Loss of Cyclin C or CDK8 provides ATR inhibitor resistance by suppressing transcription-associated replication stress.

The protein kinase ATR plays pivotal roles in DNA repair, cell cycle checkpoint engagement and DNA replication. Consequently, ATR inhibitors (ATRi) are in clinical development for the treatment of cancers, including tumours harbouring mutations in the related kinase ATM. However, it still remains unclear which functions and pathways dominate long-term ATRi efficacy, and how these vary between clinically relevant genetic backgrounds. Elucidating common and genetic-background specific mechanisms of ATRi efficacy could therefore assist in patient stratification and pre-empting drug resistance. Here, we use CRISPR-Cas9 genome-wide screening in ATM-deficient and proficient mouse embryonic stem cells to interrogate cell fitness following treatment with the ATRi, ceralasertib. We identify factors that enhance or suppress ATRi efficacy, with a subset of these requiring intact ATM signalling. Strikingly, two of the strongest resistance-gene hits in both ATM-proficient and ATM-deficient cells encode Cyclin C and CDK8: members of the CDK8 kinase module for the RNA polymerase II mediator complex. We show that Cyclin C/CDK8 loss reduces S-phase DNA:RNA hybrid formation, transcription-replication stress, and ultimately micronuclei formation induced by ATRi. Overall, our work identifies novel biomarkers of ATRi efficacy in ATM-proficient and ATM-deficient cells, and highlights transcription-associated replication stress as a predominant driver of ATRi-induced cell death.

Identification of Intrinsic Drug Resistance and Its Biomarkers in High-Throughput Pharmacogenomic and CRISPR Screens.

High-throughput drug screens in cancer cell lines test compounds at low concentrations, thereby enabling the identification of drug-sensitivity biomarkers, while resistance biomarkers remain underexplored. Dissecting meaningful drug responses at high concentrations is challenging due to cytotoxicity, i.e., off-target effects, thus limiting resistance biomarker discovery to frequently mutated cancer genes. To address this, we interrogate subpopulations carrying sensitivity biomarkers and consecutively investigate unexpectedly resistant (UNRES) cell lines for unique genetic alterations that may drive resistance. By analyzing the GDSC and CTRP datasets, we find 53 and 35 UNRES cases, respectively. For 24 and 28 of them, we highlight putative resistance biomarkers. We find clinically relevant cases such as EGFRT790M mutation in NCI-H1975 or PTEN loss in NCI-H1650 cells, in lung adenocarcinoma treated with EGFR inhibitors. Interrogating the underpinnings of drug resistance with publicly available CRISPR phenotypic assays assists in prioritizing resistance drivers, offering hypotheses for drug combinations.

Genome architecture and stability in the Saccharomyces cerevisiae knockout collection.

Despite major progress in defining the functional roles of genes, a complete understanding of their influences is far from being realized, even in relatively simple organisms. A major milestone in this direction arose via the completion of the yeast Saccharomyces cerevisiae gene-knockout collection (YKOC), which has enabled high-throughput reverse genetics, phenotypic screenings and analyses of synthetic-genetic interactions1-3. Ensuing experimental work has also highlighted some inconsistencies and mistakes in the YKOC, or genome instability events that rebalance the effects of specific knockouts4-6, but a complete overview of these is lacking. The identification and analysis of genes that are required for maintaining genomic stability have traditionally relied on reporter assays and on the study of deletions of individual genes, but whole-genome-sequencing technologies now enable-in principle-the direct observation of genome instability globally and at scale. To exploit this opportunity, we sequenced the whole genomes of nearly all of the 4,732 strains comprising the homozygous diploid YKOC. Here, by extracting information on copy-number variation of tandem and interspersed repetitive DNA elements, we describe-for almost every single non-essential gene-the genomic alterations that are induced by its loss. Analysis of this dataset reveals genes that affect the maintenance of various genomic elements, highlights cross-talks between nuclear and mitochondrial genome stability, and shows how strains have genetically adapted to life in the absence of individual non-essential genes.