WO2019049144A1 - Compositions for inhibition of the methyltransferase setd6 - Google Patents

Abstract

Described herein are synthetic polypeptide inhibitors of the SET-domain-containing protein 6 (SETD6) methyltransferase, and polypeptide chimeras thereof including at least one cell penetrating peptide. Pharmaceutical compositions and uses thereof for treatment of diseases and conditions associated with overexpression of SETD6 are also described.

Description

COMPOSITIONS FOR INHIBITION OF THE METHYLTRANSFERASE SETD6

CROSS-REFERENCE TO RELATED APPLICATIONS

Benefit is claimed to Israel Patent Application No. 254361, filed September 6, 2017, the contents of which are incorporated by reference herein in their entirety.

FIELD

This disclosure relates to synthetic polypeptide inhibitors of the SET-domain- containing protein 6 (SETD6) methyltransferase, and polypeptide chimeras thereof including at least one cell penetrating peptide. Pharmaceutical compositions and uses thereof for treatment of diseases and conditions associated with overexpression of SETD6 are also described.

BACKGROUND

Epigenetic regulation, in concert with genetic alterations, plays a critical role in the pathologies of various human diseases, including the development of cancers. One of the key mechanisms for modulating epigenetic programs involves the addition and removal of methyl groups from lysine residues of histone as well as many non-histone substrates. A lysine residue in a given protein can accept up to three methyl groups, thereby forming mono-, di-, and tri-methylated derivatives (mel, me2 and me3). Methylation of lysine residues in target proteins is performed by protein lysine (K) methyltransferases (PKMTs). There are over 60 candidate members of this enzyme family, the vast majority of which contain a conserved SET domain that is responsible for the enzymatic activity.

In recent years many studies have linked PKMT deregulation to a wide range of different pathologies, eliciting the possibility that the proteins involved in disease suppression or pathogenesis are methylated and that abnormalities in the physiological placement of this chemical mark have fundamental roles in these processes. This altered methylation state was studied in depth in the context of histones, however in recent years it became clear, that also non-histone protein methylation has a key role in the regulation of many human pathologies including cancer. The large number of enzymes involved in attaching methyl groups on lysine residues argues for the presence of numerous protein substrates in addition to the small number that has already been characterized. The large number of PKMTs also implies that there is a high degree of substrate specificity, as well as a need for specificity in inhibitory agents. As such, the development of highly specific and potent inhibitors that modulate the enzymatic activity and substrate specificity of the different PKMTs is a major challenge for the pharmaceutical industry. The present disclosure relates to the development of inhibitors of SETD6, a PKMT which was previously determined to be a key regulator of proliferation and inflammatory processes.

SUMMARY

Described herein is the observation that a RelA-derived peptide and mutants thereof have a cytotoxic effect. Accordingly, described herein is a RelA-derived peptide having an amino acid sequence at least 70% identical to SEQ ID NO: 1, or a fragment, derivative or analog thereof. In particular embodiments, the described polypeptides are fused at the N- or C- terminal end to at least one cell penetrating peptide. Pharmaceutical compositions including the described polypeptides, and polypeptide-encoding nucleic acids are also described.

Additionally provided are therapeutic methods and uses of the described polypeptides for treatment or prevention of a disease or condition characterized by aberrant

overexpression of SET -domain-containing protein 6 (SETD6), including cancer.

The foregoing and other objects, features, and advantages will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a viability assay using peptides at 8uM in HeLa cells. HeLa cells were plated at 25,000 cells/well, supplemented with indicated concentrations of the relevant peptide in serum free medium for 4h, after which cells were washed and fresh complete medium was added overnight. PrestoBlue was added for 20 min after which absorbance at 560/590nm was measured. RelAneg represents a sequence of the RelA protein that is not predicted to be methylated by SETD6. Results are shown for peptides presented in two orientations of the Rl l-NLS CPP and the RelA sequence. Peptides RelAKA-NLS-Rl 1 and RelAKM-NLS-Rl l were tested here only in one orientation. Results were normalized to the control cells, treated the same but with no peptide. Standard error represent triplicates.

Figure 2 shows a dose dependent viability assay of peptides in increasing concentrations in HeLa cells. HeLa cells were plated at 25,000 cells/well, supplemented with indicated concentrations of the relevant peptide in serum free medium for 4h, after which cells were washed and fresh complete medium was added overnight. PrestoBlue was added for 20 min after which absorbance at 560/590nm was measured. Peptides presented are all in one orientation, with the RelA sequence at the N-terminal of the peptide. Besides the 4 peptides presented in this Figure, the RelAKM-Rl l mutant was also tested and was found to be non-effective. Results were normalized to the control cells, treated the same but with no peptide. Standard error represent triplicates.

Figure 3 shows peptide effects on MDA-MB-231 cells. Cells grown overnight in a 48-wells plate were incubated with the different peptides at a final concentration of 6 uM for 24 hours, before performing a PrestoBlue viability assay.

Figure 4 shows peptide effects on U87 cells. Mortality was observed in all cells tested for RelA-NLS-Rl 1 and RelAKA-NLS-Rl 1. Cells grown overnight in a 48-wells plate were incubated with 4.3 μιη RelAKA-NLS-Rl 1 and 4.7 μιη for the other 3 peptides for 24 hours, before performing a PrestoBlue viability assay.

Figure 5 shows that RelA NLS Rl 1 has a toxic effect on B 16 cells, while RelAKR does not have any effect. Living cell percent as shown is calculated from comparison to untreated wells per peptide concentration. After 24 hours of incubation PrestoBlue dyeing was performed. All raw fluorescence data had the average blank (not shown) subtracted.

Figure 6 shows that RelA NLS Rl 1 has a higher toxic effect on HepG2 cells than NLS Rl 1 RelAKR, while both have a toxic effect. Shown is living cell percent (compared to untreated wells) per peptide concentration. After 24 hours of incubation (full method described herein) PrestoBlue dyeing was performed. All raw fluorescence data had the average blank (not shown here) subtracted.

Figures 7A-7C show the effects of VP22 RelA peptide on wound healing migration assay in HeLa cells. Fig. 7A shows the VP22 (SEQ ID NO: 17) or the VP22-RelA (SEQ ID NO: 16) peptides that were used in the shown experiments. Fig. 7B: Cells were plated at 25,000 cells/well, supplemented with 40uM of the VP22 or VP22-RelA peptides in serum free medium for 4h, after which cells were washed and fresh complete medium was added overnight. A scratch was performed across the center of the well, and pictures were taken. 24 h after which pictures were taken again. Fig 7C: Migration distance was quantified using ImageJ and results were normalized to cells treated with the negative control peptide VP22. Standard error represent six repeats of each condition.

