In the absence of Panx3 or following peptide inhibition of the Panx3-BCL6 interaction, the abundance of BCL6 is significantly reduced, NFKB activity is increased, and Nox4 is selectively upregulated, driving chronic H2O2 generation and elevating blood pressure. In this system, imbalance in endogenous ROS generation drives oxidative stress and cardiovascular disease.

Oxidative stress is a critical determinate of cardiovascular health. 02" has well- established roles driving hypertension(Dikalova et al., 2010), atherosclerosis (Vendrov et al., 2007), and ischemia/reperfusion injury (Chouchani et al., 2016). Obesity-related imbalances of peroxynitrite (ONOO") in endothelium have been shown to impair endothelial cell function and drive hypertension (Ottolini et al., 2020). Circulating levels of 02" and H2O2 are increased in hypertensive patients, though the abundance of both ROS were effectively reduced when antihypertensive therapies successfully attenuate blood pressure (Prabha et al., 1990). However, redox signaling is a tenet of normal cardiovascular physiology. H2O2 in particular has been recognized as a major redox signaling molecule involved in many beneficial cell processes (Cseko et al., 2004; Gao et al., 2003; Saeedi Saravi et al., 2020; Yamaguchi K, 1994). Perhaps it is not surprising that administration of general antioxidants have been ineffective at reducing oxidative stress-related cardiovascular diseases (Ward et al., 2005). It is possible that healthy physiological systems require a balance, not an ablation, of oxidant generation and antioxidant defenses to avoid injurious effect. Indeed, ROS are mediators of a variety of cellular processes in the vasculature including but not limited to nitric oxide (NO)-mediated dilation, and 02" production downstream of renin-angiotensin-aldosterone signaling. Here we uncover a novel mechanism of redox dysregulation which develops following disruption of the Panx3/BCL6 interaction, involving persistent overproduction of H2O2. Initial studies in human patients hint that maintenance of the Panx3-BCL6 interaction may hold clinical relevance as a potential therapeutic target for resistant hypertension (Figure 1A-1B). Developing clinical strategies to target specific ROS imbalance may prove to be more effective than general antioxidants administration for cardiovascular diseases.

Our evidence implicates oxidative dysfunction of resistance arteries and blood pressure is due to H2O2, likely by Nox4. However, although the majority of reactive species produced by Nox4 appear to be H2O2, it does not completely preclude effects due to superoxide (02‘) (Nisimoto et al., 2014). We observe a significant increase in 3 -nitrotyrosine in the circulation (Figure 7B) and aortic wall (Figure 13B) following loss of endothelial Panx3. 3NT protein adducts are canonically generated following tyrosine nitration by ONOO". H2O2 is insufficient to directly drive production of ONOO", but nitric oxide (NO) reacts rapidly with 02" to generate ONOO", which may suggest dysregulation of 02" following genetic deletion of endothelial Panx3. However, it is unclear if this occurs downstream of disrupting the Panx3-BCL6 interaction because tyrosine nitration can alternatively be generated when H2O2 and nitrite (N02‘) react with heme-containing proteins (Grzelak et al., 2001), such as endothelial a -globin (Keller et al., 2022; Straub et al., 2012).

In the vasculature, H2O2 has been shown to induce contradictory vasoactive effects based on dose, duration, and vascular tone. Ex vivo systems across multiple species and vascular beds demonstrate exposure to low levels (<10'⁴M H2O2) strengthens basal tone and induces a mild constriction, likely mediated by thromboxane A2/prostaglandin H2 (Cseko et al., 2004; Gao et al., 2003; Yamaguchi K, 1994). Higher concentrations (>10'⁴M H2O2) induce a potent dilatory response following a brief constriction (Cseko et al., 2004; Gao et al., 2003; Yamaguchi K, 1994), with many works suggesting a role for H2O2 as an endothelium-derived hyperpolarization factor (EDHF) (Larsen et al., 2008; Yada et al., 2003). Constitutive endothelial-specific overexpression of Nox4 has been reported to lower blood pressure (Ray et al., 2011). While Nox4 expression was not quantified, vascular H2O2 was increased 7-fold in this system via xylenol orange assay, suggesting a dramatic increase in Nox4 abundance in their system (Ray et al., 2011). In contrast, interruption of the Panx3-BCL6 interaction, either from our genetic model or following BCLiP (SEQ ID NO. 2) administration, results in mild, but significant increase in expression and abundance of H2O2-producing Nox4 (Figure 6N, Figure 7A, Figure 8J). This was associated with catalase-sensitive impairments in ACh- induced dilation (Figure 41, Figure 7E) and systemic hypertension (Figure 4A-4B) following genetic deletion of endothelial Panx3. Thus, interruption of the Panx3-BCL6 interaction induces a mild elevation of intracellular H2O2, as the catalase-sensitive dose response to ACh was significantly impaired. Interestingly, arteries from EC Panx3^^A mice also exhibit reduced dilation to NS309 (Figure 4L), suggesting that H2O2 imbalances impair conductance through small- and intermediate-conductance Ca²⁺-activated K⁺ channels (SK, IK). This finding is not in alignment with reports that exposure to H2O2 promotes conductance through SK/IK channels, though the discrepancy may be due to high doses of H2O2 (>10'⁴M) used in the study (Behringer et al., 2013).

Indeed, much of the literature on the effects of H2O2 involve bath applied exposure to exogenous H2O2, often provided at supraphysiological levels. Recent advances in chemogenetic H2O2 probes have been used to demonstrate endogenous imbalance of H2O2 can drive distinct localized cellular effects in endothelial cells as compared to application of exogenous H2O2, even when intracellular H2O2 abundance is perturbed to the same degree (Saeedi Saravi et al., 2020). For instance, when the H2O2 generating probe was targeted to caveolae at the plasma membrane, H2O2 generation caused a rapid increase in inhibitory eNOS phosphorylation at Thr⁴⁹⁵ which was not observed following bath exposure to the same concentration of exogenous H2O2 (Saeedi Saravi et al., 2020). The work presented here establishes EC Panx3^^A mice and BCLiP (SEQ ID NO. 2) administration as models of redox imbalance which could be used for future studies uncovering role of mild H2O2 elevation generated from cellular dysfunction rather than exogenous addition.

BCLiP (SEQ ID NO. 2), a novel BCL6 mimetic peptide in accordance with the presently disclosed subject matter, was generated as an orthogonal approach to inhibit the Panx3-BCL6 interaction from our genetic model. BCLiP (SEQ ID NO. 2) exposure reduced the ability of PANX3 to immunoprecipitate with BCL6 from human cells (Figure 8C). After five days of IP administration to wildtype mice, BCLiP (SEQ ID NO. 2) exposure successfully recapitulates every mechanistic step of our model: including (1) reduction in BCL6 protein, but not mRNA (Figure 6A-6B, Figure 8D-8E), (2) increased NFKB activity (Figure 6J-6K, Figure 8F-6G), (3) specific upregulation of Nox4 (Figure 6L-6N, Figure 8H-6J) and (4) elevation of blood pressure (Figure 4A-6B, Figure 8K-6M). Further refinement of the dosing schedule and route of administration can likely escalate the effect of BCLiP (SEQ ID NO. 2) on blood pressure. While the use of the BCLiP peptide (SEQ ID NO. 2) enabled us to corroborate our findings from our genetic model, we recognize its various limitations. First, we cannot draw conclusions about the participation of the L328-P344 region of BCL6, (SEQ ID NO. 10), in the endogenous Panx3-BCL6 interactions from our current findings. Moreover, given that BCLiP (SEQ ID NO. 2) mimics a region of BCL6 involved in interaction with MAPK, BCLiP (SEQ ID NO. 2) could potentially compete with BCL6 for MAPK binding. However, inhibition of BCL6 binding MAPK would protect BCL6 from targeted degradation. Our evidence contradicts this prediction, as BCLiP (SEQ ID NO. 2) exposure is associated with reduced BCL6 protein abundance (Figure 8E). In addition, BCLiP (SEQ ID NO. 2) indiscriminately interrupts Panx3-BCL6 interactions, thus we cannot preclude the possibility of BCLiP (SEQ ID NO. 2) additionally inducing oxidative stress in other cell types, such as lymphocytes from which the Panx3-BCL6 interaction was first reported (Miles et al., 2005). Endogenous Nox4 expression is largely restricted to the vasculature and the kidney (Thul et al., 2017), which would limit these confounding effects. Despite these limitations, BCLiP (SEQ ID NO. 2) administration phenocopies genetic inhibition of the Panx3-BCL6 interaction.

