Fanconi Anemia DNA Repair Research Articles

Proximity Proteomics Reveals USP44 Forms a Complex with BRCA2 in Neuroblastoma Cells and Is Required to Prevent Chromosome Breakage.

The enzyme ubiquitin-specific protease 44 (USP44) is a deubiquitinating enzyme with identified physiological roles as a tumor suppressor and an oncogene. While some binding partners and substrates are known for USP44, the identification of other interactions may improve our understanding of its role in cancer. We therefore performed a proximity biotinylation study that identified products of several known cancer genes that are associated with USP44, including a novel interaction between BRCA2 and USP44. We expressed a fusion protein that linked USP44 and mutant Escherichia coli biotin ligase BioID in SH-SY5Y neuroblastoma cells. Control experiments were performed using BioID alone. In duplicate experiments, cells were pulsed with biotin and biotinylated proteins were isolated under denaturing conditions and the proteins were identified by mass spectrometry. The resulting list of proteins were analyzed using Enrichr and cross-referenced with the COSMIC Cancer Gene Census. We validated the association with BRCA2 using immunoprecipitation. The role of USP44 in the Fanconi anemia DNA repair pathway was investigated using chromosome analysis of wild-type or Usp44-knockout cells after exposure to mitomycin C. We identified 146 proteins that were selectively retrieved by the USP44 construct and compared with cells expressing the BioID ligase alone, including 15 gene products encoded by genes on tier 1 of the COSMIC Cancer Gene Census, including BRCA2. The association between USP44 and BRCA2 was validated through immunoprecipitation. We tested the functional role of USP44 in the Fanconi anemia DNA repair pathway through chromosome breakage analysis and found that cells lacking USP44 had a significant increase in chromosome breaks and radial chromosomes. We found that high BRCA2 transcript was correlated with poor survival in neuroblastoma, likely due to its tight association with proliferation in these tumors. Our results identified novel potential binding partners and potential substrates for USP44, including several with direct roles in cancer pathogenesis. Our results identified a novel association between BRCA2 and USP44, and a previously unknown role for USP44 in the Fanconi anemia DNA repair pathway that may contribute to its role in cancer.

NuRD chromatin remodeling is required to repair exogenous DSBs in the Caenorhabditis elegans germline.

Organisms rely on coordinated networks of DNA repair pathways to protect genomes against toxic double-strand breaks (DSBs), particularly in germ cells. All repair mechanisms must successfully negotiate the local chromatin environment in order to access DNA. For example, nucleosomes can be repositioned by the highly conserved Nucleosome Remodeling and Deacetylase (NuRD) complex. In Caenorhabditis elegans, NuRD functions in the germline to repair DSBs - the loss of NuRD's ATPase subunit, LET-418/CHD4, prevents DSB resolution and therefore reduces fertility. In this study, we challenge germlines with exogenous DNA damage to better understand NuRD's role in repairing DSBs. We find that let-418 mutants are hypersensitive to cisplatin and hydroxyurea: exposure to either mutagen impedes DSB repair, generates aneuploid oocytes, and severely reduces fertility and embryonic survival. These defects resemble those seen when the Fanconi anemia (FA) DNA repair pathway is compromised, and we find that LET-418's activity is epistatic to that of the FA component FCD-2/FANCD2. We propose a model in which NuRD is recruited to the site of DNA lesions to remodel chromatin and allow access for FA pathway components. Together, these results implicate NuRD in the repair of both endogenous DSBs and exogenous DNA lesions to preserve genome integrity in developing germ cells.

Enhanced Genomic Stability in Monomorphic Post-Transplant Lymphoproliferative Disorders Is Driven By Distinct Mechanisms Stratified By EBV Status

