Molecular modulators of cyclin-dependent kinase 4/6 inhibitor response in experimental glioma identified through genome-wide CRISPR-Cas9 screening

BACKGROUND: Glioblastoma harbors frequent alterations in the retinoblastoma pathway, providing a genetic rationale for therapeutic targeting with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. The NOA-20 trial did not reveal a progression-free survival benefit of CDK4/6 inhibition plus radiation therapy in newly diagnosed, O6-methylguanine DNA methyltransferase (MGMT)-unmethylated glioblastoma. In fact, CDK4/6 inhibitor monotherapy has not demonstrated efficacy in solid tumors. We aimed at discovering response modulators to CDK4/6 inhibition, paving the way for rational combination therapies. METHODS: We conducted genome-wide CRISPR-Cas9 screens in human glioma cell lines and stem-like cells (LN229, LN18, LNZ308, T98G, and GS-9) under CDK4/6 inhibition, employing knockout (Brunello library) and activation strategies (Calabrese library), followed by genetic and pharmacological validation of selected candidate genes in vitro and ex vivo (primary cultures) as well as the investigation of 1 functionally instructed combination therapy in vivo. RESULTS: Loss of AMBRA1 and gain of function of CCNE1 reduced sensitivity to CDK4/6 inhibition in glioma cells, whereas disruption of checkpoint kinase 1 (CHEK1) or FAM122A resulted in synthetic lethality in combination with CDK4/6 inhibition. AMBRA1-deficient glioma cells exhibited increased sensitivity to CHK1 inhibition, revealing a context-specific vulnerability. Combined inhibition of CHK1 and CDK4/6 led to synergistic antiglioma activity in vitro, ex vivo, and in vivo. CONCLUSIONS: Our data identify AMBRA1, CCNE1, CHEK1, and FAM122A as potential molecular modifiers of CDK4/6 inhibition response in experimental glioma and provide a biological rationale for combinatorial targeting with CDK4/6 inhibition in glioblastoma.