Vicky Li

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.

Supplementary Table S1 shows key mtDNA mutations identified through whole-exome sequencing and targeted mtDNA sequencing.

Supplementary Table S2 shows nuclear genome variants identified in NCI-237-R PDX through whole-exome sequencing.

Supplementary Figure S1 shows validation of key genetic features in NCI-237 and metabolic characterization of thyroid cancer cell lines. Supplementary Figure S2 shows growth curves and depletion/enrichment plots for pooled CRISPR-Cas9 screens. Supplementary Figure S3 shows validation of complex I impairment conferring sensitivity to LDH inhibition. Supplementary Figure S4 shows metabolic profiling of LDH inhibition in NCI-237 cells. Supplementary Figure S5 shows the validation of NADH-oxidizing enzymes and that NAD+ regeneration rescues LDH inhibition. Supplementary Figure S6 shows that LDH inhibition impairs NCI-237-R PDX proliferation.

Supplementary Table S3 shows the results of our genome-wide and targeted CRISPR-Cas9 screens.

A metabolic hallmark of cancer identified by Warburg is the increased consumption of glucose and secretion of lactate, even in the presence of oxygen. Although many tumors exhibit increased glycolytic activity, most forms of cancer rely on mitochondrial respiration for tumor growth. We report here that Hürthle cell carcinoma of the thyroid (HTC) models harboring mitochondrial DNA-encoded defects in complex I of the mitochondrial electron transport chain exhibit impaired respiration and alterations in glucose metabolism. CRISPR–Cas9 pooled screening identified glycolytic enzymes as selectively essential in complex I–mutant HTC cells. We demonstrate in cultured cells and a patient-derived xenograft model that small-molecule inhibitors of lactate dehydrogenase selectively induce an ATP crisis and cell death in HTC. This work demonstrates that complex I loss exposes fermentation as a therapeutic target in HTC and has implications for other tumors bearing mutations that irreversibly damage mitochondrial respiration.Significance:HTC is enriched in somatic mtDNA mutations predicted to affect complex I of the electron transport chain (ETC). We demonstrate that these mutations impair respiration and induce a therapeutically tractable reliance on aerobic fermentation for cell survival. This work provides a rationale for targeting fermentation in cancers harboring irreversible genetically encoded ETC defects.See related article by Gopal et al., p. 1904.This article is highlighted in the In This Issue feature, p. 1749

Supplementary Table S1 shows key mtDNA mutations identified through whole-exome sequencing and targeted mtDNA sequencing.

Supplementary Table S2 shows nuclear genome variants identified in NCI-237-R PDX through whole-exome sequencing.

Supplementary Table S1 shows key mtDNA mutations identified through whole-exome sequencing and targeted mtDNA sequencing.

Supplementary Table S3 shows the results of our genome-wide and targeted CRISPR-Cas9 screens.