FANCD2 Monoubiquitination Research Articles

Patient-derived C-terminal mutation of FANCI causes protein mislocalization and reveals putative EDGE motif function in DNA repair

AbstractFanconi anemia (FA) is a rare familial genome instability syndrome caused by mutations in FA genes that results in defective DNA crosslink repair. Activation of the FA pathway requires the FA core ubiquitin ligase complex-dependent monoubiquitination of 2 interacting FA proteins, FANCI and FANCD2. Although loss of either FANCI or FANCD2 is known to prevent monoubiquitination of its respective partner, it is unclear whether FANCI has any additional domains that may be important in promoting DNA repair, independent of its monoubiquitination. Here, we focus on an FA-I patient-derived FANCI mutant protein, R1299X (deletion of 30 residues from its C-terminus), to characterize important structural region(s) in FANCI that is required to activate the FA pathway. We show that, within this short 30 amino acid stretch contains 2 separable functional signatures, a nuclear localization signal and a putative EDGE motif, that is critical for the ability of FANCI to properly monoubiquitinate FANCD2 and promote DNA crosslink resistance. Our study enable us to conclude that, although proper nuclear localization of FANCI is crucial for robust FANCD2 monoubiquitination, the putative FANCI EDGE motif is important for DNA crosslink repair.

Bcr/Abl Interferes with the Fanconi Anemia/BRCA Pathway: Implications in the Chromosomal Instability of Chronic Myeloid Leukemia Cells

Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic system caused by the expression of the BCR/ABL fusion oncogene. Although it is well known that CML cells are genetically unstable, the mechanisms accounting for this genomic instability are still poorly understood. Because the Fanconi anemia (FA) pathway is believed to control several mechanisms of DNA repair, we investigated whether this pathway was disrupted in CML cells. Our data show that CML cells have a defective capacity to generate FANCD2 nuclear foci, either in dividing cells or after DNA damage. Similarly, human cord blood CD34+ cells transduced with BCR/ABL retroviral vectors showed impaired FANCD2 foci formation, whereas FANCD2 monoubiquitination in these cells was unaffected. Soon after the transduction of CD34+ cells with BCR/ABL retroviral vectors a high proportion of cells with supernumerary centrosomes was observed. Similarly, BCR/ABL induced a high proportion of chromosomal abnormalities, while mediated a cell survival advantage after exposure to DNA cross-linking agents. Significantly, both the impaired formation of FANCD2 nuclear foci, and also the predisposition of BCR/ABL cells to develop centrosomal and chromosomal aberrations were reverted by the ectopic expression of BRCA1. Taken together, our data show for the first time a disruption of the FA/BRCA pathway in BCR/ABL cells, suggesting that this defective pathway should play an important role in the genomic instability of CML by the co-occurrence of centrosomal amplification and DNA repair deficiencies.

Genomic Instability in Mice Is Greater in Fanconi Anemia Caused by Deficiency of Fancd2 than Fancg

Fanconi anemia (FA) results from mutations in the FANC genes and is characterized by bone marrow failure, birth defects, and a high incidence of cancer. FANCG is a part of the FA core complex that is responsible for monoubiquitination of FANCD2 and FANCI. The precise role of the FA pathway is not well understood, although it may be involved in homologous recombination (HR), nonhomologous end joining, and translesion synthesis (TLS). Fancd2(-/-) mice have a more severe phenotype than Fancg(-/-), and other FA core complex-deficient mice, although both Fancg and Fancd2 belong to the same FA pathway. We hypothesized that Fancd2 deficiency results in a more severe phenotype because Fancd2 also has a FA pathway-independent function in the maintenance of genomic integrity. To test this hypothesis, we determined the level of DNA damage and genomic instability in Fancd2(-/-), Fancg(-/-), and wild-type controls. Fancd2(-/-) mice displayed a higher magnitude of chromosomal breakage and micronucleus formation than the wild-type or Fancg(-/-) mice. Also, DNA strand breaks were increased in Fancd2(-/-) but not in Fancg(-/-) mice. In addition, Fancd2(-/-) mice displayed an elevated frequency of DNA deletions, resulting from HR at the endogenous p(un) locus. In contrast, in Fancg(-/-) mice, the frequency of DNA deletions was decreased. Thus, Fancd2 but not Fancg deficiency results in elevated chromosomal/DNA breakage and permanent genome rearrangements. This provides evidence that Fancd2 plays an additional role in the maintenance of genomic stability than Fancg, which might explain the higher predisposition to cancer seen in the Fancd2(-/-) mice.

