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WO2025193649A1 - Utilisation d'un agent de dégradation du p300/cbp utilisée pour le traitement du cancer - Google Patents

Utilisation d'un agent de dégradation du p300/cbp utilisée pour le traitement du cancer

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Publication number
WO2025193649A1
WO2025193649A1 PCT/US2025/019293 US2025019293W WO2025193649A1 WO 2025193649 A1 WO2025193649 A1 WO 2025193649A1 US 2025019293 W US2025019293 W US 2025019293W WO 2025193649 A1 WO2025193649 A1 WO 2025193649A1
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WIPO (PCT)
Prior art keywords
compound
cbp
prostate cancer
cancer
subject
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PCT/US2025/019293
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English (en)
Inventor
Zhixiang Chen
Jie Luo
Abhijit PAROLIA
Shaomeng Wang
Arul Chinnaiyan
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Regents Oe University Of Michigan
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Regents Oe University Of Michigan
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Publication of WO2025193649A1 publication Critical patent/WO2025193649A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4166—1,3-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. phenytoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This disclosure relates to a CBP/p300 degrader compound, methods of using the compound to degrade CBP/p300, and methods to treat diseases and disorders, such as cancer.
  • the androgen receptor (AR) is the primary lineage-specific transcription factor in prostatic epithelial cells. Dai, C., Heemers, H. & Sharifi, N. Androgen Signaling in Prostate Cancer. Cold Spring Harb Perspect Med 7, doi:10.1101/cshperspect.a030452 (2017). Targeting AR has emerged as a promising therapeutic approach, yet the development of resistance to anti-androgen treatments highlights the critical need for new therapeutic strategies. Watson, P. A., Arora, V. K. & Sawyers, C. L. Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer. Nat Rev Cancer 15, 701-711 , doi:10.1038/nrc4016 (2015).
  • AR cancer-specific enhanceosome the AR neo- enhanceosome
  • the transcriptional coactivators p300 and CBP are histone acetyltransferases.
  • Ortega, E. et al. Transcription factor dimerization activates the p300 acetyltransferase.
  • a key AR cofactor, BRD4 has been recognized as a druggable target in PCa.
  • HATs Paralog histone acetyltransferases
  • Also provided herein are methods of treating prostate cancer in a subject comprising administering a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, to the subject.
  • Figs. 1a-c show p300 is a determinant cofactor of the activated AR enhanceosome in prostate cancer.
  • Fig. 1a Immunoblot analysis of key histone marks in four pairs of matched prostate cancer (T) and benign adjacent tissues (N) . Quantitation and fold change (FC) of the respective histone marks is provided to the right.
  • Fig. 1b Cumulative DepMap CRISPR knockout essentiality scores of histone acetyltransferases and deacetylases in AR+ prostate cancer cell lines. Aggregated z-scores for each gene derived from LNCaP and VCaP DepMap screens.
  • Figs. 2a-e show degradation of p300/CBP selectively represses epigenetic marks in the AR enhancesome more effectively and distinctly compared to bromodomain inhibition.
  • Fig. 2a Structure of Compound A and schematic of p300 and CBP domains.
  • Compound A-targeted bromodomain (BRD) is highlighted by the arrow.
  • Key domains of p300/CBP proteins are noted.
  • Taz1 TAZ zinc finger domain; Kix: Kinase-inducible domain (KID) interacting domain; PHD: Plant homeodomain; HAT: Histone acetyltransferase domain; IBID: N-terminal interferon-binding domain.
  • Fig. 2b Immunoblot analysis of p300 and CBP in VCaP cells treated with 100nM Compound A for indicated time durations.
  • Fig. 2c TMT (tandem mass tag) mass spectrometry assay to evaluate effects of Compound A (1 OOnM, 4hrs) on the proteome of VCaP cells.
  • Data plotted Iog2 of the fold change (FC) versus DMSO control against - Iog2 of the p-value per protein from n 3 independent experiments. All t-tests performed were two-tailed t-tests assuming equal variances.
  • Fig. 2d Venn diagrams of genome-wide changes of H2BK5ac and H2BK20ac ChlP-seq peaks after Compound A (100nM 4hrs) or GNE-049 (1uM 4hrs) treatments in VCaP cells.
  • Fig. 2e Immunoblots of labeled proteins and histone marks in VCaP cells treated with 100nM Compound A for indicated time durations.
  • Figs. 3a-c show p300/CBP degradation relative to bromodomain inhibition leads to repression of key oncogenic gene programs and targets.
  • Fig. 3a Volcano plot of gene expression in VCaP cells treated for 24 hours with either 100nM Compound A or 1uM GNE-049. Genes CCND1, CITED2, and NKX3-1, which are exclusively suppressed by Compound A, are highlighted.
  • Fig. 3b Cuantitative-PCR (qPCR) of NKX3-1, CCND1, CITED2 and Myc expressions in VCaP cells pre-treated with 100uM thalidomide for 1 hr, then treated with Compound A or GNE-409 for 4hrs.
  • Figs. 4a-e show p300/CBP degradation inhibits tumor growth in preclinical in vivo models of CRPC and synergizes with enzalutamide without evident toxicities.
  • Figs. 5a-c show p300 is the key factor for H2BNTac and activated ERG cistrome in prostate cancer cells.
  • Fig. 5a Bar charts depicting proportion of FOXA1, SMARCA4, p300, and BRD4 peaks that map to all AR peaks located in non-promoter regions.
  • Fig. 5b Distribution of genomic locations based on ChlP-seq peak analysis: p300 only binding sites, AR/p300 co-bound sites, AR only binding sites, BRD4/AR co-bound sites, and BRD4 only binding sites.
  • Fig. 5c ChlP-seq (ERG, FOXA1, p300, BRD4, MED1, H3K27ac and H2BK20ac) and ATAC-seq readdensity heatmaps at ERG/p300 co-bound and ERG only binding sites in VCaP cells.
  • Figs. 6a-b show characterizing the on-target degradation effects of Compound A.
  • Fig. 6b Schematic of a methyl Compound A structure (Compound A-me).
  • Fig. 7 shows immunoblots of H2BK5ac, H2BK20ac and H2BK120ac in VCaP cells treated with 100nM Compound A for noted time durations.
  • Figs. 8a-b show Compound A inhibits histone acetylation at AR enhanceosome without affecting chromatin accessibility and AR/FOXA1 chromatin distribution.
  • Fig. 8a Venn diagrams depicting genome-wide changes of H3K27ac ChlP-seq peaks in VCaP cells treated with 100nM Compound A or 1uM GNE-049 for 4hrs.
