Shukla, Sanjeev

AbstractBackgroundThe E‐cadherin, α‐ and β‐Catenin interaction at the cell adherens junction plays a key role in cell adhesion; alteration in the expression and function of these genes are associated with disease progression in several solid tumors including prostate cancer. The membranous β‐Catenin is dynamically linked to the cellular cytoskeleton through interaction with α‐Catenin at amino acid positions threonine 120 (T120) to 151 of β‐Catenin. Nuclear presence of α‐Catenin modulates the sensitivity of cells to DNA damage. The objective of this study is to determine the role of α‐Catenin and protein kinase D1 (PrKD1) in DNA damage response.MethodsProstate cancer cells; LNCaP, LNCaP (Sh‐PrKD1; silenced PrKD1), C4‐2 and C4‐2 PrKD1 were used for various sets of experiments to determine the role of DNA damage in PrKD1 overexpression and silencing cells. These cells were treated with compound‐10 (100 nM) and Etoposide (30 µM), total cell lysates, cytosolic and nuclear fractions were prepared to observe various protein expressions. We performed single cell gel electrophoresis (COMET assay) to determine the etoposide induce DNA damage in C4‐2 and C4‐2 PrKD1 cells. The animal experiments were carried out to determine the tolerability of compound‐10 by mice and generate preliminary data on efficacy of compound‐10 in modulating the α‐Catenin and PrKD1 expressions in inhibiting tumor progression.ResultsPrKD1, a novel serine threonine kinase, phosphorylates β‐Catenin T120. In silico analysis, confirmed that T120 phosphorylation alters β‐ to α‐Catenin binding. Forced expression of PrKD1 in prostate cancer cells increased β‐ and α‐Catenin protein levels associated with reduced etoposide induced DNA damage. Downregulation of α‐Catenin abrogates the PrKD1 mitigation of DNA damage. The in vitro results were corroborated in vivo using mouse prostate cancer patient derived xenograft model by inhibition of PrKD1 kinase activity with compound‐10, a selective PrKD inhibitor, demonstrating decreased total β‐ and α‐Catenin protein levels, and β‐Catenin T120 phosphorylation.ConclusionsAlteration in DNA damage response pathways play major role in prostate cancer progression. The study identifies a novel mechanism of α‐Catenin dependent DNA damage mitigation role for PrKD1 in prostate cancer.

Abstract Bromodomains (BD) are epigenetic readers of histone acetylation involved in chromatin remodeling and transcriptional regulation of protooncogene cellular myelocytomatosis (c- Myc) and other genes. Because c-Myc cannot be directly targeted by small molecular inhibitors due to disordered alpha helical structure, epigenetic targeting of c-Myc by BD inhibitors is an attractive therapeutic strategy for diseases such as prostate cancer (PC) associated with increased c-Myc upregulation with advancing disease. We studied the efficacy of MT1, a novel bivalent BD inhibitor that is 100-fold more potent than the first in class BD inhibitor JQ1, at inhibiting PC growth. We tested the effect on viability by MT-1 on PC cell lines, 3D spheroids derived from clinically annotated drug resistant patient derived xenografts (PDX), mice PDX models and corroborated the molecular mechanism of MT1 down regulation of Myc leading to downstream Myc-dependent up regulation of Protein Kinase D1 (PrKD) substrate phosphorylation by western blot. MT-1 inhibited growth of PC in castration sensitive (LNCaP) and resistant PC cells (PC-3). MT-1 treatment upregulated PrKD expression and phosphorylation of known PrKD substrates: threonine 120 (Thr-120) residues in beta-catenin and the serine 216 in Cell Division Cycle 25 (CDC25C) in PC-3 cells. Moreover, MT-1 was effective in inhibition of 3D spheroids growth at IC 50 between 0.27-0.92µM in Abiraterone, Enzalutamide, Docetaxel, Cabazitaxel metastatic castrate resistant PCa patient-derived tumor 3D spheroids. Additionally, MT1 was effective in inhibiting the tumor growth in PDX mice model. A combined intra-peritoneal administration of MT-1 with another c-Myc inhibitor (3JC48-3), an obligate c-Myc and MYC-associated protein X (MAX) heterodimerization inhibitor, increased the efficacy of inhibiting the PDX growth in mice. This study provides strong pre-clinical in vitro and in vivo evidence for advancing MT- 1 as a novel c-Myc targeting drug in PC. The MT-1 drug development will likely be highly impactful as c-Myc is dysregulated in three fourths of men with advanced PC. Citation Format: Sanjeev Shukla, Mohammed Al-Toubat, Allison H. Feibus, Ahmed Elshafei, Carlos Riveros, Nathalie Meurice, Justyna Gleba, Jonathan Chardon-Robles, Adam M. Kase, John A. Copland III, Joachim L. Petit, Teruko Osumi, K.C. Balaji. Bromodomain inhibitor: MT1 and its potential role in modulation of prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2475.