Bromodomains (BD) are epigenetic readers of histone acetylation involved in chromatin remodeling and transcriptional regulation of several genes including protooncogene cellular myelocytomatosis (c-Myc). c-Myc is difficult to target directly by agents due to its disordered alpha helical protein structure and predominant nuclear localization. The epigenetic targeting of c-Myc by BD inhibitors is an attractive therapeutic strategy for prostate cancer (PC) associated with increased c-Myc upregulation with advancing disease. MT-1 is a bivalent BD inhibitor that is 100-fold more potent than the first-in-class BD inhibitor JQ1. MT-1 decreased cell viability and causes cell cycle arrest in G0/G1 phase in castration-sensitive and resistant PC cell lines in a dose-dependent fashion. The inhibition of c-Myc function by MT-1 was molecularly corroborated by the de-repression of Protein Kinase D1 (PrKD) and increased phosphorylation of PrKD substrate proteins: threonine 120, serine 11, and serine 216 amino acid residues in β-Catenin, snail, and cell division cycle 25c (CDC25c) proteins, respectively. The treatment of 3D cell cultures derived from three unique clinically annotated heavily pretreated patient-derived PC xenografts (PDX) mice models with increasing doses of MT-1 demonstrated the lowest IC50 in tumors with c-Myc amplification and clinically resistant to Docetaxel, Cabazitaxel, Abiraterone, and Enzalutamide. An intraperitoneal injection of either MT-1 or in combination with 3jc48-3, an inhibitor of obligate heterodimerization with MYC-associated protein X (MAX), in mice implanted with orthotopic PC PDX, decreased tumor growth. This is the first pre-clinical study demonstrating potential utility of MT-1 in the treatment of PC with c-Myc dysregulation.
Cyclin-dependent kinase 12 (CDK12) belongs to the cyclin-dependent kinase (CDK) family of serine/threonine protein kinases. In contrast to CDKs which promote cell cycle progression, CDK12 is a transcriptional regulator of various cellular functions, most importantly cellular response to DNA damage and stress. Genomic alterations in CDK12 have been detected in up to 7% of patients with metastatic castration-resistant prostate cancer (CRPC). Phosphoproteomic studies have revealed that Protein Kinase D1 (PrKD1), another member of the serine/threonine kinase family, is the only kinase known to phosphorylate CDK12 at serine 681 (s681) and serine 685 (s685). While there is an increasing body of literature on CDK12 downstream signaling, there is almost no published data on upstream effectors or regulation of CDK12. Using site-directed mutagenesis, we generated CDK12 s681a and s681a+s685 mutants and transfected them into stable C4-2 and C4-2-PrKD1 cell lines to evaluate the interaction between CDK12 and PrKD1. Immunoprecipitation and immunoblotting revealed that endogenous CDK12 is stable in C4-2 cells with known levels of PrKD1 expression. However, stable transfection of C4-2-PrKD1 cells with wild-type CDK12 resulted in the degradation of nascent CDK12, whereas the non-phosphorylated mutants (s681 and s681+s685) remained stable. Our results suggest that PrKD1-mediated phosphorylation of s681and/or s685 residues affect CDK12 stability in C4-2-PrKD1 cells. We confirmed this finding by treating C4-2-PrKD1 cells with MG-132, a 26 S proteasome inhibitor, and repeating the experiment. Proteasomal inhibition rescued nascent transfected CDK12 levels even after PrKD1 phosphorylation. Our study demonstrates that CDK12 degradation might be mediated by PrKD1 phosphorylation at s681 and/or s685. Because PrKD1 and CKD12 are dysregulated across cancers, the results herein presented have potential implications for the treatment of not only prostate cancer but also other human malignancies. Citation Format: Victor Chalfant, Carlos Riveros, Sanjeev Shukla, Teruko Osumi, K Balaji. Signaling of cyclin-dependent kinase 12 (CDK12) in prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1622.
The proto-oncogene c-Myc is a transcription factor that is upregulated in several human cancers. Despite its key role, therapeutic targeting of c-Myc remains a challenge because of a disordered protein tertiary structure. The basic helical structure and zipper protein of c-Myc forms an obligate heterodimer with its partner MAX to function as a transcription factor. An attractive strategy is to inhibit MYC/MAX dimerization to decrease c-Myc transcriptional function. Several methods have been described to inhibit MYC/MAX dimerization including small molecular inhibitors and proteomimetics. We used NOD SCID gamma mice (NGS mice) that are immunodeficient, non-obese diabetic laboratory mice (Jackson Laboratories, Cat Number J000077451) carrying patient-derived xenograft (PDX) following implantation of grade IV adenocarcinoma of the prostate from a patient into the flanks of each mouse. For maximum tolerable dose studies, we used C57BL/6 wild type mice (Jackson Laboratories, Cat Number J000664). We performed a maximum dose tolerability study with the drug 3JC48-3 up to 100 mg/kg to determine limiting toxicity. We monitored the rate of growth for 2 weeks and measured the final volume of the tumors. Immunoprecipitation studies were performed using the prostate cancer cell line, PC-3, that were treated with the drug 3JC48-3 at different doses (0, 5, 10, and 20uM) for 24 hours and subsequently lysed. We found 3JC48-3 decreases prostate cancer growth and viability in a dose-dependent fashion in vitro. We confirmed the inhibition of MYC/MAX dimerization by 3JC48-3 using immunoprecipitation experiments. Normal mice and mice with patient-derived prostate cancer xenografts (PDX) tolerated intraperitoneal injections of 3JC48-3 up to 100 mg/kg body weight without dose-limiting toxicity. Preliminary results in these PDX mouse models suggest that 3JC48-3 may be effective in decreasing the rate of tumor growth. Our study demonstrates that 3JC48-3 is a potent MYC/MAX heterodimerization inhibitor that decreases prostate cancer growth and viability in association with upregulation of both PrKD1 expression and kinase activity. In addition, 3JC48-3 is well tolerated in prostate cancer PDX mice models and may be a candidate drug for further preclinical development. Citation Format: Carlos Riveros, Victor Chalfant, Sanjeev Shukla, Teruko Osumi, KC Balaji. c-Myc inhibition in prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1625.
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