BCL6 stabilization is crucial to redox balance though this has not been studied in endothelium. During germinal center B cell development, BCL6 is required to drive B cell proliferation and survival despite genotoxic and oxidative damage (Dent et al., 1997; Fukuda et al., 1997; Nakagawa et al., 2021; Ye et al., 1997). Whole mouse genetic knockout of BCL6 results in premature cardiovascular death due to myocarditis and pulmonary vasculitis by mechanisms that still are not understood (Ye et al., 1997). Studies of BCL6 in endothelium have been limited to its role in angiogenesis, where BCL6 has been reported to inhibit vascular sprouting and branching (Buchberger et al., 2017), possibly through BCL6-associated zinc finger protein (BAZF)-mediation of VEGF signaling and downregulation of notch signaling (Ohnuki et al., 2012). The current study presents the first evidence of a homeostatic role for repression by BCL6 in endothelium to prevent vascular oxidative stress, as well as a novel regulatory mechanism of BCL6 activity via interactions with Panx3. The transcriptional activity of BCL6 is canonically regulated through its targeted degradation, which is accomplished by multiple MAPK phosphorylation sites (including S333 and S343) within PEST motifs driving recruitment to a SCF ubiquitin ligase complex containing FBXO11 (Niu et al., 1998). Minor alterations in the balance of BCL6 expression and degradation have been reported to significantly alter the transcriptome (Duan et al., 2012). In endothelium, the expression of Panx3 promotes BCL6 stability (Figure 6A-6B), suggesting that the Panx3- BCL6 perinuclear interaction inhibits BCL6 degradation. Here, BCL6 protein is significantly reduced, and NFKB activity (Figure 6J-6K) and expression of Nox4 (Figure 6N) are significantly increased. Mutual antagonism between BCL6 and NFKB has been described in other cell types (Barish et al., 2010; Perez-Rosado et al., 2008), but these data are the first description of this negative feedback regulation of vascular NFKB. BCL6 has previously been suggested to regulate vascular Nox4 expression: spontaneously hypertensive rats (SHR) exhibit increased Nox4 expression and reduced BCL6 expression as compared to Wistar-Kyoto rats (WKY) (D. Chen et al., 2019). Furthermore lentiviral expression of BCL6 was able to rescue Nox4 expression and reduce systemic blood pressure in SHR (D. Chen et al., 2019), further supporting that stabilization of Panx3-BCL6 has potential as antihypertensive therapy. Overall, the present data suggests BCL6 promotes homeostatic redox signaling; in the vasculature, interactions with Panx3 maintain BCL6 expression.

This study also elucidates novel roles and characteristics of endogenous Panx3, for which there is little described in the literature. Vascular Panx3 appears to exert its effect solely through this protein-protein interaction. Although it is noncanonical for a channel-based protein to have channel-independent functions, there are examples from other large-pore channels, including connexins and innexins where this has been documented (Johnstone et al., 2010, 2012; Miao et al., 2020). Unlike Panxl, many characteristics of Panx3 appear to vary based on cell type and expression system. Many of the previous works reporting functional roles (dye transfer (Celetti et al., 2010; Ishikawa et al., 2011; Penuela et al., 2007; Whyte- Fagundes et al., 2018), ATP release (Ishikawa et al., 2011; Iwamoto et al., 2010; Whyte- Fagundes et al., 2018), calcium store release (Ishikawa et al., 2011; Iwamoto et al., 2010)) for Panx3 rely on exogenous overexpression systems, which can dramatically alter Panx3 subcellular distribution (Penuela et al., 2007, 2009) and potentially even Panx3 oligomeric state. The endothelial-specific Panx3 knockout mouse model enabled us to study the function of endogenous Panx3 despite its low expression level. For example, we found Panx3 does not contribute to endothelial purinergic signaling (Figure 5A-5B) or IP3-mediated calcium store release (Figure 5C) in intact endothelium. In the vasculature, Panx3 is not sensitive to deglycosylation by PNGase F (Figure 2D), and Panx3 is retained in the membrane of the Golgi apparatus (Figure 2A, Figure 10A), suggesting that Panx3 exhibits minimal, if any, glycosylation in this tissue. The Panx3 structural conformation is another parameter for which the field has little direct evidence. In order to generate our predicted Panx3 structure, we levied AlphaFol d2 computational structural predictions with the known cryo-EM structure of Panxl and the Panx3 amino acid sequence. Panxl has been well-documented as a heptameric large- pore ion channel (Michalski et al., 2020; Nielsen et al., 2020). However, the precise structure of Panx3 has yet to be described. Our crosslinking studies were insufficient to determine the exact Panx3 oligomeric state, though resolution of the dimer suggests that the Panx3 oligomer may be more plastic that the Panxl heptamer (Figure 5G). As a comparison, chemical crosslinking of Panx2 was previously used to demonstrate octameric oligomerization (Ambrosi et al., 2010), which sets precedence that pannexin isoforms may exhibit variation in oligomeric state. Together, these data suggest Panx3 to be a highly plastic channel protein. In endothelium, there is no direct evidence for channel functionality; instead, we posit that Golgi- localized Panx3 exerts oxidative effects on the cardiovascular system through protein-protein interactions with BCL6.

REFERENCES

All references listed herein including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries (e.g., GENBANK® database entries and all annotations available therein) are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or compositions employed herein.

Alinikula, J., Nera, K.-P., Junttila, S., & Lassila, O. (2011). Alternate pathways for Bcl6- mediated regulation of B cell to plasma cell differentiation. European Journal of Immunology, 41(8), 2404-2413. https://doi.org/10.1002/eji.201141553

Ambrosi, C., Gassmann, O., Pranskevich, J. N., Boassa, D., Smock, A., Wang, J., Dahl, G, Steinem, C., & Sosinsky, G. E. (2010). Pannexinl and Pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other. The Journal of Biological Chemistry, 285(32), 24420-24431. https://doi.org/10.1074/jbc.M110.115444

Axelsson, M. A., Karlsson, N. G, Steel, D. M., Ouwendijk, J., Nilsson, T., & Hansson, G. C. (2001). Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins. Glycobiology, 11(8), 633-644. https://doi.org/10.1093/glycob/l E8.633

Bachert, C., Lee, T. H., & Linstedt, A. D. (2001). Lumenal endosomal and Golgi -retrieval determinants involved in pH-sensitive targeting of an early Golgi protein. Molecular Biology of the Cell, 72(10), 3152-3160. https://doi.org/10.1091/mbc.12.10.3152

Bailey, T. L., Johnson, J., Grant, C. E., & Noble, W. S. (2015). The MEME Suite. Nucleic Acids Research, 43 ), W39-49. https://doi.org/10.1093/nar/gkv416

Barish, G. D., Yu, R. T., Karunasiri, M., Ocampo, C. B., Dixon, J., Benner, C., Dent, A. L., Tangirala, R. K., & Evans, R. M. (2010). Bcl-6 and NF-kappaB cistromes mediate opposing regulation of the innate immune response. Genes & Development, 24(24), 2760-2765. https://doi.org/10.1101/gad.1998010 Behringer, E. J., Shaw, R. L., Westcott, E. B., Socha, M. J., & Segal, S. S. (2013). Aging impairs electrical conduction along endothelium of resistance arteries through enhanced Ca2+- activated K+ channel activation. Arteriosclerosis, Thrombosis, and Vascular Biology, 33(8), 1892-1901. https://doi.org/10.1161/ATVBAHA.113.301514

Billaud, M., Chiu, Y.-H., Lohman, A. W., Parpaite, T., Butcher, J. T., Mutchler, S. M., DeLaho, L. J., Artamonov, M. V, Sandilos, J. K., Best, A. K., Somlyo, A. V, Thompson, R. J., Le, T. H., Ravichandran, K. S., Bayliss, D. A., & Isakson, B. E. (2015). A molecular signature in the pannexinl intracellular loop confers channel activation by the al adrenoreceptor in smooth muscle cells. Science Signaling, 5(364), ral7. https://doi.org/10.1126/scisignal.2005824

