Cell-cycle checkpoint inhibitors as a novel anti-cancer strategy in Glioblastoma multiforme

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Glioblastoma (GBM), is the most common and most lethal primary malignant brain tumor in adults. A challenge in identifying new therapies is the diversity in behavior of GBM genotypes. To address this, the Beroukhim lab conducted a comprehensive molecular characterization and drug screen on 78 GBM cell lines spanning the major molecular classes of GBM. Cell lines with disruption of the tumor suppressor gene TP53 showed poor responses to most compounds, as is true of TP53-mutant tumors in general. TP53 is the most commonly mutated gene in cancer, so compounds with activity in TP53 mutant cells could provide helpful insights for a large number of people with treatment-resistant cancers. Fortunately, we identified a CHK1/2 inhibitor (CHKi) that was more effective in TP53 mutant cells. This inhibitor was most effective when the tumor suppressor gene CDKN2A had also been lost—the genetics of about 10% of GBM patients. Checkpoint kinases (CHK) are crucial mediators of the DNA damage response (DDR). They maintain genomic integrity by providing cells time to repair DNA damage before dividing or initiate apoptosis if the damage is irreparable. Several CHKis are currently undergoing clinical trials, with some already showing promising activity. Therefore, we will investigate this apparent synthetic lethal relationship between CHKi and two of the most commonly inactivated tumor suppressors across all cancers including GBMs. Our specific questions are:1. Does joint disruption of TP53 and CDKN2A generate susceptibility to CHK1/2 inhibitors? We will generate isogenic models for TP53 and CDKN2A and the combination by applying CRISPR-CAS9 technology in GBM cell lines, to determine whether joint loss of TP53 and CDKN2A sensitizes the cells to CHKi. Our readouts will be changes in cell viability, proliferation, apoptosis, cell cycle progression and the DDR. These experiments will have implications for deciding whom to target with CHKi, aiding in design and interpretation of future clinical trials.2. Evaluate resistance mechanisms to CHK1/2 inhibition. Cancers often acquire resistance to initially effective targeted therapies. Identifying these resistance mechanisms can indicate combined treatment approaches to extend response times. Here, we will take three approaches. First, we will test if resistance can be gained by slowing down the cell cycle. Second, we will perform a genome-scale open reading frame (ORF) screen on CHKi sensitive cells to identify genes whose expression generates resistance. Third, we will generate naturally arising models of CHKi resistance and evaluate how they gained it by characterization their changes in expression and development of new genetic alterations.This project will evaluate a novel approach to treating tumors with mutations in some of the most frequently altered genes in the cancer genome TP53 and CDKN2A. The prevalence of these genetic alterations across cancers indicates that this work could have a profound impact.