Figures 8A-8B show a dose-dependent viability assay of peptides in increasing concentrations in HeLa cells (Fig. 8A) and MDA-MB-231 cells (Fig. 8B). Cells were plated at 20,000 cells/well, supplemented with indicated concentrations of the VP22 or VP22-RelA peptides in serum free medium for 4h, after which cells were washed and fresh complete medium was added overnight. PrestoBlue was added for 20 min after which absorbance at 560/590nm was measured. Control cells were treated the same but with no peptide. Standard error represent triplicates.

Figures 9A-9B: are bar graphs showing the results of tumor size measurements of tumors that were formed by injection of mouse 4T1 breast cancer cells into balb/c mice breast tissue. A decrease in tumor volume following treatment with RelAKA-NLS-Rl 1 peptide. As shown in the figures, differences were more evident at the end of the treatment (Figure 9A, day 15) than at the end of the experiment (Figure 9B, day 18); the presented graphs show average measured volume with outliners removed. At the end of the injection period (Fig. 9A), there was a significant difference between the control and the 20 mg/kg group (P=0.02) but not the 5 mg/kg group (P=0.17). At the end of the experiment (Fig. 9B), there was a significant difference between the control and the 20 mg/kg group (P=0.03), but not the 5 mg kg group (p=0.25). For control and 5mg/kg experiments, n=6; for 20mg/kg experiments, n=5. Statistical difference was determined using a one way anova test.

Figure 10 shows that treatment with RelAKA-NLS-Rl 1 peptide succeeded in decreasing tumor growth rate; tumor volume was plotted to show average change over time. Undetectable or unmeasurable tumors were given a volume of 1 mm³ to include these mice into the calculation. For control and assays at 5mg/kg- n=6, for assays at 20mg/kg-n=5.

Figure 11 shows that tumor growth was suppressed in both of the tested 20 mg/kg treatment groups. Tumor size was measured using a caliper. Difference at the end point were nearly 3.5 fold for the RelAKA NLS Rl 1 peptide at 20 mg/kg, and nearly 2 fold for the RelA NLS Rl 1 peptides, compared to the saline group. Differences were statistically checked using a single factor anova, and the groups were not found to be significantly different. Bars indicate standard error. n=6 for all groups but the saline control (n=5).

Figure 12 shows that the 20 mg/kg treatment groups for RelA and RelAKA peptides resulted in lower median and average values than saline-treated and 5mg/kg RelAKA-treated groups; Dot plot representing all tumor volumes received herein. The X symbol marks the average tumor volume and the square box represents the median. A high variance was apparent in all groups, but mostly in the saline and 5mg/kg RelAKA groups.

Figure 13 shows that the 20 mg/kg RelAKA NLS Rl 1 treatment groups' tumors were 40% lighter than the saline treatment group; tumor weights were recorded for each of the different treatment groups.

Figure 14 shows that the 20 mg/kg RelAKA NLS Rl 1 group demonstrated weight gain similar to untreated and naive mice; body weight gain was calculated as percent of initial weight. Mice that were not injected with MDA cells were used as a reference and were named the naive group.

BRIEF DESCRIPTION OF THE DESCRIBED SEQUENCES

The nucleic and/or amino acid sequences provided herewith are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file named 3145_3_2_Seqlist_ST25, created September 5, 2018, about 12.3 KB, which is incorporated by reference herein. In the accompanying Sequence Listing:

SEQ ID NO: 1 is the combined RelA peptide.

SEQ ID NO: 2 is the RelA K310 peptide fragment (RelA).

SEQ ID NO: 3 is the RelA K310A peptide fragment.

SEQ ID NO: 4 is the RelA K310R peptide fragment.

SEQ ID NO: 5 is the RelA K310M peptide fragment.

SEQ ID NO: 6 is the NLS cell penetrating peptide.

SEQ ID NO: 7 is the Rl l cell penetrating peptide.

SEQ ID NO: 8 is the NLS-R11 cell penetrating peptide chimera.

SEQ ID NO: 9 is the Rl l-NLS cell penetrating peptide chimera.

SEQ ID NO: 10 is the combined RelA-Rl l C-terminal chimera.

SEQ ID NO: 11 is the combined Rl 1-RelA N-terminal chimera.

SEQ ID NO: 12 is the combined Rl 1-NLS RelA-N-terminal cell penetrating peptide chimera.

SEQ ID NO: 13 is the combined RelA-NLS-Rl 1-C-terminal cell penetrating peptide chimera.

SEQ ID NO: 14 is the RelA-K310-Rll C-terminal chimera.

SEQ ID NO: 15 is the RelA-K310A-Rl l C-terminal chimera.

SEQ ID NO: 16 is the RelA-K310R-Rl 1 C-terminal chimera.

SEQ ID NO: 17 is the RelA-K310M-Rl l C-terminal chimera.

SEQ ID NO: 18 is the Rl l-RelA-K310 N-terminal chimera.

SEQ ID NO: 19 is the Rl l-RelA-K310A N-terminal chimera.

SEQ ID NO: 20 is the Rl l-RelA-K310R N-terminal chimera.

SEQ ID NO: 21 is the Rl l-RelA-K310M N-terminal chimera. SEQ ID NO: 22 is the Rl 1-NLS RelA-K310 N-terminal cell penetrating peptide chimera.

SEQ ID NO: 23 is the Rl 1-NLS RelA-K310A N-terminal cell penetrating peptide chimera.

SEQ ID NO: 24 is the Rl 1-NLS RelA-K310R N-terminal cell penetrating peptide chimera.

SEQ ID NO: 25 is the Rl 1-NLS RelA-K310M N-terminal cell penetrating peptide chimera.

SEQ ID NO: 26 is the RelA-K310 NLS Rl l C-terminal cell penetrating peptide chimera.

SEQ ID NO: 27 is the RelA-K310A NLS Rl 1 C-terminal cell penetrating peptide chimera.

SEQ ID NO: 28 is the RelA-K310R NLS Rl 1 C-terminal cell penetrating peptide chimera.

SEQ ID NO: 29 is the RelA-K310M NLS Rl 1 C-terminal cell penetrating peptide chimera.

SEQ ID NO: 30 is the Rl l-negative control for the N-terminal fusion with Rl l. SEQ ID NO: 31 is the negative control for an N-terminal chimera of Rl 1-NLS. SEQ ID NO: 32 is the negative control for a C-terminal chimera of Rl 1-NLS. SEQ ID NO: 33 is the RelA-noK310-Rl 1 C-terminal fusion.