Billaud, M., Lohman, A. W., Straub, A. C., Looft-Wilson, R., Johnstone, S. R., Araj, C. A., Best, A. K., Chekeni, F. B., Ravichandran, K. S., Penuela, S., Laird, D. W., & Isakson, B. E. (2011). Pannexinl regulates al-adrenergic receptor- mediated vasoconstriction. Circulation Research, 109(1), 80-85. https://doi.org/10.1161/CIRCRESAHA.110.237594

Bocharnikov, A. V, Keegan, J., Wacleche, V. S., Cao, Y., Fonseka, C. Y., Wang, G., Muise, E. S., Zhang, K. X., Arazi, A., Keras, G., Li, Z. J., Qu, Y., Gurish, M. F., Petri, M., Buyon, J. P., Putterman, C., Wofsy, D., James, J. A., Guthridge, J. M., ... Accelerating Medicines Partnership (AMP) RA/SLE Network. (2019). PD-lhiCXCR5- T peripheral helper cells promote B cell responses in lupus via MAF and IL-21. JCI Insight, 4(20). https://doi.org/10.1172/jci.insight.130062

Buchberger, E., Payrhuber, D., El Harchi, M., Zagrapan, B., Scheuba, K., Zommer, A., Bugyik, E., Dome, B., Kral, J. B., Schrottmaier, W. C., Schabbauer, G, Petzelbauer, P., Grbger, M., Bilban, M., & Brostjan, C. (2017). Inhibition of the transcriptional repressor complex Bcl- 6/BCoR induces endothelial sprouting but does not promote tumor growth. Oncotarget, 5(1), 552-564. https://doi.org/10.18632/oncotarget.13477

Cardenas, M. G, Yu, W., Beguelin, W., Teater, M. R., Geng, H., Goldstein, R. L., Oswald, E., Hatzi, K., Yang, S.-N., Cohen, J., Shaknovich, R., Vanommeslaeghe, K., Cheng, H., Liang, D., Cho, H. J., Abbott, J., Tam, W., Du, W., Leonard, J. P., ... Melnick, A. M. (2016). Rationally designed BCL6 inhibitors target activated B cell diffuse large B cell lymphoma. The Journal of Clinical Investigation, 126(9), 3351-3362. https://doi.org/10.1172/JCI85795

Celetti, S. J., Cowan, K. N., Penuela, S., Shao, Q., Churko, J., & Laird, D. W. (2010).

Implications of pannexin 1 and pannexin 3 for keratinocyte differentiation. Journal of Cell Science, 123(Pt 8), 1363-1372. https://doi.org/10.1242/jcs.056093

Chang, C. C., Ye, B. H., Chaganti, R. S., & Dalla-Favera, R. (1996). BCL-6, a POZ/zinc-finger protein, is a sequence-specific transcriptional repressor. Proceedings of the National Academy of Sciences of the United States of America, 93(14), 6947-6952. https://doi.org/10.1073/pnas.93.14.6947

Chekeni, F. B., Elliott, M. R., Sandilos, J. K., Walk, S. F., Kinchen, J. M., Lazarowski, E. R., Armstrong, A. J., Penuela, S., Laird, D. W., Salvesen, G. S., Isakson, B. E., Bayliss, D. A., & Ravichandran, K. S. (2010). Pannexin 1 channels mediate “find-me” signal release and membrane permeability during apoptosis. Nature, 467(7317), 863-867. https://doi.org/10.1038/nature09413

Chen, D., Zang, Y.-H., Qiu, Y., Zhang, F., Chen, A.-D., Wang, J. -J., Chen, Q., Li, Y.-H., Kang, Y.-M., & Zhu, G.-Q. (2019). BCL6 Attenuates Proliferation and Oxidative Stress of Vascular Smooth Muscle Cells in Hypertension. Oxidative Medicine and Cellular Longevity, 2019, 5018410. https://doi.org/10.1155/2019/5018410

Cheng, T. M.-K., Blundell, T. L., & Femandez-Recio, J. (2007). pyDock: electrostatics and desolvation for effective scoring of rigid-body protein-protein docking. Proteins, 68(2), 503-515. https://doi.org/10.1002/prot.21419

Chen, Y. L., Baker, T. M., Lee, F., Shui, B., Lee, J. C., Tvrdik, P., Kotlikoff, M. I., & Sonkusare, S. K. (2021). Calcium Signal Profiles in Vascular Endothelium from Cdh5-GCaMP8 and Cx40-GCaMP2 Mice. Journal of Vascular Research, 55(3), 159-171. https://doi.org/10.1159/000514210

Choi, J., & Crotty, S. (2021). Bcl6-Mediated Transcriptional Regulation of Follicular Helper T cells (TFH). Trends in Immunology , 42(4), 336-349. https://doi.Org/10.1016/j.it.2021.02.002

Chouchani, E. T., Pell, V. R., James, A. M., Work, L. M., Saeb-Parsy, K., Frezza, C., Krieg, T., & Murphy, M. P. (2016). A Unifying Mechanism for Mitochondrial Superoxide Production during Ischemia-Reperfusion Injury. Cell Metabolism, 23(2), 254-263. https://doi.Org/10.1016/j.cmet.2015.12.009

Cox, A. G., Winterboum, C. C., & Hampton, M. B. (2010). Measuring the redox state of cellular peroxiredoxins by immunoblotting. Methods in Enzymology, 474, 51-66. https://doi.org/! 0.1016/S0076-6879(l 0)74004-0

Cseko, C., Bagi, Z., & Koller, A. (2004). Biphasic effect of hydrogen peroxide on skeletal muscle arteriolar tone via activation of endothelial and smooth muscle signaling pathways. Journal of Applied Physiology (Bethesda, Md. : 1985), 97(3), 1130-1137. https://doi.org/10.1152/japplphysiol.00106.2004

Deb, D., Rajaram, S., Larsen, J. E., Dospoy, P. D., Marullo, R., Li, L. S., Avila, K., Xue, F., Cerchietti, L., Minna, J. D., Altschuler, S. J., & Wu, L. F. (2017). Combination Therapy Targeting BCL6 and Phospho-STAT3 Defeats Intratumor Heterogeneity in a Subset of NonSmall Cell Lung Cancers. Cancer Research, 77(11), 3070-3081. https://doi.org/10.1158/0008-5472.CAN-15-3052

DeLalio, L. J., Billaud, M., Ruddiman, C. A., Johnstone, S. R., Butcher, J. T., Wolpe, A. G., Jin, X., Keller, T. C. S., Keller, A. S., Riviere, T., Good, M. E., Best, A. K., Lohman, A. W., Swayne, L. A., Penuela, S., Thompson, R. J., Lampe, P. D., Yeager, M., & Isakson, B. E. (2019). Constitutive SRC-mediated phosphorylation of pannexin 1 at tyrosine 198 occurs at the plasma membrane. The Journal of Biological Chemistry, 294(VT), 6940-6956. https://doi.org/10.1074/jbc.RA118.006982

DeLalio, L. J., Keller, A. S., Chen, J., Boyce, A. K. J., Artamonov, M. V, Askew-Page, H. R., Keller, T. C. S., Johnstone, S. R., Weaver, R. B., Good, M. E., Murphy, S. A., Best, A. K., Mintz, E. L., Penuela, S., Greenwood, I. A., Machado, R. F., Somlyo, A. V, Swayne, L. A., Minshall, R. D., & Isakson, B. E. (2018). Interaction Between Pannexin 1 and Caveolin-1 in Smooth Muscle Can Regulate Blood Pressure. Arteriosclerosis, Thrombosis, and Vascular Biology, 38(9), 2065-2078. https://doi.org/10.1161/ATVBAHA.118.311290

DeLalio, L. J., Masati, E., Mendu, S., Ruddiman, C. A., Yang, Y., Johnstone, S. R., Milstein, J. A., Keller, T. C. S., Weaver, R. B., Guagliardo, N. A., Best, A. K., Ravichandran, K. S., Bayliss, D. A., Sequeira-Lopez, M. L. S., Sonkusare, S. N., Shu, X. H., Desai, B., Barrett, P. Q., Le, T. H., ... Isakson, B. E. (2020). Pannexin 1 channels in renin-expressing cells influence renin secretion and blood pressure homeostasis. Kidney International, 98(3), 630- 644. https://doi.Org/10.1016/j.kint.2020.04.041

Dent, A. L., Shaffer, A. L., Yu, X., Allman, D., & Staudt, L. M. (1997). Control of inflammation, cytokine expression, and germinal center formation by BCL-6. Science (New York, N.Y.), 276(5312), 589-592. https://doi.org/10.1126/science.276.5312.589

Deweindt, C., Albagli, O., Bemardin, F., Dhordain, P., Quief, S., Lantoine, D., Kerckaert, J. P., & Leprince, D. (1995). The LAZ3/BCL6 oncogene encodes a sequence-specific transcriptional inhibitor: a novel function for the BTB/POZ domain as an autonomous repressing domain. Cell Growth & Differentiation : The Molecular Biology Journal of the American Association for Cancer Research, 6(12), 1495-1503.

