Defective Fanconi Anemia Research Articles

Chromatin Remodeler DEK Is a Sensor to Replication Stress and Contributes to HSC Defects in Fanconi Anemia

Hematopoietic stem cells (HSCs) have self-renewal capacity and response for life-long hematopoiesis. Any interference with normal DNA replication leads to replication stress, which drives function decline in HSCs. However, the response of HSCs to replication stress remains largely unknown. Here, draw a transcriptional and epigenetic landscape of HSCs under replication stress. We found that HSCs required opened chromatin to counteract replication stress, along with altered H3K27 acetylation and H3K4 trimethylation. The increased chromatin accessibility was mainly attributed to the reduced chromatin remodelers DEK, while DEK overexpressed HSCs confronted with a strong replication challenge, shown by increased γ-H2AX. Excessive DEK protein impeded proliferation and impaired HSC maintenance (>50% reduction), along with reduced oxidative phosphorylation and protein synthesis. Notably, we found that DEK protein was abnormally accumulated in BM CD34 + cells in Fanconi anemia, which arises from mutations in FA genes essential for replication stress tolerance. Inhibition of DEK substantially recovered HSC proliferation capacity in vitro and engraftment in vivo. At the molecular level, we identified that ATF2 directly promoted the transcription of DEK, especially relying on phosphorylated ATF2 (ser69/71) rather than phosphorylated ATF2 (ser490/498). Rad3-related (ATR) induced the phosphorylation of ATF2 at ser490/498 and mediated the reduction of DEK under replication stress. Nonetheless, knock-down of FA genes (FANCD2) in HSCs resulted in increased phosphorylated ATF2 (ser69/71), which might contribute to DEK accumulation in FA. Collectively, our findings uncover the landscape of HSC response to replication stress, reveal a novel ATR-ATF2-DEK axis helping HSCs counteract replication stress, and suggest inhibition of DEK as a therapeutic utility in FA.

Dysregulated Proteostasis Restrains the Developmental HSC Expansion in Fanconi Anemia

Hematopoietic stem cells (HSCs) require precise regulation of protein synthesis, processing, and degradation. Protein homeostasis (Proteostasis) is particularly critical during development, relying on chaperone proteins to maintain folding capacity in a highly proliferative HSC pool. Even modest changes in synthesis or degradation lead to the accumulation of unfolded proteins and compromise fetal liver HSC pool formation. Fanconi anemia (FA) is a multisystem disorder that manifests with bone marrow failure (BMF) early in life. Previous reports from our lab and others suggest that pre-natal HSC depletion in FA is a critical factor for the early onset of clinical BMF. To identify the cellular and molecular mechanisms underlying prenatal HSC defects in FA, we performed single-cell transcriptome profiling in both Fancd2 +/+and Fancd2 -/- FL samples. Gene set enrichment analysis (GSEA) of differentially expressed genes revealed significantly increased expression of c-Myc target genes in Fancd2 -/ - HSCs (normalized enrichment score, NES: 2.04, p<10 -5 FDR<10 -5), matched by an increase in c-Myc in Fancd2 -/- LT-HSC (CD150 + CD48 - Lineage - Sca-1 + c-KIT +) at the protein level (1.7-fold (p<0.05). Our sc-RNA Seq data further showed an increase in ribosome biogenesis (NES: 1.8, p<10 -5, FDR<10 -3) and translation (NES: 2.2, p<10 -5, FDR<10 -5) in Fancd2 -/- HSC, functionally corroborated by increased protein synthesis rates across Fancd2 -/- FL HSPC subpopulations (1.3-fold in LT- HSCs, p<0.01) using the in vivo O-Propargyl-puromycin (OPP) assay. Not surprisingly, the increased protein synthesis rates exceed the physiologic protein folding capacity, leading to elevated levels of unfolded proteins in Fancd2 -/- FL HSPCs by Tetraphenylethene maleimide (TPE-MI) stain. The aggregated misfolded proteins (aggresomes) in Fancd2 -/- FL HSPCs were also observed by immunofluorescence microscopy. Revisiting our scRNA Seq data, the observed proteotoxic stress was also reflected in the GSEA enrichment for unfolded protein response (UPR; NES: 1.30, p=0.01, FDR=0.01) as well as heat shock stress response (NES:1.7,p<0.0001, FDR=0.09), and coincided with upregulation of response to heat shock factor-1 ( Hsf-1; NES:1.86,p<0.0001, FDR=0.02) in Fancd2 -/- FL HSCs. Because chaperone supplementation has been shown to enhance HSC protein folding capacity, we serially injected the bile acid TUDCA (a chemical chaperone) into the pregnant dam. This not only significantly reduced unfolded protein levels, but indeed rescued FL HSC numbers in Fancd2 -/- littermates. Our observations in aggregate indicate that dysregulated proteostasis is a previously unrecognized pathogenic process constraining fetal Fancd2 -/- hematopoiesis. Protection of the HSPC proteome appears to be a new, non-canonical function of FANCD2, without immediate connection to the FA pathway and DNA crosslink repair. Importantly, we demonstrate the successful pharmacological rescue of the fetal Fancd2 -/- HSPC pool.

Concepts of multi-level dynamical modelling: understanding mechanisms of squamous cell carcinoma development in Fanconi anemia.