  • Fig. 8b ChlP-seq read-density heatmaps of AR and FOXA1 peaks within F0XA1/AR shared cis- regulatory elements in VCaP cells treated with 100nM Compound A and 1uM AU-15330 for 4hrs.
  • Figs. 9a-c show CBPD-409 significantly suppresses AR transactivation activity.
  • Fig. 9a GSEA plots for mTORCI, FOXA1, Myc and ERG signal pathways using the fold change rank- ordered gene signature from VCaP cells treated with Compound A for 24hrs.
  • Fig. 9b ChlP-seq tracks of AR, H3K27ac, H2BK20ac and Pol II within the TMPRSS2 gene locus in VCaP cells treated with or without 1uM GNE-049 for 4hrs.
  • Fig. 9c Average Pol II ChlP-seq coverage profiles of AR downregulated genes (AR_DN), AR activated genes (ARJJP) and random genes in VCaP cells treated with 100nM Compound A or 1uM GNE-049 for 4hrs.
  • Figs. 10a-d show Compound A targets a unique set of cell cycle-related genes to suppress proliferation.
  • Fig. 10a Venn diagrams showing overlaps between genes suppressed by Compound A and those suppressed by GNE-049 or CCS1477 in VCaP cells. Data obtained from RNA-Seq with two independent samples for each condition.
  • Fig. 10c GSEA plots for G2M checkpoint, AR, E2F, and Myc signaling pathways, based on rank- ordered fold change gene signatures in VCaP cells: comparison of 24hrs treatment with 100nM Compound A vs. 1uM GNE-049.
  • DEG differentially expressed gene.
  • Fig. 10d qPCR of KLK3, CITED2, CCND1 and NKX3-1 expression in LNCaP cells treated with Compound A, GNE-049 or CCS1477 for 24hrs.
  • Figs. 11 a-g show Compound A exhibits superior cytotoxicity in AR-positive prostate cancer cells over other p300/CBP inhibitors.
  • Figs. 12a-c show Compound A monotherapy in intact VCaP tumor model.
  • Fig. 12b Bar charts of endpoint (day 33) intact VCaP xenograft tumor weights, (two-sided t-test).
  • Figs. 13a-b show Compound A efficacy in VCaP-CRPC tumor model.
  • Fig. 13a Individual tumor image of vehicle, Enza, Compound A and Enza+ Compound A groups from VCaP-CRPC study.
  • Figs. 14a-c show Compound A doesn't exhibit evident toxicity in CD1 mice.
  • Fig. 14b Liver function and kidney function tests from CD1 mice study.
  • ALT alanine transaminase
  • AST aspartate transaminase
  • TBili total bilirubin
  • BUN blood urea nitrogen
  • Fig. 15 shows rank-order plot of AUG (area under the curve) values for CBPD-409 from PRISM drug screening assays of cancer cell lines with Compound A - for 52 sensitive cancer cell lines (AUC ⁇ 0.3).
  • p300/CBP are requisite histone acetyltransferases (HATs) to define the active AR enhanceosome and hyperacetylate histone H2B N-terminus (H2BNTac) to drive unique oncogenic transcriptome to promote the cell proliferation.
  • HATs histone acetyltransferases
  • H2BNTac hyperacetylate histone H2B N-terminus
  • Compound A which has the following structure: or a pharmaceutically acceptable salt thereof.
  • Compound A is sometimes alternatively referred to as CBPD-409. These names are used interchangeably throughout this disclosure.
  • Compound A can be present as a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salt refers to salts of a compound which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue side effects, such as, toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio.
  • compositions described herein include those derived from suitable inorganic and organic acids and bases. These salts can be prepared in situ during the final isolation and purification of the compound.
  • acid addition salts can be prepared by 1) reacting the purified compound in its free-base form with a suitable organic or inorganic acid and 2) isolating the salt thus formed.
  • acid addition salts might be a more convenient form for use and use of the salt amounts to use of the free basic form.
  • Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, o
  • base addition salts can be prepared by 1) reacting the purified compound in its acid form with a suitable organic or inorganic base and 2) isolating the salt thus formed.
  • base addition salt might be more convenient and use of the salt form inherently amounts to use of the free acid form.
  • Salts derived from appropriate bases include alkali metal (e.g., sodium, lithium, and potassium), alkaline earth metal (e.g., magnesium and calcium), ammonium and N + (Ci-4alkyl)4 salts.
  • alkali metal e.g., sodium, lithium, and potassium
  • alkaline earth metal e.g., magnesium and calcium
  • ammonium and N + (Ci-4alkyl)4 salts e.g., sodium, lithium, and potassium
  • Basic addition salts include pharmaceutically acceptable metal and amine salts.
  • Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum.
  • the sodium and potassium salts are usually preferred.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
  • Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like.
  • Suitable amine base addition salts are prepared from amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use.
  • Compound A can act as a degrader of CBP/p300.
  • CBP/p300 coactivator family in humans is composed of two closely related transcriptional co-activating proteins that interact with numerous transcription factors and act to increase the expression of their target genes.
  • CBP/p300 serve as critical transcriptional coactivators of AR (Comuzzi, et al. J. Pathol. 2004, 204 (2), 159-166.; Waddell, et al. Cancers 2021, 13 (12), 2872.; Chen, et al. Theranostics 2022, 12 (11), 4935-4948.), and these proteins are highly expressed in human prostate cancer and their expressions are positively associated with AR signaling in prostate cancer (Comuzzi, et al. J. Pathol. 2004, 204 (2), 159-166.; Debes, et al. Cancer Res. 2003, 63 (22), 7638-7640.; Welti, et al. Cancer Discov. 2021, 11 (5), 1118-1137.).
  • the disclosure provides a method of degrading CBP/p300 comprising contacting CBP/p300 with Compound A or salt thereof in an amount effective to decrease CBP/p300 activity.
  • the contacting occurs in vitro.
  • the contacting occurs in vivo.
  • the contacting comprises administering Compound A or salt thereof to a subject in need thereof.
  • the terms "patient” and "subject” may be used interchangeably and mean animals, such as dogs, cats, cows, horses, and sheep (i.e., non-human animals) and humans.
  • the patient is a mammal (e.g., human).
  • the subject suffers from cancer.
  • the cancer is adrenal cancer, blood cancer, or prostate cancer. In various cases, the cancer is prostate cancer. In various cases, the prostate cancer is AR+ prostate cancer. In various cases, the prostate cancer is castration-resistant prostate cancer. In various cases, the cancer is metastatic. In various cases, the metastatic cancer is castration-resistant prostate cancer. In various cases, the cancer is enzalutamide resistant. [0080] In some cases, Compound A reduces mRNA levels of AR, KLK3 (which encodes prostate specific antigen (PSA)), and/or c-Myc genes. In various cases, Compound A reduces mRNA levels of each of AR, KLK3, and c-Myc genes. In various cases, Compound A suppresses AR signaling and/or c-Myc expression.