SEQ ID NO: 34 is the VP22-RelA peptide.

SEQ ID NO: 35 is the VP22 peptide.

SEQ ID NO: 36 is the NLS- Rl l-RelA-K310R N-terminal cell penetrating peptide chimera.

DETAILED DESCRIPTION

I. Abbreviations

CPP Cell penetrating peptide

NLS Nuclear localization sequence

PKMT Protein lysine (K) methyltransferase

SETD6 SET-domain-containing protein 6 II. Terms

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." "Consisting essentially of indicates a

composition, method, or process that includes only those listed features as the active or essential elements, but can include non-active elements in addition. "About" before a numerical value includes minor variations, within 5% of the listed value. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." In case of conflict, the present specification, including explanations of terms, will control. In addition, all the materials, methods, and examples are illustrative and not intended to be limiting.

Administration: The introduction of a composition into a subject by a chosen route.

Administration of an active compound or composition can be by any route known to one of skill in the art. Administration can be local or systemic. Examples of local administration include, but are not limited to, topical administration, subcutaneous administration, intramuscular administration, intrathecal administration, intrapericardial administration, intra-ocular administration, or topical ophthalmic administration In addition, local administration includes routes of administration typically used for systemic administration, for example by directing intravascular administration to the arterial supply for a particular organ. Thus, in particular embodiments, local administration includes intra-arterial administration and intravenous administration when such administration is targeted to the vasculature supplying a particular organ. Local administration also includes the

incorporation of active compounds and agents into implantable devices or constructs, such as vascular stents or other reservoirs, which release the active agents and compounds over extended time intervals for sustained treatment effects. Systemic administration includes any route of administration designed to distribute an active compound or composition widely throughout the body via the circulatory system. Thus, systemic administration includes, but is not limited to intra- arterial and intravenous administration. Systemic administration also includes, but is not limited to, topical administration, subcutaneous administration, intramuscular administration, or administration by inhalation, when such administration is directed at absorption and distribution throughout the body by the circulatory system.

Antagonist: A molecule or compound that tends to nullify the action of another, or in some instances that blocks the ability of a given chemical to bind to its receptor or other interacting molecule, preventing a biological response. Antagonists are not limited to a specific type of compound, and may include in various embodiments peptides, antibodies and fragments thereof, and other organic or inorganic compounds (for example,

peptidomimetics and small molecules). As used herein, "inhibitor" is synonymous with "antagonist".

Apoptosis: The process by which cells are programmed to die or lose viability.

Commonly triggered by cytochrome leakage from the mitochondria and accompanied by signaling cascades (caspases and other proteins) resulting in: decreased mitochondrial and energy potential via the electron transport system, an build up of reactive oxygen species and free radical and loss of membrane integrity.

Cancer: The product of neoplasia is a neoplasm (a tumor or cancer), which is an abnormal growth of tissue that results from excessive cell division. A tumor that does not metastasize is referred to as "benign." A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant." Neoplasia is one example of a proliferative disorder. A "cancer cell" is a cell that is neoplastic, for example a cell or cell line isolated from a tumor.

Examples of hematological tumors include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and

erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin' s lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,

myelodysplastic syndrome, hairy cell leukemia and myelodysplasia. Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers (such as small cell lung carcinoma and non-small cell lung carcinoma), ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma,

hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma and retinoblastoma).

Cell Penetrating Peptide (CPP): A short polypeptide, typically less than or equal to

40 amino acids long, with the ability to translocate across the cell membrane and gain access to the cytoplasm, along with any molecule linked to the CPP. As used herein, CPPs are synonymous with and encompasses the peptides and sequences known in the art as "protein transduction domain (PTD)," "Trojan Protein," and "membrane translocating sequence (MTS)." Typical CPPs are positively charged, with amino acids such as arginine and lysine predominating in their sequence. Particular non-limiting examples of CPPs include polylysine or polyarginine peptides, for example between 4 and 17 amino acids, penetratin, TAT, SynB l, SynB3, PTD-4, PTD-5, FHV-Coat (35-49), BMV Gag (7-25), HTLV-II Rex (4- 16), D-Tat, R9-Tat, Transportan, MAP, SBP, NLS, FBP, MPG, Rl l, VP22, antp, Pep-1, and Pep-2.

Chemotherapeutic agent: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth such as psoriasis. In one embodiment, a chemotherapeutic agent is an agent of use in treating a breast cancer, an ovarian cancer, or another tumor, such as an anti-neoplastic agent. In one embodiment, a chemotherapeutic agent is a radioactive compound. One of skill in the art can readily identify a chemotherapeutic agent of use (see for example, Slapak and Kufe, Principles of Cancer Therapy). Non-limiting examples of chemotherapeutic agents include cisplatin, paclitaxel, docetaxel, doxorubicin, epirubicin, topotecan, irinotecan, gemcitabine, iazofurine, gemcitabine, etoposide, vinorelbine, tamoxifen, valspodar, cyclophosphamide, methotrexate, fluorouracil, mitoxantrone and vinorelbine. Combination chemotherapy is the administration of more than one agent to treat cancer, including a combination of the peptides described herein and one or more chemotherapeutic agent.

Chimera: A nucleic acid sequence, amino acid sequence, or protein that comprises nucleic acid sequence, amino acid sequence, or protein from two or more sources, for example amino acid sequence from two or more different species or two or more polypeptides from the same species. In general, chimeric sequences are the result of genetic engineering. The RelA-derived peptides fused to at least one CPP described herein are an example of a polypeptide chimera.

Contacting: Placement in direct physical association. Includes both in solid and liquid form. Contacting can occur in vitro with isolated cells or in vivo by administering to a subject.

Effective amount of a composition: A quantity of a composition, including the isolated peptides described herein, sufficient to achieve a desired effect in a subject being treated. An effective amount of a composition can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount of the composition will be dependent on the composition applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the composition.

Encode: A polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof.

Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked, for example the expression of the described RelA or RelA chimeric polypeptides can be operably linked to expression control sequences. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

Functional fragments and variants of a polypeptide: Included are those fragments and variants of the described RelA polypeptides and RelA-CPP chimeras that maintain one or more functions of the parent polypeptides. It is recognized that the gene or cDNA encoding a polypeptide can be considerably mutated without materially altering one or more the polypeptide' s functions. First, the genetic code is well-known to be degenerate, and thus different codons encode the same amino acids. Second, even where an amino acid substitution is introduced, the mutation can be conservative and have no material impact on the essential functions of a protein. Third, part of a polypeptide chain can be deleted without impairing or eliminating all of its functions. Fourth, insertions or additions can be made in the polypeptide chain; for example adding epitope tags, without impairing or eliminating its functions. Other modifications that can be made without materially impairing one or more functions of a polypeptide include, for example, in vivo or in vitro chemical and biochemical modifications or the incorporation of unusual amino acids. Such modifications include, for example, methylation, acetylation, carboxylation, phosphorylation, glycosylation, ubiquination, labeling, e.g., with radionucleides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art. A variety of methods for labeling polypeptides and labels useful for such purposes are well known in the art, and include radioactive isotopes such as ³²P, ligands which bind to or are bound by labeled specific binding partners (e.g. , antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands. Functional fragments and variants can be of varying length.

