Spencer D. Shelton

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

Mitochondrial DNA (mtDNA) mutations are frequent in cancer, yet their precise role in cancer progression remains debated. To functionally evaluate the impact of mtDNA variants on tumor growth and metastasis, we developed an enhanced cytoplasmic hybrid (cybrid) generation protocol and established isogenic human melanoma cybrid lines with wild-type mtDNA or pathogenic mtDNA mutations with partial or complete loss of mitochondrial oxidative function. Cybrids with homoplasmic levels of pathogenic mtDNA reliably established tumors despite dysfunctional oxidative phosphorylation. However, these mtDNA variants disrupted spontaneous metastasis from primary tumors and reduced the abundance of circulating tumor cells. Migration and invasion of tumor cells were reduced, indicating that entry into circulation is a bottleneck for metastasis amid mtDNA dysfunction. Pathogenic mtDNA did not inhibit organ colonization following intravenous injection. In heteroplasmic cybrid tumors, single-cell analyses revealed selection against pathogenic mtDNA during melanoma growth. Collectively, these findings experimentally demonstrate that functional mtDNA is favored during melanoma growth and supports metastatic entry into the blood.

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.