  • PSA prostate specific antigen
  • Another aspect of the disclosure provides a method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of Compound A or salt thereof.
  • the terms “treating”, “treat” or “treatment” and the like can include preventative (e.g., prophylactic) and palliative treatment.
  • the term "therapeutically effective” refers to the amount of a compound or a pharmaceutically acceptable salt thereof that, in either a monotherapy of Compound A or as a combination therapy of a Compound A and an additional therapeutic agent, is sufficient to treat cancer. Effectiveness of a therapy can be assessed by any means suitable for the particular cancer.
  • the patient can respond to the therapy as measured by at least a stable disease (SD), as determined by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 protocol (Eisenhauer et al., EurJ Cancer, 2009; 45(2):228-247).
  • SD stable disease
  • RECIST Response Evaluation Criteria in Solid Tumors
  • the stable disease is neither sufficient shrinkage to qualify for partial response (PR) nor sufficient increase to qualify for progressive disease (PD).
  • the disease or disorder is cancer.
  • the cancer is adrenal cancer, blood cancer, or prostate cancer.
  • the cancer is prostate cancer.
  • the prostate cancer is AR+ prostate cancer.
  • the prostate cancer is castration-resistant prostate cancer.
  • cancer is metastatic.
  • the metastatic cancer is castration-resistant prostate cancer.
  • the cancer is enzalutamide resistant.
  • a further aspect of the disclosure provides the use of Compound A or a pharmaceutically acceptable salt thereof in combination with enzalutamide in the treatment of a disease or disorder associated with aberrant CBP/p300 activity in a subject.
  • Enzalutamide is marketed as XTANDI® for treatment of castration resistant or castration sensitive prostate cancer. See XTANDI® prescribing information updated 11/2023 at https://www.accessdata.fda.gov/drugsatfda_docs/label/2023/213674s010, 203415s022lbl.pdf.
  • the disease or disorder is cancer.
  • the cancer is adrenal cancer, blood cancer, or prostate cancer.
  • the cancer is prostate cancer.
  • the prostate cancer is AR+ prostate cancer. In various cases, the prostate cancer is castration-resistant prostate cancer. In various cases, the cancer is metastatic. In various cases, the metastatic cancer is castration-resistant prostate cancer. In various cases, the cancer is enzalutamide resistant. [0085] Also contemplated is the use of Compound A, or salt thereof for the manufacture of a medicament in the treatment of cancer.
  • the cancer is adrenal cancer, blood cancer, or prostate cancer.
  • the cancer is prostate cancer.
  • the prostate cancer is AR+ prostate cancer.
  • prostate cancer is castration-resistant prostate cancer. In various cases, the cancer is metastatic. In various cases, the metastatic cancer is castration-resistant prostate cancer. In various cases, the cancer is enzalutamide resistant.
  • a method of degrading p300 and CBP comprising contacting each of p300 and CBP with an effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • a method of treating prostate cancer in a subject comprising administering a therapeutically effective amount of Compound A to the subject or a pharmaceutically acceptable salt thereof.
  • a method of treating an enzalutamide resistant tumor in a subject comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • a method of treating multiple myeloma in a subject comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • a method of treating a neuroblastoma in a subject comprising administering to the subject a therapeutically effective amount of Compound A, or a pharmaceutically acceptable salt thereof.
  • a method of depleting CBP and p300 in a subject comprising administering to the subject a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof.
  • DMAP 4-dimethylaminopyridine
  • DMF N,N’-Dimethylformamide
  • DMSO dimethyl sulfoxide
  • DIBAL diisobutylaluminum hydride
  • DIPEA N, N -Diisopropylethylamine
  • EtsN triethylamine
  • EtOAc ethyl acetate
  • HCI hydrochloric acid
  • KOH potassium hydroxide
  • MeCN acetonitrile
  • Mel methyl iodide
  • MeOH methanol
  • NaBH(OAc)3 sodium triacetoxyborohydride
  • RuPhos ruthenium-based ligand (Phospha-methyl-di(tert- butyl)phenylphosphine); RuPhos Pd G2, second-generation ruthenium-phosphine ligand; f-BuONa, sodium tert- butoxide; TFA, trifluoroacetic acid; THF, tetrahydrofuran.
  • Step 1 Synthesis of 1 ,1 '-(3-iodo-6,7-dihydro-1 H-pyrazolo[4,3-c]pyridine-1 ,5(4H)-diyl)bis(ethan-1 -one) and 1,1'- (3-iodo-6,7-dihydro-2H-pyrazolo[4,3-c]pyridine-2,5(4H)-diyl)bis(ethan-1-one) (2)
  • Step 2 Synthesis of 1-(3-iodo-1 ,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)ethan-1 -one (3)
  • Step 3 Synthesis of 1-(3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5- yl)ethan-1-one and 1-(3-iodo-2-(tetrahydro-2H-pyran-2-yl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5- yl)ethan-1-one (4)
  • Step 4 Synthesis of 1-(3-(7-(difluoromethyl)-3,4-dihydroquinolin-1 (2H)-yl)-1-(tetrahydro-2H-pyran-2-yl)-1 , 4,6,7- tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)ethan-1-one and 1-(3-(7-(difluoromethyl)-3,4-dihydroquinolin-1 (2H)-yl)- 2-(tetrahydro-2H-pyran-2-yl)-2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)ethan-1-one (6)
  • Step 5 Synthesis of 1-(3-(6-bromo-7-(difluoromethyl)-3,4-dihydroquinolin-1 (2H)-yl)-1-(tetrahydro-2H-pyran-2-yl)-
  • Steps 6-7 Synthesis of 1 -(3-(7-(difluoromethyl)-6-(1 -methyl-1 H-pyrazol-4-yl)-3,4-dihydroquinolin-1 (2H)-yl)- 1 ,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)ethan-1-one (9)
  • Step 8 Synthesis of c/s-4-(tosyloxy)cyclohexane-1-carboxylate (11)
  • Step 9 Synthesis of frans-4-(5-acetyl-3-(7-(difluoromethyl)-6-(1 -methyl-1 H-pyrazol-4-yl)-3,4-dihydroquinolin- 1 (2H)-yl)-4,5,6,7-tetrahydro-1 H-pyrazolo[4,3-c]pyridin-1-yl)cyclohexane-1 -carboxylate (12)
  • Step 10 Synthesis of frans-4-(5-acetyl-3-(7-(difluoromethyl)-6-(1 -methyl-1 H-pyrazol-4-yl)-3,4-dihydroquinolin- 1 (2H)-yl)-4,5,6,7-tetrahydro-1 H-pyrazolo[4,3-c]pyridin-1-yl)cyclohexane-1-carbaldehyde (13)
  • Step 11 Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(piperazin-1-yl)isoindoline-1 , 3-dione (15) [0099] DIPEA (1.44 mL) was added to a mixture of 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1, 3-dione (14, 570 mg, 1.0 eq) and ferf-butyl piperazine-1 -carboxylate (769 mg, 2.0 eq) in DMSO (10 mL). The mixture was heated at 110 °C for 18 h.
