Although major clinical conditions in FA result from defects in DNA damage and repair machinery, recent discoveries suggest that other cellular pathways are also compromised and contribute to the disease progression (Table 1
). It has long been appreciated, indeed, that some FA
genes are involved in additional cytoprotective pathways (Figure 2
), such as defense from reactive oxygen species (ROS)-induced cell death [50
], mitochondrial homeostasis [51
], and defense from pro-inflammatory cytokine-induced apoptosis [52
]. Intriguingly, many of the noncanonical functions of FA proteins discussed below are not restricted to HSCs and some of these processes may be independent of the DNA damage response (DDR).
Defective mitochondria are one of the sources of pro-inflammatory signaling pathways, through the production of ROS and an associated increment of oxidative stress [53
]. A large amount of research has been dedicated to uncovering how FA signaling affects mitochondrial functions.
Oxidative stress: Oxidative stress is generally defined as an imbalance that favors the production of ROS over antioxidant defenses; the majority of ROS are produced by mitochondrial respiration. FA cells (e.g., FANCA
mutant fibroblasts) have impaired electron transport in Complexes I and III, leading to changes in the relative ATP/AMP ratio, which results in a decreased respiration capacity, mitochondrial membrane potential, and oxygen uptake [54
]. This is also supplemented by inactivation of essential enzymes of the energy production pathway. From a molecular point of view, FANCA, FANCC, and FANCG are found to interact with cytochrome P450-redox related activities and to respond to oxidative damage [58
]. In addition, Mukhopadhyay and colleagues identified FANCG protein in mitochondria, as well as its interaction with the mitochondrial peroxidase peroxiredoxin3 (PRDX3) [60
]. In FA cells, however, PRDX3 is mislocalized and thioredoxin-dependent peroxidase activity strongly deregulated. More recently, a mitochondrial localization signal (MLS) on FANCG has been identified; in eight FA patients, indeed, a single nucleotide change (C.65G>C) leads to the conversion of the amino acid arginine at the 22 positions of the MLS into proline (p.Arg22Pro) [61
]. This mutant protein (R22P) fails to localize to the mitochondria and protect them from oxidative stress; on the contrary, this mutant is still able to participate to the formation of the FA core complex in the nucleus and is also resistant to ICL agents. More interestingly, in R22P stable cells, there is also an iron deficiency of FA protein FANCJ, an iron‒sulfur (Fe‒S)-containing helicase involved in DNA repair [61
]. This suggests, for the first time, that oxidative stress-mediated mitochondrial dysfunction causes, per se, defective FANCJ, leading to genomic instability.
Metabolism: Reflecting the crucial role of the mitochondria in aerobic ATP production, normal metabolism is altered in FA cells and probably complementary pathways are involved in prevailing the energetic defect. In the first systematic work about FA metabolism, glycolysis emerged as the main source when aerobic metabolism was reduced by unproductive mitochondrial electron transport complexes [62
]. However, glycolysis remains insufficient to satisfy FA cells’ energy requirements. Since energetic metabolism plays an essential role during HSCs’ differentiation into lymphocytes, this could, at least in part, explain a defective metabolic maturation in the bone marrow during the exit from the homeostatic quiescent state.