Selected Resistance to Topoisomerase II Inhibitors Correlates with the Over Expression of FANCF In a Fanconi Anemia/BRCA DNA Repair Pathway Independent Manner.

AbstractAbstract 3372Enhanced expression of the Fanconi Anemia (FA)/BRCA DNA repair pathway correlates with melphalan-resistance in multiple myeloma (MM) cell lines. Continued investigation demonstrated a bortezomib sensitive RelB/p50-mediated regulation of the FA/BRCA pathway contributed to the observed melphalan resistance.(Yarde et al 2009) The FA/BRCA pathway represents a co-dependent DNA damage response pathway involving thirteen loss of function complementation groups cloned from FA patients. The key functional event of this pathway is the interdependent mono-ubiquitination (Ub) of FANCD2 and FANCI (ID complex) by the E3 Ub-ligase activity of the FA core complex a multimer consisting of 8 FA (FANCA, B, C, E, F, G, L and M) and three non-FA proteins (FAAP100, FAAP24 and HES1). Formation of the core complex and mono-Ub of the ID complex appears to revolve around the flexible adapter protein FANCF. Nuclear localization of the core complex components requires binding of FANCA/G and FANCC/E subcomplexes to the C- terminal domain (CTD) and NTD domains of FANCF, respectively. This complex associates with FANCM:FAAP24 at sites of interstand crosslinks (ICL) via the FANCM-binding domain of FANCF, culminating in ID complex mono-Ub, recruitment of BRCA1, BRCA2/FANCD1, FANCJ and FANCN, and homologous recombination (HR) repair. Reduced function of this pathway has been associated with increased genomic instability, cancer susceptibility, and increased sensitivity to DNA cross-linking agents in FA. However, as predicted by the role of the FA/BRCA pathway in DNA repair, enhanced expression of the FA/BRCA pathway has been shown to play an important role in resistance to agents requiring HR for ICL repair. We next examined expression of this pathway in models of resistance to DNA damaging agents not predicted to utilize FA/BRCA activity. We screened 8226/Dox40 doxorubicin resistant and 8226/MR20 mitoxantrone resistant MM cell lines for expression of the 12 FA/BRCA pathway members with quantitative PCR (qPCR) using customized micro-fluidic cards. Interestingly, in these models of topoisomerase (topo) II inhibitor resistance qPCR demonstrated a 2.6 (p<0.05) and 1.7 (p<0.05) fold over expression of FANCF mRNA relative to drug sensitive RPMI8226 cells. Importantly, mRNA expression of other the eleven FA/BRCA pathway constituents was not increased relative to sensitive cells. To further characterize the relationship between FANCF and doxorubicin resistance, we examined mRNA and protein expression of FANCF in RMPI8226, 8226/Dox6 and 8226/Dox40 MM cell lines (representing progressive levels of doxorubicin resistance). FANCF qPCR demonstrated a 2 and 4.7 fold increased in mRNA expression in the 8226/Dox6 and 8226/Dox40 cell lines, respectively (p= 0.103 and p= 0.034) suggesting that increasing expression of FANCF correlated with increasing dox resistance. A similar doxorubicin resistance- dependent increase in FANCF protein was demonstrated by Western blot analysis of these cell lines. Consistent with mRNA results, FANCD2 or FANCG protein levels remained unchanged in the doxorubicin resistant versus sensitive cell lines These observations suggest that FANCF may contribute to topoII inhibitor-mediated DNA double strand break repair, a process that primarily thought to involve non-homologous end joining (NHEJ) independent of the FA/BRCA pathway. To determine if FANCF expression alone could facilitate doxorubicin resistance, pQCXIP-control or pQCXIP-FANCF constructs were expressed in RPMI8226 sensitive MM cells. MTT assays demonstrated a greater than 2 fold resistance to doxorubicin in FANCF over expressing cells at 48 and 96 hours (IC50: 1.33 ×10−6 and 5.3×10−9M) as compared to control cells (3.26×10−6 and 1.13×10−8M). Taken together, these results indicate that the flexible adaptor protein FANCF may participate in doxorubicin resistance independently of other FA/BRCA members. However, future studies will be needed to elucidate the nature of FANCF in doxorubicin resistance. Disclosures:No relevant conflicts of interest to declare.