Fanconi anemia (FA) is a rare disease (incidence of 1:300,000) primarily based on the inheritance of pathogenic variants in genes of the FA/BRCA (breast cancer) pathway. These variants ultimately reduce the functionality of different proteins involved in the repair of DNA interstrand crosslinks and DNA double-strand breaks. At birth, individuals with FA might present with typical malformations, particularly radial axis and renal malformations, as well as other physical abnormalities like skin pigmentation anomalies. During the first decade of life, FA mostly causes bone marrow failure due to reduced capacity and loss of the hematopoietic stem and progenitor cells. This often makes hematopoietic stem cell transplantation necessary, but this therapy increases the already intrinsic risk of developing squamous cell carcinoma (SCC) in early adult age. Due to the underlying genetic defect in FA, classical chemo-radiation-based treatment protocols cannot be applied. Therefore, detecting and treating the multi-step tumorigenesis process of SCC in an early stage, or even its progenitors, is the best option for prolonging the life of adult FA individuals. However, the small number of FA individuals makes classical evidence-based medicine approaches based on results from randomized clinical trials impossible. As an alternative, we introduce here the concept of multi-level dynamical modelling using large, longitudinally collected genome, proteome- and transcriptome-wide data sets from a small number of FA individuals. This mechanistic modelling approach is based on the "hallmarks of cancer in FA", which we derive from our unique database of the clinical history of over 750 FA individuals. Multi-omic data from healthy and diseased tissue samples of FA individuals are to be used for training constituent models of a multi-level tumorigenesis model, which will then be used to make experimentally testable predictions. In this way, mechanistic models facilitate not only a descriptive but also a functional understanding of SCC in FA. This approach will provide the basis for detecting signatures of SCCs at early stages and their precursors so they can be efficiently treated or even prevented, leading to a better prognosis and quality of life for the FA individual.

Lnk/Sh2b3 Deficiency Restores Hematopoietic Stem Cell Function and Genome Integrity in Fanconi Anemia

▪Although initially identified over 85 years ago, Fanconi Anemia (FA) remains a fatal genetic disease. Patients with FA exhibit developmental malformations, bone marrow failure, and increased cancer susceptibility. Twenty-one FA genes cooperate in a genome stability pathway that is essential for repair of DNA interstrand crosslinks (ICLs) and attenuation of replication stress. Cells derived from FA patients are hypersensitive to ICL-inducing agents such as Mitomycin C (MMC), a hallmark of FA. In addition, FA hematopoietic stem and progenitor cells (HSPCs) show growth retardation and increased apoptosis culminating in hematopoietic stem cell (HSC) exhaustion and bone marrow failure. The poor growth of HSPCs also poses a significant obstacle in gene therapy for FA patients. Moreover, unrepaired DNA damage increases genome instability and promotes leukemia and cancer. Of note, interventions to mitigate HSPC defects in FA do not exist, aside from allogeneic bone marrow transplantation.Remarkably, we reveal here that loss of the adaptor protein Lnk (also called Sh2b3) suppresses FA associated phenotypes without further accelerating neoplastic transformation. LNK is a negative regulator of HSC self-renewal in part by attenuating cytokine signaling, in particular thrombopoietin (TPO)-activated JAK2 signaling in HSCs. Fancd2-/-;Lnk-/- mice exhibit increased phenotypic HSCs in comparison to wildtype (WT) animals. The reconstituting ability of Fancd2-/-;Lnk-/- HSCs is also largely restored to WT levels in serial transplantation assays. In addition, Lnk deficiency restored the growth and survival of FA HSPCs in ex vivo culture. More importantly, Lnk deficiency mitigated chromosomal aberrations associated with FA upon proliferation. To determine if Lnk deficiency mitigates ICL repair, we examined MMC sensitivity in primary HSPCs that were dually null for Lnk and FancD2. Our data suggest that LNK does not play an overt role in ICL repair. Rather, Lnk deficiency in HSPCs mitigated replication stress by stabilizing replication forks and reduces genome instability associated with FA. Hence, our data suggest that Lnk deficiency alleviates replication stress-induced DNA damage and genome instability, thereby restoring cell proliferation and survival of Fancd2-/- HSPCs ex vivo and HSC functions in vivo.Last and importantly, we began to test if LNK inhibition restores human FA HSPCs. We first generated FA-like HSPCs by knocking down FANCD2 in normal umbilical cord blood (UCB) CD34+ cells. Our results demonstrated that shRNA-mediated depletion of LNK in human CD34+ cells rescued progenitor cell numbers and growth in FA-like HSPCs. Take together, our findings reveal a previously unappreciated role of cytokine/JAK signaling in alleviating replication stress and promoting replication-associated genome maintenance. Our studies also illuminate replication stress as a major underlying origin of bone marrow failure in FA patients and have implications for therapeutic strategies to treat FA associated bone marrow failure. DisclosuresNo relevant conflicts of interest to declare.

Targeting CX3CR1 Suppresses the Fanconi Anemia DNA Repair Pathway and Synergizes with Platinum.

