© 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 2657-2662 doi:10.1242/jcs.204909

CELL SCIENCE AT A GLANCE Emerging functions of the pathway at a glance Rhea Sumpter, Jr1,*,‡ and Beth Levine1,2

ABSTRACT proteins of the FA pathway to selective autophagy of viruses and Fanconi anemia (FA) is a rare disease, in which homozygous or mitochondria. Finally, we discuss how perturbations in FA protein- compound heterozygous inactivating mutations in any of 21 mediated selective autophagy may contribute to inflammatory as well lead to genomic instability, early-onset bone marrow failure and as genotoxic stress. increased cancer risk. The FA pathway is essential for DNA damage KEY WORDS: Selective autophagy, Fanconi anemia, Mitophagy, response (DDR) to DNA interstrand crosslinks. However, proteins of Inflammasome, Virophagy, DNA damage response the FA pathway have additional cytoprotective functions that may be independent of DDR. We have shown that many FA proteins Introduction participate in the selective autophagy pathway that is required for Fanconi anemia (FA), first described by Guido Fanconi in 1927, is a the destruction of unwanted intracellular constituents. In this Cell rare and potentially devastating disease associated with inactivating Science at a Glance and the accompanying poster, we briefly review homozygous or compound heterozygous mutations − except in the the role of the FA pathway in DDR and recent findings that link case of the for Fanconi anemia complementation group (FANC) B (FANCB), which is X -linked − in any of 21 1Center for Autophagy Research, Department of Internal Medicine, University of FA genes (Garaycoechea and Patel, 2014). Affected patients suffer Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390, USA. 2Howard Hughes Medical Institute, University of Texas Southwestern Medical from bone marrow failure (with a 90% prevalence in the first decade Center, Dallas, TX 75390, USA. of life), increased cancer susceptibility − particularly to acute *Present address: Department of Immunology, St. Jude Children’s Research myelogenous leukemia and squamous cell carcinomas of the upper Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA. digestive and urogenital tracts, and premature gonadal failure. ‡Author for correspondence ([email protected]) Although some patients are phenotypically normal, congenital Journal of Cell Science

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difficult to quantify. Unrepaired ICLs, ultimately, lead to DNA Box 1. FA proteins and proteins associated with FA breakage and chromosomal rearrangements, and promote the featuring in this article development and/or progression of cancer (see below) (Ceccaldi Official protein names are given in bold et al., 2016). BRAC1: breast cancer susceptibility gene 1 (also known as FANCS) Detection of ICLs by DDR surveillance proteins results in the BRAC2: breast cancer susceptibility gene 2 (also known as FACD, activation of an eight-member FA core complex (consisting of FANCD1) FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL BRIP1: BRCA1 interacting protein C-terminal 1 (also known as FANCJ) and FANCM) that directs the E3 ligase activity of FANCL ERCC4: excision repair 4, endonuclease catalytic subunit (also known to monoubiquitylate and thereby activate the DNA-binding as FANCQ) heterocomplex between Fanconi anemia group D2 protein FAAP20: FA core complex associated protein 20 (FANCD2) and FANCI (see poster). The active FANCD2 FAN1: FANCD2 and FANCI-associated nuclease 1 −FANCI complex, in turn, orchestrates the concerted downstream FANCA: Fanconi anemia complementation group (FA comp group) actions of the FA pathway and accessory proteins to resolve the ICL A protein ’ FANCB: FA comp group B protein lesion (Boisvert and Howlett, 2014; Kim and D Andrea, 2012; FANCC: FA comp group C protein Mamrak et al., 2016; Renaudin et al., 2016) (see poster). Thus, FA FANCD2: FA comp group D2 protein (also known as FACD, FANCD) proteins are crucial factors in the maintenance of genomic integrity; FANCE: FA comp groupE in fact, the FA pathway is the only known mechanism of repair for FANCF: FA comp group F ICLs that, if left unrepaired, promote genotoxic stress, genomic FANCG: FA comp group G instability and tumorigenesis. FANCI: FA comp group I protein FANCL: FA comp group L protein (also known as E3 ubiquitin-protein ligase FANCL) Other nuclear functions of the FA pathway in genome FANCM: FA comp group M protein maintenance and DDR MAD2L2: mitotic arrest-deficient-2-like 2 (also known as REV7, FANCV) In addition to its canonical role in ICL repair, accumulating PALB2: partner and localizer of BRCA2 (also known as FANCN) evidence indicates that subsets of FA proteins also participate in RAD51: RAD51 recombinase (also known as FANCR) additional pathways that are crucial to maintain genomic integrity RAD51C: RAD51 paralog C (also known as FANCO) (see poster) (reviewed in detail by Ceccaldi et al., 2016). For SLX4: