CCSG Pilot: Elucidating How a Truncating ATR Mutation Leads to Genomic Instability

ABSTRACT: Ataxia Telangiectasia and Rad3-Related (ATR) is a phosphatidylinositol 3-kinase-related kinase (PIKK) critical for genomic stability and replication fidelity. ATR is variably mutated in cancers, with genetic alterations found in up to a third of malignancies. The focus of this proposal is the ATR-I774Yfs*5 mutation which is overrepresented in solid tumors defective in the DNA mismatch repair (MMR) pathway. This frameshift mutation yields a truncated ATR protein of 778 amino acids (roughly the first third of the protein) and is found in at least 11 different tumor types, most commonly in colorectal carcinoma (CRC). We found that expression of ATR-I774Yfs*5 in a series of benign and malignant colorectal cell lines by transfection resulted in the unexpected accumulation of micronuclei suggesting that this mutation causes DNA damage and genomic instability. Despite this, cell viability was not diminished, leading us to hypothesize that ATR-I774Yfs*5 drives colorectal oncogenesis by destabilizing the genome in a non-lethal manner. We propose to elucidate how truncated ATR expression leads to micronuclei formation using two distinct stable expression systems: (1) heterozygous expression of ATR-I774Yfs*5 by CRISPR-Cas9 engineering in HCT116 colorectal carcinoma cells, and (2) φc31-mediated stable integration of ATR-I774Yfs*5 into a panel of colorectal epithelial and colorectal carcinoma cell lines. We will also study in-house CRC PDX models natively harboring the ATR- I774Yfs*5 mutation. Having documented the presence of micronuclei in each of these systems, we will establish that truncated ATR functions with dominant negative physiology to interfere with cell cycle checkpoint arrest and DNA replication to promote genomic instability and mutagenesis. TRANSLATIONAL POTENTIAL: Results from these experiments will enable the discovery of synthetic lethality therapeutic approaches based on inhibiting critical cellular responses to altered ATR function (e.g. PARP inhibition or translesion synthesis inhibition). This will introduce new targeted therapeutic possibilities to solid tumors harboring the ATR-I774Yfs*5 mutation. INTERPROGRAMMATIC COLLABORATIONS: This project will strengthen interactions between MCO, TO and CP because it involves fertile collaborations between investigators from each program. Indeed, this project could not be done without these interactions. SRF USAGE: • Flow Cytometry and Immune Monitoring: cell viability (Annexin V, propidium iodide) and cell cycle (BrdU) analysis • Biostatistics and Bioinformatics: experimental design, power analysis, statistical interpretation of data, and grant and manuscript preparation • Biospecimen Procurement and Translational Pathology Shared Resource Facility: preparation of paraffin-embedded sections and histologic analysis of 3-d spheroids FUTURE FUNDING: Critiques from a recent NIH R01 submission indicated that preliminary data using a transfection approach was confounded by possible overexpression artifact. Therefore by defining the impact of the ATR-I774Yfs*5 mutation on DNA replication and genomic instability through stable expression systems that mimic the physiologic condition as we are proposing to do here, we will be more favorably positioned for an R01 submission and/or multi-investigator NIH award (e.g. GI program project).