Heterologous: A type of sequence that is not normally (i.e. in the wild-type sequence) found adjacent to a second sequence. In one embodiment, the sequence is from a different genetic source, such as a virus or organism, than the second sequence. Chimeric polypeptides, such as those described herein are often composed of heterologous sequences.

Hyperproliferative disease: A disease or disorder characterized by the uncontrolled proliferation of cells. Hyperproliferative diseases include, but are not limited to malignant and non-malignant tumors and psoriasis.

Injectable composition: A pharmaceutically acceptable fluid composition comprising at least one active ingredient, for example, a protein, peptide, or antibody. The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally comprise minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, pH buffering agents and the like. Such injectable compositions that are useful for use with the compositions of this disclosure are conventional; appropriate formulations are well known in the art.

Isolated: A biological component (such as a nucleic acid molecule, protein or organelle) that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

Linker: One or more nucleotides or amino acids that serve as a spacer between two molecules, such as between two nucleic acid molecules or two peptides, for example between a RelA polypeptide as described herein and one or more CPPs, and between CPPs in those polypeptides with multiple CPPs present.

Neoplasia, malignancy, cancer and tumor: A neoplasm is an abnormal growth of tissue or cells that results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as "benign." A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant." Malignant tumors are also referred to as "cancer." Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this disclosure are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Pharmaceutical agent: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or a cell. Incubating includes exposing a target to an agent for a sufficient period of time for the agent to interact with a cell. Contacting includes incubating an agent in solid or in liquid form with a cell.

Polypeptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. The term polypeptide or protein as used herein encompasses any amino acid sequence and includes modified sequences such as

glycoproteins. The term polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.

The term polypeptide fragment refers to a portion of a polypeptide which exhibits at least one useful epitope. The phrase "functional fragments of a polypeptide" refers to all fragments of a polypeptide that retain an activity, or a measurable portion of an activity, of the polypeptide from which the fragment is derived

Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Preventing or treating a disease: Preventing a disease refers to inhibiting the full development of a disease, for example inhibiting the development of myocardial infarction in a person who has coronary artery disease or inhibiting the progression or metastasis of a tumor in a subject with a neoplasm. Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell.

Radiation Therapy (Radiotherapy): The treatment of disease (e.g., cancer or another hyperproliferative disease or condition) by exposure of a subject or their tissue to a radioactive substance. Radiation therapy is the medical use of ionizing radiation as part of cancer treatment to control malignant cells. Radiotherapy may be used for curative or adjuvant cancer treatment. It is used as palliative treatment where cure is not possible and the aim is for local disease control or symptomatic relief.

Recombinant: A nucleic acid that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g. , by genetic engineering techniques.

Sequence identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Methods of alignment of sequences for comparison are well known in the art, for example the NCBI Basic Local Alignment Search Tool (BLAST), which is available from several sources, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at the NCBI website, together with a description of how to determine sequence identity using this program.

Subject: Living multi-cellular organisms, including vertebrate organisms, a category that includes both human and non-human mammals.

Therapeutically effective amount: A quantity of compound sufficient to achieve a desired effect in a subject being treated. An effective amount of a compound may be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount will be dependent on the compound applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the compound. For example, a therapeutically effective amount of an active ingredient can be measured as the concentration (moles per liter or molar-M) of the active ingredient (such as a small molecule, peptide, protein, or antibody) in blood (in vivo) or a buffer (in vitro) that produces an effect. Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transfected host cell. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses.

III. Overview of Several Embodiments

Described herein is the observation that some RelA-derived peptide and mutants thereof have a cytotoxic effect. Accordingly, described herein is a RelA-derived peptide having an amino acid sequence at least 70% identical to SEQ ID NO: 1, or a fragment, derivative, or analog thereof. In particular embodiments, the described isolated polypeptides include a polypeptide at least 70% identical to an amino acid sequence set forth as SEQ ID NOs 2-5.

In some embodiments, the described polypeptides include at least one cell penetrating peptide (CPP) fused to the isolated polypeptide at either the N- or C-terminal ends of the isolated polypeptide. Such polypeptides are referred to herein, inter alia, as isolated chimeric polypeptides.

In particular embodiments of the isolated chimeric polypeptides, the CPP is fused to the isolated polypeptide through at least one linking amino acid. Particular examples of the described chimeric polypeptides include an amino acid sequence at least 70% identical to SEQ ID NOs: 10-13, or a fragment, derivative or analog thereof, including polypeptides at least 70% identical to an amino acid sequence set forth as SEQ ID NOs 14-29.

In particular embodiments, the described isolated polypeptides and isolated chimeric polypeptides further include an acetyl group at the N- or C-terminal ends.

Additionally described herein are isolated nucleic acids, such as an expression vector, including a nucleic acid sequence encoding the described isolated polypeptides or chimeric polypeptides.

In particular embodiments, the described isolated polypeptides, chimeric

polypeptides, or isolated nucleic acids are used in therapeutic methods such as in the preparation of a medicament for treatment or prevention of a disease or condition characterized by aberrant overexpression of SET-domain-containing protein 6 (SETD6). Such diseases or conditions include a SETD6-associated cancer including breast cancer, glioblastoma, cervical cancer, and leukemia. In further embodiments, the described compositions can be used in a treatment regimen including an additional chemotherapeutic agent, surgery, or radiotherapy.

IV. SETD6 Inhibitor and Pro-Proliferative Peptides

RelA (also known as p65), a known member of the nuclear factor kappa-light-chain- enhancer of activated B cells (NF-κΒ) transcription factor complex family, is also known to interact with SETD6. Described herein are isolated synthetic polypeptides derived from the RelA-SETD6 interacting sequence having an enhanced ability to inhibit SETD6 function. The described synthetic polypeptides include variants and fragments of the following polypeptide including variable position Xi: RKRTYETFXiSIMKKS (SEQ ID NO: 1); wherein in particular embodiments, Xi can be K, A, R, or M. Particular embodiments of the isolated synthetic polypeptides include polypeptides having sequences set forth herein as SEQ ID Nos 2-5.