structure-specific endonuclease subunit SLX4 (also known as FANCP) example, heterodimers of ubiquitylated FANCD2 and FANCI (and, UBE2T: ubiquitin-conjugating 2 T (also known as FANCT) additionally, the FA core complex and accessory proteins) localize XRCC2: X-ray repair cross-complementing 2 (also known as FANCU) to stalled replication forks (Lossaint et al., 2013), where they protect nascent (single-stranded) DNA from degradation by nucleases (Schlacher et al., 2011, 2012) and suppress inappropriate new origin firing and entry into mitosis (Chaudhury et al., 2013; Chen et al., defects are often associated with FA, e.g. skeletal and skin 2015). These functions prevent a destabilization or collapse of the abnormalities (Neveling et al., 2009). Although FA is a rare replication fork and, thus, any resulting genomic instability disease, inherited and somatic mutations or epigenetic silencing of (Ceccaldi et al., 2016). Members of the FA pathway are also FA genes are also found in many cancers. These include breast and required for the suppression of non-homologous end-joining ovarian cancers in patients with mutations of the inherited breast (Renaud et al., 2016), the correct segregation of cancer susceptibility gene 1 (BRCA1), or breast cancer during cytokinesis (Chan et al., 2009; Naim and Rosselli, 2009), as susceptibility gene 2 (BRCA2), as well as pancreatic, brain and well as for numerous other forms of non-ICL-directed DDR, other cancers that are associated with a loss-of-function of FA genes including nucleotide excision repair (Kelsall et al., 2012), (D’Andrea, 2010; Mamrak et al., 2016) (see Box 1). In this Cell translesion synthesis (Haynes et al., 2015), homologous Science at a Glance article and accompanying poster, we will briefly recombination (Haynes et al., 2015) and alternative end joining review the role of the FA pathway in DNA damage response (DDR) (Nguyen et al., 2014). Thus, FA pathway proteins participate in and then explore a recently described function of FA proteins in diverse non-canonical (i.e. non-ICL repair) roles in concert with selective autophagy and its implications for our understanding of the other pathways that are involved in genomic stability and DDR to pathophysiology of FA. ameliorate many forms of genotoxic stress.

Role of the FA pathway in DDR to of interstrand crosslinks Clues to a DDR-independent role for FANCC in cytoprotection The FA pathway is required for the DDR to interstrand crosslinks Although hypersensitivity to DNA damage is the best known (ICLs), highly toxic lesions in which adjacent bases on opposite phenotype of cells that are deficient in FA-gene function (Ceccaldi DNA strands are covalently linked (Mamrak et al., 2016; et al., 2016), it has long been appreciated that, at least some, Michl et al., 2016). It has been estimated that each human cell has genes of the FA pathway play additional cytoprotective roles, such to repair ∼10 ICLs/day (Grillari et al., 2007) and that as few as 20- as protection from cell death induced by ROS (Schindler and 40 unresolved ICL lesions can lead to cell death (Clauson et al., Hoehn, 1988), proinflammatory cytokines (Haneline et al., 1998; 2013). ICLs are generated during treatment with certain Whitney et al., 1996) or growth factor withdrawal (Cumming chemotherapeutic agents (e.g. nitrogen mustards, Mitomycin C, et al., 1996). Previous studies have offered some insight into or Cisplatin) (Lopez-Martinez et al., 2016). However, even in possible mechanisms underlying the cytoprotective effects of the absence of exogenous insults, endogenous sources, such as the FA protein FANCC. For example, FANCC-mediated reactive oxygen species (ROS) and their peroxidated intermediates, protection against proinflammatory cytokine-induced cell death as well as reactive aldehydes, induce- ICLs (Clauson et al., is correlated with its biochemical interactions with signal 2013; Lopez-Martinez et al., 2016), although their relative transducer and activator of transcription 1 (STAT1) (Pang et al., contributions to the burden of ICLs in a given cell population are 2000), protein kinase R (PKR; officially known as EIF2AK2) (Pang Journal of Cell Science

2658 CELL SCIENCE AT A GLANCE Journal of Cell Science (2017) 130, 2657-2662 doi:10.1242/jcs.204909 et al., 2002) and stress-inducible 70 FANCC is required for virophagy (HSPA1A) (Pang et al., 2002, 2001b). FANCC-mediated We first confirmed that FANCC is not required for non-selective protection against growth factor withdrawal-induced cell death is starvation-induced autophagy but is required for Sindbis virophagy associated with its interaction and activation of the xenobiotic- and in murine embryonic fibroblasts (MEFs) because the colocalization ROS-detoxifying enzyme glutathione S-transferase P1 (GSTP1) of mCherry-labeled Sindbis virus capsids and autophagosomes is (Cumming et al., 2001). reduced in Fancc−/− MEFs (Sumpter et al., 2016). We also found Some of the earliest work on FANCC indicated