Cytoplasmic FANCA-FANCC Complex Interacts and Stabilizes the Cytoplasm-dislocalized Leukemic Nucleophosmin Protein (NPMc)

Eight of the Fanconi anemia (FA) proteins form a core complex that activates the FA pathway. Some core complex components also form subcomplexes for yet-to-be-elucidated functions. Here, we have analyzed the interaction between a cytoplasmic FA subcomplex and the leukemic nucleophosmin (NPMc). Exogenous NPMc was degraded rapidly in FA acute myeloid leukemia bone marrow cells. Knockdown of FANCA or FANCC in leukemic OCI/AML3 cells induced rapid degradation of endogenous NPMc. NPMc degradation was mediated by the ubiquitin-proteasome pathway involving the IBR-type RING-finger E3 ubiquitin ligase IBRDC2, and genetic correction of FA-A or FA-C lymphoblasts prevented NPMc ubiquitination. Moreover, cytoplasmic FANCA and FANCC formed a cytoplasmic complex and interacted with NPMc. Using a patient-derived FANCC mutant and a nuclearized FANCC, we demonstrated that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability. Finally, depletion of FANCA and FANCC in NPMc-positive leukemic cells significantly increased inflammation and chemoresistance through NF-κB activation. Our findings not only reveal the molecular mechanism involving cytoplasmic retention of NPMc but also suggest cytoplasmic function of FANCA and FANCC in NPMc-related leukemogenesis.

Convergence of Rad6/Rad18 and Fanconi Anemia Tumor Suppressor Pathways upon DNA Damage

Extremely high cancer incidence associated with patients with Fanconi anemia (FA) suggests the importance of the FA signaling pathway in the suppression of non-FA human tumor development. Indeed, we found that an impaired FA signaling pathway substantially contributes to the development of non-FA human tumors. However, the mechanisms underlying the function of the FA pathway remain less understood. Using RNA interfering approach in combining with cell proliferation and reporter assays, we showed that the function of FA signaling pathway is at least partly mediated through coupling with hRad6/hRad18 signaling (HHR6 pathway). We previously reported that FANCD2 monoubiquitination, a hallmark of the FA pathway activation, can be regulated by HHR6. Here we found that hRad18 can also regulate activation of the FA pathway. More importantly, we found that FANCD2 is capable of modulating activity of DNA translesion synthesis polymerase eta, an effector of HHR6 pathway. These results provide novel insights into how the FA pathway is intertwined with HHR6 pathway to maintain chromosomal stability and suppress the development of human cancer, representing an important conceptual advance in the field of FA cancer research.

Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.

A Genetic Screen Identifies FAN1, a Fanconi Anemia-Associated Nuclease Necessary for DNA Interstrand Crosslink Repair

The Fanconi anemia (FA) pathway is responsible for interstrand crosslink repair. At the heart of this pathway is the FANCI-FAND2 (ID) complex, which, upon ubiquitination by the FA core complex, travels to sites of damage to coordinate repair that includes nucleolytic modification of the DNA surrounding the lesion and translesion synthesis. How the ID complex regulates these events is unknown. Here we describe a shRNA screen that led to the identification of two nucleases necessary for crosslink repair, FAN1 (KIAA1018) and EXDL2. FAN1 colocalizes at sites of DNA damage with the ID complex in a manner dependent on FAN1's ubiquitin-binding domain (UBZ), the ID complex, and monoubiquitination of FANCD2. FAN1 possesses intrinsic 5'-3' exonuclease activity and endonuclease activity that cleaves nicked and branched structures. We propose that FAN1 is a repair nuclease that is recruited to sites of crosslink damage in part through binding the ubiquitinated ID complex through its UBZ domain.