Dikalova, A. E., Bikineyeva, A. T., Budzyn, K., Nazarewicz, R. R., McCann, L., Lewis, W., Harrison, D. G, & Dikalov, S. I. (2010). Therapeutic targeting of mitochondrial superoxide in hypertension. Circulation Research, 107( ), 106-116. https://doi.org/10.1161/CIRCRESAHA.109.214601

Duan, S., Cermak, L., Pagan, J. K., Rossi, M., Martinengo, C., di Celle, P. F., Chapuy, B., Shipp, M., Chiarle, R., & Pagano, M. (2012). FBXO11 targets BCL6 for degradation and is inactivated in diffuse large B-cell lymphomas. Nature, 457(7379), 90-93. https : //doi . org/10.1038/nature 10688

Duy, C., Yu, J. J., Nahar, R., Swaminathan, S., Kweon, S.-M., Polo, J. M., Valls, E., Klemm, L., Shojaee, S., Cerchietti, L., Schuh, W., Jack, H.-M., Hurtz, C., Ramezani-Rad, P., Herzog, S., Jumaa, H., Koeffler, H. P., de Alboran, I. M., Melnick, A. M., ... Miischen, M. (2010). BCL6 is critical for the development of a diverse primary B cell repertoire. The Journal of Experimental Medicine, 207(6), 1209-1221. https://doi.org/10.1084/jem.20091299

Fukuda, T., Yoshida, T., Okada, S., Hatano, M., Miki, T., Ishibashi, K., Okabe, S., Koseki, H., Hirosawa, S., Taniguchi, M., Miyasaka, N., & Tokuhisa, T. (1997). Disruption of the Bcl6 gene results in an impaired germinal center formation. The Journal of Experimental Medicine, 186(3), 439-448. https://doi.Org/10.1084/jem.186.3.439

Gao, Y.-J., Hirota, S., Zhang, D.-W., Janssen, L. J., & Lee, R. M. K. W. (2003). Mechanisms of hydrogen-peroxide-induced biphasic response in rat mesenteric artery. British Journal of Pharmacology, 138(6), 1085-1092. https://doi.org/10.1038/sj.bjp.0705147

Geng, H., Brennan, S., Milne, T. A., Chen, W.-Y., Li, Y., Hurtz, C., Kweon, S.-M., Zickl, L., Shojaee, S., Neuberg, D., Huang, C., Biswas, D., Xin, Y., Racevskis, J., Ketterling, R. P., Luger, S. M., Lazarus, H., Tailman, M. S., Rowe, J. M., ... Melnick, A. M. (2012). Integrative epigenomic analysis identifies biomarkers and therapeutic targets in adult B- acute lymphoblastic leukemia. Cancer Discovery, 2(11), 1004-1023. https://doi.org/10.1158/2159-8290.CD-12-0208

Gensous, N., Schmitt, N., Richez, C., Ueno, H., & Blanco, P. (2017). T follicular helper cells, interleukin-21 and systemic lupus erythematosus. Rheumatology (Oxford, England), 56(4), 516-523. https://doi.org/! 0.1093/rheumatology/kew297

Good, M. E., Chiu, Y.-H., Poon, I. K. H., Medina, C. B., Butcher, J. T., Mendu, S. K., DeLalio, L. J., Lohman, A. W., Leitinger, N., Barrett, E., Lorenz, U. M., Desai, B. N., Jaffe, I. Z., Bayliss, D. A., Isakson, B. E., & Ravichandran, K. S. (2018). Pannexin 1 Channels as an Unexpected New Target of the Anti-Hypertensive Drug Spironolactone. Circulation Research, 122(4), 606-615. https://doi.org/10.1161/CIRCRESAHA.117.312380

Grzelak, A., Balcerczyk, A., Mateja, A., & Bartosz, G. (2001). Hemoglobin can nitrate itself and other proteins. Biochimica et Biophysica Acta, 1528(2-3), 97-100. https: //doi . org/10.1016/s0304-4165(01 )00176-3

Halliwill, K. D., Quigley, D. A., Kang, H. C., Del Rosario, R., Ginzinger, D., & Balmain, A. (2016). Panx3 links body mass index and tumorigenesis in a genetically heterogeneous mouse model of carcinogen-induced cancer. Genome Medicine, 5(1), 83. https://doi.org/10.1186/sl3073-016-0334-8

Hatzi, K., & Melnick, A. (2014). Breaking bad in the germinal center: how deregulation of BCL6 contributes to lymphomagenesis. Trends in Molecular Medicine , 20(6), 343-352. https://doi.Org/10.1016/j.molmed.2014.03.001

Heink, S., Yogev, N., Garbers, C., Herwerth, M., Aly, L., Gasperi, C., Husterer, V., Croxford, A. L., Moller-Hackbarth, K., Bartsch, H. S., Sotlar, K., Krebs, S., Regen, T., Blum, H., Hemmer, B., Misgeld, T., Wunderlich, T. F., Hidalgo, J., Oukka, M., ... Kom, T. (2017). Trans-presentation of IL-6 by dendritic cells is required for the priming of pathogenic TH 17 cells. Nature Immunology, 75(1), 74-85. https://doi.org/10.1038/ni.3632

Hurtz, C., Chan, L. N., Geng, H., Ballabio, E., Xiao, G, Deb, G, Khoury, H., Chen, C.-W., Armstrong, S. A., Chen, J., Ernst, P., Melnick, A., Milne, T., & Miischen, M. (2019). Rationale for targeting BCL6 in MLL-rearranged acute lymphoblastic leukemia. Genes & Development, 33(17-18), 1265-1279. https://doi.org/10.1101/gad.327593.119

Hurtz, C., Hatzi, K., Cerchietti, L., Braig, M., Park, E., Kim, Y., Herzog, S., Ramezani-Rad, P., Jumaa, H., Muller, M. C., Hofmann, W.-K., Hochhaus, A., Ye, B. H., Agarwal, A., Druker, B. J., Shah, N. P., Melnick, A. M., & Miischen, M. (2011). BCL6-mediated repression of p53 is critical for leukemia stem cell survival in chronic myeloid leukemia. The Journal of Experimental Medicine, 205(11), 2163-2174. https://doi.org/10.1084/jem.20110304 Ishikawa, M., Iwamoto, T., Nakamura, T., Doyle, A., Fukumoto, S., & Yamada, Y. (2011).

Pannexin 3 functions as an ER Ca(2+) channel, hemi channel, and gap junction to promote osteoblast differentiation. The Journal of Cell Biology, 193(7), 1257-1274. https://doi.org/10.1083/jcb.201101050

Ishikawa, M., Williams, G. L., Ikeuchi, T., Sakai, K., Fukumoto, S., & Yamada, Y. (2016).

