Abstract C166: Development of BLX-3030, a potent and selective inhibitor of CDK9 for the treatment of N-MYC driven NeuroEndocrine Prostate Cancers (NEPC)
Authors: Vankayalapati Hariprasad , Li Zhaoliang , Lin Chenyu , Medley Kyle , Bearss David J 
Published: December 2023

Abstract
Background: Cell lineage reprogramming in cancer cells is a major mechanism for cancers to develop drug resistance and escape targeted therapy. The use of new potent targeted therapies in prostate cancers has led to the development of highly aggressive cancers, including NEPC. Gene amplification of N-MYC and C-MYC oncogenes leads to its overexpression in NEPC and mCRPC patients. This overexpression is highly prevalent in these lethal subtypes of cancers, detected in 2% of all Prostate Cancers (PCa) and over 10-17% of mCRPC patients. N-MYC is a transcriptional target of CDK9, which acts as an oncogenic driver, sufficient to transform human-derived prostate cancer cells to take on NEPC phenotypic changes. This results in a more aggressive disease identified in late-stage human PCa patients. Additionally, N-MYC and C-MYC are required for tumor drug resistance and its downregulation, through CDK9 inhibition, lowers tumor growth. Our efforts have demonstrated that targeting both N-MYC and C-MYC as a driver of NEPC and mCRPC with a highly selective CDK9 inhibitor (BLX-3030) is a viable approach for therapeutic intervention of mCRPC and NEPC.
Materials and Methods: We employed our MolecuLernTM fragment-based design strategies, in vitro cellular efficacies in single agent and combination experiments. Other assays are FACS/apoptosis, cell migration, live cell imaging, immunofluorescence. In vivo 22Rv1, LASCPC-0 and C4-2 cell engraftment mouse models, 7-day tox, PK, selectivity, NanoBRET, and ADME-Tox, in vitro safety and secondary pharmacological assays.
Results: We synthesized a series of novel small molecule CDK9 agents that reversibly bind to CDK9, competitively block the ATP site with an IC50 of 4.2 nM in kinase and 23 nM in target engagement assays, elicit pharmacological responses in N-MYC, C-MYC, and other prostate cancer cells in vitro and in vivo. We detected the expression of CDK9 and N-MYC in LASCPC-01, 22Rv1, C4-2 and C4-2B cell lines. BLX-3030 exhibited potent cellular efficacy in PCa cells (IC50 of 22 to 590 nM) and in N-MYC expressed cells (IC50 of 76, 470, and 132 nM), respectively. BLX-3030 reduced pCDK9, C-MYC, Mcl-1, N-MYC and its downstream RNA Pol II (Ser-2) and pAR (Ser-81) in a dose dependent manner. Additionally, we performed FACS/apoptosis, cell migration, and live cell imaging, including immunofluorescence. We also conducted three separate in vivo efficacy mouse models (LASCPC-01, 22RV1, and C4-2) which demonstrated &gt;55 to 70% Tumor Growth Inhibition. Several in vitro ADME-Tox, hERG, P450, safety panel screen, in vivo MTD and dose range finding experiments in SD rats and beagle dogs were conducted.
Conclusion: In summary, a series of BLX-3030 and its analogs display a potency in MYC-driven PCa, with ideal PK properties in rodent, canine species ranging 28-83 % F, with low clearance, and &gt;3.0 hr half-life. Within this profile, BLX-3030 was selected as a candidate that had the appropriate developable properties. Our plans for IND enabling studies and future clinical trial plans will also be discussed.
Citation Format: Hariprasad Vankayalapati, Zhaoliang Li, Chenyu Lin, Kyle Medley, David J Bearss. Development of BLX-3030, a potent and selective inhibitor of CDK9 for the treatment of N-MYC driven NeuroEndocrine Prostate Cancers (NEPC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C166.