Identification of KIAA1018/FAN1, a DNA Repair Nuclease Recruited to DNA Damage by Monoubiquitinated FANCD2

DNA interstrand crosslinks (ICLs) are highly toxic because they block the progression of replisomes. The Fanconi Anemia (FA) proteins, encoded by genes that are mutated in FA, are important for repair of ICLs. The FA core complex catalyzes the monoubiquitination of FANCD2, and this event is essential for several steps of ICL repair. However, how monoubiquitination of FANCD2 promotes ICL repair at the molecular level is unknown. Here, we describe a highly conserved protein, KIAA1018/MTMR15/FAN1, that interacts with, and is recruited to sites of DNA damage by, the monoubiquitinated form of FANCD2. FAN1 exhibits endonuclease activity toward 5' flaps and has 5' exonuclease activity, and these activities are mediated by an ancient VRR_nuc domain. Depletion of FAN1 from human cells causes hypersensitivity to ICLs, defects in ICL repair, and genome instability. These data at least partly explain how ubiquitination of FANCD2 promotes DNA repair.

91 Analysis of BRIP1 in italian male breast cancer patients

The Fanconi anemia (FA) molecular network consists of 15 “FANC” proteins, of which 13 are associated with mutations in patients with this cancer-prone chromosome instability disorder. Whereas historically the common phenotype associated with FA mutations is marked sensitivity to DNA interstrand crosslinking agents, the literature supports a more global role for FANC proteins in coping with diverse stresses encountered by replicative polymerases. We have attempted to reconcile and integrate numerous observations into a model in which FANC proteins coordinate the following physiological events during DNA crosslink repair: (a) activating a FANCM–ATR-dependent S-phase checkpoint, (b) mediating enzymatic replication-fork breakage and crosslink unhooking, (c) filling the resulting gap by translesion synthesis (TLS) by error-prone polymerase(s), and (d) restoring the resulting one-ended double-strand break by homologous recombination repair (HRR). The FANC core subcomplex (FANCA, B, C, E, F, G, L, FAAP100) promotes TLS for both crosslink and non-crosslink damage such as spontaneous oxidative base damage, UV-C photoproducts, and alkylated bases. TLS likely helps prevent stalled replication forks from breaking, thereby maintaining chromosome continuity. Diverse DNA damages and replication inhibitors result in monoubiquitination of the FANCD2–FANCI complex by the FANCL ubiquitin ligase activity of the core subcomplex upon its recruitment to chromatin by the FANCM–FAAP24 heterodimeric translocase. We speculate that this translocase activity acts as the primary damage sensor and helps remodel blocked replication forks to facilitate checkpoint activation and repair. Monoubiquitination of FANCD2–FANCI is needed for promoting HRR, in which the FANCD1/BRCA2 and FANCN/PALB2 proteins act at an early step. We conclude that the core subcomplex is required for both TLS and HRR occurring separately for non-crosslink damages and for both events during crosslink repair. The FANCJ/BRIP1/BACH1 helicase functions in association with BRCA1 and may remove structural barriers to replication, such as guanine quadruplex structures, and/or assist in crosslink unhooking.

Genetic inactivation of the Fanconi anemia gene FANCC identified in the hepatocellular carcinoma cell line HuH-7 confers sensitivity towards DNA-interstrand crosslinking agents