Simple SummaryChemotherapeutics exerting their antiproliferative actions by introducing DNA crosslinks, such as platinum drugs, are used to treat numerous cancers. Unfortunately, their therapeutic potential is limited due to adverse side effects and acquired resistance, the latter often associated with enhanced DNA repair capacity. Thus, targeting DNA repair is a promising strategy to lower effective doses and associated side effects, and to restore sensitivity to treatment. The C-X3-C motif chemokine receptor 1 (CX3CR1) is an emerging anticancer target which expression correlates with worse overall survival in cancer patients undergoing DNA damaging treatments. Here we show for the first time that the clinical-phase small molecule inhibitor KAND567 targeting CX3CR1 augments the efficacy of DNA crosslinking chemotherapeutics in cancer cell lines, including platinum resistant models, by interference of the Fanconi anemia DNA repair pathway. Hence, the interplay between CX3CR1 and FA repair provides novel potential therapeutic opportunities in cancers treated with DNA crosslinking agents.The C-X3-C motif chemokine receptor 1 (CX3CR1, fractalkine receptor) is associated with neoplastic transformation, inflammation, neurodegenerative diseases and aging, and the small molecule inhibitor KAND567 targeting CX3CR1 (CX3CR1i) is evaluated in clinical trials for acute systemic inflammation upon SARS-CoV-2 infections. Here we identify a hitherto unknown role of CX3CR1 in Fanconi anemia (FA) pathway mediated repair of DNA interstrand crosslinks (ICLs) in replicating cells. FA pathway activation triggers CX3CR1 nuclear localization which facilitates assembly of the key FA protein FANCD2 into foci. Interfering with CX3CR1 function upon ICL-induction results in inability of replicating cells to progress from S phase, replication fork stalling and impaired chromatin recruitment of key FA pathway factors. Consistent with defective FA repair, CX3CR1i results in increased levels of residual cisplatin-DNA adducts and decreased cell survival. Importantly, CX3CR1i synergizes with platinum agents in a nonreversible manner in proliferation assays including platinum resistant models. Taken together, our results reveal an unanticipated interplay between CX3CR1 and the FA pathway and show for the first time that a clinical-phase small molecule inhibitor targeting CX3CR1 might show benefit in improving responses to DNA crosslinking chemotherapeutics.

MiRNA-200C expression in Fanconi anemia pathway functionally deficient lung cancers

The Fanconi Anemia (FA) pathway is essential for human cells to maintain genomic integrity following DNA damage. This pathway is involved in repairing damaged DNA through homologous recombination. Cancers with a defective FA pathway are expected to be more sensitive to cross-link based therapy or PARP inhibitors. To evaluate downstream effectors of the FA pathway, we studied the expression of 734 different micro RNAs (miRNA) using NanoString nCounter miRNA array in two FA defective lung cancer cells and matched control cells, along with two lung tumors and matched non-tumor tissue samples that were deficient in the FA pathway. Selected miRNA expression was validated with real-time PCR analysis. Among 734 different miRNAs, a cluster of microRNAs were found to be up-regulated including an important cancer related micro RNA, miR-200C. MiRNA-200C has been reported as a negative regulator of epithelial-mesenchymal transition (EMT) and inhibits cell migration and invasion by promoting the upregulation of E-cadherin through targeting ZEB1 and ZEB2 transcription factors. miRNA-200C was increased in the FA defective lung cancers as compared to controls. AmpliSeq analysis showed significant reduction in ZEB1 and ZEB2 mRNA expression. Our findings indicate the miRNA-200C potentially play a very important role in FA pathway downstream regulation.

Identification of metabolic changes leading to cancer susceptibility in Fanconi anemia cells.

Fanconi anemia (FA) is a chromosomal instability disorder of bone marrow associated with aplastic anemia, congenital abnormalities and a high risk of malignancies. The identification of more than two dozen FA genes has revealed a plethora of interacting proteins that are mainly involved in repair of DNA interstrand crosslinks (ICLs). Other important findings associated with FA are inflammation, oxidative stress response, mitochondrial dysfunction and mitophagy. In this work, we performed quantitative proteomic and metabolomic analyses on defective FA cells and identified a number of metabolic abnormalities associated with cancer. In particular, an increased de novo purine biosynthesis, a high concentration of fumarate, and an accumulation of purinosomal clusters were found. This was in parallel with decreased OXPHOS and altered glycolysis. On the whole, our results indicate an association between the need for nitrogenous bases upon impaired DDR in FA cells with a subsequent increase in purine metabolism and a potential role in oncogenesis.

Fancd2-deficient hematopoietic stem and progenitor cells depend on augmented mitochondrial translation for survival and proliferation.

Members of the Fanconi anemia (FA) protein family are involved in multiple cellular processes including response to DNA damage and oxidative stress. Here we show that a major FA protein, Fancd2, plays a role in mitochondrial biosynthesis through regulation of mitochondrial translation. Fancd2 interacts with Atad3 and Tufm, which are among the most frequently identified components of the mitochondrial nucleoid complex essential for mitochondrion biosynthesis. Deletion of Fancd2 in mouse hematopoietic stem and progenitor cells (HSPCs) leads to increase in mitochondrial number, and enzyme activity of mitochondrion-encoded respiratory complexes. Fancd2 deficiency increases mitochondrial protein synthesis and induces mitonuclear protein imbalance. Furthermore, Fancd2-deficient HSPCs show increased mitochondrial respiration and mitochondrial reactive oxygen species. By using a cell-free assay with mitochondria isolated from WT and Fancd2-KO HSPCs, we demonstrate that the increased mitochondrial protein synthesis observed in Fancd2-KO HSPCs was directly linked to augmented mitochondrial translation. Finally, Fancd2-deficient HSPCs are selectively sensitive to mitochondrial translation inhibition and depend on augmented mitochondrial translation for survival and proliferation. Collectively, these results suggest that Fancd2 restricts mitochondrial activity through regulation of mitochondrial translation, and that augmented mitochondrial translation and mitochondrial respiration may contribute to HSC defect and bone marrow failure in FA.