Pannexin 3 and connexin 43 modulate skeletal development through their distinct functions and expression patterns. Journal of Cell Science, 129(5), 1018-1030. https://doi.org/10.1242/jcs.176883

Iwamoto, T., Nakamura, T., Doyle, A., Ishikawa, M., de Vega, S., Fukumoto, S., & Yamada, Y. (2010). Pannexin 3 regulates intracellular ATP/cAMP levels and promotes chondrocyte differentiation. The Journal of Biological Chemistry, 285(24), 18948-18958. https://doi.org/10.1074/jbc.M110.127027

Johnstone, S. R., Best, A. K., Wright, C. S., Isakson, B. E., Errington, R. J., & Martin, P. E. (2010). Enhanced connexin 43 expression delays intra-mitotic duration and cell cycle traverse independently of gap junction channel function. Journal of Cellular Biochemistry, 110(3), 772-782. https://doi.org/10.1002/jcb.22590

Johnstone, S. R., Kroncke, B. M., Straub, A. C., Best, A. K., Dunn, C. A., Mitchell, L. A., Peskova, Y., Nakamoto, R. K., Koval, M., Lo, C. W., Lampe, P. D., Columbus, L., & Isakson, B. E. (2012). MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation. Circulation Research, 111(2), 201-211. https://doi.org/10.1161/CIRCRESAHA.112.272302

Keller, T. C. S., Lechauve, C., Keller, A. S., Broseghini-Filho, G. B., Butcher, J. T., Askew Page, H. R., Islam, A., Tan, Z. Y., DeLaho, L. J., Brooks, S., Sharma, P., Hong, K., Xu, W., Padilha, A. S., Ruddiman, C. A., Best, A. K., Macal, E., Kim-Shapiro, D. B., Christ, G, ... Isakson, B. E. (2022). Endothelial alpha globin is a nitrite reductase. Nature Communications, 13(3), 6405. https://doi.org/10.1038/s41467-022-34154-3

Khan, S., Tavema, F., Rohlenova, K., Treps, L., Geldhof, V., de Rooij, L., Sokol, L., Pircher, A., Conradi, L.-C., Kalucka, J., Schoonjans, L., Eelen, G, Dewerchin, M., Karakach, T., Li, X., Goveia, J., & Carmeliet, P. (2019). EndoDB: a database of endothelial cell transcriptomics data. Nucleic Acids Research, 47 V) ), D736-D744. https://doi.org/10.1093/nar/gky997

Kim, J., Yoo, J.-Y., Suh, J. M., Park, S., Kang, D., Jo, H., & Bae, Y. S. (2019). The flagellin- TLR5-Nox4 axis promotes the migration of smooth muscle cells in atherosclerosis. Experimental & Molecular Medicine, 51(7), 1-13. https://doi.org/10.1038/sl2276-019- 0275-6

Krull, J. E., Wenzl, K., Hartert, K. T., Manske, M. K., Sarangi, V., Maurer, M. J., Larson, M. C., Nowakowski, G. S., Ansell, S. M., McPhail, E., Habermann, T. M., Link, B. K., King, R. L., Cerhan, J. R., & Novak, A. J. (2020). Somatic copy number gains in MYC, BCL2, and BCL6 identifies a subset of aggressive altemative-DH/TH DLBCL patients. Blood Cancer Journal, 10(11), 117. https://doi.org/10.1038/s41408-020-00382-3

Kurosu, T., Fukuda, T., Miki, T., & Miura, O. (2003). BCL6 overexpression prevents increase in reactive oxygen species and inhibits apoptosis induced by chemotherapeutic reagents in B- cell lymphoma cells. Oncogene, 22(29), 4459-4468. https://doi.org/10.1038/sj.onc.1206755

Larsen, B. T., Gutterman, D. D., Sato, A., Toyama, K., Campbell, W. B., Zeldin, D. C., Manthati, V. L., Falck, J. R., & Miura, H. (2008). Hydrogen peroxide inhibits cytochrome p450 epoxygenases: interaction between two endothelium-derived hyperpolarizing factors. Circulation Research, 102(1), 59-67. https://doi.org/10.1161/CIRCRESAHA.107.159129

Li, Q., Zhou, L., Wang, L., Li, S., Xu, G, Gu, H., Li, D., Liu, M., Fang, L., Wang, Z., Han, S., & Zheng, B. (2020). Bcl6 modulates innate immunity by controlling macrophage activity and plays critical role in experimental autoimmune encephalomyelitis. European Journal of Immunology, 50(4), 525-536. https://doi.org/10.1002/eji.201948299

Liu, X., Lu, H., Chen, T., Nallaparaju, K. C., Yan, X., Tanaka, S., Ichiyama, K., Zhang, X., Zhang, L., Wen, X., Tian, Q., Bian, X.-W., Jin, W., Wei, L., & Dong, C. (2016). Genome- wide Analysis Identifies Bcl6-Controlled Regulatory Networks during T Follicular Helper Cell Differentiation. Cell Reports, 14(7), 1735-1747. https://doi.Org/10.1016/j.celrep.2016.01.038

Lohman, A. W., Leskov, I. L., Butcher, J. T., Johnstone, S. R., Stokes, T. A., Begandt, D., DeLalio, L. J., Best, A. K., Penuela, S., Leitinger, N., Ravichandran, K. S., Stokes, K. Y., & Isakson, B. E. (2015). Pannexin 1 channels regulate leukocyte emigration through the venous endothelium during acute inflammation. Nature Communications, 6, 7965. https://doi.org/10.1038/ncomms8965

Madapura, H. S., Nagy, N., Ujvari, D., Kallas, T., Krohnke, M. C. L., Amu, S., Bjorkholm, M., Stenke, L., Mandal, P. K., McMurray, J. S., Keszei, M., Westerberg, L. S., Cheng, H., Xue, F., Klein, G., Klein, E., & Salamon, D. (2017). Interferon y is a STAT1 -dependent direct inducer of BCL6 expression in imatinib-treated chronic myeloid leukemia cells. Oncogene, 36(32), 4619-4628. https://doi.org/10.1038/onc.2017.85

Manea, A., Manea, S. A., Gafencu, A. V, Raicu, M., & Simionescu, M. (2008). AP-1 -dependent transcriptional regulation of NADPH oxidase in human aortic smooth muscle cells: role of p22phox subunit. Arteriosclerosis, Thrombosis, and Vascular Biology, 28(5), 878-885. https://doi.org/10.1161/ATVBAHA.108.163592

Marullo Rossella, Ahn Haelee, Cardenas Mariano, & Melnick Ari. (2016). The transcription factor BCL6 is a rational target in non-small cell lung cancer (NSCLC). Cancer Research, 76(14 Supplement), 1271-1271.

Miao, G, Godt, D., & Montell, D. J. (2020). Integration of Migratory Cells into a New Site In Vivo Requires Channel -Independent Functions of Innexins on Microtubules. Developmental Cell, 54(4), 501-515. e9. https://doi.Org/10.1016/j.devcel.2020.06.024

Michalski, K., Syrjanen, J. L., Henze, E., Kumpf, J., Furukawa, H., & Kawate, T. (2020). The Cryo-EM structure of pannexin 1 reveals unique motifs for ion selection and inhibition. ELife, 9. https://doi.org/10.7554/eLife.54670

Miles, R. R., Crockett, D. K., Lim, M. S., & El enitoba- Johnson, K. S. J. (2005). Analysis of BCL6-interacting proteins by tandem mass spectrometry. Molecular & Cellular Proteomics : MCP, 4(12), 1898-1909. https://doi.org/10.1074/mcp.M500112-MCP200

Mirdita, M., Schiitze, K., Moriwaki, Y., Heo, L., Ovchinnikov, S., & Steinegger, M. (2022). ColabFold: making protein folding accessible to all. Nature Methods, 19(6), 679-682. https://doi.org/10.1038/s41592-022-01488-l

Misawa, T., SoRelle, J. A., Choi, J. H., Yue, T., Wang, K.-W., McAlpine, W., Wang, J., Liu, A., Tabeta, K., Turer, E. E., Evers, B., Nair-Gill, E., Poddar, S., Su, L., Ou, F., Yu, L., Russell, J., Ludwig, S., Zhan, X., ... Beutler, B. (2020). Mutual inhibition between Prkd2 and Bcl6 controls T follicular helper cell differentiation. Science Immunology, 5(43). https://doi.org/10.1126/sciimmunol.aaz0085