that at least some that virophagy of a herpes simplex virus type 1 (HSV-1) mutant that of its cytoprotective roles might be independent of its role in DDR. lacks a 20 amino acid region of the infected cell protein 34.5 For example, already in 1996, Yamashita et al. demonstrated that (ICP34.5) neurovirulence gene product − rendering it incapable of cells of FA patients that endogenously express the naturally binding to the essential autophagy protein Beclin 1 and inhibiting occurring FANCC mutant c.67delG (which results in the use of autophagy − is impaired in Fancc−/− MEFs. Ultrastructural analysis an alternative start codon and the deletion of 54 N-terminal amino of the cytoplasm of wild-type MEFs revealed that HSV-1 acids) were indistinguishable from FA patient cells that harbor a null nucleocapsids are primarily located within autolysosomes and in mutation in FANCC with respect to their hypersensitivity to the process of being degraded. In contrast, in Fancc−/− MEFs, intact mitomycin C (MMC)-induced DNA damage (Yamashita et al., HSV-1 nucleocapsids and enveloped virions can be found free in the 1996). However, patients that carry the c.67delG mutant have a cytoplasm, suggesting a failure of their targeting to autophagosomes/ milder clinical course than patients with null mutations in FANCC autolysosomes (see poster). Similar to other previously identified (i.e. mutations that abrogate both its DDR and cytoprotective roles), virophagy factors, such as sequestosome 1 (SQSTM1) (Orvedahl suggesting that FANCC has other disease-modulating functions that et al., 2010) and SMAD-specific E3 ubiquitin protein ligase 1 are independent of its role in DDR (Neveling et al., 2009). Indeed, (SMURF1) (Orvedahl et al., 2011), FANCC (as well as FANCA) in a previous study, the FANCC c.67delG mutant was shown to interacted biochemically with the Sindbis virus capsid protein. rescue interferon-γ (IFNγ)-induced STAT1 activation, which is Additionally, both FANCC and Sindbis virus capsid protein can be correlated with resistance to cell death induced by IFNγ and/or immunoprecipiated within LC3-positive membrane structures, which tumor necrosis factor-α (TNFα), to the same extent as wild-type are indicative of autophagosomes. FANCC (Pang et al., 2001a). Taken together, these in vitro results Importantly, Fancc−/− mice are more susceptible to death after and clinical observations hint at the existence of a clinically intracerebral inoculation with both the neuronotropic RNA virus important additional function or functions of FANCC beyond its (Sindbis) and the neuronotropic DNA virus (HSV-1) (Sumpter role in DDR. et al., 2016). This increased susceptibility to lethal CNS infection is associated with increased neuronal cell death (as measured by Identification of FA proteins as putative selective autophagy TUNEL staining; see poster) and increased viral antigen loads, but factors not with raised levels of infectious virus titers. These phenotypes in Autophagy is a phylogenetically conserved cellular housekeeping mice with deletion of a selective autophagy factor (FANCC) are pathway that plays pleiotropic roles in cellular and organismal consistent with those observed in previous studies of mice with homeostasis (Levine and Kroemer, 2008). In contrast to general neuronal deficiency of Atg5 − a core component of the autophagy (e.g. starvation-induced) autophagy, which is thought to be machinery (Orvedahl et al., 2010; Yordy et al., 2012). These results nonspecific, a specialized form of autophagy that has been termed suggest that the autophagy pathway exerts its pro-survival role selective autophagy, specifically targets unwanted cytoplasmic during viral infections of the CNS by removing excess viral protein contents (e.g. viruses, intracellular bacteria, damaged mitochondria products and preventing cell death due to proteotoxicity, i.e. damage and endoplasmic reticulum, lipid droplets, peroxisomes) for of cellular functions due to protein misfolding. engulfment by double-membraned vesicles (the so-called Taken together, the in vitro and in vivo studies with Sindbis virus autophagosomes) and then delivered to lysosomes for destruction and HSV-1 (see poster) indicate that FANCC is an adaptor protein (Khaminets et al., 2016). Although our understanding of the during virophagy that functions in antiviral host defense in the CNS mechanism(s) of selective autophagy has progressed significantly in (Sumpter et al., 2016). The precise mechanism by which FANCC recent years, much remains to be learned about this process before it and, potentially, additional FA proteins function to deliver viral can be harnessed for clinical use, for instance, to treat infectious cargoes for autophagic destruction remains to be elucidated, as do diseases, cancer and conditions related to aging. the precise mechanisms by which FANCC protects mice in vivo To this end, we performed a high-content, image-based, genome- against lethal neuronotropic viral