Abstract 3118: The proteasome inhibitor Bortezomib rescues clinically identified unstable missense variants of the DNA mismatch repair protein Msh2

Msh2 is required for DNA mismatch repair in eukaryotes. Deleterious mutations in human MSH2 account for approximately half of the alleles associated with Hereditary Nonpolyposis Colorectal Cancer (HNPCC) or Lynch Syndrome. Previously, we characterized clinically-identified MSH2 missense mutations, using yeast as a model system and found that the most common cause of defective DNA mismatch repair was low levels of the variant Msh2 proteins. Here, we measured the half-lives of the Msh2 variants and found that increased protein turnover was responsible for the low cellular levels. Additionally, restoring levels with increased gene dosage fully restored mismatch repair function in 10 of the 21 variants in a DNA polymerase proofreading defective strain background in which mismatch repair is essential. Using immunoprecipitation, we detected high molecular weight ubiquitinated species of unstable Msh2 variants when over-expressed. Additionally, genetic and chemical inhibition of the proteasome stabilized low-level Msh2 missense variants. We identified San1, an ubiquitin ligase known to target misfolded nuclear proteins, as responsible for targeting the MSH2 missense variants. Deletion of San1 restored protein levels for all but one variant, but did not elevate wild-type Msh2 levels. Additionally, mismatch repair function was significantly improved when the Msh2 variants that had displayed high-copy restoration of function were expressed in a san1α strain. Taken together, these results indicate that the ubiquitin-mediated proteasome degradation pathway is the major mechanism for increased turnover of the clinically identified Msh2 variants. Human MutSα, comprised of Msh2 and Msh6, is also regulated by the ubiquitin-mediated pathway, underscoring the conservation of regulation as well as function for mismatch repair in eukaryotes. Additionally, it is well established that mismatch repair defective tumors are resistant to standard chemotherapeutic regimens. Of therapeutic significance, the clinically approved drug Bortezomib partially restored levels and mismatch repair function in two different msh2 missense variants. In this regard, Bortezomib may prove to be a valuable adjuvant chemotherapeutic for individuals with particular alleles of MSH2. Our results provide the foundation for an innovative therapeutic regime for certain mismatch repair defective cancers that are refractory to conventional chemotherapies. Currently we are testing the rescue phenotype in human