Moon, P. M., Penuela, S., Barr, K., Khan, S., Pin, C. L., Welch, I., Attur, M., Abramson, S. B., Laird, D. W., & Beier, F. (2015). Deletion of Panx3 Prevents the Development of Surgically Induced Osteoarthritis. Journal of Molecular Medicine (Berlin, Germany), 93(8), 845-856. https: //doi . org/10.1007/s00109-015- 1311 - 1

Moriyama, M., Yamochi, T., Semba, K., Akiyama, T., & Mori, S. (1997). BCL-6 is phosphorylated at multiple sites in its serine- and proline-clustered region by mitogen- activated protein kinase (MAPK) in vivo. Oncogene, 14(20), 2465-2474. https://doi.org/10.1038/sj.onc.1201084

Nakagawa, R., Toboso-Navasa, A., Schips, M., Young, G, Bhaw-Rosun, L., Llorian-Sopena, M., Chakravarty, P., Sesay, A. K., Kassiotis, G, Meyer-Hermann, M., & Calado, D. P. (2021). Permissive selection followed by affinity-based proliferation of GC light zone B cells dictates cell fate and ensures clonal breadth. Proceedings of the National Academy of Sciences of the United States of America, 118(2). https://doi.org/10.1073/pnas.2016425118 Nielsen, B. S., Toft-Bertelsen, T. L., Lolansen, S. D., Anderson, C. L., Nielsen, M. S., Thompson, R. J., & MacAulay, N. (2020). Pannexin 1 activation and inhibition is permeant-selective. The Journal of Physiology, 598(2), 361-379. https://doi.org/10.1113/JP278759

Nisimoto, Y., Diebold, B. A., Cosentino-Gomes, D., & Lambeth, J. D. (2014). Nox4: a hydrogen peroxide-generating oxygen sensor. Biochemistry, 53(31), 5111-5120. https://doi.org/10.1021/bi500331y

Nisimoto, Y., Jackson, H. M., Ogawa, H., Kawahara, T., & Lambeth, J. D. (2010). Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain. Biochemistry, 49(11), 2433-2442. https://doi.org/10.1021/bi9022285

Niu, H., Ye, B. H., & Dalla-Favera, R. (1998). Antigen receptor signaling induces MAP kinase- mediated phosphorylation and degradation of the BCL-6 transcription factor. Genes & Development, 72(13), 1953-1961. https://doi.org/10.1101/gad.12.13.1953

Nurieva, R. I., Chung, Y., Martinez, G. J., Yang, X. O., Tanaka, S., Matskevitch, T. D., Wang, Y.-H., & Dong, C. (2009). Bcl6 mediates the development of T follicular helper cells. Science (New York, N.Y.), 325(5943), 1001-1005. https://doi.org/10.1126/science.1176676

Ohnuki, H., Inoue, H., Takemori, N., Nakayama, H., Sakaue, T., Fukuda, S., Miwa, D., Nishiwaki, E., Hatano, M., Tokuhisa, T., Endo, Y., Nose, M., & Higashiyama, S. (2012). BAZF, a novel component of cullin3 -based E3 ligase complex, mediates VEGFR and Notch cross-signaling in angiogenesis. Blood, 779(11), 2688-2698. https://doi.org/10.1182/blood- 2011-03-345306

Ottolini, M., Hong, K., Cope, E. L., Daneva, Z., DeLalio, L. J., Sokolowski, J. D., Marziano, C., Nguyen, N. Y., Altschmied, J., Haendeler, J., Johnstone, S. R., Kalani, M. Y., Park, M. S., Patel, R. P., Liedtke, W., Isakson, B. E., & Sonkusare, S. K. (2020). Local Peroxynitrite Impairs Endothelial Transient Receptor Potential Vanilloid 4 Channels and Elevates Blood Pressure in Obesity. Circulation, 747(16), 1318-1333. https://doi.org/! 0.1161/CIRCULATIONAHA.119.043385

Penuela, S., Bhalla, R., Gong, X.-Q., Cowan, K. N., Celetti, S. J., Cowan, B. J., Bai, D., Shao, Q., & Laird, D. W. (2007). Pannexin 1 and pannexin 3 are glycoproteins that exhibit many distinct characteristics from the connexin family of gap junction proteins. Journal of Cell Science, 720(Pt 21), 3772-3783. https://doi.org/10.1242/jcs.009514

Penuela, S., Bhalla, R., Nag, K., & Laird, D. W. (2009). Glycosylation regulates pannexin intermixing and cellular localization. Molecular Biology of the Cell, 20(20), 4313-4323. https://doi.org/10.1091/mbc.e09-01-0067

Perez-Rosado, A., Artiga, M., Vargiu, P., Sanchez-Aguilera, A., Alvarez-Barrientos, A., & Piris, M. (2008). BCL6 represses NFkappaB activity in diffuse large B-cell lymphomas. The Journal of Pathology , 214(4), 498-507. https://doi.org/10.1002/path.2279

Polo, J. M., Juszczynski, P., Monti, S., Cerchietti, L., Ye, K., Greally, J. M., Shipp, M., & Melnick, A. (2007). Transcriptional signature with differential expression of BCL6 target genes accurately identifies BCL6-dependent diffuse large B cell lymphomas. Proceedings of the National Academy of Sciences of the United States of America, 104(9), 3207-3212. https://doi.org/10.1073/pnas.0611399104

Prabha, P. S., Das, U. N., Koratkar, R., Sagar, P. S., & Ramesh, G. (1990). Free radical generation, lipid peroxidation and essential fatty acids in uncontrolled essential hypertension. Prostaglandins, Leukotrienes, and Essential Fatty Acids, 41(1), 27-33. https://doi.org/10.1016/0952-3278(90)90127-7

Ray, R., Murdoch, C. E., Wang, M., Santos, C. X., Zhang, M., Alom-Ruiz, S., Anilkumar, N., Ouattara, A., Cave, A. C., Walker, S. J., Grieve, D. J., Charles, R. L., Eaton, P., Brewer, A. C., & Shah, A. M. (2011). Endothelial Nox4 NADPH oxidase enhances vasodilatation and reduces blood pressure in vivo. Arteriosclerosis, Thrombosis, and Vascular Biology, 37(6), 1368-1376. https://doi.org/! 0.1161/ATVB AHA.110.219238 Redemann, S., Lantzsch, I., Lindow, N., Prohaska, S., Srayko, M., & Muller-Reichert, T. (2018). A Switch in Microtubule Orientation during C. elegans Meiosis. Current Biology : CB, 25(18), 2991-2997.e2. https://doi.Org/10.1016/j.cub.2018.07.012

Roel-Touris, J., Jimenez-Garcia, B., & Bonvin, A. M. J. J. (2020). Integrative modeling of membrane-associated protein assemblies. Nature Communications, 77(1), 6210. https://doi.org/10.1038/s41467-020-20076-5

Saeedi Saravi, S. S., Eroglu, E., Waldeck-Weiermair, M., Sorrentino, A., Steinhorn, B., Belousov, V., & Michel, T. (2020). Differential endothelial signaling responses elicited by chemogenetic H2O2 synthesis. Redox Biology, 36, 101605. https://doi.Org/10.1016/j.redox.2020.101605

Sewastianik, T., Szydlowski, M., Jablonska, E., Bialopiotrowicz, E., Kiliszek, P., Gomiak, P., Polak, A., Prochorec-Sobieszek, M., Szumera-Cieckiewicz, A., Kaminski, T. S., Markowicz, S., Nowak, E., Grygorowicz, M. A., Warzocha, K., & Juszczynski, P. (2016). FOXO1 is a TXN- and p300-dependent sensor and effector of oxidative stress in diffuse large B-cell lymphomas characterized by increased oxidative metabolism. Oncogene, 35(46), 5989-6000. https://doi.org/10.1038/onc.2016.126

Seyfert, V. L., Allman, D., He, Y., & Staudt, L. M. (1996). Transcriptional repression by the proto-oncogene BCL-6. Oncogene, 72(11), 2331-2342.

