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CDK4/6 small molecule inhibitors represent a milestone in treating hormone receptor-positive breast cancer. PALOMA (Palbociclib: Ongoing Trials in the Management of Breast Cancer) studies have confirmed prolonged progression-free survival after CDK4/6 inhibitor and anti-estrogen therapy. Notably, disease progression is observed in a subset of patients receiving Palbociclib-Fulvestrant treatment (Turner et al., NEJM 2018). Circulating tumor DNA from PALOMA-3 patients revealed genetic mutations correlating with patient response (O'Leary et al., Cancer Discov, 2018). Compensations in CDK4/6i resistance include circumvention of CDK4/6 substrate regulation, CCNE1/2 amplification (Herrera-Abreu et al., Cancer Res. 2016), and compensatory CDK2 activity. The importance of compensatory CDK2 cancer re-wiring is highlighted by recently reported, highly efficacious CDK2/4/6i (Freeman-Cook et al., in review). Thus, mechanistic understanding of cancer cell and compensatory CDK2 activity is imperative to improve patient outcome. Acute, temporal phenotypes in CDK2-signaling have been observed by live single-cell tracking microscopy in human breast epithelial and ER+ breast cancer cells (Arora & Moser et al., in review). In extending these studies, we utilize first-in-class small molecule inhibitors against CDK2 combined with high content imaging approaches to illuminate cancer cell CDK2 signaling and identify potential synthetic lethality. We report biomarkers parsed in cycling populations as quantifiable phenotypes and identify disengaged control of several processes including S- and G2/M licensing after CDK2i. Combined with internal -omics studies, CDK2i selective molecules support a positive regulatory role of CDK2 on CCNB1-PLK1 signaling before mitotic entry. We combine our high-content techniques with proximity labeling to quantify alternative CDK-Cyclin complex formation following pharmacologic perturbation. We hypothesize that dysregulated CDK-Cyclin complex formation elicits unintended CDK2 kinase activity, revealing potential synthetic lethality with inhibition of centrosome duplication pathways in ER+ breast cancers. Citation Format: Tim Sen Wang, Jon Almaden, Chen Shen, John Lapek, Sherry Niessen, Nichol Miller, Stephen Dann, Todd VanArsdale. Identifying synthetic lethality with novel CDK2 small molecule inhibitors via integration of high-content microscopy and -omics level platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1954.

The PI3K pathway, which regulates cell growth, proliferation and survival, is activated in many types of human tumors by mutational activation of PI3Kα, loss of function of PTEN or activation of receptor tyrosine kinases. Inhibition of key signaling proteins in the pathway, such as PI3K, AKT and mTOR, therefore represents a high value targeting strategy for diverse cancers. PF-04691502 is a dual-specificity inhibitor of PI3K and mTOR which shows potent and selective activity in in vitro biochemical, cell and xenograft models. In in vitro biochemical assays PF-04691502 inhibited recombinant PI3Kα, β, γ and δ isoforms with Ki's of 1.2-2.2 nM and recombinant mTOR with a Ki of 9.1 nM. PF-04691502 demonstrated a high degree of selectivity for inhibition of PI3K family kinases as shown by lack of activity against a panel of >75 protein kinases, including the Class III PI3K hVps34. PF-04691502 also inhibited transformation of avian fibroblasts mediated by PI3K γ, δ, mutant PI3Kα E545K or membrane-localized AKT with IC50's of ∼100nM. In cell assays PF-04691502 inhibited PI3K/mTOR signaling in SKOV3 ovarian cancer cells with PI3Kα mutations and in U87MG glioblastoma cells with PTEN alteration, as indicated by reduced levels of phosphorylation of AKT(T308), AKT(S473) and S6 ribosomal proteins. Functional studies for anti-proliferative effects suggest PF-04691502 has broad efficacy across tumor types. In SKOV3 and U87MG xenograft models PF-04691502 treatment resulted in dose-dependent tumor growth inhibition (TGI) with maximum TGI of ∼70% at the maximum tolerated dose of 10 mg/kg, by once daily oral dosing. Inhibition of AKT(S473) phosphorylation and S6RP(S235/236)/PRAS40(T246)/4EBP1(T37/46) phosphorylation were used as quantitative and qualitative pharmacodynamic (PD) endpoints, respectively; a clear pharmacokinetic (PK)/PD relationship was established in both models after multiple dose oral administration. In the U87MG xenograft model AKT(S473) phosphorylation was inhibited with an estimated EC50 of 5.7 nM (free plasma concentration) based on PK/PD modeling. The free plasma Area Under Curve was estimated to be 850 nM*hr for 70% TGI at 10mg/kg and was found to be similar in the SKOV3 model. The projected human efficacious dose of 10 mg once daily oral dosing provides Caverage steady state exposure of 22.4 nM (free plasma concentration) which is sufficient for 50-80% inhibition of pAKT S473, and corresponds to 74% TGI. Phase 1 clinical trials of PF-04691502 as a single agent are planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4479.