Morphology: Oxidative phosphorylation (OXPHOS) impairment is not the only mitochondrial damage in FA cells. Several reports demonstrate alterations in mitochondrial morphology; mitochondria appear swollen with matrix rarefaction, altered cristae, and reticulum fragmentation. For example, mitochondria in FANCD2 mutant cells show wall ruptures, thinner walls and cristae, and smaller sizes [63
], as well as mitochondria from FANCG−/−
fibroblasts displaying frequent elongation and irregular shapes [60
]. Two opposing coordinated processes, fusion and fission, determine mitochondrial content and structure and are essential for maintaining ordinary mitochondrial function and regulating mitochondrial morphology [64
]. Mitochondrial fission involves the recruitment of GTPase dynamin-related protein (DRP1) from the cytosol to the mitochondrial membranes to catalyze the fission reaction [65
]. Shyamsunder and colleagues found an accumulation of DRP1 in the mitochondria of FANCA and FANCC-deficient cells, which positively affects mitochondria fission [66
Mitophagy: A specific cellular process called mitophagy removes damaged and old mitochondria through double-membraned vesicles known as autophagosomes. In contrast to bulk autophagy, which removes parts of the cytoplasm nonspecifically, mitophagy is one of the forms of selective autophagy that precisely removes unnecessary cytoplasmic contents (e.g., bacteria, mitochondria, and endoplasmic reticulum) [67
]. In 2016, Sumpter and colleagues, using a CRISPR/Cas9-mediated approach in HeLa cells, FANCC mutant fibroblasts of FA patients, and bone marrow-derived macrophages from Fancc
-deficient mice, described the role of FANCC, as well as of other FA genes, in mitophagy [68
]. Moreover, through siRNA experiments, FANCF and FANCL have also been found to be required in mitophagy, providing a novel role for these FA proteins. In detail, FANCC is recruited to the mitochondria and interacts with the E3 ligase PARKIN, a key enzyme that regulates mitochondrial degradation by mitophagy in a mitochondrial damage-dependent manner. FANCC
-deficient cells, indeed, show accumulation of damaged mitochondria, suggesting a defect in the mitophagy process [68
]. This is in line with a previous study that found mitochondrial fission as a precondition for mitophagy defects in FA. The blockade of this process may allow autophagy to remove dysfunctional mitochondria [66
]. Interestingly, the function of FANCC and FANCA in mitophagy seems to be genetically separate from their role in DDR [69
]. Indeed, the hypomorphic mutants of FANCC (c.67delG) and FANCA (p.Arg951Gln/Trp, p.His913Pro) are not functional in DNA repair but preserve the mitochondria functions. Patients with these mutations have a mild clinical disease, suggesting the importance of FA-mediated mitochondria removal in improving the disease course.
Biosynthesis: Homeostasis of mitochondrial mass is maintained by a balance between mitophagy and mitochondrial biogenesis. A set of DNA-binding core proteins involved in mtDNA maintenance and transcription forms the mitochondrial nucleoid. The most frequently identified components of this complex, essential for mitochondrion biosynthesis, are ATAD3 and translation mitochondrial factor of elongation (TUFM). ATAD3 is an ATPase that plays a central role in nucleoid organization, as it associates with both the inner membrane and the mitochondrial ribosome, and also binds to D-loop sequences of mtDNA [70
]. Intriguingly, by a proteomic approach, FANCD2 has been found to be associated with both Atad3 and Tufm and, through its localization at the mitochondrion, regulates Atad3/Tufm expression [71
], thus providing the first evidence for FANCD2 as a crucial player of mitochondrial biosynthesis. Intriguingly, by a proteomic approach, FANCD2 has been found to be associated with the nucleoid complex components Atad3 and Tufm and, through its localization at the mitochondrion, regulates Atad3/Tufm expression [71
], providing the first evidence for FANCD2 as a crucial player of mitochondrial biosynthesis.
One of the features of FA cells is the increase in inflammation markers and hypersensitivity to pro-inflammatory cytokine-induced apoptosis such as TNF-α [76
], IL-6 [80
], and IL-1β [81
]. Of course, persistent DNA damage is well known to excite the production of inflammatory mediators [82
], which probably impact on bone marrow activity. Activation of toll-like receptors in FANCA and FANCC-deficient monocytes abnormally increases IL-1β expression, which negatively affects HSCs’ self-replication [52
]. Interestingly, hampering the action of the inflammatory cytokine interleukin 1 beta (IL-1β), using IL-1 receptor blockade, avoids bone marrow failure [84
]. However, how DNA damage plays a role in this pathway is not well understood. Of course, parallel noncanonical pathways could contribute to the progression of BMF in FA through ROS production. In FANCC−/−
primary bone-marrow-derived macrophages (BMDMs), indeed, an impaired bacterial lipopolysaccharide (LPS)-mitophagy is associated with ROS-dependent inflammasome hyperactivation and higher IL-1β secretion, resulting in the persistence of pathogen-associated and danger associated molecular patterns (PAMPs and DAMPs) [68
]. Moreover, a range of intricate secondary effects likely account for some of the inflammatory susceptibilities of FA HSCs. Using isogenic cells derived from patients and from nullizygous mice carrying inactivating mutations in the FANCC
gene, Pang and colleagues discovered that FANCC protects against proinflammatory cytokine-induced cell death by interacting with signal transducer and activator of transcription 1 (STAT1) [85
] and stress-inducible heat shock protein 70 (HSPA1A) [86
]. Mutations in the FANCA
, and FANCG
genes, indeed, markedly increase the interaction between eukaryotic translation initiation factor 2-alpha kinase 2 (PKR) and FANCC, leading to the hypersensitivity of BM progenitor cells to growth repression mediated by IFN-γ and TNF-γ [87
]. However, further studies are necessary to confirm that these effects are truly independent of induced DNA damage.