Sommars, M. A., Ramachandran, K., Senagolage, M. D., Futtner, C. R., Germain, D. M., Allred, A. L., Omura, Y., Bederman, I. R., & Barish, G. D. (2019). Dynamic repression by BCL6 controls the genome-wide liver response to fasting and steatosis. ELife, 8. https : //doi . org/ 10.7554/ eLife .43922

Straub, A. C., Lohman, A. W., Billaud, M., Johnstone, S. R., Dwyer, S. T., Lee, M. Y., Bortz, P. S., Best, A. K., Columbus, L., Gaston, B., & Isakson, B. E. (2012). Endothelial cell expression of haemoglobin a regulates nitric oxide signalling. Nature, 491(7424), 473-477. https : //doi . org/ 10.1038/nature 11626

Sultan, M., Nearing, J. T., Brown, J. M., Huynh, T. T., Cruickshank, B. M., Lamoureaux, E., Vidovic, D., Dahn, M. L., Fernando, W., Coyle, K. M., Giacomantonio, C. A., Langille, M. G. I., & Marcato, P. (2021). An in vivo genome- wide shRNA screen identifies BCL6 as a targetable biomarker of paclitaxel resistance in breast cancer. Molecular Oncology, 75(8), 2046-2064. https://doi.org/! 0.1002/1878-0261.12964

Thul, P. J., Akesson, L., Wiking, M., Mahdessian, D., Geladaki, A., Ait Blal, H., Alm, T., Asplund, A., Bjork, L., Breckels, L. M., Backstrbm, A., Danielsson, F., Fagerberg, L., Fall, J., Gatto, L., Gnann, C., Hober, S., Hjelmare, M., Johansson, F., ... Lundberg, E. (2017). A subcellular map of the human proteome. Science (New York, N.Y.), 356(6340). https : //doi . org/ 10.1126/sci ence . aal 3321

Vendrov, A. E., Hakim, Z. S., Madamanchi, N. R., Rojas, M., Madamanchi, C., & Runge, M. S. (2007). Atherosclerosis is attenuated by limiting superoxide generation in both macrophages and vessel wall cells. Arteriosclerosis, Thrombosis, and Vascular Biology, 27(12), 2714- 2721. https://doi.org/10.1161/ATVBAHA.107.152629

Venkatadri, R., Sabapathy, V., Dogan, M., Mohammad, S., Harvey, S. E., Simpson, S. R., Grayson, J. M., Yan, N., Perrino, F. W., & Sharma, R. (2022). Targeting Bcl6 in the TREX1 D18N murine model ameliorates autoimmunity by modulating T-follicular helper cells and germinal center B cells. European Journal of Immunology , 52(5), 825-834. https://doi.org/10.1002/eji.202149324

Wakefield, C. B., Lee, V. R., Johnston, D., Boroumand, P., Pillon, N. J., Sayedyahossein, S., O’Donnell, B. L., Tang, J., Sanchez-Pupo, R. E., Barr, K. J., Gros, R., Flynn, L., Borradaile, N. M., Klip, A., Beier, F., & Penuela, S. (2022). Pannexin 3 deletion reduces fat accumulation and inflammation in a sex-specific manner. International Journal of Obesity (2005), 46 4), 726-738. https://doi.org/10.1038/s41366-021-01037-4

Walker, S. R., Liu, S., Xiang, M., Nicolais, M., Hatzi, K., Giannopoulou, E., Elemento, O., Cerchietti, L., Melnick, A., & Frank, D. A. (2015). The transcriptional modulator BCL6 as a molecular target for breast cancer therapy. Oncogene, 34(9), 1073-1082. https://doi.org/10.1038/onc.2014.61

Ward, N. C., Hodgson, J. M., Croft, K. D., Burke, V., Beilin, L. J., & Puddey, I. B. (2005). The combination of vitamin C and grape-seed polyphenols increases blood pressure: a randomized, double-blind, placebo-controlled trial. Journal of Hypertension, 23(2), 427- 434. https://doi.org/10.1097/00004872-200502000-00026

Whyte-Fagundes, P., Kurtenbach, S., Zoidl, C., Shestopalov, V. I., Carlen, P. L., & Zoidl, G. (2018). A Potential Compensatory Role of Panx3 in the VNO of a Panxl Knock Out Mouse Model. Frontiers in Molecular Neuroscience, 11, 135. https://doi.org/10.3389/finmol.2018.00135

Williams, C. J., Headd, J. J., Moriarty, N. W., Prisant, M. G., Videau, L. L., Deis, L. N., Verma, V., Keedy, D. A., Hintze, B. J., Chen, V. B., Jain, S., Lewis, S. M., Arendall, W. B., Snoeyink, J., Adams, P. D., Lovell, S. C., Richardson, J. S., & Richardson, D. C. (2018). MolProbity: More and better reference data for improved all-atom structure validation. Protein Science : A Publication of the Protein Society, 27(1), 293-315. https://doi.org/10.1002/pro.3330

Xu, L., Chen, Y., Dutra-Clarke, M., Mayakonda, A., Hazawa, M., Savinoff, S. E., Doan, N., Said, J. W., Yong, W. H., Watkins, A., Yang, H., Ding, L.-W., Jiang, Y.-Y., Tyner, J. W., Ching, J., Kovalik, J.-P., Madan, V., Chan, S.-L., Miischen, M., ... Koeffler, H. P. (2017). BCL6 promotes glioma and serves as a therapeutic target. Proceedings of the National Academy of Sciences of the United States of America, 114(15), 3981-3986. https://doi.org/10.1073/pnas.1609758114

Yada, T., Shimokawa, H., Hiramatsu, O., Kajita, T., Shigeto, F., Goto, M., Ogasawara, Y., & Kajiya, F. (2003). Hydrogen peroxide, an endogenous endothelium-derived hyperpolarizing factor, plays an important role in coronary autoregulation in vivo. Circulation, 107(7), 1040-1045. https://doi.org/10.1161/01.cir.0000050145.25589.65

Yamaguchi K. (1994). Oxygen Transport to Tissue XVI (Springer).

Yang, Y., Delalio, L. J., Best, A. K., Macal, E., Milstein, J., Donnelly, I., Miller, A. M., McBride, M., Shu, X., Koval, M., Isakson, B. E., & Johnstone, S. R. (2020). Endothelial Pannexin 1 Channels Control Inflammation by Regulating Intracellular Calcium. Journal of Immunology (Baltimore, Md. : 1950), 204(11), 2995-3007. https://doi.org/10.4049/jimmunol.1901089

Ye, B. H., Cattoretti, G, Shen, Q., Zhang, J., Hawe, N., de Waard, R., Leung, C., Nouri-Shirazi, M., Orazi, A., Chaganti, R. S., Rothman, P., Stall, A. M., Pandolfi, P. P., & Dalla-Favera, R. (1997). The BCL-6 proto-oncogene controls germinal-centre formation and Th2-type inflammation. Nature Genetics, 16(2), 161-170. https://doi.org/10.1038/ng0697-161

Ye, B. H., Lista, F., Lo Coco, F., Knowles, D. M., Offit, K., Chaganti, R. S., & Dalla-Favera, R. (1993). Alterations of a zinc finger-encoding gene, BCL-6, in diffuse large-cell lymphoma. Science (New York, N.Y.), 262(5134), 747-750. https://doi.org/10.1126/science.8235596

It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMS What is claimed is:

1. An inhibitor of an oncogenic nuclear sequence-specific transcriptional repressor, optionally B-Cell lymphoma 6 (BCL6), the inhibitor comprising a peptide that promotes degradation of the oncogenic nuclear sequence-specific transcriptional repressor and/or BCL6 in vitro and/or in vivo.

2. The inhibitor of claim 1, wherein the inhibitor comprises a BCL6 inhibitory peptide (BCLiP), wherein the BCLiP comprises a peptide comprising an amino acid sequence that mimics amino acids L328-P344 , (SEQ ID NO. 10), of native human BCL6 (SEQ ID NO. 1), optionally wherein the BCLiP comprises SEQ ID NO. 2 or 10.

3. The inhibitor of any of the above claims, wherein the inhibitor comprises a conserved region containing degradation-targeting phosphorylation sites S333 and S343 of native human BCL6 (SEQ ID NO. 1).

4. The inhibitor of any of the above claims, wherein the inhibitor comprise an about 17 amino acid sequence, optionally SEQ ID NO. 2 or 10, optionally a peptide sequence having about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identity to SEQ ID NO. 2 or 10, optionally a biologically active fragment and/or homolog of any of the foregoing sequences including SEQ ID NO. 2 or 10.