  • Step 12 Synthesis of 5-(4-(((1 r,4r)-4-(5-acetyl-3-(7-(difluoromethyl)-6-(1-methyl-1 H-pyrazol-4-yl)-3,4- dihydroquinolin-1 (2H)-yl)-4,5,6,7-tetrahydro-1 H-pyrazolo[4,3-c]pyridin-1-yl)cyclohexyl)methyl)piperazin-1-yl)-2- (2, 6-dioxopiperidin-3-yl)isoindoline-1 , 3-dione (Compound A, CBPD-409)
  • Step 1 Synthesis of ferf-butyl 4-(5-acetyl-3-(7-(difluoromethyl)-6-(1 -methyl-1 H-pyrazol-4-yl)-3,4- dihydroquinolin-1 (2H)-yl)-4,5,6,7-tetrahydro-1 H-pyrazolo[4,3-c]21 midazol-1-yl)piperidine-1 -carboxylate (18)
  • CS2CO3 (460 mg, 3.0 eq) was added to a solution of compound 9 (200 mg, 1.0 eq) and tert-butyl 4- ((methylsulfonyl)oxy)piperidine-1 -carboxylate (264 mg, 2.0 eq) in DMF (6 mL). The mixture was heated at 80 °C for 18 h.
  • Step 2 Synthesis of 1-(3-(7-(difluoromethyl)-6-(1 -methyl-1 H-pyrazol-4-yl)-3,4-dihydroquinolin-1 (2H)-yl)-1- (piperidin-4-yl)-1 ,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]21 midazol-5-yl)ethan-1-one (19)
  • Step 3 Synthesis of (3aS,4S,6aR)-4-(5-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1 -methyl-1 H-pyrazol-4-yl)-3,4- dihydroquinolin-1 (2H)-yl)-4, 5, 6, 7-tetrahydro-1 H-pyrazolo[4,3-c]22midazol-1-yl)pi peridin-1 -yl)-5- oxopentyl)tetrahydro-1 H-thieno[3,4-d]22midazole-2(3H)-one (21, GNE-049-Biotin)
  • reaction mixture was directly purified by pre-HPLC (35-100% MeCN/FW in 65 min) to give product (3aS,4S,6aR)-4-(5-(4-(5-acetyl-3-(7-(difluoromethyl)-6-(1 -methyl-1 H- pyrazol-4-yl)-3,4-dihydroquinolin-1 (2H)-yl)-4,5,6,7-tetrahydro-1 H-pyrazolo[4,3-c]22midazol-1-yl)piperidin-1-yl)-5- oxopentyl)tetrahydro-1 H-thieno[3,4-d]22midazole-2(3H)-one (21, GNE-049-Biotin): UPLC-MS: 1.50 min, purity > 95%; MS (ESI) m/z calcd.
  • H2BNTac H2BNTac in Pea tissues.
  • This set which includes H2BK5ac, H2BK12ac, H2BK16ac, and H2BK20ac, is known to mark active intergenic enhancer regions.
  • p300 is shown to be a determinant cofactor of the activated AR enhanceosome in prostate cancer. (Fig. 1 a).
  • H2BK5ac 82.3%) and H2BK20ac (76%) sites are co-localized with p300 peaks.
  • H3K18ac 46.7%)
  • H3K27ac 46.3%) sites showed overlap with p300, further supporting the specific dependency of H2BNTac on p300.
  • HDACs histone deacetylases
  • p300 As an essential coactivator, p300 predominantly co-localizes with transcription factors at specific chromatin regions, playing a pivotal role in the regulation of gene transcription. Exploration of the interplay between p300 and AR and ERG, which are the two most critical oncogenic transcription factors in PCa cells, in AR + /ERG + VCaP cells. Tomlins, S. A. et al. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 310, 644-648, doi : 10.1126/science.1117679 (2005).
  • ChlP-seq peaks of PCa master transcription factors (AR, ERG and FOXA1) and transcriptional cofactors (SMARCA4, p300 and BRD4) were categorized into quartiles (Q1-Q4) and then assessed their presence at binding sites of AR and ERG. As expected, majority of the strongest AR and ERG peaks (Q4 peaks) are occupied by pioneer transcription factor FOXA1 (Parolia, A. et al. Distinct structural classes of activating FOXA1 alterations in advanced prostate cancer. Nature 571, 413-418, doi: 10.1038/s41586-019-1347-4 (2019). Sahu, B. et al.
  • the AR cistrome was then categorized into p300- dependent and p300-independent groups, which are defined by the co-occupancy of AR and p300. Notably, although the intensity of AR peaks was similar between the two groups, 50% of AR cistrome which are p300 dependent, exhibit high activity, with BRD4, MED1, H2BK20ac and H3K27ac enrichment and high chromatin accessibility, yet the p300 independent AR cistrome are clearly inactive (Fig. 5a). By analyzing the superenhancer scores, it was found that the super-enhancers are more abundant on AR/p300 co-occupied sites, which further suggests that p300 is the determinant for activity of AR enhanceosome. The same analysis by intercepting p300 and ERG binding sites demonstrate that p300 acts as critical factor for ERG activity as well (Fig. 5a-b).
  • CRISPR-Cas9 and shRNA were used to target p300 in LNCaP and 22Rv1 cells.
  • targeting p300 is enough to suppress various histone acetylation and cell proliferation, but targeting both p300 and its paralog, GBP, could reach the enhanced suppression effects on histone acetylation and cell proliferation (Fig. 1c).
  • targeting both p300 and CBP is required to inhibit histone acetylation and cell growth in 22Rv1 cells (Fig. 5c), suggesting the functional compensation of two paralogs and the necessity to target both paralogs in order to reach the optimal suppression effects.
