Fanconi anemia (FA) is a generally autosomal recessive orphan disease caused by germline loss of function mutations in any one of more than 20 genes (Table 1) associated with the FA DNA repair pathway, although rare cases display X-linked or dominant negative inheritance [1]. FA affects 1 in 160,000 individuals, with a variable life expectancy of 20 years ^[2][3][2,3]. The FA pathway functions classically in the repair of DNA (interstrand crosslinks) ICLs [1]. ICLs are covalent adducts between DNA strands which block transcription and replication and can lead to double strand DNA breaks [4]. Continuous removal of ICLs is required for sustained cell survival. ICLs form as a response to either exogenous or endogenous crosslinkers. Exogenous crosslinkers include chemotherapeutics such as mitomycin C, cisplatin, melphalan, psoralens, tobacco use, broiled meat and alcohol consumption ^[5][6][7][5,6,7]. Endogenous crosslinkers include natural byproducts of mitochondrial and fat metabolism, metabolism of tobacco smoke or ingested alcohol, all of which can produce reactive oxygen species (ROS) [8] and aldehydes ^[9][10][9,10]. Cells from individuals with FA are highly sensitive to DNA crosslinkers, resulting in increased chromosomal abnormalities and accumulation of cells arrested in the G2/M phase of the cell cycle ^[5][11][5,11]. Individuals with FA are unable to properly repair ICLs, and exhibit congenital defects and progressive bone marrow failure, often early in life. Moreover, patients with FA demonstrate increased susceptibility to human papillomavirus (HPV) and SV40 infection ^[12][13][12,13]. Bone marrow failure in FA can evolve into leukemia ^{[9][14][15][16][17][18]}[9,14,15,16,17,18] and persons with FA have a markedly elevated frequency of early aggressive keratinocyte-based squamous cell carcinomas (SCCs) of the head and neck, esophagus, anogenital tract, and skin with advancing age compared to the general population ^{[1][19][20][21][22][23][24][25]}[1,19,20,21,22,23,24,25]. Other non-canonical activities reported for the FA pathway include the stabilization of stalled replication forks [26], control of mitosis and cytokinesis ^[27][28][27,28], suppression of non-homologous end-joining (NHEJ) [29], clearance of damaged mitochondria by mitophagy [30], clearance of viruses by autophagy [30], regulation of the HPV life cycle ^{[13][21][24][31][32][33][34][35]}[13,21,24,31,32,33,34,35], maintenance of cellular redox reactions [36] and metabolic regulation ^[37][38][39][37,38,39].

2. Mitochondrial Activities Play a Role in Oncogenesis

Mitochondria are cellular organelles responsible for oxygen-dependent energy metabolism and are a main source and target of ROS formation. The transport of ADP, phosphate and protons across the inner membrane normally accelerates the rate of electron transport and most of the oxygen consumed by the respiratory electron chain is reduced to water. mtDNA encodes some but not all of the respiratory enzyme subunits that are essential for oxidative phosphorylation and rRNA and tRNA needed for mitochondrial protein synthesis. Nuclear DNA encodes the remaining mitochondrial proteins ^[40][86]. Mutations in mtDNA are thought to significantly stimulate oxidative phosphorylation, support neoplastic transformation, and fulfill the sustained bioenergetic demands of cancer cells ^[41][87]. Meta-analysis of 20 different cancer types from 859 non-FA patients showed that 66% harbored mutations in mtDNA ^[42][88]. Included in this analysis were adult leukemia (9/24) 38% and head and neck cancers (337/467) 72%, which harbored mutations in mtDNA ^[42][88]. Although the role of these mutations in cancer and metastasis remains unknown, they may increase energy metabolism and ROS generation and support cell survival. Several studies have uncovered links between FA gene products and mitochondrial dysfunction ^{[38][43][44][45][46][47][48]}[38,89,90,91,92,93,94], and oxidative stress-derived mitochondrial dysfunction in combination with decreased scavenging of endogenous aldehydes [10], increased lipid peroxidation ^[49][95] and impaired ATP production ^[38][44][50][38,90,96], have emerged as metabolic phenotypic hallmarks of FA ^[44][90]. More detailed FA metabolic studies are needed to determine required compensatory reprogramming such as increased lactic fermentation which provides metabolic energy, intermediates for proliferative anabolism, and more potent tumor-supporting microenvironments by acidification and negative regulation of immune cell function ^[51][97]. FA has recently been identified as a mitochondrial disease (MD) given these and other connections between FA proteins and impaired mitochondrial activities ^[52][98]. MDs are a group of disorders associated with mutations in nuclear and mitochondrial DNA and consequent impaired oxidative phosphorylation. MDs are characterized by a number of clinical pathologies including short stature, exercise intolerance and hypertrophic or dilated cardiomyopathy ^[53][99]. Although FA shares limited clinical similarities with MDs aside from short stature, mitochondrial dysfunction is a hallmark in both instances ^[52][98].