disease. wide screen to identify host factors involved the selective autophagy of Sindbis virus (virophagy) (Orvedahl et al., 2011). This screen FANCC is required for mitophagy in a DDR-independent identified genes that blocked the colocalization of a red fluorescent manner Sindbis virus capsid protein with microtubule-associated protein 1 While by definition selective autophagy is associated with exquisite light chain 3 alpha (MAP1LC3A, also known as LC3) conjugated to cargo selectivity, there is also extensive evidence for shared green fluorescent protein (GFP-LC3), a marker of autophagosomes. mechanisms that are involved in the selective targeting of diverse To our surprise, three FA genes, FANCC, FANCF and FANCL that, intracellular cargoes (Khaminets et al., 2016). For example, the E3 like the other FA genes, had no known association with autophagy, ligase Parkin plays an important role in both the removal of were confirmed positives in the virophagy screen. We also found a damaged mitochondria (mitophagy) (Pickrell and Youle, 2015) and high degree of overlap between the positives from the virophagy the autophagic targeting of the intracellular bacterium screen and the genes that are required for another type of selective Mycobacterium tuberculosis (Manzanillo et al., 2013). Moreover, autophagy – Parkin-mediated autophagy of damaged mitochondria as noted above, there is extensive overlap between genes (including (a form of mitophagy) – suggesting a conservation of cellular factors FANCC, FANCF and FANCL) that are required for selective required for the selective autophagy of seemingly unrelated virophagy and mitophagy − another form of selective autophagy cytoplasmic cargoes. (Orvedahl et al., 2011). Journal of Cell Science

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The concept that FA proteins can function in mitophagy is formation, which is consistent with a model in which failure of attractive as a potential mechanism that contributes to the FANCC-mediated mitophagy during inflammasome activation cytoprotective functions of FANCC (discussed above) and a results in mtROS-driven hyperactivation of the inflammasome previously described role for FA proteins in mitochondrial quality and associated hypersecretion of IL-1β (see poster). control. Kumari et al. found that FA-deficient cells have a number of mitochondrial defects, including elevated levels of mitochondrial Additional FA proteins are required for mitophagy ROS, decreased mitochondrial membrane potential, decreased ATP The role for FA proteins in Parkin-mediated mitophagy seems to production, impaired oxygen uptake and abnormal mitochondrial extend to multiple family members. At least in siRNA knockdown morphology (Kumari et al., 2014), although the molecular basis of studies, the core complex proteins FANCF, FANCL and FANCA the latter phenotype has not been described yet. (the most commonly mutated protein in FA patients) are required, We confirmed a role for FANCC in mitophagy by using CRISPR/ along with FANCD2, BRCA1 and BRCA2, which have no known Cas9-mediated deletion in HeLa cells (see poster), FANCC mutant direct functions outside the nucleus (see poster) (Sumpter et al., fibroblasts of FA patients and bone marrow-derived macrophages 2016). Moreover, another group recently found that cells from (BMDMs) from Fancc-deficient mice (Sumpter et al., 2016). patients that carry FANCA (or FANCC) mutations have impaired Furthermore, we observed the accumulation of damaged levels of both basal and mitochondrial uncoupling agent-induced mitochondria in post-mitotic tissues (i.e. brain and heart) of aged mitophagy (Shyamsundar et al., 2016). Fancc−/− mice (see poster) (Sumpter et al., 2016), a finding that is Currently, it is impossible to conclude whether the different consistent with a defect in mitochondrial quality control in vivo.We FA proteins function directly in mitophagy or indirectly, i.e. as a result could also show that FANCC (as well as FANCA) interacted of other defined cellular functions, including the nuclear roles biochemically with the E3 Parkin and translocated discussed above. Although it conceptually more straightforward to to mitochondria (see poster) in a Parkin- and mitochondrial damage- postulate direct roles for FA proteins that are already known to have dependent manner (Sumpter et al., 2016). cytoplasmic functions, the possibility of direct roles for other FA The role of FANCC in Parkin-mediated mitophagy appears to be proteins without any known cytoplasmic functions warrants further genetically distinct from its role in DDR. Specifically, the c.67delG investigation. A mitochondrial localization of some of these proteins FANCC mutant, which − as mentioned above − is not functional in has been described, although, except for a role in mitophagy, no DDR but fully functional in protecting cells against cytokine- mitochondrial function is currently known. For example, FANCD2 induced death, can completely restore Parkin-mediated