  • HATi histone acetyltransferase inhibitors
  • RNA-Pol2 ChlP-seq shows that Compound A not only suppresses histone modifications at AR binding sites, but also dislodges RNA-Pol2 from AR target genes loci, which could not be achieved by GNE-049 (Fig. 9c). Importantly, Compound A only inhibits RNA-Pol2 loading on AR activated genes, without affecting AR suppressed genes and other random genes. Nascent RNA production by 5-ethy ny luridine sequencing (EU-seq) was monitored and found out that Compound A pronouncedly and preferentially represses AR target genes nascent RNA production. Notably, the substantial decrease of nascent RNAs produced at AR binding sites, which are considered as AR controlled enhancer RNAs (eRNAs), supports that targeting p300/CBP disrupts the activity of AR enhanceosome.
  • eRNAs AR controlled enhancer RNAs
  • Compound A suppresses p300/CBP bromodomain dependent and independent oncogenic activities
  • VCaP cells were treated with CBPD-409 and cereblon ligand thalidomide, which competitively inhibit the p300/CBP degradation by proteosome system. Strikingly, thalidomide fully recovers the expression of CITED2, NKX3-1 and CCND1, but minimally rescues Myc expression, which is reliant on p300/CBP bromodomain activity (Fig. 3b & Fig. 10d).
  • Compound A exhibits enhanced cytotoxicity in AR positive prostate cancer cells
  • Compound A Compared to GNE-049 and CCS-1477, Compound A exhibits higher potency to suppress AR positive cell growth (Fig. 3c). Importantly, compared to other reported p300/CBP degraders, Compound A also exhibits the enhanced cytotoxicity to suppress AR+ PCa cells growth (Fig. 11 d).
  • the PROTAC-inactive Compound A-Me exhibited similar suppression effects to GNE-049 in LNCaP cells.
  • Compound A cytotoxicity was addressed in a panel of more than 120 human-derived cancer or normal cell lines from 22 different lineages.
  • AR positive prostate cancer cells display highest sensitivity to Compound A.
  • AR negative prostate cancer cells, neuroendocrine prostate cells and human non- neoplastic prostatic cells are all resistant to Compound A, which suggests p300/CBP preferentially play the pivotal role in concert with AR.
  • the multiple myeloma and neuroblastoma cells which have been characterized as p300/CBP dependent, are also sensitive to Compound A.
  • VCaP cells were subcutaneously implanted in SCID mice, which underwent castration two weeks post-implantation once the tumors reached 200mm 3 , to induce disease regression. Subsequently, castration-resistant tumors regrew to 200 mm 3 , at which points the treatments commenced.
  • the Response Evaluation Criteria in Solid Tumors (RECIST) were applied for tumor stratification: Progressive Disease (PD);, defined as at least a 20% increase in tumor size; Stable Disease (SD), defined as an increase of less than 20% and a decrease of less than 30%; and Partial Response (PR), defined as at least a 30% decrease in tumor size.
  • PD Progressive Disease
  • SD Stable Disease
  • PR Partial Response
  • the vehicle and Enza groups exhibited 100% PD; the Compound A group showed 83% PD, 11% SD, and 6% PR; and the Compound A + Enzalutamide group showed 17% PD, 33% SD, and 50% PR.
  • Compound A is potent to suppress VCaP-CRPC tumor growth and exhibits enhanced efficacy when combining with enzalutamide, triggering disease regression in more than 60% of animals (11 out of 18) (Fig. 4a), without affecting animal body weights.
  • Compound A+enzalutamide demonstrate synergy and degrade p300/CBP which inhibits CRPC tumor growth and without any apparent toxicities. Concordant with in vitro results, the IHC staining and western blot confirm the robust downregulation of p300/CBP, AR, Myc, Ki67, H3K27ac and H2BK20ac after 5 days of treatments.
  • Compound A The only noted side effect of Compound A is defect in germ cell maturation and testicular atrophy, yet such side effect is reversible after halt the Compound A treatment.
  • the toxicity of Compound A in human primary cells was evaluated by treating human primary CD3+ Pan T cells, human primary NK cells and immortalized human B cells (GM24694) with Compound A or BRD4 degrader ZBC-260. In contrast to the strong inhibition effects of ZBC-260, Compound A does not exhibit significant cytotoxicity in all three human cells.
  • H2BNT N-terminal histone H2B
  • H2BNTac which includes the recently characterized H2BK5ac, H2BK12ac, H2BK16ac, and H2BK20ac, is known to mark active intergenic enhancer regions and solely dependent on histone acetyltransferases p300/CBP1.
  • sensitivity to Compound A strongly correlated with p300 genetic dependency as assessed by CRISPR knockout studies, reinforcing the specificity of Compound A for targeting enhancer-dependent cancers. Additionally, sensitivity was closely associated with dependency on multiple components of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, further underscoring the importance of histone hyperacetylation pathways in these cancer subtypes.
  • SAGA Spt-Ada-Gcn5 acetyltransferase
  • LNCaP_Parental LNCaP_EnzR
  • All cells were genotyped to confirm their identity at the University of Michigan Sequencing Core and tested routinely for Mycoplasma contamination.
  • LNCaP, 22RV-1, CWR-R1, PC-3, and DU145 were grown in Gibco RPMI-1640 + 10% FBS (ThermoFisher).
  • VCaP was grown in Gibco DMEM Glutamax + 10% FBS (ThermoFisher).
  • GNE-049, CCS1477, A485, JQAD1, enzalutamide, carfilzomib, and thalidomide were purchased from Selleck Chemicals.
  • Dcbpl was purchased from MedChemExpress.
  • H3K27ac (Diagenode: C15410196); p300 (Abeam: ab14984); H2BK20ac (Cell Signaling Technology: 34156S); H2BK5ac (Cell Signaling Technology: 12799S); AR (Millipore: 06-680); FOXA1 (Thermo Fisher Scientific: PA5-27157); BRD4 (Diagenode: C15410337); H3K18ac (Active Motif: 39755); MED1 (Active motif: 61065); ERG (Cell Signaling Technology: 97249S); RNA Pol II (Active motif: 39097); H3K18ac (Active Motif: 39755); CCND1
  • a total of 4000 cells per well were plated into poly-D lysine coated 96-well plates (Corning) in their respective culture medium and incubated at 37 °C in an atmosphere of 5% CO2. After 24 hours incubation, a serial dilution of compounds was prepared and added to the plate with each 6 replicates of each concentration. The cells were further incubated for 120 hours, and the CellTiter-Glo assay (Promega) was performed according to the manufacturer's instruction to determine cell proliferation. The luminescence signal from each well was read by the Infinite M1000 Pro plate reader (Tecan). The results were analyzed by GraphPad Prism software (GraphPad Software).
