Abstract 5813: Pharmacodynamic biomarker strategies for CDK9 inhibition

Abstract

Abstract Cyclin-dependent kinase 9 (CDK9) is a promising target for cancer therapy due to its regulation of RNA polymerase II (RPB1) and key downstream anti-apoptosis proteins such as myeloid cell leukemia-1 (MCL-1). TP-1287 is a novel oral prodrug of alvocidib, a potent CDK9 inhibitor, and is currently under clinical investigation in patients with advanced solid tumors (clinicaltrials.gov, NCT03604783). TP-1287 is converted to alvocidib by alkaline phosphatases. CDK9 mediates phosphorylation of RPB1 at Ser2 and subsequently drives transcription of key oncogenic signaling genes, such as MCL-1. MCL-1 is a critical factor for survival of tumor types including acute myeloid leukemia (AML), multiple myeloma (MM) and myelodysplastic syndromes (MDS). MCL-1 is also expressed in normal peripheral blood mononuclear cells (PBMCs), thus it was hypothesized that CDK9 inhibition can be assessed clinically using PBMCs as a surrogate biomarker at multiple time points. We hypothesized that alvocidib, the pharmaceutically active form of TP-1287, would modulate CDK9 signaling pathways in cancer cells, resulting in tumor growth inhibition. In cell viability assays using CellTiter-Glo to evaluate the in vitro anti-tumor activity of alvocidib, we observed an IC50 of around 100 nM in AML and MM cell lines: 53-78 nM in MOLM-13, MV-4-11, and HL-60 AML cell lines and 31-223 nM in NCI-H929 and RPMI-8226 MM cell lines. In flow cytometry analysis, alvocidib reduced phosphorylation of RPB1 (p-RPB1) and MCL-1 protein expression in RPMI-8226 and MV-4-11 cells in a dose and time-dependent manner. In a pharmacodynamic study using the RPMI-8226 xenograft model after a single oral administration of TP-1287, we observed a substantial decrease of p-RPB1 and MCL-1 in tumor tissues followed by induction of cleaved caspase-3, an indicator of apoptosis. Furthermore, in an in vivo efficacy study using the RPMI-8226 model, TP-1287 achieved tumor growth inhibition (%TGI), ranging from 17.3% at 0.5 mg/kg (QD) to 86.6% at 15 mg/kg (Q7D) at day 22 after treatment. We hypothesized that PBMCs could serve as a surrogate tissue to measure CDK9 inhibition. To assess this approach in PBMCs, we performed ex vivo assays using human PBMCs from multiple healthy donors. Alvocidib demonstrated dose-dependent p-RPB1 and MCL-1 inhibition after 24 hr treatment. Preliminary data from the Phase 1 trial with TP-1287 showed p-RPB1 suppression in PBMCs from multiple patients, suggesting the successful execution of this assay and evidence of target engagement in the clinical setting. Taken together, TP-1287 demonstrated potent cell and tumor growth inhibition in multiple hematological cell lines, including AML and MM. Furthermore, a newly established flow cytometry system for p-RPB1 and MCL-1 to evaluate CDK9 inhibition in human PBMCs was developed, which could be useful as a surrogate biomarker for TP-1287 in clinical trials and warrants further investigation. Citation Format: Yuta Matsumura, Ethika Tyagi, Clifford J. Whatcott, Jason M. Foulks, Adam Siddiqui-Jain, David J. Bearss, Steven L. Warner. Pharmacodynamic biomarker strategies for CDK9 inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5813.