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Mutations in polymerases Pold1 and Pole exonuclease domains in humans are associated with increased cancer incidence, elevated tumor mutation burden (TMB) and response to immune checkpoint blockade (ICB). Although ICB is approved for treatment of several cancers, not all tumors with elevated TMB respond. Here we generated Pold1 and Pole proofreading mutator mice and show that ICB treatment of mice with high TMB tumors did not improve survival as only a subset of tumors responded. Similarly, introducing the mutator alleles into mice with Kras/p53 lung cancer did not improve survival, however, passaging mutator tumor cells in vitro without immune editing caused rejection in immune-competent hosts, demonstrating the efficiency by which cells with antigenic mutations are eliminated. Finally, ICB treatment of mutator mice earlier, before observable tumors delayed cancer onset, improved survival, and selected for tumors without aneuploidy, suggesting the use of ICB in individuals at high risk for cancer prevention., Highlights: Germline somatic and conditional Pold1 and Pole exonuclease domain mutations in mice produce a mutator phenotype. Spontaneous cancers arise in mutator mice that have genomic features comparable to human tumors with these mutations.ICB treatment of mutator mice with tumors did not improve survival as only a subset of tumors respond. Introduction of the mutator alleles into an autochthonous mouse lung cancer model also did not produce immunogenic tumors, whereas passaging mutator tumor cells in vitro caused immune rejection indicating efficient selection against antigenic mutations in vivo . Prophylactic ICB treatment delayed cancer onset, improved survival, and selected for tumors with no aneuploidy.

Mitochondrial function is important for both energetic and anabolic metabolism. Pathogenic mitochondrial DNA (mtDNA) mutations directly impact these functions, resulting in the detrimental consequences seen in human mitochondrial diseases. The role of pathogenic mtDNA mutations in human cancers is less clear; while pathogenic mtDNA mutations are observed in some cancer types, they are almost absent in others. We report here that the proofreading mutant DNA polymerase gamma ( PolG D256A ) induced a high mtDNA mutation burden in non-small-cell lung cancer (NSCLC), and promoted the accumulation of defective mitochondria, which is responsible for decreased tumor cell proliferation and viability and increased cancer survival. In NSCLC cells, pathogenic mtDNA mutations increased glycolysis and caused dependence on glucose. The glucose dependency sustained mitochondrial energetics but at the cost of a decreased NAD+/NADH ratio that inhibited de novo serine synthesis. Insufficient serine synthesis, in turn, impaired the downstream synthesis of GSH and nucleotides, leading to impaired tumor growth that increased cancer survival. Unlike tumors with intact mitochondrial function, NSCLC with pathogenic mtDNA mutations were sensitive to dietary serine and glycine deprivation. Thus, mitochondrial function in NSCLC is required specifically to sustain sufficient serine synthesis for nucleotide production and redox homeostasis to support tumor growth, explaining why these cancers preserve functional mtDNA., In Brief: High mtDNA mutation burden in non-small-cell lung cancer (NSCLC) leads to the accumulation of respiration-defective mitochondria and dependency on glucose and glycolytic metabolism. Defective respiratory metabolism causes a massive accumulation of cytosolic nicotinamide adenine dinucleotide + hydrogen (NADH), which impedes serine synthesis and, thereby, glutathione (GSH) and nucleotide synthesis, leading to impaired tumor growth and increased survival., Highlights: Proofreading mutations in Polymerase gamma led to a high burden of mitochondrial DNA mutations, promoting the accumulation of mitochondria with respiratory defects in NSCLC.Defective respiration led to reduced proliferation and viability of NSCLC cells increasing survival to cancer.Defective respiration caused glucose dependency to fuel elevated glycolysis.Altered glucose metabolism is associated with high NADH that limits serine synthesis, leading to impaired GSH and nucleotide production.Mitochondrial respiration defects sensitize NSCLC to dietary serine/glycine starvation, further increasing survival.