Also described herein are synthetic fusions of RelA and the cell penetrating peptide VP22, which are observed to promote cellular proliferation. An example of such fusion peptides is set forth herein as SEQ ID NO: 34.

In particular embodiments, the described synthetic polypeptides (inhibitory or pro- proliferative) are fused to at least one cell penetrating peptide (CPP), which can facilitate passage of the isolated RelA-derived polypeptide from the external environment and into the cellular interior. As described herein, numerous examples of CPPs exist, and which in fusion with a RelA-derived synthetic polypeptide can be used to produce the chimeric RelA-CPP polypeptides described herein. The RelA-CPP chimeras can include one, two, three, or more CPPs from the same or multiple sources, which can be fused to the RelA-derived polypeptide at the C- and/or N-terminus of the polypeptide. Particular examples of CPPs for use in the described chimeric peptides include the Rl 1 CPP and the NLS CPP. In those embodiments where multiple CPPs are fused to the RelA-derived polypeptides, the multiple CPPs can be fused to the RelA peptide in all possible orientations. For example, if the Rl 1 and NLS CPPs are fused to the N-terminus of RelA, the orientation (N to C terminal) of peptide segments can be Rl 1, NLS, and RelA; or can be NLS, Rl 1, and RelA. It will be appreciated that similar variations in orientation are possible in embodiments wherein the multiple CPPs are fused to the C terminus of the RelA peptide. Particular examples of RelA-derived peptides that are fused to one or more CPPs (also referred to herein as "chimeras") are peptides that are set forth herein as SEQ ID Nos 10-29. As described above, the RelA-derived pro-proliferative peptide is fused to the VP22 CPP. In some embodiments, the at least one CPP is fused directly to the RelA-derived synthetic peptide and/or to other CPPs in the polypeptide chimera. In other embodiments, the at least one CPP is fused to the RelA-derived peptide and/or to other CPPs in the polypeptide chimera by way of a peptide linker, which can be one or more amino acids linking the CPP(s) and/or RelA-derived peptide. Non-limiting examples of linking amino acids or peptides for use in the described polypeptide chimeras include one or more isoleucine and/or tryptophan residues.

The described polypeptides can be produced by any method known to the art. In particular embodiments the polypeptides are chemically synthesized. In other embodiments, the polypeptides are produced by and purified from a suitable prokaryotic, fungal, plant, or animal cell host into which a suitable polypeptide-expression vector has been introduced. Methods of protein isolation and purification are also standard (e.g. methods of affinity chromatography, size exclusion chromatography and the like).

Variants, fragments, and analogs of the described polypeptides are included in the current disclosure. Such polypeptides include polypeptides that share about 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity with the described RelA-derived peptides and chimeras thereof, including the RelA-CPP derived peptides disclosed herein. Other exemplary variants include peptides that differ by only one, two or three amino acids from those set forth herein. In particular embodiments, the variation from those sequences expressly described herein can be conservative substitutions that one of skill will not expect to significantly alter the shape or charge of the polypeptide.

The described polypeptides also include those polypeptides that share 100% sequence identity to those indicated, but which differ in post-translational or post- synthesis modifications from the native sequence. For example, the described synthetic polypeptides can be acetylated at the N- or C-terminal ends of the polypeptide. Other examples include conjugation of a palmitic acid group at either terminus of the peptide or other modifications common in the art of polypeptide synthesis (see for examples sigmaaldrich.com/technical- documents/articles/biology/peptide-modifications-n-terminal-internal-and-c-terminal.html).

In particular embodiments, the described RelA-derived peptides and chimeric peptides are provided as a discrete biomolecule. In other embodiments, the described polypeptides are a domain of a larger polypeptide, such as an independently-folded structural domain, or an environment-accessible functional domain.

Also provided herein are nucleic acids encoding the described RelA-derived polypeptides and chimeric polypeptides, including variations due to codon degeneracy and particular nucleic acid sequences optimized for the codon bias of bacterial, animal, and plant cells.

In particular embodiments, the described nucleic acid sequences are contained within a DNA cloning and/or expression plasmid as are standard in the art. It will be appreciated that any standard expression plasmid can be used to express one or more of the described RelA-derived polypeptides and chimeric polypeptide-encoding nucleic acids, as discussed herein. Such plasmids will minimally contain an origin of replication, selection sequence (such as, but not limited to, an antibiotic resistance gene), and expression control sequences operably linked to the RelA-derived polypeptide or chimeric polypeptide-encoding nucleic acid. In particular embodiments, the expression plasmids include post-translational sequences (e.g. signal sequences to direct polypeptide processing and export) that are encoded in-frame with the RelA-derived polypeptides or chimeric polypeptide-encoding nucleic acids.

Particular non- limiting examples of bacterial expression plasmids include IPTG- inducible plasmids, arabinose-inducible plasmids and the like. Other non-limiting examples of expression induction include light induction, temperature induction, nutrient-induction, and autoinduction, and mammalian- specific DNA expression plasmids. Custom-made expression plasmids are commercially available from suppliers such as New England Biolabs (Ipswich, MA) and DNA 2.0 (Menlo Park, CA). In a particular embodiment, a RelA-derived polypeptide or chimeric polypeptide-expressing plasmid can be designed for specific localized induction in response to a local cellular micro environment, such as the local environment of a particular cancer type.

V. Compositions for Treatment of SETD6-associated Disease

Additionally provided herein are compositions including the described isolated RelA- derived inhibitory polypeptides, chimeric polypeptides, and encoding nucleic acids, for use in treatment or prevention of a disease or condition characterized by overexpression of SET- domain-containing protein 6 (SETD6). Exemplary uses include the preparation of a medicament for the described treatment or prevention, as well as methods of treatment by administering the described compositions in a therapeutically effective amount to a subject in need thereof.

SETD6 expression is associated with a variety of cancer types, and modulates oncogenesis and cancer progression in a variety of ways. In particular embodiments, the described compositions can be used in treatment for cancers associated with SETD6 overexpression, including but not limited to breast cancer, melanoma, prostate cancer, cervical cancer, glioblastoma multiforme, colon cancer, ovarian cancer, lung cancer, and leukemia, including chronic lymphoblastic leukemia.

In particular embodiments, the therapeutic compositions described herein can be supplied in any pharmaceutically acceptable composition. In such embodiments, one or more RelA-derived polypeptide or chimeric polypeptide-expressing nucleic are provided in a pharmaceutical formulation having a therapeutically effective dose of each therapeutic agent, as described herein, and including standard pharmaceutically acceptable salts, excipients, fillers and the like.