  • a total of 4,000 cells per well were seeded in clear flat bottom 96-well plates. After 24 hours incubation, compounds were added to the cells at logarithmic dose series. One day and 6 days after seeding, cellular ATP content was measured using Cel IT! terGio (Promega). Measurements after 8 days were divided by the measurement after 1 day (that is, the TO plate) to derive fold proliferation.
  • 4,000 cells per well were seeded in clear 96-well plates (Costar no. 3513). Every 4h, phase object confluence (percentage area) for proliferation were measured.
  • Cell lysates were prepared by RIPA buffers (ThermoFisher Scientific) with 1x HaltTM Protease Inhibitor Cocktail (ThermoFisher) and denatured in the complete NuPage 1x LDS/reducing agent buffer (Invitrogen) with 15 min heating at 70 °C.
  • the protein concentration was measured by Pierce BCA Protein Assay Kit (ThermoFisher Scientific). 15-30 ug protein was loaded and separated on NuPAGE 3 to 8%, Tris-Acetate Protein Gel (ThermoFisher Scientific) or NuPAGE 4 to 12%, Bis-Tris Protein Gel (ThermoFisher Scientific) and transferred to 0.45-pm nitrocellulose membrane (Thermo Fisher Scientific).
  • the membranes were blocked by blocking buffer (Tris-buffered saline, 0.1% Tween (TBS-T), 5% non-fat dry milk) for 1 hour and then blotted with primary antibodies in 4°C overnight. After incubation with HRP-conjugated secondary antibodies, membranes were imaged on an Odyssey CLx Imager (LiCOR Biosciences). Immunoprecipitations were performed in LNCaP and VCaP cells treated as described. 600 pg of nuclear extracts isolated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific) were immunoprecipitated with SMARCC1, AR, FOXA1, or ERG antibodies according to the manufacturer's protocol. Eluted proteins were subjected to western blot or mass spectrometry analysis.
  • blocking buffer Tris-buffered saline, 0.1% Tween (TBS-T), 5% non-fat dry milk
  • HRP-conjugated secondary antibodies membranes were imaged on an
  • QIAzol Lysis Reagent QIAGEN
  • cDNA was synthesized using the extracted RNA as a template. Reverse transcription was carried out using Maxima First Strand cDNA Synthesis Kit (ThermoFisher), following the manufacturer's instructions. The resulting cDNA was then used for quantitative PCR (qPCR) using SYBRTM Green PCR Master Mix (Applied Biosystems), depending on the specific gene targets. The qPCR reactions were performed in QuantStudio 5 Real-Time PCR system (Applied Biosystems), and the data were analyzed using the AACt method to quantify gene expression levels, normalizing to the expression of GAPDH gene.
  • NKX3-1 F CCCACACTCAGGTGATCGAG (SEQ ID NO: 11) and R: GAGCTGCTTTCGCTTAGTCTT (SEQ ID NO: 12); Myc F: ATGGCCCATTACAAAGCCG (SEQ ID NO: 13) and R: TTTCTGGAGTAGCAGCTCCTAA (SEQ ID NO: 14); CCND1 F: GCTGCGAAGTGGAAACCATC (SEQ ID NO: 15) and R: CCTCCTTCTGCACACATTTGAA(SEQ ID NO: 16).
  • Short guide RNAs targeting the exons of human p300 or CBP were designed by Benchling (https://www.benchling.com/).
  • Non-targeting Ctrl sgRNA, p300 and CBP- sgRNAs were cloned into lentiCRISPR v2 plasmid based on previous report.
  • Dahlin, J. L. et al. Assay interference and off-target liabilities of reported histone acetyltransferase inhibitors. Nat Commun 8, 1527, doi:10.1038/s41467-017-01657-3 (2017).
  • LNCaP and 22Rv1 cells were transiently transfected with lentiCRISPR v2 encoding Ctrl or pair of two independent p300 or CBP-targeting sgRNAs. Forty-eight hours after transfection, cells were selected with 1 pg ml-1 puromycin for three days. Western blot was performed to identify the knock-out efficiency.
  • sgRNA sequences used are as follows: sgNC#1 :5'-GTAGCGAACGTGTCCGGCGT-3' (SEQ ID NO: 17); sgNC#2:5'-GACCGGAACGATCTCGCGTA-3' (SEQ ID NO: 18); sgp300#1 : 5’- CACCGTTCAATTGGAGCAGGCCGA-3' (SEQ ID NO: 19); sgp300#2: 5’- CACCGCATCCCTGTGTTCATTCCCA-3' (SEQ ID NO: 20); sgCBP#1: 5’- CACCGCGCGTGACCAGTCATTTGCG-3' (SEQ ID NO: 21); sgCBP#2: 5’- CACCGCAACTGTCGGAGCTTCTACG-3' (SEQ ID NO: 22).
  • the human p300 and CBP ON-TARGETplus SMARTPool siRNAs were ordered from Horizon Discovery. Cells were plated in a 6-well plate at the density of 300,000 cells per well. After 24 hours, cells were transfected with 30 nM of siRNAs using the RNAIMAX transfection reagents (Life Technologies) on two consecutive days. The protein was extracted on day 3 to identify efficient (>80%) knockdown of the target genes.
  • Protein Extraction Samples were taken from -80°C and homogenized in 1 mL lysis buffer (8 M urea, 100 mM Tris, pH 8.0, 1% protease inhibitor) by sonication. Any cell debris were removed by centrifugation at 15,000 rpm for 15 min at 4°C. The protein concentration was then determined by using BCA assay.
  • Protein Digestion 2 mg protein of each sample was diluted to 1 mL with lysis buffer. Disulfide bridges were reduced by 4.5 mM DTT at 37°C for 1 h. Reduced cysteine residues were alkylated by 10 mM iodoacetamide (IAA) in the dark at room temperature for 30 min. The solution was then diluted to 4 mL with 50 mM ammonium bicarbonate and subject to overnight digestion at 37°C with trypsin (Promega) using an enzyme to substrate ratio of 1 :200 (w/w). After digestion, TFA was added to 1 % final concentration. Any precipitate was removed by centrifugation at 1,780 g for 15 min.
  • IAA iodoacetamide
  • Peptide Desalting Purification of peptides is performed at room temperature on C18 reversed-phase columns. The columns were first conditioned by 100% ACN followed by 0.1% TFA, 50% ACN and then equalized by 0.1% TFA. The acidified and cleared digest were then loaded onto column. After wash by 0.1% TFA, the peptides were eluted from the column by 0.1% TFA, 50% ACN. [0147] Acetylated Peptide Enrichment: The eluent was lyophilized to dry and resuspended in IAP buffer (50 mM NaCI, 10 mM Na2HPO4, 50 mM MOPS, pH 7.2).
  • the beads were washed in pre-chilled PBS 4 times.