Lnk/Sh2b3 deficiency restores hematopoietic stem cell function and genome integrity in Fancd2 deficient Fanconi anemia

Fanconi anemia (FA) is a bone marrow failure (BMF) syndrome that arises from mutations in a network of FA genes essential for DNA interstrand crosslink (ICL) repair and replication stress tolerance. While allogeneic stem cell transplantation can replace defective HSCs, interventions to mitigate HSC defects in FA do not exist. Remarkably, we reveal here that Lnk (Sh2b3) deficiency restores HSC function in Fancd2−/− mice. Lnk deficiency does not impact ICL repair, but instead stabilizes stalled replication forks in a manner, in part, dependent upon alleviating blocks to cytokine−mediated JAK2 signaling. Lnk deficiency restores proliferation and survival of Fancd2−/− HSCs, while reducing replication stress and genomic instability. Furthermore, deletion of LNK in human FA-like HSCs promotes clonogenic growth. These findings highlight a new role for cytokine/JAK signaling in promoting replication fork stability, illuminate replication stress as a major underlying origin of BMF in FA, and have strong therapeutic implications.

Abstract 1361: Micro RNA-200C is one of the important Fanconi Anemia (FA) pathway downstream regulators in lung cancer

Abstract The Fanconi Anemia (FA) pathway is essential for human cells to maintain integrity following DNA damage. This pathway is involved in the endogenous repair of double stranded DNA breaks and homologous recombination as well as repair of DNA cross-linking caused by exogenous agents. Cancers with defective FA pathway are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. We have recently reported the detection of 22% of NSCLC to be FA functionally inactive by Fanconi Anemia Triple Staining Immunofluorescence (FATSI) test. Studies have shown involvement of certain micro RNA (miRNA) as regulatory elements in the development of lung cancer. We set out to evaluate potential involvement of miRNAs in the regulation of the Fanconi Anemia (FA) pathway. Using Nanostring counter miRNA array we screened 734 different miRNA expression in two FA defective lung cancer cells and matched control cells along with two FA pathway deficient lung tumors and matched non-tumor lung tissue samples. Selected miRNA expression were validated with real-time PCR analysis. miRNA target gene expression was analyzed through AmpliSeq RNA gene expression analysis. Among 734 different miRNAs, a cluster of microRNAs were found to be up-regulated including an important cancer related micro RNA, miR-200C. Nanostring data showed that miR-200C was increased 7.5 fold on average in the FA defective lung cancer cells as compared to control cell. An average of 22 fold increase in miR-200C was detected in the FA defective lung tumor tissues comparing to matching non-tumor tissues. AmpliSeq analysis showed the ZEB1(zinc finger E-box binding homeobox 1) mRNA expression was down regulated in10 out 10 lung tumors (100%) comparing to non-tumor tissues, and 9 out of 10 samples (90%) showed reduction in ZEB2 expression. MiRNA-200C has been reported as a negative regulator of epithelial-mesenchymal transition (EMT) and inhibiting cell migration and invasion by promoting the upregulation of E-cadherin through targeting ZEB1 and ZEB2 transcription factors. Our findings indicate that the FA pathway regulates downstream genes through regulation of miRNAs in lung cancer. MiR-200C appears to be one of the most important FA downstream regulators in lung cancer. Validation with a larger sample size will be needed to confirm our findings. Citation Format: Wenrui Duan, Shirley Tang, Li Gao, Kathleen Dotts, Andrew Fink, Arjun Kalvala, Brittany Aguila, Miguel A. Villalona-Calero. Micro RNA-200C is one of the important Fanconi Anemia (FA) pathway downstream regulators in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1361.

Involvement of FANCD2 in Energy Metabolism via ATP5\u03b1

Growing evidence supports a general hypothesis that aging and cancer are diseases related to energy metabolism. However, the involvement of Fanconi Anemia (FA) signaling, a unique genetic model system for studying human aging or cancer, in energy metabolism remains elusive. Here, we report that FA complementation group D2 protein (FANCD2) functionally impacts mitochondrial ATP production through its interaction with ATP5α, whereas this relationship was not observed in the mutant FANCD2 (K561R)-carrying cells. Moreover, while ATP5α is present within the mitochondria in wild-type cells, it is instead located mostly outside in cells that carry the non-monoubiquitinated FANCD2. In addition, mitochondrial ATP production is significantly reduced in these cells, compared to those cells carrying wtFANCD2. We identified one region (AA42-72) of ATP5α, contributing to the interaction between ATP5α and FANCD2, which was confirmed by protein docking analysis. Further, we demonstrated that mtATP5α (∆AA42-72) showed an aberrant localization, and resulted in a decreased ATP production, similar to what was observed in non-monoubiquitinated FANCD2-carrying cells. Collectively, our study demonstrates a novel role of FANCD2 in governing cellular ATP production, and advances our understanding of how defective FA signaling contributes to aging and cancer at the energy metabolism level.