Various delivery systems are known and can be used to administer polypeptides and nucleic acids as therapeutic agents. Such systems include, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the therapeutic molecule(s), construction of a therapeutic nucleic acid as part of a retroviral or other vector, and the like. Methods of introduction include, but are not limited to, intrathecal, intradermal, intramuscular, intraperitoneal (ip), intravenous (iv), subcutaneous, intranasal, epidural, and oral routes. The therapeutics can be formulated for administration by any convenient route, including, for example, infusion or bolus injection, topical, absorption through epithelial or mucocutaneous linings (e.g. , oral mucosa, rectal and intestinal mucosa, and the like) ophthalmic, nasal, and transdermal, and may be administered together with other biologically active agents. Pulmonary administration can also be employed (e.g. , by an inhaler or nebulizer), for instance using a formulation containing an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the described

pharmaceutical treatments by injection, catheter, suppository, or implant (e.g. , implants formed from porous, non-porous, or gelatinous materials, including membranes, such as sialastic membranes or fibers), and the like. In another embodiment, therapeutic agents are delivered in a vesicle, in particular liposomes.

In particular embodiments, the described polypeptides and nucleic acids can be formulated for immediate release, whereby they are immediately accessible to the surrounding environment, thereby providing an effective amount of the active agent(s), upon administration to a subject, and until the administered dose is metabolized by the subject.

In yet another embodiment, the described polypeptides and nucleic acids can be formulated in a sustained release formulation or system. In such formulations, the therapeutic agents are provided for an extended duration of time, such as 1 , 2, 3, 4 or more days, including 1-72 hours, 24-48 hours,. 16-36 hours, 12-24 hours, and any length of time in between. In particular embodiments, sustained release formulations are immediately available upon administration, and provide an effective dosage of the therapeutic

composition, and remain available at an effective dosage over an extended period of time. In other embodiments, the sustained release formulation is not immediately available within the subject and only becomes available, providing a therapeutically effective amount of the active compound(s), after the formulation is metabolized or degraded so as to release the active compound(s) into the surrounding environment.

In one embodiment, a pump may be used. In another embodiment, the sustained released formulations include polymeric materials commonly used in the art, such as in implants, gels, capsules, and the like.

In particular embodiments, the described polypeptides and nucleic acids are formulated using methods well known to those with skill in the art. For instance, in some embodiments, the compounds are formulated with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia world-wide for use in animals, and, more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions, blood plasma medium, aqueous dextrose, and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The medium may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers such as cyclodextrins, proteins such as serum albumin, hydrophilic agents such as methyl cellulose, detergents, buffers, preservatives and the like.

Examples of pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The described compositions can, if desired, also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The described compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, all in immediate and sustained-release formulations as understood in the art. The therapeutic can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.

Therapeutic preparations will contain a therapeutically effective amount of at least one active ingredient, preferably in purified form, together with a suitable amount of carrier so as to provide proper administration to the patient. The formulation should suit the mode of administration.

The ingredients of the described formulations can be supplied either separately or mixed together in unit dosage form, for example, in solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions, or suspensions, or as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Kits comprising the described RelA-derived polypeptide or chimeric polypeptides or encoding nucleic acids are accordingly also contemplated herein.

In particular embodiments of the described pharmaceutical compositions and methods of their use, the described RelA-derived polypeptide or chimeric polypeptides or encoding nucleic acid is administered to the subject as a polypeptide. In other embodiments, it is administered to the subject by way of an expression vector containing a described nucleic acid. It will be appreciated that in such embodiments, expression of the polypeptide can be constitutive or induced, as is well-known in the art. In some embodiments, inducible expression systems can allow for specific targeting of an area, such as a local tumor environment which contains an inducing signal.

In some embodiments, the described RelA-derived polypeptide or chimeric polypeptide, or encoding nucleic acid thereof, is administered to the subject in combination with other pharmaceutical agents for treatment of the disease or condition under treatment. For example, in methods for treating breast cancer the described polypeptides or nucleic acids can be combined with trastuzumab (Herceptin) therapy. In other examples of cancer treatment, the described compositions can be combined with surgery and/or radiation therapy. When provided as part of a treatment regimen or a method of treatment in combination with other therapies, the described RelA-derived polypeptide or chimeric polypeptide or encoding nucleic acids can be administered to the subject in sequence (prior to or following) or concurrently with the described compositions. Where applicable, in particular embodiments, combinations of active ingredients can be administered to a subject in a single or multiple formulations, and by single or multiple routes of administration. The amount of each therapeutic agent for use in the described compositions and methods, and that will be effective, will depend on the nature of the disorder or condition to be treated, as well as the stage of the disorder or condition. Therapeutically effective amounts can be determined by standard clinical techniques. The precise dose to be employed in the therapeutic compositions will also depend on the route of administration for use with the composition, and should be decided according to the judgment of the health care practitioner and each patient' s circumstances. The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, and severity of the condition of the host undergoing therapy.

The therapeutic compounds and compositions of the present disclosure can be formulated or administered at about the same dose throughout a treatment period, in an escalating dose regimen, or in a loading-dose regime (e.g., in which the loading dose is about two to five times the maintenance dose). In some embodiments, the dose is varied during the course of a treatment based on the condition of the subject being treated, the severity of the disease or condition, the apparent response to the therapy, and/or other factors as judged by one of ordinary skill in the art. In some embodiments long-term treatment with the drug is contemplated.

The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

EXAMPLES

Example 1: Cytotoxicity of RelA-CPP chimeric peptides

Methods

Peptides

All peptides were custom-synthesized by GL Biochem, Mimotopes, CPC, and Peptron; the sequences tested are as listed in below Table 1. All peptides assayed were acetylated at the N- terminus. Table 1. Select RelA derived peptides

PrestoBlue Cytotoxicity Assay

Peptides were dissolved to a cone, of lOmM with 5% DMSO (in DDW). Cells were grown in a 75cm2 flask with 12ml DMEM, supplemented, at a final concentration, with 10% FBS, Penicillin (lOOunits/ml) and streptomycin (100 g/ml), in humidified incubator with 5% C02 flow and 37°C. The cells were collected by trypsinization and collected in a 15ml tube at a volume of 12ml. cells were seeded in a 48-well plate at 25,000 cells/well. Cells were Incubated overnight in humidified incubator with 5% C02 flow and 37°C. The medium was removed and replaced with fresh medium containing the peptides at concentration of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 μΜ and then Incubated in humidified incubator with 5% C02 flow and 37°C, for 24h. PrestoBlue cell viability assay was perfumed according to the manufacturer's Instructions. Results

Cytotoxicity of RelA derived-CPP peptide chimeras (detailed in Table 1) were tested in vitro with the Presto Blue assay as described. As shown in Figures 1-6, cytotoxicity was tested on cell lines of cervical cancer (HeLa cells, Figures 1 and 2), breast cancer (MDA MB231 cells, Figure 3), gliobastoma (U87 cells, Figure 4), mouse melanoma (B 16 cells, Figure 5), and liver cancer (HepG2 cells, Figure 6). Several of the tested peptides demonstrated cytotoxicity, with the strongest effects against multiple cell types observed with the RelA-NLS-Rl 1 chimera (SEQ ID NO: 26), Rl 1-NLS-RelA R chimera (SEQ ID NO: 24), and the RelA KA-NLS-R11 chimera (SEQ ID NO: 27).