5. The inhibitor of any of the above claims, wherein the inhibitor comprises a cell entry tag.

6. The inhibitor of claim 5, wherein the cell entry tag is selected from the group consisting of a lipid tag, peptide sequences and any combination thereof.

7. The inhibitor of claim 5, wherein the cell entry tag comprises an N-terminal Stearyl tag.

8. The inhibitor of any of the above claims, wherein the inhibitor comprises one or more phosphorylation sites to target BCL6 protein for proteasomal degradation.

9. The inhibitor of any of the above claims, further comprising a pharmaceutically acceptable carrier.

10. The inhibitor of any of the above claims, for use in inducing degradation of an oncogenic nuclear sequence-specific transcriptional repressor and/or B-Cell lymphoma 6 (BCL6) in vitro and/or in vivo.

11. The inhibitor of any of the above claims, for use in treating a cancer and/or a tumor in a subject in need thereof.

12. The inhibitor of claim 11, wherein the cancer and/or tumor comprises a cancer and/or tumor driven by oncogenic BCL6 transcriptional activity, optionally diffuse large B Cell Lymphoma, B-acute lymphoblastic leukemia, chronic myeloid leukemia, breast cancer, nonsmall cell lung cancer, and/or glioma.

13. The inhibitor of any of the above claims for use in modeling pathological autoimmunity, modulating Pannexin (Panx) channels in vitro and/or in vivo, modulating inflammation in a subject, modulating vascular oxidative stress in a subject, and/or modulating hypertension in a subject in which said modeling and/or modulating is desired.

14. An inhibitor of a transcription factor, optionally B-Cell Lymphoma 6 (BCL6), the inhibitor comprising a peptide that blocks the interaction of the transcription factor with a pannexin channel in vitro and/or in vivo.

15. The inhibitor of claim 14, wherein the inhibitor comprises a BCL6 inhibitory peptide (BCLiP), wherein the BCLiP comprises a peptide comprising an amino acid sequence that mimics amino acids L328-P344 (SEQ ID NO. 10) of native human BCL6 (SEQ ID NO. 1), optionally wherein the BCLiP comprises SEQ ID NO. 2 or 10.

16. The inhibitor of claim 14 or claim 15, wherein the inhibitor comprises a conserved region containing degradation-targeting phosphorylation sites S333 and S343 of native human BCL6 (SEQ ID NO. 1).

17. The inhibitor of any of claims 14 to 16, wherein the inhibitor comprise an about 17 amino acid sequence, optionally SEQ ID NO. 2 or 10, optionally a peptide sequence having about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identity to SEQ ID NO. 2 or 10, optionally a biologically active fragment and/or homolog of any of the foregoing sequences including SEQ ID NO. 2 or 10.

18. The inhibitor of any of claims 14-17, wherein the inhibitor comprises a cell entry tag.

19. The inhibitor of claim 18, wherein the cell entry tag is selected from the group consisting of a lipid tag, a peptide sequence, and any combination thereof.

20. The inhibitor of claim 18 , wherein the cell entry tag comprises an N-terminal tag, optionally an N-terminal stearyl tag.

21. The inhibitor of any of claims 14 to 20, wherein the inhibitor comprises one or more phosphorylation sites to target BCL6 protein for proteasomal degradation.

22. The inhibitor of any of claims 14 to 21, wherein the pannexin channel is optionally one of three isoforms, optionally Panxl, Panx2 and/or Panx3.

23. The inhibitor of any of claims 14 to 22, wherein the pannexin channel is Panx3.

24. The inhibitor of any of claims 14 to 23, further comprising a pharmaceutically acceptable carrier.

25. The inhibitor of any of claims 14 to 24, for use in inducing degradation of an oncogenic nuclear sequence-specific transcriptional repressor and/or B-Cell lymphoma 6 (BCL6) in vitro and/or in vivo.

26. The inhibitor of any of claims 14 to 24, for use in treating a cancer and/or a tumor in a subject in need thereof.

27. The inhibitor of claim 26, wherein the cancer and/or tumor comprises a cancer and/or tumor driven by oncogenic BCL6 transcriptional activity, optionally diffuse large B Cell Lymphoma, B-acute lymphoblastic leukemia, chronic myeloid leukemia, breast cancer, nonsmall cell lung cancer, and/or glioma.

28. The inhibitor of any of claims 14 to 24, for use in modeling pathological autoimmunity, modulating Pannexin (Panx) channels in vitro and/or in vivo, modulating inflammation in a subject, modulating vascular oxidative stress in a subject, and/or modulating hypertension in a subject in which said modeling and/or modulating is desired.

29. A method of inducing degradation of an oncogenic nuclear sequence-specific transcriptional repressor and/or B-Cell lymphoma 6 (BCL6) in vitro and/or in vivo, the method comprising contacting a cell in vivo or in vitro with an inhibitor of any of claims 1-28, and/or administering to a subject an inhibitor of any of claims 1-28.

30. A method of treating a cancer and/or a tumor in a subject, the method comprising administering to a subject in need of treatment a composition comprising an inhibitor of any of claims 1-28, optionally wherein the inhibitor is BCLiP, further optionally wherein the BCLiP comprises SEQ ID NO. 2 or 10.

31. The method of claim 30, wherein the cancer and/or tumor comprises a cancer and/or tumor driven by oncogenic BCL6 transcriptional activity, optionally diffuse large B Cell Lymphoma, B-acute lymphoblastic leukemia, chronic myeloid leukemia, breast cancer, nonsmall cell lung cancer, and/or glioma.

32. A method of modeling pathological autoimmunity in a subject, the method comprising administering to a subject a composition comprising an inhibitor of any of claims 1-28, optionally wherein the inhibitor is BCLiP, further optionally wherein the BCLiP comprises SEQ ID NO. 2 or 10.

33. A method of modulating Pannexin (Panx) channels in vitro and/or in vivo, the method comprising contacting a cell in vivo or in vitro with an inhibitor of any of the above claims, and/or administering to a subject an inhibitor of any of the above claims, wherein the modulating comprises disruption of BCL6 binding with Panx, wherein the Panx is optionally one of three isoforms, optionally Panxl, Panx2 and/or Panx3.

34. A method of modulating inflammation, hypertension, and/or vascular oxidative stress in a subject, the method comprising administering to a subject a composition comprising an inhibitor of any of claims 1-28, optionally wherein the inhibitor is BCLiP, further optionally wherein the BCLiP comprises SEQ ID NO. 2 or 10.

35. Use of an inhibitor of a transcription factor, optionally B-Cell Lymphoma 6 (BCL6), for the modulation and/or mimicking of pathological autoimmunity, inflammation, hypertension, and/or vascular oxidative stress in a subject, optionally wherein the inhibitor is an inhibitor of any of claims 1-28, optionally wherein the inhibitor is BCLiP, further optionally wherein the BCLiP comprises SEQ ID NO. 2 or 10.

36. Use of an inhibitor of a transcription factor, optionally B-Cell Lymphoma 6 (BCL6), for the treatment of cancer and/or a tumor in a subject in need thereof, optionally wherein the inhibitor is an inhibitor of any of claims 1-28, optionally wherein the inhibitor is BCLiP, further optionally wherein the BCLiP comprises SEQ ID NO. 2 or 10.

37. The use of claim 36, wherein the cancer and/or tumor comprises a cancer and/or tumor driven by oncogenic BCL6 transcriptional activity, optionally diffuse large B Cell Lymphoma, B-acute lymphoblastic leukemia, chronic myeloid leukemia, breast cancer, non-small cell lung cancer, and/or glioma.

38. A method of evaluating a candidate therapeutic composition, the method comprising providing a subject modulated with an inhibitor of a transcription factor, optionally B-Cell Lymphoma 6 (BCL6); administering a candidate therapeutic composition to the subject; and assessing an effect of the candidate therapeutic composition, optionally wherein the inhibitor is an inhibitor of any of claims 1-28, optionally wherein the inhibitor is BCLiP, further optionally wherein the BCLiP comprises SEQ ID NO. 2 or 10.