Abstract 4442: micoRNA in FA defective tumor

Abstract The Fanconi Anemia (FA) pathway is essential for human cells to maintain integrity following DNA damage. This pathway is involved in the endogenous repair of double stranded DNA breaks and homologous recombination as well as repair of DNA cross-linking caused by exogenous agents. Cancers with defective FA pathway are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. We have recently reported the detection of up to 20% of NSCLC to be FA functionally inactive (lack of FANCD2 foci formation in the nucleus of proliferating cells by triple stain immunofluorescence, FATSI negative). MicroRNAs (miRs, miRNA) are small, 19-25 nucleotide, single stranded, non-coding RNA molecules that have regulatory roles in humans by targeting mRNAs via cleaved or translational repression which can affect the output of protein coding genes and cause various changes in developmental and physiological processes including cancer. It has been published that some miRNAs are tumor suppressors, and others are oncogenes. However, microRNAs and the FA pathway in relationship to lung cancer progression remain unknown. In order to elucidate the role of FA related miRs in human lung cancer development or treatment, we analyzed miR expression in lung cancer cells and patient lung tumors. Several cell lines were generated using RNAi technology with FANCD2 deficiency and confirmed via reduction in FANCD2 protein expression. Using Nanostring counter technology, microRNA array analysis was performed on FANCD2 foci positive and foci negative tumors as well as matching normal lung tissues. A cluster of microRNAs (mir-431, 377, 376c, 200c, 26a, 135a, and let-7e) were found to be up-regulated, and miR-494,205, 630, 145,671 were found to be down-regulated in the foci negative tumors samples compared to matching non-tumor lung tissue. The same group of miRs were upregulated and its corresponding group were down-regulated in the foci negative tumors as compared to foci positive tumors. These findings indicate that the FA pathway may regulate downstream genes through regulation of microRNAs in lung cancer. In order to validate this hypothesis, specific miRs were be analyzed with Real-Time Quantitative PCR via foci positive and foci negative samples (cell and patient tissue) in hopes of finding significant and consistent regulation. This study contributes to further understanding in miRNA and cancer pathology that may be applicable to futuristic lung cancer treatment. Citation Format: Shirley Tang, Li Gao, Kathleen Kathleen, Andrew Fink, Arjun Kalvala, Brittany Aguila, Gregory Otterson, Miguel Villalona-Calero, Wenrui Duan. micoRNA in FA defective tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4442. doi:10.1158/1538-7445.AM2017-4442

Rare Variants Affecting the Fanconi Anaemia DNA Repair Genes Associate with Increased Risk for AML

While there have been extensive studies to define the roles of recurrent somatic mutations in AML, the contribution of germline variants to AML initiation and progression is less well established. DNA repair disorders often predispose patients to developing myeloid malignancies. In particular, biallelic mutations affecting FANC genes cause the recessive heritable bone marrow failure syndrome Fanconi Anemia (FA), which is associated with >800-fold increased risk of progression to AML. A recent explosion of cancer predisposition studies has also revealed the importance of germlineFANC variants in elevated cancer risk (Cancer Treat Rev 2012; 38:89). To investigate the role of FANC gene variants in AML we have performed a case-control study, analyzing rare, deleterious somatic and germline variants for the 19 FANC genes in adult AML and healthy controls cohorts.Whole exome sequencing was performed on diagnosis samples from 131 adult Caucasian AML patients from two major Australian centers, and a cohort of 329 healthy females. We identified rare Tier 1 variants using a minor allele frequency (MAF) < 0.001, as reported in common dbSNP137, 1000 Genome and NHLBI-ESP project databases. Combined Annotation Dependent Depletion algorithm (CADD, Nat Genet 2014; 46: 310) >10 was used to filter for FANC gene variants with high probability of pathogenicity. Sanger sequencing of matched tumour/non-tumour DNA showed the large majority of variants tested to be germline (90%), consistent with previous studies reporting that somatic FANC genes variants are extremely rare in AML (< 1%). Overall, we identified 52 FANC gene variants in 44 cases with 34% of AML cases carrying one or more variant.For independent validation we determined the presence of somatic and germline FANC variants in the TCGA AML cohort using an identical pipeline and filtering analysis. In line with our results, we found that 36% of TCGA AML patients carry at least one germline FANC variant. We investigated known disease-causing (D-C) variants in these two AML cohorts using the FA (FAMutdb) and breast cancer (kConFab and BIC) mutation databases. We found 8 D-C FANC variants in the Australian AML cohort and 5 in the TCGA cohort, with 1 variant present in both cohorts. Moreover, the frequency of D-C variants in our cohort of females with AML (n=51) is 13.7%, while the frequency in the healthy female cohort is 4.5%, comparable to that reported in the ESP database for female European-Americans (2.1%, Hum Mol Genet 2014; 23: 6815). Accordingly, we determined that deleterious FANC germline variants confer a significant increased risk of AML (P=0.018, OR=3.3 for the Australian AML cohort).Finally, we performed mutational burden analysis to investigate enrichment of variants associated with particular FANC genes across the AML cohort. This revealed a significant enrichment of FANCL variants in AML vs healthy controls (P=0.008, Figure 1). FANCL is the enzymatic component of the FA core complex that monoubiquitinates the FANCD2/I heterodimer initiating DNA repair, and its down-regulation has been linked to AML (Oncogene 2016; doi:10.1038). Several FANCL variants, found in our AML cohort, affect the catalytic RING domain and are of particular interest. These include a D-C null variant present in 2 patients, a frame shift variant in 2 patients who presented with AML at a very early age (27 and 46 years old), and a variant affecting a critical conserved residue required for monoubiquitination of FANCD2/I.In conclusion, we show enrichment of rare potentially deleterious FANC gene mutations in AML, associated with a 3-fold increased risk of developing the disease. We hypothesise that, in hematopoietic stem/progenitor cells, these variants confer a subtle defect in interstrand cross-link repair leading to an increased accumulation of mutations and subsequent development of AML. Consistent with this there have been several reports of defective DNA damage repair and increased sensitivity to DNA damaging agents in cells from FANC carriers compared to normal controls (Nat Commun 2014; 5:5496; Mutagenesis 2009; 24:67). Importantly, it is possible to target defects in several DNA repair pathways, and our finding identifies a group of AML patients who may benefit from approaches that target defective FA and homologous recombination pathways. [Display omitted] DisclosuresGill:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 4438: Promoter hypermethylation status of Fanconi Anemia (FA) pathway genes FANCF, FANCL and FANCS in non-small cell lung cancer (NSCLC)

