PROJECT SUMMARY Mutations that inactivate DNA repair factors increase genome instability and cancer susceptibility. Predicting the phenotypic effects of mutations is confounded by pervasive polymorphisms and elusive environmental modifiers. This proposal will tackle this problem by utilizing a fundamental epigenetic mechanism that mediates gene-by- environment interactions in biological systems, protein folding. Proteins must properly fold to function; this is achieved with help from specialized proteins called protein-folding chaperones. In helping proteins fold and function, the abundant chaperone HSP90 “buffers” (that is – mitigates) the effects of deleterious mutations. Environmental stressors tax HSP90 availability within cells thus revealing cryptic phenotypes in a mutant-specific way. However, the selectivity of HSP90 for cancer mutations remains poorly defined. Addressing this question will enable preemptive identification of environmentally sensitive genotypes in the population as well as stratification of cancer patients who would benefit from the use of HSP90 inhibitors. Indeed, HSP90 mediates resistance in breast cancer cells by buffering a Breast Cancer Associated 1 (BRCA1) protein folding mutant. Targeted inhibition of HSP90 restores cancer cell sensitivity to genotoxic agents and induces genome instability. I hypothesize that HSP90 buffers a broad range of mutations in BRCA1, and in doing so renders the induction of genome instability conditional upon environmental stressors that impair HSP90 function. I will test this hypothesis by using a powerful approach based on next-generation functional genomics and classical cytogenetics methodologies to: 1) evaluate chaperone binding as a classifier of BRCA1 missense mutant severity in cancer patients, 2) determine the HSP90-buffered landscape of BRCA1 mutations, 3) determine how HSP90 influences the conditional haploinsufficiency of BRCA1 heterozygotes. Each aim will utilize highly specific HSP90 inhibitors administered at non-toxic doses to specifically target HSP90-buffered mutants and not wild- type variant controls. I will engineer non-malignant and malignant cells to express HSP90-buffered BRCA1 mutants and measure the effects of HSP90 inhibition on genome stability and cell viability. This work will not only reveal fundamental mechanisms shaping the manifestations of BRCA1 mutations, but will also establish a practical approach to improve the efficacy of BRCA1 targeted therapies using HSP90 inhibition to exacerbate the effects of HSP90-buffered genome instability mutations. This proposal will prepare me for a career as an independent scientist investigating the molecular mechanisms through which cancer mutations perturb the function of DNA repair complexes at single-molecule resolution. I have established a career committee of experts who will train me in high-throughput systems biology and functional genomics, genome instability and cancer biology, genome engineering, and single-mo...