Objective: CDK4/6 inhibition has become an important treatment option in some epithelial malignancies but its utility in the setting of NSCLC remains limited. While CDK4-based monotherapy has been generally ineffective, combinatorial regimens have shown promise. Here we sought to perform an unbiased assessment of potential combinatorial strategies in pre-clinical models of NSCLC.

Methods: We performed a CRISPR/Cas9 screen in two NSCLC cell lines (A549 and H2030) in the presence and absence of the CDK4/6 inhibitor Palbociclib and identified guide RNAs that selectively depleted in the setting of drug treatment. We subsequently performed validation and mechanistic experiments as described. CDK4 and CDK2 activity was assessed using a fluorescent reporter system.

Results: Palbociclib-sensitizing screen hits include MAPK1 (ERK2), AKT1, MAP3K2 (MEKK2), PIK3CA, MAPK14 (p38a), AKT3, PORCN, LGR4, CDK1, CDK9, and AURKB. Screen results were compared with a previously published Palbociclib sensitizing screen and shared hits were identified. Of these, CCNE1 was the highest scoring hit suggesting relevance across histologies. Interestingly, several screen hits such as ERK2, PIK3CA, VCP and ELAVL1 have reported roles in CCNE1 transcription or post-translational stabilization suggesting a central role for CCNE1 in determining drug sensitivity. Western blot analyses demonstrate that CCNE1 is rapidly upregulated following Palbociclib exposure in a panel of NSCLC cell lines and immunofluorescence shows strong post-treatment CCNE1 expression in human A549 xenografts in vivo. CDK2/4 fluorescent reporter assays show partial restoration of CDK2 activity within 48 hours of Palbociclib treatment. Genetic validation experiments using dox-inducible shRNA constructs targeting CCNE1 show that CCNE1 knockdown sensitizes cells to Palbociclib treatment and enhances senescence induction. Pharmacologic and genetic co-inhibition of CDK2 improves Palbociclib efficacy yet such co-inhibition further elevates CCNE1 levels and CDK2 suppression remains incomplete based the fluorescence reporting system. Furthermore, knockdown of CCNE1 in conjunction with CDK2/4/6 inhibition shows improved growth inhibition compared to drug treatment alone. Based on this data, we hypothesize that targeting signaling pathways upstream of CCNE1 transcription or those which affect CCNE1 stabilization and turnover may be more efficacious than combined CDK inhibition.

Conclusion: CCNE1 is a critical mediator of sensitivity to CDK4/6 inhibition and Palbociclib treatment results in rapid compensatory increases in CCNE1 levels. Despite drug treatment, NSCLC cells retain CDK2 activity which is only partially inhibited by combined CDK 2/4/6 inhibition. Screen results suggest several potential approaches for co-targeting of CCNE1 to enhance drug sensitivity.

Citation Format: Matthew Bott, Maria Skamagki, Nelson Salgado, Francisco Sanchez-Rivera, Scott Lowe. Druggable CRISPR/Cas9 screen identifies Cyclin E1 (CCNE1) as a key mediator of sensitivity to CDK4/6 inhibition in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5990.