Dissecting Resistance to CDK4/6 Inhibition in Experimental Glioma via Genome-wide CRISPR-Cas9 Screening

CDK4/6 inhibitors exhibit therapeutic potential in several tumor entities. Their clinical efficacy in glioblastoma might be limited by intrinsic and acquired resistance mechanisms. Understanding underlying genetic and molecular factors of this resistance might inform the design of effective therapeutic strategies. Genome-wide CRISPR-Cas9 screens offer a powerful approach to systematically identify resistance drivers and synthetic lethal interactions, paving the way for rational combination therapies.We conducted genome-wide CRISPR-Cas9 knockout and activation screens in human glioma cell lines LN229 and GS9 under CDK4/6 inhibition using Brunello and Calabrese libraries. Top hits were validated using functional assays. Furthermore, we conducted bulk RNA sequencing of treated and untreated cells for transcriptomic insights into drug response modifiers and molecular pathways involved.Functional genomics identified Ambra1 and CCNE1 as key drivers of resistance. Both Ambra1 knockout and CCNE1 overexpression, conferred resistance across both long-term and stem-like glioma models. Additionally, knockout of CHEK1 and FAM122A uncovered synthetic lethal interactions with CDK4/6 inhibition. Functional validation confirmed that glioma cells rely on CHEK1 or FAM122A for cell cycle progression, with cell line specific sensitivity. Transcriptomic analyses consistently highlighted alterations in pathways associated with cell cycle regulation, DNA replication, and the Fanconi anemia pathway. Of note, several differentially expressed genes overlapped with screen hits, supporting the robustness of our findings. Notably, pharmacological inhibition of CHEK1, both in vitro and ex vivo, enhanced sensitivity to CDK4/6 inhibition in a sequence-dependent manner, indicating a critical role of the sequence of drug administration.