Abstract 5650: RP-6306, a novel PKMYT1 inhibitor, demonstrates synthetic lethality as monotherapy and in combination with gemcitabine in CCNE1 amplified cancer cells
Authors: Fourtounis Jimmy , Martino John , Stocco Rino , Baruah Prasamit , Duffy Nicole , Gallo David , Fournier Sarah , Li JingJing , Li Li , Aguado Elia , Petrone Adam , Roulston Anne , Mamane Yael , Morris Stephen , Szychowski Janek , Papp Robert , Zinda Mike , Marshall C. Gary 
Published: June 2022

Abstract
Cyclin E1, the protein product of the CCNE1 gene, complexes with CDK2 and is a key regulator of the G1-S transition in cycling cells. CCNE1 amplification has been associated with increased replication stress and genomic instability associated with tumorigenesis. The serine-threonine protein kinase family member PKMYT1 negatively regulates the G2-Mitosis transition by phosphorylating and inactivating CDK1. The aim of the current study was to evaluate the impact of RP-6306, a novel and selective PKMYT1 inhibitor, on the CCNE1 amplified human breast cancer cell line, HCC1569. RP-6306 is a highly potent PKMYT1 inhibitor that displays single digit nM potency in an in vitro enzyme assay. RP-6306 dose-dependently inhibited the phosphorylation of CDK1 on Thr14 in HCC1569 cells and had no impact on the Tyr15 phospho-site of CDK1 that is regulated by family member Wee1. Cell-based assays showed increased phosphorylation of replication stress and pre-mitotic entry biomarkers in RP-6306 treated HCC1569 cells. Micronuclei and Caspase-3 were detected in a dose-dependent manner in HCC1569 cells treated with RP-6306 indicating the onset of genomic instability and apoptosis in these cells. Growth assays confirmed irreparable damage and proliferation defects in cells treated with RP-6306. Experiments to evaluate the combination of RP-6306 with gemcitabine, an S-phase specific pyrimidine analog that inhibits DNA synthesis showed profound synergistic growth defects in HCC1569 cells. In vivo, RP-6306 inhibition of Thr14 phosphorylation of CDK1 in HCC1569 tumors was directly proportional to free circulating plasma levels and resulted in significant inhibition of tumor growth in a dose and time dependent manner. In combination with gemcitabine, RP-6306 demonstrated tumor regression and superior efficacy compared to single agent treatment of either agent alone in multiple CCNE1 amplified models, including HCC1569 and OVCAR3. Our studies show that inhibition of PKMYT1 kinase activity impairs the growth of CCNE1 amplified cancer cell lines both in vitro and in vivo and stands to benefit cancer patients with CCNE1 amplification. A Phase I clinical trial (NCT04855656- Mythic Study) is currently underway to evaluate RP-6306 in patients with advanced solid tumors.
Citation Format: Jimmy Fourtounis, John Martino, Rino Stocco, Prasamit Baruah, Nicole Duffy, David Gallo, Sarah Fournier, JingJing Li, Li Li, Elia Aguado, Adam Petrone, Anne Roulston, Yael Mamane, Stephen Morris, Janek Szychowski, Robert Papp, Mike Zinda, C. Gary Marshall. RP-6306, a novel PKMYT1 inhibitor, demonstrates synthetic lethality as monotherapy and in combination with gemcitabine in CCNE1 amplified cancer cells [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 5650.