BackgroundInactivation of the Fanconi anemia (FA) pathway through defects in one of 13 FA genes occurs at low frequency in various solid cancer entities among the general population. As FA pathway inactivation confers a distinct hypersensitivity towards DNA interstrand-crosslinking (ICL)-agents, FA defects represent rational targets for individualized therapeutic strategies. Except for pancreatic cancer, however, the prevalence of FA defects in gastrointestinal (GI) tumors has not yet been systematically explored.ResultsA panel of GI cancer cell lines was screened for FA pathway inactivation applying FANCD2 monoubiquitination and FANCD2/RAD51 nuclear focus formation and a newly identified FA pathway-deficient cell line was functionally characterized. The hepatocellular carcinoma (HCC) line HuH-7 was defective in FANCD2 monoubiquitination and FANCD2 nuclear focus formation but proficient in RAD51 focus formation. Gene complementation studies revealed that this proximal FA pathway inactivation was attributable to defective FANCC function in HuH-7 cells. Accordingly, a homozygous inactivating FANCC nonsense mutation (c.553C > T, p.R185X) was identified in HuH-7, resulting in partial transcriptional skipping of exon 6 and leading to the classic cellular FA hypersensitivity phenotype; HuH-7 cells exhibited a strongly reduced proliferation rate and a pronounced G2 cell cycle arrest at distinctly lower concentrations of ICL-agents than a panel of non-isogenic, FA pathway-proficient HCC cell lines. Upon retroviral transduction of HuH-7 cells with FANCC cDNA, FA pathway functions were restored and ICL-hypersensitivity abrogated. Analyses of 18 surgical HCC specimens yielded no further examples for genetic or epigenetic inactivation of FANCC, FANCF, or FANCG in HCC, suggesting a low prevalence of proximal FA pathway inactivation in this tumor type.ConclusionsAs the majority of HCC are chemoresistant, assessment of FA pathway function in HCC could identify small subpopulations of patients expected to predictably benefit from individualized treatment protocols using ICL-agents.

Abstract LB-102: The p21Cip1/Waf1 cyclin-dependent kinase inhibitor is required for the activation of the FA-BRCA pathway

Abstract Fanconi anemia (FA) is a rare recessive disorder characterized in part by pronounced cancer susceptibility. The FA proteins, and the protein products of the major breast cancer susceptibility genes BRCA1 and BRCA2, function cooperatively in the FA-BRCA pathway, to repair damaged DNA and to prevent cellular transformation. A major step in the activation of the FA-BRCA pathway is the mono-ubiquitination of the FANCD2 and FANCI proteins, targeting these proteins to discrete chromatin-associated nuclear foci. Importantly, the regulation of FANCD2 (and FANCI) mono-ubiquitination remains poorly understood. To gain further insight into this important post-translational modification step, here we have examined the roles of p53 tumor suppressor as well its downstream target p21Cip1/Waf1 in the regulation of the mono-ubiquitination of FANCD2. In this study we have used the isogenic HCT116 p53+/+, p53−/−, p21+/+, and p21−/− cell lines, to determine that p21, and not p53, plays a key role in the regulation of the mono-ubiquitination of FANCD2 following exposure to DNA damaging agents. For example, robust FANCD2 mono-ubiquitination is observed in HCT116 p21+/+ cells 2 h following exposure to 20 J/m2 UV irradiation, while this effect is markedly attenuated in p21−/− cells. Using immunofluorescence microscopy we also demonstrate that p21 is necessary for the efficient recruitment of FANCD2 to nuclear foci following exposure to UV irradiation. Furthermore, preliminary protein-protein interaction analyses in COS-7 cells demonstrate that FLAG-p21 and 6xHis/V5-FANCD2 physically interact. Interestingly, despite a failure to efficiently activate the mono-ubiquitination of FANCD2 following exposure to DNA damaging agents, p21−/− cells are not hypersensitive to the cytotoxic effects of the DNA crosslinking agent mitomycin C (MMC). However, elevated levels of distinct chromosome aberrations, including telomere associations and dicentric chromosomes, are observed in p21−/− cells compared with p21+/+ cells following exposure to MMC. Taken together, our results demonstrate an important upstream role for the p21 cyclin-dependent-kinase inhibitor in the activation of the FA-BRCA pathway. A greater understanding of the regulation of this important tumor suppressor pathway will lead to improved diagnostic and therapeutic approaches to FA and enhance our understanding of the elevated cancer susceptibility of FA patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-102.