Discussion

We used the sequence of the RelA peptide, whose crystal structure in a complex with SETD6 has been solved, and designed several RelA subpeptides, which were fused to several cell-penetrating peptides (CPPs) to enable penetration across cellular membranes.

Viability assays revealed that some of the CPPs conferred on the RelA chimeric peptides the ability to induce cell death effect in diverse cancer cell lines, including HeLa cells (Cervical cancer), MDA-MB-231 (Breast cancer), and U87 cells (Glioblastoma brain cancer).

Moreover, a single amino acid substitution of the lysine residue (RelA K310, known as SETD6 methylation site) increased the cytotoxic effect of the peptide and reduced the concentration required for efficient cell death.

Example 2: Pro-proliferative effect of VP22-RelA Fusion Peptides

This example describes the pro-proliferative activity of the RelA peptide fused to the cell penetrating peptide VP22.

All methods are as described above unless otherwise noted.

The effect of VP22-RelA on cellular proliferation and migration was determined. Cellular migration was determined through a wound healing assay of HeLa cells treated with the VP22-RelA peptide, or the control peptide VP22. Cells were grown to confluence. The resultant monolayer was then scratched, and treated with VP22-RelA or VP22 control for 24 hours, and imaged at 0 and 24 hours after the scratch was produced. Treatment with the VP22-RelA peptide showed an accelerated rate of cellular migration in comparison to untreated (Fig. 7). The effect of VP22-RelA on cellular proliferation was measured by PrestoBlue viability assays in HeLa (Cervical cancer) and MDA-MB-231 (Breast cancer) cells. The presented results show a similar trend of proliferation activation by the VP22-RelA peptide as observed in the wound healing assay. This activation was not observed in the control cells, or when the cells were treated with the VP22 negative control peptide (Fig. 8).

These results imply that treatment of cells with the RelA peptide fused to the CPP VP22, leads to accelerated proliferation.

Example 3: In-vivo evaluation of RelA-CPP chimeric peptide efficacy in reduction of cancer progression and tumor growth

This example demonstrates the in vivo effects of RelA chimeric peptides on tumor growth and progression.

To assess the effect of the RelAKA-NLS-Rl 1 peptide on breast cancer tumors in vivo, mouse 4T1 breast cancer cells, were injected into balb/c mice breast tissue and tumors were allowed to grow with or without addition of RelAKA-NLS-Rl 1.

Each balb/C mouse was injected with 2xl0⁵ cells into the breast tissue. Tumors were allowed to grow for a period of 4 days without any treatment. At the end of this initial growth period, daily injections of peptides and controls were performed subcutaneously for 10 days at 5 and 20 ^ concentrations (until day 15 of the experiment). A total of 18 mice were used (6 per treatment condition). 18 days from the beginning of the experiment (3 days after the last injection) mice were sacrificed and tumor volume and weight; and liver, spleen, and lung weight were determined. The results of this experiment are summarized in Table 2 below, and Figures 9 -10.

Mouse tumor volume was measured by caliper at 4 different time points. The volume was calculated using the formula V=LxW². The time points are the date the tumors were first measurable, the last day of peptide injections, the day before that, and the date of euthanization. Mouse #3 in the 20 mg kg group was euthanized before the end of the experiment due to a wound in the neck injection location. Table 2- Tumor volumes at various time points

Discussion

Despite intensive treatment, both RelAKA-NLS-Rl l treated groups gained weight, though the 20mg/kg group gained the least. This was likely due to the effect of the peptide dose on the tumor and its effect on metabolism, or possibly due to the animal size directly. An increase in tumor growth rate was observed in the period between the last injections to the end of the experiment. While the control tumors grew 1.6 times in volume after the last injections, the 5 mg/kg and 20 mg/kg peptide-treated mice had an increase of 1.8 and 2.9 fold respectively, indicating an inhibitory effect of the peptides on tumor growth.

Liver and lung weight-to-body-weight ratios remained unchanged by the treatment or the injection of 4T1 cells, indicating a low toxicity in subcutaneous injections. Control and low dose (5 mg kg) treatment mouse had increased spleen weight-to-body- weight ratio in compared to naive mice. Treatment with a higher dose (20 mg kg) was shown to decrease this ratio to values closer to those received by naive mice. Intravenous injections were potentially lethal as 3 out of the 6 treated mice died, and were therefore removed from the current statistical analysis. Mice treated with subcutaneous injections were vital and lively looking throughout the experiment.

Example 4: In vivo effects of RelAKA-NLS-Rll and RelA-NLS-Rll on breast cancer tumors

This example demonstrates the effect of the RelA inhibitory chimeras on breast cancer tumors using a mouse model.

In order to determine the effect of peptides RelAKA-NLS -R 11 and RelA-NLS-Rl 1 on breast cancer tumors in vivo. MDA-MB 231 cells were injected into breast tissue of nude mice, with each mouse receiving 2xl0⁵ cells. Tumors were allowed to grow for a period of 3 days. At the end of the growth period, RelAKA-NLS-Rl l and RelA-NLS-Rl l were injected subcutaneously daily for 10 days at 5 and 20 concentrations. At the end of the 10 day injection period, mice were sacrificed. Tumor size and weight; and liver, spleen, and lung weight were measured. Results are shown in Figures 11-14 and in Table 3, below.