Abstract Gene promoter methylation is an epigenetic mechanism used by cells to control gene expression. Over recent decades, scientists have made various discoveries linking DNA methylation to several adverse outcomes, including human cancers. The Fanconi Anemia (FA) pathway is involved in homologous recombination, one of the major mechanisms of DNA repair. This pathway is essential for human cells to maintain integrity following DNA damage. Cancers with defective FA pathways are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. The FA pathway contains at least 19 genes, and some of the members have been implicated in susceptibility to a number of cancers by genetic or epigenetic alterations. Promoter methylation in FA genes is thought to play a role in the occurrence of cancer. Recently we screened 139 non-small cell lung cancer (NSCLC) formalin-fixed, paraffin-embedded (FFPE) tumors for FANCD2 foci formation by FA triple stain immunofluorescence (FATSI) analysis. Among the 104 evaluable tumors, 23 (22%) were FANCD2 foci negative. Since epigenetic inactivation can be one of the mechanisms for FA functional deficiency in these tumors, we evaluated 39 NSCLC samples (21 foci positive and 18 foci negative; 21 adenocarcinomas, 17 squamous cell carcinomas, 1 large cell carcinoma) for FANCF, FANCL and FANCS (BRCA1) promoter methylation. Human lung tumor tissue samples were obtained from The Tissue Procurement Shared Resources of the Ohio State University after IRB approval. Genomic DNA and total RNA samples were isolated from frozen lung tumor and matching non-tumor tissues. The promoter methylation status of FANCF, FANCL and FANCS was evaluated using methylation-specific PCR (MS-PCR). Among the 18 FATSI negative tumors, promoter methylation was found in FANCF (1 adenocarcinoma), FANCL (1 adenocarcinoma) and FANCS (1 adenocarcinoma). Among the 21 FATSI positive tumors, no promoter methylation was detected in FANCF or FANCL. Promoter methylation in FANCS was found in 1 (squamous cell carcinoma) of 21 FATSI positive tumors. The above observations suggest that epigenetic alterations, specifically methylation, can be one of the factors that contribute to FA functional deficiency in NSCLC patients. These findings may have clinical implications, since these tumors may be more sensitive to cross-link based therapy. However, an important caveat is that these changes may not be stable and could revert during treatment. Further studies in FA gene expression are needed to determine the impact of FA gene promoter methylation on FA repair foci formation. Citation Format: Andrew Fink, Arjun Kalvala, Li Gao, Kathleen Dotts, Brittany Aguila, Shirley Tang, Gregory A. Otterson, Miguel A. Villalona-Calero, Wenrui Duan. Promoter hypermethylation status of Fanconi Anemia (FA) pathway genes FANCF, FANCL and FANCS in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4438.

KIT Blockade Is Sufficient to Sustain Donor Hematopoietic Stem Cell Engraftment in Fanconi Anemia Mice

Background: Fanconi anemia (FA) is the most common cause of inherited bone marrow failure (BMF). Currently, the only curative option for the BMF in FA is an allogenic hematopoietic stem cell transplant (HSCT). However, due to the underlying DNA repair defect, FA patients poorly tolerate alkylating chemotherapy or irradiation based conditioning, which is necessary for donor engraftment. However, this results in significant short and long term morbidity/mortality and augments the inherent increased risk of malignancies in FA patients. To overcome the adverse effects associated with alkylating conditioning agents, alternate experimental approaches exploiting the inherent hematopoietic stem cell (HSC) defect in FA are of utmost clinical necessity.Objective: To develop a safe KIT blocking antibody (KIT-Ab) based HSCT conditioning regimen for FA that does not involve chemotherapy or irradiation.Method: High purity KIT-Ab was made from the ACK2 hybridoma and its specificity to KIT binding was validated using mast cell assay. Baseline peripheral blood cells and the bone marrow hematopoietic stem and progenitor cell (HSPC) compartment (Lin-Kit+Sca+ and Lin-Kit+Sca+CD150+CD48- cells) of FANCA-/- and FANCD2-/- murine models were analyzed. Mechanistic studies using sorted FA bone marrow HSPC were performed ex vivo. This was followed by definitive primary and secondary transplants experiments following injection of KIT-Ab.Results: Several features of FA hematopoietic stem/progenitor cells (HSPC) suggested their susceptibility to KIT-Ab blockade-mediated killing: (a) Expression of KIT was significantly lower in FANCA-/- HSPC, while expression of its ligand was higher in bone marrow stroma; (b) Moreover, genes associated with apoptosis/senescence, stress and inflammatory signaling that were upregulated in WT-HSPC following KIT-Ab blockade, were upregulated in FANCA-/- HSPC at baseline; (c) Furthermore, FANCA-/- HSPC demonstrated increased susceptibility to KIT-Ab mediated apoptosis and had a reduced proliferative capacity. In-vivo studies following ACK2 injection showed a marked reduction of colony-forming units (CFU-C) from both FANCA-/- and FANCD2-/- mice one week following injection, when compared to WT mice (48% and 76% decrease in CFU-C, respectively). Based on these findings, we evaluated the role of ACK2 as a sole HSCT conditioning regimen in FANCA-/- and FANCD2-/- mice. Indeed, definitive HSCT in both FANCA-/- and FANCD2-/- mice using KIT-Ab based conditioning resulted in donor HSC engraftment with multi-lineage chimerism, which progressively increased to 22-24% by 4-months, and was sustained in secondary transplants. Overall, we show that KIT-blockade alone is an adequate non-genotoxic HSPC-targeted conditioning in FA mice, and its clinical translation could circumvent the extensive transplant-related morbidity/mortality in this disease. DisclosuresNo relevant conflicts of interest to declare.