  • the peptide solution was then added into the vial containing motif antibody beads and incubated on a rotator for 4 h at 4°C. After incubation, the beads were washed with pre-chilled IAP buffer 3 times and HPLC grade water 4 times. The enriched peptides were eluted from the beads by 0.1% TFA.
  • NanoLC Nanoflow UPLC Ultimate 3000 nano UHPLC system (ThermoFisher Scientific, USA);
  • Nanocolumn trapping column (PepMap C18, 100A, 100 pm x 2 cm, 5 pm) and an analytical column (PepMap C18, 100A, 75 pm x 50 cm, 2 pm)
  • Mobile phase A: 0.1% formic acid in water; B: 0.1% formic acid in 80% acetonitrile.
  • the full scan was performed between 300-1,650 m/z at the resolution 60,000 at 200 m/z, the automatic gain control target for the full scan was set to 3e6.
  • the MS/MS scan was operated in Top 20 mode using the following settings: resolution 15,000 at 200 m/z; automatic gain control target 1e5; maximum injection time 19ms; normalized collision energy at 28%; isolation window of 1 .4 Th; charge sate exclusion: unassigned, 1 , > 6; dynamic exclusion 30 s.
  • RNA extraction was performed as previously described. Following extraction, ribosomal RNA (rRNA) was depleted from the total RNA samples using the RiboErase module of the KAPA RNA HyperPrep Kit (Roche). The rRNA-depleted RNA was then applied for library preparation. The procedure was conducted following the protocol provided with the KAPA RNA HyperPrep Kit. The prepared libraries were validated for quality using the Agilent 2100 Bioanalyzer. The libraries were then sequenced on an Illumina HiSeq 2500, utilizing a paired-end sequencing strategy (2 x 100 nucleotide read length with sequence coverage to 15-20M paired reads).
  • RNA-Seq data was handled via kallisto (version 0.46.1) [kallisto], R package EdgeR (edgeR_3.39.6 ) was used to generated normalized and filtered read counts (counts >10) [edgeR], Differential expression was performed using Limma-Voom (limma_3.53.10) [ limma], R package fgsea (fgsea_1 .24.0) was used for Gene Set Enrichment Analyis (GSEA) [fgsea]. Further R packages tidyverse, gtable, gplots, ggplot2 and EnhancedVolcano (EnhancedVolcano_1.15.0) were also used for generating figures. R version 4.2.1.
  • Chromatin immunoprecipitation experiments were conducted by using the Ideal ChlP-seq Kit for Transcription Factors (for AR, FOXA1, p300, ERG, BRD4 and MED1 ChlP-seq) or Histones (For H3K27ac, H2BK5ac, H3K20ac and RNA Pol II ChlP-seq) (Diagenode) following manufacturer's protocol.
  • ChlP-seq libraries were prepared from purified ChIP samples (1-10 ng) as described previously. Xiao, L. et al. Targeting SWI/SNF ATPases in enhancer-addicted prostate cancer. Nature 601, 434-439, doi : 10.1038/s41586-021 -04246-z (2022). Libraries were quantified and quality checked using the Bioanalyzer 2100 (Agilent) and sequenced on the Illumina HiSeq 2500 Sequencer (125-nucleotide read length).
  • ChlP-Seq data analysis started with trimming using Trimmomatic version 0.39 (settings TruSeq3-PE- 2.fa:2:30: 10, minlen 50) 66 .
  • BWA was used to align reads to hg38 (GRCh38) human genome reference ("bwa mem” command with options -5SP -TO, version 0.7.17-r1198-dirty). Alignments were filtered using samtools (quality score cutoff of 20) and picard MarkDuplicates (removed duplicates).
  • MACS2 was used for peak calling with narrowpeak setting for narrow peaks and a second set of parameters for histone peaks (e.g., H3K27Ac, - broad -B -cutoff-analysis -broad-cutoff 0.05 -max-gap 500).
  • bedtools was used to remove blacklisted regions of the genome from the peaks list (Encode's exclusion list ENCFF356LFX.bed).
  • UCSC's tool wigtoBigwig was used for conversion to bigwig formats.
  • ATAC-seq was conducted as previously described. Xiao, L. et al. Targeting SWI/SNF ATPases in enhancer-addicted prostate cancer. Nature 601, 434-439, doi: 10.1038/s41586-021 -04246-z (2022). Briefly, 50,000 cancer cells were lysed using CER-I cytoplasmic lysis buffer from the NE-PER kit (Invitrogen), incubated for 5 minutes on ice with occasional gentle mixing. After centrifugation at 1,300g for 5 minutes at 4°C, the nuclear pellets were isolated.
  • the nuclei were then treated with 50 pl of 1 x TD buffer and 2 pl Tn5 enzyme for 30 minutes at 37°C, using the Nextera DNA Library Preparation Kit. After Transposition, samples were immediately purified using a Qiagen minElute column and subjected to PCR amplification with NEBNext High-Fidelity 2X PCR Master Mix (NEB). Optimal PCR cycles were determined via qPCR to avoid over-amplification. The amplified libraries were further purified using a Qiagen minElute column and SPRI beads (Beckman Coulter). Finally, the ATAC-seq libraries were sequenced on the Illumina HiSeq 2500 platform, utilizing a 125-nucleotide paired-end read length.
  • NEB NEBNext High-Fidelity 2X PCR Master Mix
  • RNA sequencing was conducted using the Click-iTTM Nascent RNA Capture Kit (Invitrogen). Cells were treated with or without CBPD-409 for 2 and 4 hours, with 0.5mM 5-Ethynyl Uridine (EU) added to the medium 45 minutes before cell harvest. After treatment, cells were washed with PBS, harvested, and lysed with the kit's lysis buffer. RNA isolation was performed using QIAzol reagent and RNeasy Mini spin column (Qiagen), following previously described methods. For ribosomal RNA depletion, 5pg of total RNA were processed using the RibominusTM Eukaryote System v2 (ThermoFisher).
  • the rRNA-depleted, EU-labeled RNA (250-500ng) was then incubated with 0.25mM biotin azide in the Click-iT® reaction cocktail for 30 minutes with gentle vortexing.
  • Biotin-conjugated EU-RNA was precipitated using UltraPureTM Glycogen (1 pL, ThermoFisher), 7.5 M ammonium acetate (50 pL, Sigma-Aldrich), and chilled 100% ethanol (700 pL) at -70°C overnight.
  • the sample was then centrifuged at 13,000 x g for 20 minutes at 4°C, and the RNA pellet was washed with 75% ethanol.
  • 100- 200ng of the purified EU-RNA was used for library preparation and sequencing, as detailed in the RNA-seq section.