Abstract SY28-01: Targeting DNA repair pathways in cancer therapy

Abstract SY28-01: Targeting DNA repair pathways in cancer therapy

Abstract 1318: Proteasome and Fanconi Anemia/BRCA pathways as prognostic determinants of glioblastoma response to radiation and temozolomide: a proposed clinical trial

Abstract OBJECTIVE: Despite the standard regimen of maximal resection followed by concurrent ionizing radiation (IR) and temozolomide (TMZ), long-term survivors of patients afflicted with glioblastoma (GBM) remain rare. We sought to identify molecular determinants of resistance to these agents using a combined functional genomic and bioinformatic approach. METHODS: Parallel RNAi screens were performed to identify gene silencings that modulate IR and TMZ resistance. A viability-based screen was performed in multiple glioma lines with the Hannon-Elledge library of 74,705 short hairpin RNAs targeting nearly all human genes. A focused screen was also performed to identify modulators of the Fanconi Anemia (FA)/BRCA pathway, a pathway we previously identified as a determinant of IR and TMZ resistance. The most promising candidates were confirmed by independent RNAi and pharmacologic inhibition in vitro and in vivo xenografts. Correlations between relative expression levels of the candidate genes and patient survival were made using data from seven series of clinically annotated GBM gene expression profiles. RESULTS: PSMA1, a core subunit of the 20S proteasome, was a top hit in both screens. Proteasome inhibition by either RNAi or pharmacologic inhibition sensitized GBM cell lines to IR and TMZ in both in vitro and in vivo xenograft models. This inhibition also resulted in decreased FANCD2 monoubiquitination, a marker for FA pathway activation. Using our collated set of 531 patients, we observed that patients with low (&amp;lt; mean) PSMA1 showed higher OS vs. those with high expression, median OS = 14.8 vs. 12.2 months (log-rank P = 0.017). This correlation was recapitulated with several other proteasome genes. Since recent studies revealed distinct GBM subtypes, we tested whether the prognostic value of proteasome gene expression was subtype-specific. We found that the difference was only significant in the proneural subtype, median OS = 16.5 vs. 9.4 mo. (P = 0.012). Realizing that proteasome inhibition exerts phenotypes beyond FA pathway inhibition, we determined the extent to which FA/BRCA expression impacts prognosis. High FA/BRCA expression correlated with lower OS only for proneural tumors (P = 0.015); this observation suggested that proteasome function impacts tumor survival, in large part, by modulating the FA/BRCA pathway. CONCLUSION: Proteasome inhibition sensitizes GBM cells to IR and TMZ and results in impaired FA pathway activation. Our combined functional genomic and bioinformatic analysis suggests a clinical trial: patients with proneural gliomas with high FA/BRCA expression should be treated with proteasome inhibitors such as bortezomib to augment standard therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1318.

MHF1-MHF2, a Histone-Fold-Containing Protein Complex, Participates in the Fanconi Anemia Pathway via FANCM

FANCM is a Fanconi anemia nuclear core complex protein required for the functional integrity of the FANC-BRCA pathway of DNA damage response and repair. Here we report the isolation and characterization of two histone-fold-containing FANCM-associated proteins, MHF1 and MHF2. We show that suppression of MHF1 expression results in (1) destabilization of FANCM and MHF2, (2) impairment of DNA damage-induced monoubiquitination and foci formation of FANCD2, (3) defective chromatin localization of FA nuclear core complex proteins, (4) elevated MMC-induced chromosome aberrations, and (5) sensitivity to MMC and camptothecin. We also provide biochemical evidence that MHF1 and MHF2 assemble into a heterodimer that binds DNA and enhances the DNA branch migration activity of FANCM. These findings reveal critical roles of the MHF1-MHF2 dimer in DNA damage repair and genome maintenance through FANCM.

A Histone-Fold Complex and FANCM Form a Conserved DNA-Remodeling Complex to Maintain Genome Stability

FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here, we show that FANCM forms a conserved DNA-remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human.