mitophagy that is not monoubiquitylated (and, therefore, not targeted by the FA in FANCC knockout cells (Sumpter et al., 2016). Although these core complex) is constitutively present in immunoprecipitated results strongly suggest that the DDR function of FANCC is mitochondria (Sumpter et al., 2016), and a fraction of BRCA1 is dispensable for Parkin-mediated mitophagy, further studies are located at the mitochondrial matrix (Coene et al., 2005). required to rule out other potential nuclear functions of FANCC in mitophagy. A general role for DDR pathways in mitochondrial quality control and mitophagy? Defective FANCC-mediated mitophagy and suppression of In recent years, evidence has mounted that defects in multiple non- mtROS results in aberrant inflammasome signaling FA DDR pathways result in defective mitophagy, leading to Damaged mitochondria produce mitochondrial reactive oxygen accumulation of damaged mitochondria and concomitant increased species (mtROS) that activate the NACHT, LRR and PYD domain- intracellular oxidative stress (Drake et al., 2017). These defects containing protein 3 (NLRP3) inflammasome pathway, resulting in include mutations in the ataxia-telangiectasia (A-T) double-strand the caspase-1-mediated cleavage of pro-IL-1β (pro-interleukin-1β) break repair pathway (Valentin-Vega et al., 2012), the Cockayne followed by secretion of the potent proinflammatory cytokine syndrome B transcription-coupled repair pathway (Scheibye- interleukin-1β (IL-1β) (Zhou et al., 2011), and mitophagy Knudsen et al., 2012) and the xeroderma pigmentosum group A downregulates inflammasome signaling by removing mtROS nucleotide excision repair pathway (Fang et al., 2014). Interestingly, (Zhong et al., 2016) (see poster). Given the emerging the A-T serine/threonine kinase (ATM) has also been shown to be interrelationships between mitophagy, mtROS and inflammasome required for pexophagy, the removal of peroxisomes (which are also activation, coupled with previous reports that link FANCC a source of intracellular ROS) by selective autophagy (Zhang et al., deficiency and increased mitochondrial ROS production (Kumari 2015). This suggests that, like FANCC, ATM is also required for the et al., 2014) or hyperactivation of inflammasome signaling (Garbati removal of diverse substrates by the selective autophagy pathway. et al., 2013), we sought to determine whether a failure to clear As with FA proteins, more studies are needed to determine the mtROS-producing damaged mitochondria is responsible for precise molecular mechanisms by which other non-FA, DDR enhanced inflammasome activation in FANCC-deficient cells. pathway proteins function in selective autophagy. Indeed, primary BMDMs from Fancc−/− mice, primed with the pathogen-associated molecular pattern (PAMP) lipopolysaccharide Physiological consequences of defects in mitophagy (a component of the cell wall of Gram-negative bacteria) and then A causal connection between mitophagy and tumor suppression is treated with extracellular ATP (to mimic the ‘danger’ signal from strongly supported by the observations that mitophagy-associated nearby dying cells) (de Zoete et al., 2014), results in increased proteins, including the E3 ubiquitin-protein ligase parkin (PARK2), mtROS generation, hypersecretion of IL-1β, and generation of more Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) and and larger apotosis-associated speck-like protein containing a Bcl-2/adenovirus E1B 19-kDa-interacting protein 3-like (BNIP3L) containing a caspase recruitment domain (PYCARD, hereafter have been linked with cancer (Chourasia et al., 2015). Pathogenic referred to as ASC) specks (i.e. markers of inflammasome assembly, alterations in mitochondrial homeostasis are thought to contribute to overlying damaged mitochondria; see poster) (Sumpter, 2016). In tumorigenesis through a variety of mechanisms. These include agreement, addition of MitoTEMPO, a mtROS-specific free radical changes in mitochondrial metabolic pathways to support tumor cell scavenger, dramatically reduces IL-1β secretion and ASC speck metabolism (metabolic reprogramming), as well as pro-tumor Journal of Cell Science

2660 CELL SCIENCE AT A GLANCE Journal of Cell Science (2017) 130, 2657-2662 doi:10.1242/jcs.204909 changes in cell signaling due to oxidative post-translational protein Cell science at a glance modifications (Vyas et al., 2016). Mitochondria-derived ROS also A high-resolution version of the poster and individual poster panels are available for directly or indirectly oxidize cellular macromolecules (e.g. proteins, downloading at http://jcs.biologists.org/lookup/doi/10.1242/jcs.204909.supplemental lipids, DNA), ultimately leading to increased genotoxic stress References (Drake et al., 2017; Vyas et al., 2016). Thus, a potential role for Auerbach, A. D. (2009). Fanconi anemia and its diagnosis. Mutat. Res. 668, 4-10. mitophagy in the pathogenesis of cancers in FA and other DDR- Boisvert, R. A. and