Abstract 1784: Pre-clinical characterization of G1T28-1, a novel CDK 4/6 inhibitor for protection of bone marrow from cytotoxic chemotherapies

Abstract

Abstract G1T28-1 is a clinical stage, small molecule inhibitor of cyclin dependent kinases 4/6 (CDK4/6). Hematopoietic stem and progenitor cells (HSPC) require CDK4/6 for proliferation, and CDK4/6 inhibition allows the transient arrest of HSPC in the G1 phase of the cell cycle. This arrest may reduce the sensitivity of HSPC to DNA damaging chemotherapies by limiting G1 to S-phase progression in the setting of unrepaired DNA damage. Reducing HSPC death may reduce chemotherapy-induced myelosuppression (CIM), the major dose-limiting toxicity of most cytotoxic anti-cancer agents. G1T28-1 was specifically designed with high potency, exquisite selectivity, and favorable pharmacology to induce a predictable and well-defined transient arrest of HSPC as compared to less potent and selective CDK4/6 inhibitors. Biochemical profiling demonstrates that G1T28-1 is a competitive inhibitor of CDK4/6 at low nanomolar concentrations, and that G1T28-1 is highly selective for CDK4/cyclin D1 and CDK6/cyclin D3 as compared to CDK2/cyclin A or CDK2/cyclin E. G1T28-1 induces a clean G0/G1 arrest in CDK4/6 dependent cell lines in vitro. Since the down-stream target of CDK4/6 is the retinoblastoma protein (Rb), we investigated the ability of G1T28-1 to inhibit Rb phosphorylation. In CDK4/6 dependent cell lines, G1T28-1 exposure fully blocks Rb phosphorylation by 16 hours, while no effect on Rb in CDK4/6-independent cells is observed. To determine the duration and reversibility of G1T28-1's effects on cells in culture, Rb competent cells were treated with G1T28-1 for 24 hours, and then the drug was washed out. While cells were arrested at 24 hours, they re-entered the cell cycle by 16 hours after washout and maintained normal cell cycle kinetics thereafter. To demonstrate that the G0/G1 arrest induced by G1T28-1 decreases DNA damage and apoptosis following exposure to chemotherapeutic agents, Rb competent cells were pre-treated with G1T28-1 or vehicle control for 16 hours followed by incubation with various chemotherapies. Cells were then assayed for γ-H2AX formation and caspase activation. Treatment with G1T28-1 prior to DNA damaging agents attenuates DNA damage as measured by γ-H2AX formation and decreased caspase 3/7 activation in a dose-dependent manner, indicating a decrease in chemotherapy-induced apoptosis. In Rb null cancers, such as small cell lung cancer (SCLC), G1T28-1 may effectively protect HSPCs without affecting the chemotherapy's efficacy. In vitro testing with SCLC cell lines confirmed that chemotherapy efficacy was unaffected by the addition of G1T28-1. In summary, G1T28-1 is a novel potent and selective CDK4/6 inhibitor that induces a transient and reversible G0/G1 cell cycle arrest in CDK4/6-sensitive cells CDK4/6-dependent cells, thereby decreasing CIM. However, the anti-tumor activity of chemotherapy in CDK4/6-resistant cells is unaffected by G1T28-1. Citation Format: John E. Bisi, Hannah S. White, Jessica A. Sorrentino, Patrick J. Roberts, Jay C. Strum. Pre-clinical characterization of G1T28-1, a novel CDK 4/6 inhibitor for protection of bone marrow from cytotoxic chemotherapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1784. doi:10.1158/1538-7445.AM2015-1784