Fanconi Anemia pathway heterogeneity revealed by cisplatin and oxaliplatin treatments

Genetic or epigenetic inactivation of the pathway formed by the Fanconi Anemia (FA) proteins occurs in several cancer types, including head and neck squamous cell carcinomas (HNSCC), rendering the affected tumors potentially hypersensitive to DNA crosslinking agents. However, the cytotoxicity of other commonly used cancer therapeutics in cells with FA pathway defects remains to be defined. Here, we focused on the effects of cisplatin and oxaliplatin in a panel of HNSCC and fibroblast cell lines. We found that FANCC- and FANCD2-mutant cells were unexpectedly more sensitive to platinum drugs than FANCA-mutant cells, and mono-ubiquitination of FANCD2, which is mediated by the FANCA and FANCC containing FA core complex was not required for platinum resistance. Interestingly, platinum hypersensitivity could be dissociated from mitomycin C hypersensitivity suggesting different underlying mechanisms. FANCD2 or RAD51 subnuclear foci were not useful as biomarkers of platinum hypersensitivity of FANCC/FANCD2-mutant cells. Our data add to an emerging body of evidence indicating that the FA pathway is not linear and that several protein subcomplexes with different functions exist. It will be important to establish biomarkers that can predict the sensitivity of tumors with specific FA defects to chemotherapeutic agents.

XPF-ERCC1 participates in the Fanconi anemia pathway of cross-link repair.

Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81(-/-), Ercc1(-/-), and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair.

A Novel Upregulation of Fanconi Anemia/BRCA DNA Repair Pathway Is Observed Upon Co-Culture of Bone Marrow Stroma with Multiple Myeloma Cell Lines: A Potential New Participant(s) in Environment Mediated Drug Resistance.

Abstract 2798Poster Board II-774Multiple Myeloma (MM) is an incurable malignancy of mature clonal B cells. The refractory nature of MM has long been attributed to the acquisition of drug resistance. Traditionally, mechanisms of drug resistance have been defined by acquired changes in the expression or function of specific gene products. To this end, we have recently demonstrated that selected resistance to the cytotoxic agent melphalan correlated with increased expression of components of the Fanconi Anemia (FA)/BRCA DNA repair pathway and a concomitant increase in repair of DNA interstrand cross-links (ICLs).(Hazlehurst et al Cancer Res 2003; Chen et al Blood 2005) Further, the exogenous expression of specific FANC components in RPMI 8226 cell lines enhanced ICL repair, favored the release from melphalan-induced S-phase delay, and rendered these cells partially resistant to melphalan treatment. Together, these results suggest a causal relationship between increased expression of FA DNA repair components, increased DNA repair, and acquired resistance to melphalan. Over the past decade a large body of evidence has emerged demonstrating that in addition to drug resistance mechanisms intrinsic to the cancer cell, there exist dynamic, de novo mechanisms coordinated by the tumor microenvironment resulting in an environment-mediated drug resistance (EM-DR). As such, we examined the potential role of the microenvironment in regulating the FA/BRCA DNA repair pathway. FA pathway protein expression was evaluated with anti-sera to FANCD1/BRCA2, FANCC, FANCD2, FANCI, FANCG and BRCA1 in drug sensitive RPMI 8226 cells and melphalan resistant 8226/LR5 cells in co-culture with the HS-5 bone marrow stromal cell line. With these preliminary results we present three novel findings. First, we demonstrate that expression of FA/BRCA pathway components is regulated by intracellular interactions in both MM cells and bone marrow stromal cells (BMSCs). Second, we show that the acquisition of drug resistance alters FANC protein expression profiles upon co-culture. Third, in the HS-5 BMSCs, mono-ubiquitinated FANCD2 is observed in the absence of detectable FANCG. In RPMI 8226 cells, Western blot analysis demonstrated an acute (within 30minutes) and prolonged (up to 48hours) time-dependent increase in expression of FANCD2/BRCA2, FANCC, FANCD2, and BRCA1 upon incubation with BMSCs relative to MM cells incubated alone. However, no appreciated increases in FANCI or FANCG were noted under the same conditions. Incubation of 8226/LR5s with HS-5 BMSCs demonstrated a slightly different up-regulation of FA/BRCA pathway protein expression with addition of increased FANCI expression and no increase in FANCD2 or FANCC expression. We also examined FANC protein expression in the HS-5 cells. Interestingly, in the BMSCs significant differences were noted in the FANC expression profiles. Co-culture of RPMI 8226 cells with HS-5 cells demonstrated only modest elevations in FANCD2; however, co-culture with drug-resistant 8226/LR5s resulted in increased levels of FANCD2, FANCI and BRCA1. These data indicate that different tumor cells may alternately influence FA/BRCA-mediated DNA repair and potentially drug resistance in juxtaposed bone marrow stroma. Curiously, we also observed mono-ubiquitinated FANCD2 in the absence of any detectable levels of FANCG protein under co-culture conditions. As the FA/BRCA DNA repair pathway has been associated with cell cycle progression, we evaluated cell cycle kinetics under the co-culture conditions. The results of BrdU analysis demonstrated that the observed changes in FA/BRCA protein expression in MM and BMSC could not be fully explained by cell-cycle distribution. Therefore, within this report we demonstrate for the first time that microenvironmental interactions can modulate the FA/BRCA DNA repair pathway in MM and BMSCs. These results suggest that the FA/BRCA DNA damage repair pathway may be an important modulatory component of EM-DR. Importantly, the potential de novo drug resistance likely involves both the MM tumor cell and adjacent stromal cells. Current and future studies will attempt to examine a causal relationship between increased FANC expression and melphalan (and other drug) resistance seen in co-culture conditions, as well as to identify specific signaling molecules and mechanisms controlling the enhanced expression in both cell models. Disclosures:No relevant conflicts of interest to declare.