  • the sequencing reads were mapped to the reference genome (GRCh38.p14) downloaded from Gencode using Burrows-Wheeler Alignment Tool (bwa mem) with default parameters.
  • the resulting sam files were then converted to bam format and sorted using Samtools; bigwig files were then generated using bamCoverage of deeptools with RPKM normalization method.
  • Quantification of gene and enhancer expression levels was conducted with FeatureCounts.
  • AR binding sites at non-promoter regions were defined as AR enhancers.
  • the coordinates of AR enhancers were based on ChlP-seq peaks of AR and peaks that overlap with gene promoters (n-3kb of TSS) were excluded. Normalized enhancer expression levels in TPM were used for data visualization.
  • Meta profile plots were generated with the plotProfile function of deeptools, in which the inputs were merged bigwig files from duplicate libraries using UCSC BigwigMerge tool.
  • the read density heatmaps and enrichment plots were all created using the software Deeptools.
  • the reference Point parameter was set to +/- 2.5kb for histone signals and +/- 1 kb for other signals. Other settings included using ‘skipzeros', ‘averagetype mean', and ‘plotype se'.
  • the Encode blacklist ENCFF356LFX was used. Non-promoter regions were selected based on annotation by R package ChlPseeker with +/- 1 kb windows to gene regions.
  • Regions were selected from the overlap comparison of ChlP-Seq peaks as peaks only in AR sample, or peaks found both in the AR sample and the P300 sample.
  • ChlP-seg peaks guartile categorization and Heatmaps
  • Immunohistochemistry (IHC) and immunofluorescence (IF) were carried out on 4-micron formalin-fixed, paraffin-embedded (FFPE) tissue slices on the Ventana ULTRA automated slide stainer platform.
  • the antigen retrieval was done by heating tissues for cell conditioning media 1 and 2 with primary antibody incubation done.
  • Anti-rabbit or anti-mouse secondary antibodies wherever applicable were used to develop the immune complex.
  • IHC OmniMap and UltraMap Universal DAB RTU detection kit were used while for IF, Ventana FITC, and Red 610 RTU detection kit were used to develop the signal. An additional step of counterstaining by DAPI kit was used. Below table contains data on the essential reagent.
  • Compound A was dissolved in 100% PEG400 freshly before administration to mice.
  • Enzalutamide was prepared in 1% carboxymethyl cellulose (CMC) with 0.25% Tween-80 and homogenized by sonication. Both Compound A and enzalutamide were administrating to mice by oral gavage.
  • CMC carboxymethyl cellulose
  • mice Six-eight weeks old male SCID mice were acquired from the breeding colony at the University of Michigan. They were used to establish subcutaneous tumors on both sides of dorsal flanks. Tumor sizes were measured weekly using digital calipers, applying the formula (TT/6) (Length x Width 2 ). After the study finished, the mice were euthanized, and the tumors were harvested and weighed. The University of Michigan's IACUC approved these procedures.
  • mice received a subcutaneous injection of 3 million VCaP cells in a mix of serum-free medium and 50% Matrigel (BD Biosciences). Once tumors grew to about 200 mm 3 , the mice were grouped randomly and received either Compound A (3 mg/kg) or a control treatment through oral gavage three times weekly for five weeks.
  • Compound A 3 mg/kg
  • mice were treated with either 3 mg/kg Compound A or a control, via oral gavage three times weekly, and optionally with or without 10 mg/kg enzalutamide orally five times weekly for five weeks.
  • Prostate patient-derived xenograft models [0168] The patient-derived xenograft (PDX) MDA-PCa-146-12 from the University of Texas M. D. Anderson Cancer Center, which was developed from a CRPC patient as described before. Palanisamy, N. et al. The MD Anderson Prostate Cancer Patient-derived Xenograft Series (MDA PCa PDX) Captures the Molecular Landscape of Prostate Cancer and Facilitates Marker-driven Therapy Development. Clin Cancer Res 26, 4933-4946, doi:10.1158/1078-0432. CCR-20-0479 (2020). This line is positive for AR.
  • PDX patient-derived xenograft
  • the prostate cancer sample used for WA74 PDX development was collected during a rapid autopsy case as part of the Michigan Legacy Tissue Program (MTLP). For this line, metastases were excised from the intestinal mesentery and immediately placed into cold DMEM. Within 2-3 hours, tumor chunks were implanted into subcutaneous pockets of male NSG mice. Mice were monitored weekly. Out of 5 mice, only one animal showed tumor growth after 10 months. This tumor was harvested and sequentially passaged in both NSG and CB17SCID mice. This WA74 PDX line bears BRCA2 somatic mutation and TMPRSS2-ERG fusion.
  • the PDXs were propagated in male SCID mice. This involved the surgical implantation of 2 mm 3 tumor pieces, encapsulated in 100% Matrigel, into the flanks of the mice. After the tumor grew to 200 mm 3 , the mice were randomized into different treatment groups. These groups were administered either Vehicle, 10mg/kg enzalutamide (5 times per week) or 3 mg/kg Compound A (3 times per week) with 10mg/kg enzalutamide by oral gavage for 4 weeks.
  • Serum chemistry analysis [0174] Serum was obtained for biochemical analysis through a standardized collection and separation process. More than 200 piL of blood was drawn into serum separator tubes and tubes were set aside to allow the blood to clot for over 30 minutes. Following the clotting period, the tubes were centrifuged at 1800-3000g for 10 minutes. The cell-free serum was then extracted from the tubes for analysis at IVAC.
  • Alcian blue staining was performed using the Alcian Blue Stain Kit (pH 2.5, Abeam) following the manufacturer's protocol. Tissue sections on slides were first incubated overnight at 58°C, then deparaffinized with xylene and rehydrated through a series of ethanol solutions (100%, 70%) and water, each for 5 minutes. The slides were then treated with acetic acid solution for 3 minutes, followed by incubation in Alcian blue stain (pH 2.5) for 30 minutes at room temperature. After staining, the slides were rinsed in acetic acid for 1 minute and washed three times with water, 2 minutes each. Nuclear Fast Red was applied as a counterstain for 5 minutes, followed by washing, dehydration in ethanol and xylene, and mounting with EcoMount (Thermo Fisher).

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Abstract

La présente divulgation concerne un composé qui peut dégrader le coactivateur transcriptionnel p300/CBP et est utile dans le traitement de maladies et de troubles associés, notamment le cancer de la prostate.
PCT/US2025/019293 2024-03-11 2025-03-11 Utilisation d'un agent de dégradation du p300/cbp utilisée pour le traitement du cancer Pending WO2025193649A1 (fr)

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