Introduction: We previously showed that HIV associated EBV negative (-) diffuse large B-cell lymphomas (DLBCL) of germinal center origins (GCB) have enhanced genomic stability and increased expression of DNA repair genes compared to their HIV negative counterparts (Maguire et al 2019). Given that immune deficiency can be induced virally or therapeutically, including the administration of immunosuppressive drugs post solid organ or hematopoietic stem cell transplant, we questioned whether these observations are specific to HIV or if they represent a more global immune impaired signature. Here we assessed the genomic stability of monomorphic DLBCL post-transplant lymphoproliferative disorders (PTLD) and the potential role of DNA repair. Methods: A total of 49 monomorphic DLBCL PTLDs (26 EBV(-), 23 EBV positive (+)) and 170 immune competent (IC) DLBCL (EBV status unknown) were studied. All were pathologically reviewed and samples with <60% tumor content were macro-dissected before RNA/DNA extraction. Cell of origin (COO) profiles were assessed using the DLBCL90 molecular assay which can distinguish primary mediastinal B-cell lymphoma (PMBL) from GCB, Activated B-cell (ABC) and unclassified (UNC) DLBCL (Ennishi et al 2019). NanoString gene expression profiling (GEP) was performed using the PanCancer Pathways panel and analyzed using NanoString nSolver Advanced Analysis. Copy number (CN) analysis was performed on a subset of samples (45 PTLD, 41 DLBCL) using the OncoScan CN assay and analyzed using Chromosome Analysis Suite (ChAS) v4.1. All p-values listed were Benjamini-Hochberg adjusted and Mann-Whitney tested for the GEP and CN data respectively. Results: In contrast to IC-DLBCL which are GCB enriched (50%), COO profiling revealed PTLD to be ABC enriched (43%). CN analyses revealed that the median CN/sample for PTLD (0.57, p<0.001), EBV(+)PTLD (0.25, p<0.001) and EBV(-)PTLD (0.80, p = 0.050) tumors were significantly less aberrant than IC-DLBCL (1.01). The data also showed that EBV(+)PTLD are less aberrant than EBV(-)PTLD tumors (0.25 vs 0.80, p<0.001). GEP pathway analysis identified DNA repair as downregulated in PTLD and EBV(+)PTLD but upregulated in EBV(-)PTLD compared IC-DLBCL. Nine DNA repair genes ( RAD51, CCNA2a, CHEK1b, POLR2Da, PCNAa, WHSC1a, GTF2H3a, ATM c, EP300 c) were differentially expressed (DE) in EBV(-)PTLD compared to IC-DLBCL (7 increased, 2 decreased, p ≤ 0.0425): where the most overexpressed gene RAD51, a critical mediator of Homologous Recombination (HR) and the Fanconi Anemia (FA) DNA repair pathways, was not DE in PTLD or EBV(+)PTLD compared to IC-DLBCL. Five a genes were increased in PTLD (p ≤ 0.0204) but not EBV(+)PTLD, 1 b was decreased in EBV(+)PTLD (p = 0.01) but not PTLD and 2 c were reduced in PTLD regardless of EBV status compared to IC-DLBCL (p ≤ 0.0024). Twenty-8 genes were DE in EBV(+)PTLD compared to IC-DLBCL. Of these, 23 were downregulated (p ≤ 0.0418) and included HR, FA and mismatch repair genes ( CHEK1, CHEK2, BRAC1/FANCS, FANCA, FANCB, FANCE, MSH2, MSH6). All but 2/23 were similarly expressed in PTLD compared to IC-DLBCL of which 11 were significant (p ≤ 0.0356). The five upregulated genes ( ZBTB32, DDB2, NBN, POLB, RBX1, p ≤ 0.0092) were also upregulated in PTLD (p ≤ 0.0042) but not EBV(-)PTLD compared to IC-DLBCL. Two additional genes ( SKP1, FBXW7) were also identified as increased in EBV(+)PTLD and PTLD (p ≤ 0.0204) compared to IC-DLBCL. In contrast, SKP1 was increased (p = 2.7E-4) and FBXW7 was decreased (p = 0.3610) in EBV(-)PTLD compared to IC-DLBCL. Together the protein products of RBX1, SKP1 and FBXW7 comprise the functional cores of a complete UPS complex, the SCF FBXW7 E3 ubiquitin ligase which have been reported to regulate the DNA damage response and promote genomic stability. Conclusions: Like HIV associated DLBCL, these results show that PTLD is more genomically stable than IC-DLBCL, and that genomic stability is further enhanced in EBV(+)PTLD which have increased expression of UPS related genes. In contrast, EBV(-)PTLD have increased expression of DNA damage repair genes, including RAD51. In summary, monomorphic DLBCL PTLD tumors have enhanced genomic stability that appears to be mediated by two distinct mechanisms stratified by EBV status. Further work is required to assess if this stratification of mechanisms is unique to PTLD or reflects a more global immune impaired EBV related signature.