FANCD2 Localizes to Cross-Linked Induced Nuclear Foci That Are Distinct From Those to Which αaII Spectrin and the Cross-Link Repair Protein, XPF, Co-Localize, Indicating An Involvement of These Proteins in Different Steps of the DNA Interstrand Cross-Link Repair Process.

Abstract 3196Poster Board III-133The hereditary bone marrow failure disorder, Fanconi anemia (FA), is characterized by a markedly increased incidence of acute myelogenous leukemia, diverse congenital abnormalities and a defect in ability to repair DNA interstrand cross-links. We have previously shown that in FA cells there is a deficiency in the structural protein nonerythroid a spectrin (aSpII), which is involved in repair of DNA interstrand cross-links and binds to cross-linked DNA. aSpII co-localizes in nuclear foci with FANCA and the cross-link repair protein, XPF, after normal human cells are damaged with a DNA interstrand cross-linking agent. One of the FA proteins which is thought to play an important role in the repair of DNA interstrand cross-links is FANCD2, which is known to form nuclear foci after cross-link damage. The present study was undertaken in order to get a better understanding of the relationship between aSpII and FANCD2, whether they interact with each other during the DNA repair process and co-localize in damage-induced nuclear foci. Immunofluorescence microscopy was carried out to determine whether these proteins co-localized in nuclear foci after cells were damaged with a DNA interstrand cross-linking agent, 8-methylpsoralen plus UVA light (8-MOP) or mitomycin C (MMC). Time course measurements showed that FANCD2 foci were first visible at 2 hours after damage and increased up to 16 hours and were still present at 72 hours after damage. This time course of foci formation correlated with levels of monoubiquitination of FANCD2. Measurement of gH2AX foci formation showed that the time course of foci formation was similar to that of FANCD2 measured up to 72 hours post damage. In contrast, aSpII foci were first visible between 8-10 hours after damage. The number of these foci peaked at 16 hours and by 24 hours foci were no longer observed. Co-localization studies showed that there was little co-localization of the FANCD2 and aSpII foci over this time course. This indicates that these two proteins may be involved in different steps in the DNA interstrand cross-link repair process. Based on models that have been proposed for the role of FANCD2 in the repair of DNA interstrand cross-links, we propose that, after DNA damage, FANCD2 localizes at DNA replication forks stalled at sites of interstrand cross-links and aids in the assembly of proteins at this site. This is followed by localization of aSpII and XPF and other proteins involved in the initial incision steps in DNA interstrand cross-link repair where they play a role in the unhooking of the cross-link. FANCD2 is then involved in subsequent steps in the repair process, which involve homologous recombination. Thus two proteins, FANCD2 and aSpII, both of which have been shown to be critical for the DNA interstrand cross-link repair process may be involved in different or distinct steps in this repair process. Deficiencies in these proteins would impact on DNA interstrand cross-link repair and, as we have shown for aIISp, would have an adverse effect on the genomic stability of FA cells. . DisclosuresNo relevant conflicts of interest to declare.