Table 3- Tumor volumes at various time points

20mg/kg 2.5 2.4 14.4 3.4 3.1 32.674 4.2 3.1 40.362 sc-1

20mg kg 2.8 2.2 13.552 2.5 2.5 15.625 2.5 2.5 15.625 sc-2

20mg/kg 1 1 1 3.7 2.4 21.312 4.5 2.7 32.805 sc-3

20mg kg 1 1 1 3 2.9 25.23 2 2 8 sc-4

20mg kg 1 1 1 2.4 1.6 6.144 2.2 1.9 7.942 sc-5

20mg kg 1 1 1 1 1 1 1 1 1 sc-6

RelA 20mg kg 3.1 27.9 5.5 4.9 132.055 5.3 3.7 72.557

-NLS-R sc-1

11 20mg/kg 1 3.9 3.2 39.936 4.3 3.3 46.827 sc-2 ^{1 1}

20mg kg 1 1 1 4.3 3.2 44.032 3.2 2.7 23.328 sc-3

20mg kg 1 1 1 1 1 1 1 1 1 sc-4

20mg/kg 1 1 1 2.6 2.5 16.25 3.4 3.1 32.674 sc-5

20mg kg 1 1 1 1 1 1 2.4 2.2 11.616 sc-6

Discussion

The data in Table 2 show a nearly 3.5 -fold difference in tumor volume in the 20 mg/kg group for the RelAKA NLS Rl 1 peptide, and nearly 2-fold difference in tumor volume for the RelA NLS Rl l peptide administered at the same concentration. This result further supports the in vivo results presented in the previous example using 4T1 breast cancer cells on balb/c mice. A cytotoxic effect, which could be expected of anti-cancer treatments, was not apparent when organ-body weight ratio was measured. The relative weights of the spleen, the lungs and the liver remained similar to those of the naive mice, which were not injected with cells, and also similar to those of the saline treatment group- which indicates the peptides had no adverse effect on these vital organs. Like the tumor volume, the tumor weights were also low, allowing a high variance. Despite this, a decrease of nearly 40% was observed in tumor volume for the 20 mg kg RelAKA NLS Rl l treatment. The 20 mg/kg RelAKA NLS Rl l treatment group was shown to gain weight similarly to the naive and the saline-treated groups, but not the 5 mg/kg RelAKA NLS Rl 1 treatment or the 20 mg kg RelA NLS Rl 1 treatment groups. The naive group and the saline groups gained weight in a similar rate, implying that the low weight gain in the 5 mg/kg RelAKA NLS Rl l treatment group and the 20 mg/kg RelA NLS Rl l treatment group is due to the treatment itself. This effect is not shown in the 20 mg/kg RelAKA NLS Rl l treatment group which further shows its lack of toxicity and adverse effects on the mouse body. The 20 mg/kg RelAKA NLS Rl 1 treatment group showed the highest success in this experiment, with a nearly 3.5-fold smaller tumor, weighing 40% less, and showing no adverse effects in either of the tested parameters.

Example 5: In vivo effects of RelAKA-NLS-Rll and RelA-NLS-Rll on various well- developed tumors

This example demonstrates the effect of the RelA inhibitory chimeras on cancer tumors or various origin, using a mouse model. Tumors are allowed to grow to larger size and be present for a greater length of time prior to treatment than in previous examples.

Female nude mice at the age of 7 weeks will be anesthetized by an Isoflurane delivery system (SomnoSuite®, Kent scientific). Mice will be injected with 2-15xl0⁶ MDA MB231, U87, or U251, B 16, Hela, 4T1 or HepG2 cells in a 60% serum-free DMEM 40% GelTrex (ThermoFisher) (V/V ratios). Mice will be kept on a heating pad until recovery.

Developing tumors will be measured using a caliper to calculate tumor volume. Upon reaching an average tumor volume of 100mm³, 1-100 mg kg, preferably 5-25 mg/kg peptides will be injected subcutaneously on a 5-times-a-week basis for a period of 3 weeks. Mouse weight and tumor volume will be monitored twice each week. Mice will be compared to a negative control group- injected with saline only, and a naive group for organ weight comparison.

Following the 3 -weeks treatment, mice will be euthanized and tumors will be extracted. Tumor weight will be recorded. The liver, lungs and spleen will also be removed, and weight will be recorded, to observe any toxic effect.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

Claims

1. An isolated polypeptide inhibitor of SET-domain-containing protein 6 (SETD6) methyltransferase, consisting essentially of an amino acid sequence set forth herein as SEQ ID NO: 1, or a functional fragment or variant thereof.

2. An isolated polypeptide inhibitor of SET-domain-containing protein 6 (SETD6) methyltransferase, comprising an amino acid sequence set forth herein as SEQ ID NOs 2-5, or a functional fragment or variant thereof.

3. The isolated polypeptide of claim 1 or claim 2, further comprising at least one cell penetrating peptide (CPP) fused to the isolated polypeptide at either the N- or C-terminal ends of the isolated polypeptide, thereby producing an isolated chimeric polypeptide.

4. The isolated chimeric polypeptide of claim 3, wherein the CPP is fused to the isolated polypeptide through at least one linking amino acid.

5. The isolated chimeric polypeptide of claim 3, comprising an amino acid sequence at least 98% identical to SEQ ID NOs: 10-13, or a functional fragment or variant thereof.

6. The isolated chimeric polypeptide of claim 3, comprising a polypeptide at least 98% identical to an amino acid sequence set forth as SEQ ID NOs 14-29, or a functional fragment or variant thereof.

7. The isolated polypeptide of any one of claims 1-2 or the isolated chimeric polypeptide of any one of claims 3-6, further comprising an Acetyl group at the N- or C- terminal ends of the isolated polypeptide or the isolated chimeric polypeptide.

8. An isolated nucleic acid comprising a nucleic acid sequence encoding the isolated polypeptide of any one of claims 1-2 or the isolated chimeric polypeptide of any one of claims 3-6.

9. An expression vector comprising the isolated nucleic acid of claim 8.

10. A composition comprising the isolated polypeptide of any one of claims 1-2 or the isolated chimeric polypeptide of any one of claims 3-7, for use in treatment or prevention of a disease or condition characterized by aberrant overexpression of SET-domain- containing protein 6 (SETD6).

11. The composition of claim 10, wherein the disease or condition is a SETD6- associated cancer.

12. The composition of claim 11, wherein the SETD6-associated cancer is selected from the group consisting of breast cancer, glioblastoma, cervical cancer, colon cancer, ovarian cancer, lung cancer, and leukemia.

13. A composition comprising the isolated nucleic acid of claim 8 or expression vector of claim 9, for use in treatment or prevention of a disease or condition characterized by aberrant overexpression of SET -domain-containing protein 6 (SETD6).

14. The composition of claim 13, wherein the disease or condition is a SETD6- associated cancer.

15. The composition of claim 14, wherein the SETD6-associated cancer is selected from the group consisting of breast cancer, glioblastoma, cervical cancer, colon cancer, ovarian cancer, lung cancer, and leukemia.

16. The composition of any one of claims 10-15 for use in a treatment regimen including an additional chemotherapeutic agent, surgery, or radiotherapy.