Howlett, N. G. (2014). The Fanconi anemia ID2 complex: associated diseases associated with the accumulation of damaged dueling saxes at the crossroads. Cell Cycle 13, 2999-3015. mitochondria warrants further exploration. Brosh, R. M., Jr, Bellani, M., Liu, Y. and Seidman, M. M. (2017). Fanconi Anemia: a DNA repair disorder characterized by accelerated decline of the hematopoietic Aberrant IL-1β signaling, which is another consequence of stem cell compartment and other features of aging. Ageing Res. Rev. 33, 67-75. defective mitophagy (Zhou et al., 2011), has been implicated in a Ceccaldi, R., Sarangi, P. and D’Andrea, A. D. (2016). The Fanconi anaemia wide range of pathophysiologies, including autoinflammatory pathway: new players and new functions. Nat. Rev. Mol. Cell Biol. 17, 337-349. Chan, K. L., Palmai-Pallag, T., Ying, S. and Hickson, I. D. (2009). Replication diseases (Zhong et al., 2016), cancer (Zitvogel et al., 2012), stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat. Cell Biol. metabolic disorders (de Zoete et al., 2014) and senescence (Salama 11, 753-760. et al., 2014). Drugs targeting the inflammasome pathway are already Chaudhury, I., Sareen, A., Raghunandan, M. and Sobeck, A. (2013). FANCD2 used to treat some of these diseases (Shao et al., 2015). A crucial regulates BLM complex functions independently of FANCI to promote replication fork recovery. Nucleic Acids Res. 41, 6444-6459. unanswered question is to what extent aberrant inflammasome Chen, Y.-H., Jones, M. J. K., Yin, Y., Crist, S. B., Colnaghi, L., Sims, R. J., III, activation, in addition to genotoxic stress, contributes to bone Rothenberg, E., Jallepalli, P. V. and Huang, T. T. (2015). ATR-mediated marrow failure, cancer susceptibility and developmental phosphorylation of FANCI regulates dormant origin firing in response to replication − stress. Mol. Cell 58, 323-338. abnormalities all of which are observed in patients that carry Chourasia, A. H., Boland, M. L. and Macleod, K. F. (2015). Mitophagy and cancer. mutations of FANCC and other FA genes (Auerbach, 2009; Cancer Metab. 3,4. Ceccaldi et al., 2016; Mamrak et al., 2016; Neveling et al., 2009). Clauson, C., Scharer, O. D. and Niedernhofer, L. (2013). Advances in understanding the complex mechanisms of DNA interstrand cross-link repair. Cold Spring Harb. Perspect. Biol. 5, a012732. Conclusions Coene, E. D., Hollinshead, M. S., Waeytens, A. A., Schelfhout, V. R., Eechaute, W. P., Shaw, M. K., Van Oostveldt, P. M. and Vaux, D. J. (2005). Phosphorylated The FA pathway is crucial not only in DDR of ICLs but it also has BRCA1 is predominantly located in the nucleus and mitochondria. Mol. Biol. Cell emerging roles in other nuclear functions (stabilization of 16, 997-1010. replication forks, cytokinesis and other types of DNA repair), as Cumming, R. C., Lightfoot, J., Beard, K., Youssoufian, H., O’Brien, P. J. and Buchwald, M. (2001). Fanconi anemia group C protein prevents apoptosis in well as in the cytoplasmic process of selective autophagy. Although hematopoietic cells through redox regulation of GSTP1. Nat. Med. 7, 814-820. there are many open questions regarding the mechanism of action of Cumming, R. C., Liu, J. M., Youssoufian, H. and Buchwald, M. (1996). FA proteins in their ‘non-canonical’ roles, the exciting emerging Suppression of apoptosis in hematopoietic factor-dependent progenitor cell links between the FA pathway and selective autophagy might lines by expression of the FAC gene. Blood 88, 4558-4567. D’Andrea, A. D. (2010). Susceptibility pathways in Fanconi’s anemia and breast provide a roadmap to new and unexpected therapeutic targets in cancer. N. Engl. J. Med. 362, 1909-1919. order to treat FA and diseases associated with FA gene mutations. de Zoete, M. R., Palm, N. W., Zhu, S. and Flavell, R. A. (2014). Inflammasomes. An intriguing question is why the FA pathway would be involved in Cold Spring Harb. Perspect. Biol. 6, a016287. Drake, L. E., Springer, M. Z., Poole, L. P., Kim, C. J. and Macleod, K. F. (2017). selective autophagy in addition to its role in DDR. We speculate this Expanding perspectives on the significance of mitophagy in cancer. Semin. pathway is involved because ROS can inflict the type of DNA lesion Cancer Biol. doi:10.1016/j.semcancer.2017.04.008 [Epub ahead of print]. (ICL) the FA pathway is required to repair; moreover, it acts at two Fang, E. F., Scheibye-Knudsen, M., Brace, L. E., Kassahun, H., SenGupta, T., crucial stages to protect the cell against genotoxic stress. By helping Nilsen, H., Mitchell, J. R., Croteau, D. L. and Bohr, V. A. (2014). Defective mitophagy in XPA via PARP-1 hyperactivation and NAD(+)/SIRT1 reduction. Cell to remove the main endogenous source of ROS (mtROS from 157, 882-896. damaged mitochondria), members of the FA pathway minimize the Garaycoechea, J. I. and Patel, K. J. (2014). Why does the bone marrow fail in generation of nuclear ICLs. As well as deficiencies in the direct FA Fanconi anemia? Blood 123, 