3. FA Proteins Localize to Mitochondria

Multiple FA proteins are detectable in mitochondria and regulate mitochondrial metabolic function through physical interactions with other mitochondrial proteins, such as the peroxidase peroxiredoxin-3 (PRDX3) ^[45][91], and ATP synthase (ATP5α), a subunit of the mitochondrial ATPase. In FANCA, C and G deficient cells, constitutive oxidative stress suppressed mitochondrial activities by reducing the transmembrane potential, oxygen consumption rate, ATP production, and ROS detoxification ^[45][91]. FANCA and FANCC deficient subtypes also suppressed PRDX3, a member of a family of antioxidant enzymes which regulate physiological levels of hydrogen peroxide (H₂O₂) ^[45][91]. FANCG physically interacts with PRDX3, and FANCG mutated cells harbored a distorted mitochondrial structure and reduced thioredoxin-dependent peroxidase activity ^[45][91]. Overexpression of PRDX3 restored the resistance of FANCG-deficient cells to oxidative stress, while PRDX3 downregulation increased sensitivity to mitomycin C. FANCA and C deficient subtypes also harbored decreased PRDX3 expression, indicating an as of yet unknown functional interaction between other FA proteins and PRDX3 ^[45][91]. Furthermore, a physical interaction between FANCD2 and ATP5α was reported to be essential for optimal ATP synthesis [39]. FANCD2 deficient cells harbored reduced mitochondrial ATP production due to inappropriate ATP5α localization [39]. In addition, FANCD2 localizes to mitochondria in a process mediated by the ATPase Family AAA Domain-Containing Protein 3A (ATAD3) ^[54][100], a member of the mitochondrial nucleoid complex that also includes Mitochondrial Transcription Factor A (Tfam) and Mitochondrial Tu Translation Elongation Factor (Tufm). This complex is essential for mtDNA-encoded gene transcription, translation and mitochondrial biosynthesis ^[54][55][56][100,101,102], and might therefore play a key role in the maintenance of mitochondrial activities. Indeed, genetic deletion of Fancd2 in murine hematopoietic stem and progenitor cells (HSPCs) led to a significant increase in mitochondrial number, mitochondrial protein synthesis, enzyme activity of mitochondrially encoded respiratory complexes and, consequently, OXPHOS and mtROS levels in HSPCs ^[54][100]. Increased mitochondrial number and proteins may cause an imbalance in nuclear and mitochondrially encoded factors that is further exacerbated by a significant increase in mitochondrial stress related proteins and a deregulated mitochondrial stress response in FANCD2 -deficient cells ^[54][57][100,103].

