Mitochondrial DNA is a major source of driver mutations in cancer

Abstract

Mitochondrial DNA (mtDNA) mutations are among the most common genetic events in all tumors and directly impact metabolic homeostasis. Despite the central role mitochondria play in energy metabolism and cellular physiology, the role of mutations in the mitochondrial genomes of tumors has been contentious. Until recently, genomic and functional studies of mtDNA variants were impeded by a lack of adequate tumor mtDNA sequencing data and available methods for mitochondrial genome engineering. These barriers and a conceptual fog surrounding the functional impact of mtDNA mutations in tumors have begun to lift, revealing a path to understanding the role of this essential metabolic genome in cancer initiation and progression. Here we discuss the history, recent developments, and challenges that remain for mitochondrial oncogenetics as the impact of a major new class of cancer-associated mutations is unveiled.

Keywords: cancer; genome editing; mitochondrial DNA; mutation selection.

Figure 1.. Hierarchy of variation in mtDNA genotypes.

(A) At the resolution of tissues, renal, thyroid, and colorectal cancer types have enrichment of truncating mutations with higher heteroplasmy levels, suggestive of positive selection in those tumor types. The right figure is a reproduction from [32]. (B) At the resolution of individual cells, tumors are composed of genetically diverse cell populations with distinct mtDNA genotypes. Changes in bulk mtDNA heteroplasmy over time thus reflect both changes in clonal composition, as intracellular shifts in heteroplasmy. Both processes may be subject to evolutionary selection. (C) At the resolution of mtDNA genotypes, mutations non-randomly accumulate in specific regions of the genome. Protein-truncating mutations, which have been the primary subject of interest in genomic studies, account for only 27% of variants observed in genomes. Such truncating mutations preferentially accumulate in complex I subunits, suggesting that the function of truncating mutations is shaped by their impact on respiratory function. The figures are a reproduction from [35].

Figure 2.. Single cell sequencing of mtDNA.

Most single cell analysis of mtDNA variants to date has been for the purposes of reconstructing evolutionary phylogenies, using mtDNA variants as cell-endogenous barcodes. (A) As cancer cells divide, they accumulate new somatic changes to nuclear and mitochondrial DNA that reflect their evolutionary phylogeny. (B) Evolutionarily related cells can be clustered into clones using information about the presence and heteroplasmy of heteroplasmic mtDNA variants. In htis example, a heatmap of the heteroplasmy levels of mtDNA variants reflects the evolutionary structure shown above in (A) The columns indicate each cell and rows indicate mtDNA variants. (C) Some mtDNA variants may not be silent, but rather may be associated with a particular phenotype. In this case, examination of nuclear gene expression patterns (e.g. derived from in single cell RNA-seq) may identify phenotypes associated with heteroplasmic dosage.

Figure 3.. Current approaches for editing mtDNA in animals.

(A) Random mutators of mtDNA, based on the defective mtDNA polymerase (PolgA^mut) or the mitochondrially targeted monomeric cytosine deaminase APOBEC1 [88,89] allow generation of abundant, low heteroplasmy mutations of mtDNA resulting in non-physiological forms of mitochondrial dysfunction. Heteroplasmy shifting exploits the tendency for mitochondria to rapidly degrade genomes containing DNA double strand breaks. If allele-specific double strand breaks can be introduced in the context of a cell bearing a pre-existing mtDNA mutation, the heteroplasmy of that mutation can be amplified or diminished. Several approaches to heteroplasmy shifting, with robust effects in vitro and in vivo, have been developed, including but not limited to mitochondrially targeted: restriction enzymes [81,91,92], zinc-finger nucleases [8,77] and transcription activator-like effector nucleases [76,78]. Base editing, at present consisting exclusively of engineered, dimeric DddAtox cytosine deaminase base editor (DdCBE) domains fused to TALE DNA binding domains, permit a degree of selective mutation introduction into mtDNA, in situ, for the first time. These chimeric enzymes are limited in terms of targetable sites (based on TALE DNA binding constraints, need for 5’TC’3 context, immediate off-target considerations) and the mutations they can produce (C>T or G>A only, no indels), but allow for production of many previously unobtainable genotypes at relevant levels of heteroplasmy. (B) Depiction of the impact of cumulative heteroplasmies shown in A on mitochondrial function.

Figure 4.. A model for selection of deleterious heteroplasmies in cancer cells.

(A) Unlike nuclear DNA, mtDNA is replicated both during S phase and throughout all stages of the cell cycle. While the misincorporation rate of the mitochondrial replicative polymerase (Pol γ) is considered very low, the impact of continuous replication is such that mitochondrial mutational heterogeneity is seeded throughout the body in a time-dependent fashion, in agreement with both the strand asymmetric distribution of mutations (due to mode of mtDNA replication) and type of mutations found in cancers. Cells bearing mtDNA mutations in proliferative neoplasms acquire a fitness advantage due to increased capacity for anabolism and proliferation due to enhanced mitochondrial mass, a common response to mitochondrial dysfunction. The coupling of mtDNA mutations to increases in mitochondrial mass and consequent cellular fitness provides a rational framework for the consistent selection of mitochondrial mutations by cancer cells. (B) A visual representation of the proposed framework for selection of mtDNA mutations in cancer.