Ultramutagenesis through combined Msh2 depletion and proofreading-deficient DNA polymerase ε expression enhances small molecule target identification in forward genetic screens

DNA mismatch repair (MMR) deficiency has been widely utilized in forward genetic screens to identify drug resistance mutations and elucidate the mechanisms of action of cytotoxic small molecules. However, MMR deficiency generates a characteristic mutational signature enriched for C>T transitions that precludes saturation of mutagenesis. We hypothesized that expression of proofreading deficient DNA polymerase epsilon, as has been observed in patients with biallelic MMR deficiency, would enhance mutagenesis and enable identification of new resistance mutations and cytotoxin mechanisms. Here, we combine auxin-inducible degradation of Msh2 with doxycycline-inducible expression of the proofreading-deficient Polε ^P286R mutant in murine small cell lung cancer (SCLC) cells. Simultaneous MMR deficiency and mutant Polε expression markedly increased the emergence of bortezomib-resistant clones relative to either perturbation alone and targeted sequencing of Psmb5 in bortezomib-resistant clones identified recurrent mutations previously reported in chemical mutagenesis-based resistance screens. We next applied this platform to investigate resistance to lurbinectedin, a clinically relevant therapy for SCLC. Whole-exome sequencing revealed recurrent loss-of-function alterations in nucleotide excision repair (NER) genes, most notably Ercc5 and Ercc4 , implicating NER deficiency as a major mechanism of lurbinectedin resistance and supporting a central role for NER in mediating lurbinectedin-induced cytotoxicity. Collectively, these findings establish inducible ultramutagenesis as a powerful and versatile platform for the unbiased discovery of drug resistance mechanisms and therapeutic vulnerabilities.