4. ROS May Be a Cause or Consequence of Mitochondrial Abnormalities in FA

ROS are produced by several endogenous sources that require oxygen ^[58][105]. H₂O₂ is generated by a wide variety of oxygen-dependent oxidation reactions, as well as by dismutation of superoxide ^[59][106]. Oxygen radicals, which include superoxide, oxidize macromolecules such as lipids and proteins, and can generate adducts between DNA strands ^[60][107]. Some of these negative effects can be countered by antioxidant defenses such as upregulation of the antioxidant detoxification components NADPH quinone oxidoreductase-1 and Redox factor-1 (Ref-1) ^[58][61][105,108]. Thus, oxidative stress is defined by the imbalance between oxidants and antioxidants ^[49][62][95,109]. The mitochondrial respiratory chain is the major producer of ROS, as a byproduct of ATP production facilitated by NADH oxidases ^[63][110]. Other endogenous enzymatic reactions not limited to the mitochondria which produce ROS are prostaglandin synthesis, phagocytosis, and the cytochrome P450 system ^[64][111]. In addition, exogenous agents such as metals, therapeutic agents, radiation and environmental toxins such as benzo(a)pyrene also produce ROS ^[64][65][111,112]. It has been suggested that mitochondria in FA deficient cells are shifted to a semi-resting state, where ATP production is defective and the rate of oxygen consumption is decreased due to compromised Complex I activity ^[44][66][90,113]. FANCA-deficient cells displayed defects in mitochondrial respiratory chain Complex I activity, resulting in diminished ATP production ^[66][113]. Impaired oxygen consumption and reduced mitochondrial membrane potential as a consequence of low ATP production have been identified as phenotypes of FANCA, FANCC and FANCD2 deficient cells ^[44][90]. Overexpression of superoxide dismutase 1 (SOD1) rescued oxygen uptake and respiration capacity in FA cells. Some mitochondrial enzymes responsible for ROS clearance were unable to respond to H₂O₂ in FA cells, suggesting impairment of the mitochondrial detoxifying machinery ^[44][90]. Indeed, in pathological conditions such as FA, buildup and/or failure to detoxify ROS further diminish mitochondrial activities ^[62][67][68][109,114,115]. Accumulation of ROS dissipates the transmembrane potential, which leads to lower ATP levels in FA deficient cells as compared to their genetically corrected counterparts. The low transmembrane potential and overproduction of ROS could be a result of alterations in mitochondrial morphology, including membrane thinning or rupture, abnormal shapes and mitophagy in FA cells. Treatment of the same cells with H₂O₂ further increased mitochondrial fragmentation. In turn, addition of the ROS scavenger N-acetyl-cysteine (NAC) lowered the production of ROS in FA-depleted cells ^[44][90]. Importantly, the sensitivity of FA deficient cells to crosslinking agents such as mitomycin C was reduced in the presence of ROS scavengers. Pretreatment of FA deficient cells with NAC significantly improved ATP production, restored oxygen consumption and stimulated increased resistance to MMC, albeit in the absence of rescue of abnormal mitochondrial morphologies ^[44][90]. These data suggest that some of the mitochondrially induced ROS-related phenotypes of FA deficient cells are reversible ^[44][66][90,113].

5. Increased ROS Exacerbate DNA Damage in FA

ROS at low concentrations can have beneficial roles in signal transduction ^[69][70][116,117] and organismal defense against pathogens ^[71][72][118,119]. However, ROS at high concentrations can induce oxidation and damage of DNA, protein and lipid, with subsequent pro-inflammatory cytokine production, apoptosis, autophagy and/or necrosis. Elevated ROS also induces chronic inflammation which promotes cancer initiation and may further increase cancer risk ^[73][74][120,121]. Chronic inflammation and infection (e.g., persistent HPV infection) can cause the production of chemokines and cytokines, which in turn promote cellular transformation ^[75][122], by-pass of tumor suppressor activity ^[76][123] and proliferation and angiogenesis ^[77][78][124,125]. ROS stimulates pro-inflammatory chemokine and cytokine production, which initiates a positive feedback loop resulting in further ROS accumulation and an inflammatory oncogenic environment ^[79][126]. Interestingly, ROS-dependent lipid peroxidation results in aldehyde production, especially malondialdehyde (MDA) and 4-hydroxynonenal (4HNE), both of which stimulate ICLs and hyper-mutagenicity ^[70][80][117,127]. Heightened endogenous aldehyde production via ROS and lipid peroxidation may therefore co-operate with the classical ICL repair defects to produce structural variants and chromosomal abnormalities that are hallmarks of FA ^[81][82][128,129]. Damaged mitochondria in FA cells are also more likely to rupture, inducing apoptosis at lower levels of stress than in normal cells. The FANCA, FANCC, FANCD2, FANCF, FANCL, BRCA1/FANCS and BRCA2/FANCD1 proteins aid in Parkin-mediated mitophagy, implicating this non-canonical role of FA proteins in disease pathologies ^[30][47][30,93]. Defective mitophagy, in turn, leads to the accumulation of damaged mitochondria and concomitantly increased intracellular oxidative stress ^[83][130]. Interestingly, in murine embryonic fibroblasts (MEFs), FANCC is essential for host immunity against herpes simplex virus type 1 mutant strain and Sindbis virus and plays a role in virophagy as well as autophagy [30]. Overall, these data suggest that genetic FA defects lead to the accumulation of ROS, as well as mitochondrial abnormalities with impaired antioxidant defenses ^[84][131], thus further damaging mitochondria to diminish cellular respiration and ATP synthesis.