Exhaustion in Myeloid Lineage and Very Early Defect in HSPC Pool: An Embryonic Origin of Fanconi Haematological Disorders

Fanconi anemia (FA) is a genetic disorder due to mutations in one of the sixteen FANC genes involved in DNA repair. Many FA patients develop bone marrow failure (BMF) during childhood, and FA strongly predisposes to myelodysplasia syndrome and/or acute myeloid leukaemia. The pathogenesis of the BMF remains uncompletely understood. Low hematopoietic progenitor cell (HPCs) counts observed early in life and preceeding the onset of blood cytopenia in patients led we, and other, to hypothesize that the hematopoietic development might be abnormal in the FA embryo. Indeed, unlike adult hematopoietic stem cells (HSCs) which are quiescent in the BM niche, during embryonic life HSCs are in active proliferation in sites of expansion such as fetal liver and placenta, where they get amplified and acquire properties of adult HSC .We hypothesized that in FA, the FA defect in response to the replicative stress could impair the expension of the HSC pool.In order to investigate this hypothesis, we carried out studies in Fancg-/- knock out mice and in human FA fetuses obtained with informed consent from medical abortion.In Fancg-/- mice, FACS analysis revealed a 1,5- to 3-fold deficiency in hematopoietic stem and progenitor cells (HSPC) very early during embryonic development (i.e 11.5 days of gestation - E11.5) in fetal liver (FL) and placenta (Pl) (p <0.001). In both organs, this defect persists during the whole period of amplification (until E14.5 for FL and E12.5 for Pl). In vitro clonogenic assays also demonstrated a 2- fold defect in granulocyte, erythrocyte and macrophage (GEM) progenitors both in Fancg-/- FL or Pl compared to WT (p <0.001), and 4 to 5- fold defect in more immature mixed GEM progenitors in FL (p <0.001). LTC-IC frequency of the HSC-enriched Lineage- Sca1+ AA4.1+ population (LSA) of E14.5 Fancg-/- FL comforted this later result, since it was 5-fold lower than for WT.In vivo long-term hematopoietic reconstitution (LTR) assays confirmed a deficit of the HSC enriched LSA population of E14.5 Fancg-/- FL. Indeed, although the percentage of mice reconstituted was as good as that obtained with the same number of WT LSA, the CD45 Ly5.2 chimerism was reduced (49±20% vs 84±4% for 1000 LSA injected, and 56±12% vs 87±2% for 5000 LSA). Interestingly, bone marrow analysis of mice reconstituted with Fancg-/- LSA 22 weeks after injection showed a level of CD45 Ly5.2 chimerism 3-fold lower than that found in blood, spleen and thymus, as well as a very low chimerism for myeloid GEM lineages, contrasting with a high chimerism for B and T lymphoid lineages. Moreover, we were able to demonstrate that this deficit is already present at E12.5, both in Fancg-/- FL and Pl. Indeed, no mice reconstituted with 3.105 total Fancg-/- fetal liver cells, while 100% injected with the same number of WT FL cells got reconstituted with a chimerism of 59,5±5%. For Pl, when 500 000 cells were injected, reconstitution was observed in only 1 out of 3 mice for Fancg-/- (29% chimerism), and in 3 out of 3 mice for WT (88±4% chimerism).In human FA FL of 14 weeks of gestation, we also observed a 4-fold defect of HSPC with a total lack of in vitro amplification compared to control, in agreement with the mice data.Taken together, these data demonstrate that a profound deficit of HSCs and progenitors cells is present since the earlier stages of embryonic development in FA. In addition, using organotypic cultures of E11 aortas, we could show that this defect of amplification is already present in HSCs emerging from Fancg-/- aorta, which showed a 2-fold lower rate of amplification compared to WT. More importantly, our results show for the first time exhaustion in myeloid lineage of FA, in agreement with what is observed in children with FA disease. Altogether, our work suggests a role of the FA pathway during the development of the hematopoietic system leading to a deficit of amplification of HSC. Comparison of FA HSC transcriptome with that of control HSC in FL and Pl is in progress. It should allow to identify the key pathways involved in the embryonic HSC amplification that are deregulated in FA, and hopefully getting more insights in the pathogenesis of the BMF and leukemogenesis in FA patients. DisclosuresNo relevant conflicts of interest to declare.

