Abstract B057: Preclinical development of PKMYT1 and ATR inhibitor combinations

Abstract

Abstract Background: Inactivation of the PKMYT1 kinase is synthetically lethal in tumor cells with genomic alterations leading to high replication stress, such as CCNE1 amplification frequently found in high-grade serous ovarian, uterine, and gastro-esophageal cancers. On this basis, we developed lunresertib (RP-6306), a first-in-class, potent and selective oral PKMYT1 inhibitor currently in Phase I clinical trials (NCT04855656). We have shown previously that PKMYT1 inhibition abrogates an inhibitory phosphorylation of the cyclin-dependent kinase CDK1 at threonine 14 (CDK1 pT14), leading to premature mitotic entry and catastrophic chromosome shattering specifically in cyclin E-high tumor cells, but not in cells with normal cyclin E levels. This leads to robust tumor growth inhibition by lunresertib in cyclin E-high pre-clinical models. We hypothesized that inhibition of the DNA damage response kinase ATR may further potentiate the tumor cytotoxicity of lunresertib, as ATR is known to regulate CDK1 pT14 levels in a parallel fashion to PKMYT1 through inhibition of the CDC25 family of phosphatases. Here we present the pre-clinical validation of this hypothesis. Methods: We used in vitro cell viability studies to determine the level of synergy between lunresertib and the ATR inhibitor camonsertib (RP-3500) in various engineered as well as tumor-derived models. We investigated the mechanism of action of the lunresertib/camonsertib combination using high content imaging and immunoblotting for DNA damage and cell cycle markers in cultured cells. Finally, we have evaluated the pre-clinical efficacy and tolerability of lunresertib/camonsertib combinations in vivo using mouse xenograft models. Results: In CCNE1-overexpressing cells, combining lunresertib with camonsertib led to a synergistic increase in cytotoxicity and catastrophic DNA damage at doses that did not affect wild type counterparts. Increased DNA damage resulted primarily from enhanced CDK1 dephosphorylation when lunresertib was combined with camonsertib, leading to robust and rapid induction of premature mitosis. Altogether these data suggest that camonsertib synergistically potentiates lunresertib’s mechanism of action. Consistent with that, we observed synergy between camonsertib and lunresertib in models carrying genomic biomarkers predictive of lunresertib sensitivity (CCNE1 amplification, FBXW7 loss-of-function), and not unrelated DNA repair defects (ATM or BRCA1 loss-of-function). In mouse xenograft models of tumors carrying predictive molecular alterations, the combination of lunresertib/camonsertib was well tolerated, enhanced CDK1 dephosphorylation and DNA damage, and was highly efficacious, including generating tumor regressions at doses sub-therapeutic as monotherapy. Conclusions: Lunresertib and camonsertib elicit synergistic activity in pre-clinical models of CCNE1-amplified and cyclin E-overexpressing tumors due to a complementary mechanism of action. These data provide a rationale for clinical development of PKMYT1 and ATR inhibitor combinations. Citation Format: David Gallo, Marc L. Hyer, Giovanni Martino, Adam Pertrone, Elia Aguado-Fraile, Rosie Kryczka, Jimmy Fourtounis, Rino Stocco, Stephen J. Morris, C. Gary Marshall, Anne Roulston, Michael Zinda, Michal Zimmermann. Preclinical development of PKMYT1 and ATR inhibitor combinations [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 B057.