26-34. Garbati, M. R., Hays, L. E., Keeble, W., Yates, J. E., Rathbun, R. K. and Bagby, DNA repair pathway, the failure to suppress extensive genotoxic G. C. (2013). FANCA and FANCC modulate TLR and p38 MAPK-dependent stress emanating from the cytoplasm by FA proteins might be expression of IL-1beta in macrophages. Blood 122, 3197-3205. important in the pathophysiologies associated with FA, such as Grillari, J., Katinger, H. and Voglauer, R. (2007). Contributions of DNA interstrand cancer and accelerated aging (Brosh et al., 2017; Drake et al., 2017). cross-links to aging of cells and organisms. Nucleic Acids Res. 35, 7566-7576. Haneline, L. S., Broxmeyer, H. E., Cooper, S., Hangoc, G., Carreau, M., More broadly, it is possible that other types of DNA damage linked Buchwald, M. and Clapp, D. W. (1998). Multiple inhibitory cytokines induce to ROS are also modulated by mitophagy, thus providing a common deregulated progenitor growth and apoptosis in hematopoietic cells from Fac-/- selective pressure for different DNA damage pathways to function mice. Blood 91, 4092-4098. Haynes, B., Saadat, N., Myung, B. and Shekhar, M. P. V. (2015). Crosstalk together in selective autophagy (Drake et al., 2017). between translesion synthesis, Fanconi anemia network, and repair pathways in interstrand DNA crosslink repair and Acknowledgements development of chemoresistance. Mutat. Res. Rev. Mutat. Res. 763, 258-266. We are grateful to Angela Diehl for assistance with the graphic design and Kelsall, I. R., Langenick, J., MacKay, C., Patel, K. J. and Alpi, A. F. (2012). The illustrations of the poster. Fanconi anaemia components UBE2T and FANCM are functionally linked to nucleotide excision repair. PLoS ONE 7, e36970. Competing interests Khaminets, A., Behl, C. and Dikic, I. (2016). Ubiquitin-dependent and independent The authors declare no competing or financial interests. signals in selective autophagy. Trends Cell Biol. 26, 6-16. Kim, H. and D’Andrea, A. D. (2012). Regulation of DNA cross-link repair by the Fanconi anemia/BRCA pathway. Genes Dev. 26, 1393-1408. Funding Kumari, U., Ya Jun, W., Huat Bay, B. and Lyakhovich, A. (2014). Evidence of ’ Work in B.L. s laboratory was supported by the National Institutes of Health (grant mitochondrial dysfunction and impaired ROS detoxifying machinery in Fanconi numbers: K08 AI099150 to R.S., U19 AI109725 to B.L., RO1 CA109618 to B.L.), the anemia cells. Oncogene 33, 165-172. Burroughs Wellcome Fund (Career Award for Medical Scientists to R.S.), the Levine, B. and Kroemer, G. (2008). Autophagy in the pathogenesis of disease. Cell University of Texas Southwestern Medical Center President’s Research Council 132, 27-42. (Distinguished Researcher Award to R.S.), and the Cancer Prevention and Lopez-Martinez, D., Liang, C.-C. and Cohn, M. A. (2016). Cellular response to Research Institute of Texas (grant number: RP120718 to B.L.). Deposited in PMC DNA interstrand crosslinks: the Fanconi anemia pathway. Cell. Mol. Life Sci. 73, for release after 12 months. 3097-3114. Journal of Cell Science

2661 CELL SCIENCE AT A GLANCE Journal of Cell Science (2017) 130, 2657-2662 doi:10.1242/jcs.204909

Lossaint, G., Larroque, M., Ribeyre, C., Bec, N., Larroque, C., Décaillet, C., Gari, Salama, R., Sadaie, M., Hoare, M. and Narita, M. (2014). Cellular senescence and K. and Constantinou, A. (2013). FANCD2 binds MCM proteins and controls its effector programs. Genes Dev. 28, 99-114. replisome function upon activation of s phase checkpoint signaling. Mol. Cell 51, Scheibye-Knudsen, M., Ramamoorthy, M., Sykora, P., Maynard, S., Lin, P.-C., 678-690. Minor, R. K., Wilson, D. M., III, Cooper, M., Spencer, R., de Cabo, R. et al. Mamrak, N. E., Shimamura, A. and Howlett, N. G. (2016). Recent discoveries in (2012). Cockayne syndrome group B protein prevents the accumulation of the molecular pathogenesis of the inherited bone marrow failure syndrome damaged mitochondria by promoting mitochondrial autophagy. J. Exp. Med. 209, Fanconi anemia. Blood Rev. 3, 93-99. 855-869. Manzanillo, P. S., Ayres, J. S., Watson, R. O., Collins, A. C., Souza, G., Rae, Schindler, D. and Hoehn, H. (1988). Fanconi anemia mutation causes cellular C. S., Schneider, D. S., Nakamura, K., Shiloh, M. U. and Cox, J. S. (2013). The susceptibility to ambient oxygen. Am. J. Hum. Genet. 43, 429-435. ubiquitin ligase parkin mediates resistance to intracellular pathogens. Nature 501, Schlacher, K., Christ, N., Siaud, N., Egashira, A., Wu, H. and Jasin, M. (2011). 512-516. Double-strand break repair-independent role for BRCA2 in blocking stalled Michl, J., Zimmer, J. and Tarsounas, M. (2016). Interplay between Fanconi replication fork degradation by MRE11. Cell 145, 529-542. anemia and homologous recombination pathways in genome integrity. EMBO J. Schlacher, K., Wu, H. and Jasin, M. (2012). A distinct replication fork protection 35, 909-923. pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2. Naim, V. and Rosselli, F. (2009). The FANC pathway and BLM collaborate during Cancer Cell 22, 106-116. mitosis to prevent micro-nucleation and chromosome abnormalities. Nat. Cell Biol. Shao, B. Z., Xu, Z. Q., Han, B. Z., Su, D. F. and Liu, C. (2015). NLRP3 11, 761-768. inflammasome and its inhibitors: a review. Front. Pharmacol. 