Functional Significance of Nuclear α Spectrin

Nonerythroid alpha spectrin (αIISp) interacts in the nucleus with an array of different proteins indicating its involvement in a number of diverse functions. However, the significance of these interactions and their functional importance has been a relatively unexplored area. The best documented role of nuclear αIISp is in DNA repair where it is critical for repair of DNA interstrand cross-links (ICLs), acting as a scaffold recruiting proteins to sites of damage in genomic and telomeric DNA. A deficiency in αIISp can importantly impact DNA ICL repair as is seen in cells from patients with the genetic disorder, Fanconi anemia (FA), where loss of αIISp leads to not only defects in repair of both genomic and telomeric DNA but also to telomere dysfunction and chromosome instability. This previously unexplored link between αIISp and telomere function is important in developing an understanding of maintenance of genomic stability after ICL damage. In FA cells, these defects in chromosome instability after ICL damage can be corrected when levels of αIISp are returned to normal by knocking down μ-calpain, a protease which cleaves αIISp. These studies suggest a new direction for correcting a number of the phenotypic defects in FA and could serve as a basis for therapeutic intervention. More in depth, examination of the interactions of αIISp with other proteins in the nucleus is of major importance in development of insights into the interacting key elements involved in the diverse processes occurring in the nucleus and the consequences loss of αIISp has on them.

Mutations in the Gene Encoding the E2 Conjugating Enzyme UBE2T Cause Fanconi Anemia

Fanconi anemia (FA) is a rare genetic disorder characterized by genome instability, increased cancer susceptibility, progressive bone marrow failure (BMF), and various developmental abnormalities resulting from the defective FA pathway. FA is caused by mutations in genes that mediate repair processes of interstrand crosslinks and/or DNA adducts generated by endogenous aldehydes. The UBE2T E2 ubiquitin conjugating enzyme acts in FANCD2/FANCI monoubiquitination, a critical event in the pathway. Here we identified two unrelated FA-affected individuals, each harboring biallelic mutations in UBE2T. They both produced a defective UBE2T protein with the same missense alteration (p.Gln2Glu) that abolished FANCD2 monoubiquitination and interaction with FANCL. We suggest this FA complementation group be named FA-T.

Functional Genetic Screen Identifies Increased Sensitivity to WEE1 Inhibition in Cells with Defects in Fanconi Anemia and HR Pathways.

WEE1 kinase regulates CDK1 and CDK2 activity to facilitate DNA replication during S-phase and to prevent unscheduled entry into mitosis. WEE1 inhibitors synergize with DNA-damaging agents that arrest cells in S-phase by triggering direct mitotic entry without completing DNA synthesis, resulting in catastrophic chromosome fragmentation and apoptosis. Here, we investigated how WEE1 inhibition could be best exploited for cancer therapy by performing a functional genetic screen to identify novel determinants of sensitivity to WEE1 inhibition. Inhibition of kinases that regulate CDK activity, CHK1 and MYT1, synergized with WEE1 inhibition through both increased replication stress and forced mitotic entry of S-phase cells. Loss of multiple components of the Fanconi anemia (FA) and homologous recombination (HR) pathways, in particular DNA helicases, sensitized to WEE1 inhibition. Silencing of FA/HR genes resulted in excessive replication stress and nucleotide depletion following WEE1 inhibition, which ultimately led to increased unscheduled mitotic entry. Our results suggest that cancers with defects in FA and HR pathways may be targeted by WEE1 inhibition, providing a basis for a novel synthetic lethal strategy for cancers harboring FA/HR defects.

Metastatic Breast Cancer in a Patient with Fanconi Anemia

Fanconi anemia (FA) is a rare inherited disorder characterized by progressive bone marrow failure, congenital malformations and a propensity for developing malignancies at an early age. The underlying genetic defect in FA creates a state of cellular hypersensitivity to many traditional chemotherapy agents, making the treatment of malignancies in this population particularly challenging.We describe a 42-year-old female who presented with a solitary mass in her left breast. Core biopsy revealed an invasive ductal carcinoma that did not express estrogen (ER) or progesterone receptors (PR), but did express human epidermal growth factor receptor 2 (HER2). Staging work-up revealed diffuse skeletal metastatic disease. At her initial consultation with medical oncology, she was discovered to be pancytopenic. Further history revealed a sibling with aplastic anemia and that she had undergone chromosomal breakage testing for FA in the past, which was subsequently confirmed to be positive. She underwent a bone marrow aspirate and biopsy that showed metastatic marrow infiltration by non-hematopoietic cells. In addition there was morphological evidence of dyserythropoiesis and cytogenetic abnormalities on karyotyping, features suggestive of FA. She was initially started on trastuzumab monotherapy. Low dose radiation therapy was added due to local tumor progression. Combined HER2 directed therapy was to be implemented, but was held due to a functional decline in the patient. To date, she has not received definitive genetic testing to determine which FA subgroup she belongs to.This case highlights two important aspects of FA. The first is the inherent increase in susceptibility to neoplasms in this group, including solid tumors such as breast cancer. The genes associated with FA are involved in deoxyribonucleic acid (DNA) repair pathways, including mutations in the breast cancer susceptibility gene, BRCA2. The second is the heightened sensitivity to the toxic effects of many standard chemotherapy and radiation treatments. This creates unique challenges in the treatment of malignancies in this population and stresses the importance of targeted therapies. DisclosuresNo relevant conflicts of interest to declare.