Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1–4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR–Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB–FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.

Amplification of the gene encoding cyclin E (CCNE1) is an oncogenic driver in several malignancies and is associated with chemoresistance and poor prognosis. To uncover therapeutic targets for CCNE1-amplified tumors, we undertook genome-scale CRISPR/Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here, we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumor regressions when combined with gemcitabine in models of CCNE1-amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1 overexpressing-cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the FOXM1/MYBL2/MuvB-dependent mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.

PKMYT1 is a regulator of CDK1 phosphorylation and is a compelling therapeutic target for the treatment of certain types of DNA damage response cancers due to its established synthetic lethal relationship with CCNE1 amplification. To date, no selective inhibitors have been reported for this kinase that would allow for investigation of the pharmacological role of PKMYT1. To address this need compound 1 was identified as a weak PKMYT1 inhibitor. Introduction of a dimethylphenol increased potency on PKMYT1. These dimethylphenol analogs were found to exist as atropisomers that could be separated and profiled as single enantiomers. Structure-based drug design enabled optimization of cell-based potency. Parallel optimization of ADME properties led to the identification of potent and selective inhibitors of PKMYT1. RP-6306 inhibits CCNE1-amplified tumor cell growth in several preclinical xenograft models. The first-in-class clinical candidate RP-6306 is currently being evaluated in Phase 1 clinical trials for treatment of various solid tumors.

PKMYT1 is an important regulator of CDK1 phosphorylation and is a compelling therapeutic target for the treatment of certain types of DNA damage response cancers due to its established synthetic lethal relationship with CCNE1 amplification. To date, no selective inhibitors have been reported for this kinase that would allow for investigation of the pharmacological role of PKMYT1 in the treatment of cancer. To address this need we conducted a focused screening effort that identified compound 1 as a weak PKMYT1 inhibitor. Introduction of a dimethylphenol dramatically increased potency on PKMYT1. These dimethylphenol analogs were found to exist as Type III atropisomers that could be separated and profiled as single enantiomers. Structure-based drug design aided by co-crystal structures of several analogs enabled optimization of cell-based potency and kinase selectivity. Parallel optimization of ADME properties led to the identification of potent and selective inhibitors of PKMYT1 with favorable pharmacokinetics. RP-6306 inhibits the phosphorylation of CDK1 Thr14 in vivo in tumor tissue and inhibits CCNE1-amplified tumor cell growth in several preclinical xenograft models. The first-in-class clinical candidate RP-6306 is currently being evaluated in Phase 1 clinical trials (NCT04855656) for treatment of various solid tumors.

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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.