6, 262. Neveling, K., Endt, D., Hoehn, H. and Schindler, D. (2009). Genotype-phenotype Shyamsunder, P., Esner, M., Barvalia, M., Wu, Y. J., Loja, T., Boon, H. B., correlations in Fanconi anemia. Mutat. Res. 668, 73-91. Lleonart, M. E., Verma, R. S., Krejci, L. and Lyakhovich, A. (2016). Impaired Nguyen, T. V., Riou, L., Aoufouchi, S. and Rosselli, F. (2014). Fanca deficiency mitophagy in Fanconi anemia is dependent on mitochondrial fission. Oncotarget reduces A/T transitions in somatic hypermutation and alters class switch 7, 58065-58074. recombination junctions in mouse B cells. J. Exp. Med. 211, 1011-1018. Sumpter, R., Jr, Sirasanagandla, S., Fernández, A. F., Wei, Y., Dong, X., Franco, Orvedahl, A., MacPherson, S., Sumpter, R., Jr, Tallóczy, Z., Zou, Z. and Levine, L., Zou, Z., Marchal, C., Lee, M. Y., Clapp, D. W. et al. (2016). Fanconi anemia B. (2010). Autophagy protects against Sindbis virus infection of the central proteins function in mitophagy and immunity. Cell 165, 867-881. nervous system. Cell Host Microbe 7, 115-127. Valentin-Vega, Y. A., Maclean, K. H., Tait-Mulder, J., Milasta, S., Steeves, M., Orvedahl, A., Sumpter, R., Jr, Xiao, G., Ng, A., Zou, Z., Tang, Y., Narimatsu, M., Dorsey, F. C., Cleveland, J. L., Green, D. R. and Kastan, M. B. (2012). Gilpin, C., Sun, Q., Roth, M. et al. (2011). Image-based genome-wide siRNA Mitochondrial dysfunction in ataxia-telangiectasia. Blood 119, 1490-1500. screen identifies selective autophagy factors. Nature 480, 113-117. Vyas, S., Zaganjor, E. and Haigis, M. C. (2016). Mitochondria and cancer. Cell 166, Pang, Q., Fagerlie, S., Christianson, T. A., Keeble, W., Faulkner, G., Diaz, J., 555-566. Rathbun, R. K. and Bagby, G. C. (2000). The Fanconi anemia protein FANCC Whitney, M. A., Royle, G., Low, M. J., Kelly, M. A., Axthelm, M. K., Reifsteck, C., binds to and facilitates the activation of STAT1 by gamma interferon and Olson, S., Braun, R. E., Heinrich, M. C., Rathbun, R. K. et al. (1996). Germ cell hematopoietic growth factors. Mol. Cell. Biol. 20, 4724-4735. Pang, Q., Christianson, T. A., Keeble, W., Diaz, J., Faulkner, G. R., Reifsteck, C., defects and hematopoietic hypersensitivity to gamma-interferon in mice with a Olson, S. and Bagby, G. C. (2001a). The Fanconi anemia complementation targeted disruption of the Fanconi anemia C gene. Blood 88, 49-58. group C gene product: structural evidence of multifunctionality. Blood 98, Yamashita, T., Wu, N., Kupfer, G., Corless, C., Joenje, H., Grompe, M. and ’ 1392-1401. D Andrea, A. D. (1996). Clinical variability of Fanconi anemia (type C) results from Pang, Q., Keeble, W., Christianson, T. A., Faulkner, G. R. and Bagby, G. C. expression of an amino terminal truncated Fanconi anemia complementation (2001b). FANCC interacts with to protect hematopoietic cells from IFN- group C polypeptide with partial activity. Blood 87, 4424-4432. gamma/TNF-alpha-mediated cytotoxicity. EMBO J. 20, 4478-4489. Yordy, B., Iijima, N., Huttner, A., Leib, D. and Iwasaki, A. (2012). A neuron- Pang, Q., Christianson, T. A., Keeble, W., Koretsky, T. and Bagby, G. C. (2002). specific role for autophagy in antiviral defense against herpes simplex virus. Cell The anti-apoptotic function of Hsp70 in the interferon-inducible double-stranded Host Microbe 12, 334-345. RNA-dependent protein kinase-mediated death signaling pathway requires the Zhang, J., Tripathi, D. N., Jing, J., Alexander, A., Kim, J., Powell, R. T., Dere, R., Fanconi anemia protein, FANCC. J. Biol. Chem. 277, 49638-49643. Tait-Mulder, J., Lee, J.-H., Paull, T. T. et al. (2015). ATM functions at the Pickrell, A. M. and Youle, R. J. (2015). The roles of PINK1, parkin, and peroxisome to induce pexophagy in response to ROS. Nat. Cell Biol. 17, mitochondrial fidelity in Parkinson’s disease. Neuron 85, 257-273. 1259-1269. Renaud, E., Barascu, A. and Rosselli, F. (2016). Impaired TIP60-mediated H4K16 Zhong, Z., Sanchez-Lopez, E. and Karin, M. (2016). Autophagy, NLRP3 acetylation accounts for the aberrant chromatin accumulation of 53BP1 and inflammasome and auto-inflammatory/immune diseases. Clin. Exp. Rheumatol. RAP80 in Fanconi anemia pathway-deficient cells. Nucleic Acids Res. 44, 34, 12-16. 648-656. Zhou, R., Yazdi, A. S., Menu, P. and Tschopp, J. (2011). A role for mitochondria in Renaudin, X., Koch Lerner, L., Menck, C. F. M. and Rosselli, F. (2016). The NLRP3 inflammasome activation. Nature 469, 221-225. ubiquitin family meets the Fanconi anemia proteins. Mutat. Res. Rev. Mutat. Res. Zitvogel, L., Kepp, O., Galluzzi, L. and Kroemer, G. (2012). Inflammasomes in 769, 36-46. carcinogenesis and anticancer immune responses. Nat. Immunol. 13, 343-351. Journal of Cell Science

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