Multiple cancers escape from multiple MAPK pathway inhibitors and use DNA replication stress signaling to tolerate aberrant cell cycles.

Many cancers harbor pro-proliferative mutations of the mitogen-activated protein kinase (MAPK) pathway. In BRAF-driven melanoma cells treated with BRAF inhibitors, subpopulations of cells escape drug-induced quiescence through a nongenetic manner of adaptation and resume slow proliferation. Here, we found that this phenomenon is common to many cancer types driven by EGFR, KRAS, or BRAF mutations in response to multiple, clinically approved MAPK pathway inhibitors. In 2D cultures and 3D spheroid models of various cancer cell lines, a subset of cells escaped drug-induced quiescence within 4 days to resume proliferation. These "escapee" cells exhibited DNA replication deficits, accumulated DNA lesions, and mounted a stress response that depended on the ataxia telangiectasia and RAD3-related (ATR) kinase. We further identified that components of the Fanconi anemia (FA) DNA repair pathway are recruited to sites of mitotic DNA synthesis (MiDAS) in escapee cells, enabling successful completion of cell division. Analysis of patient tumor samples and clinical data correlated disease progression with an increase in DNA replication stress response factors. Our findings suggest that many MAPK pathway-mutant cancers rapidly escape drug action and that suppressing early stress tolerance pathways may achieve more durable clinical responses to MAPK pathway inhibitors.

Genotoxic aldehyde stress prematurely ages hematopoietic stem cells in a p53-driven manner

Aged hematopoietic stem cells (HSCs) display diminished self-renewal and a myeloid differentiation bias. However, the drivers and mechanisms that underpin this fundamental switch are not understood. HSCs produce genotoxic formaldehyde that requires protection by the detoxification enzymes ALDH2 and ADH5 and the Fanconi anemia (FA) DNA repair pathway. We find that the HSCs in young Aldh2-/-Fancd2-/- mice harbor a transcriptomic signature equivalent to aged wild-type HSCs, along with increased epigenetic age, telomere attrition, and myeloid-biased differentiation quantified by single HSC transplantation. In addition, the p53 response is vigorously activated in Aldh2-/-Fancd2-/- HSCs, while p53 deletion rescued this aged HSC phenotype. To further define the origins of the myeloid differentiation bias, we use a GFP genetic reporter to find a striking enrichment of Vwf+ myeloid and megakaryocyte-lineage-biased HSCs. These results indicate that metabolism-derived formaldehyde-DNA damage stimulates the p53 response in HSCs to drive accelerated aging.

Abstract 6100: The Fanconi Anemia pathway protein complex FANCI/FANCD2 couples the DNA damage response to R-loop regulation through SRSF1-mediated mRNA export

Abstract Background: Fanconi Anemia (FA) is a rare genetic disease characterized by bone marrow failure and cancer susceptibility. FA gene mutations are also widespread somatically in non-FA cancer patients. While the FA pathway participates in DNA repair, it is also linked to R-loop metabolism and regulation. R-loops are co-transcriptional RNA:DNA hybrids that form as a result of replication-transcription collisions that are enhanced due to DNA damage, but their aberrant formation drives genomic instability and is an endogenous source of genotoxicity. However, the interplay between FA proteins, DNA repair, and R-loops remains mechanistically ill-defined. Disturbance of mRNA processing, export, and splicing has also been linked to R-loop-mediated genomic instability. The splicing factor SRSF1 participates in mRNA export through the Nuclear Export Factor 1 (NXF1). Aberrant splicing factors expression has been linked to R-loops, cancer, and myelodysplastic syndrome (MDS), which clinically resembles FA. In the present study, we study the coupling of the DNA damage response to the prevention of pathogenic R-loops via mRNA export regulation through the FA pathway and SRSF1. Methods: Cells: FA-D2 mutant, corrected, and ubiquitin-dead mutant; HeLa. siRNA transfections to reduce SRSF1 and FANCD2 protein levels. Cell survival assays against DNA damaging agents. In vitro immunoprecipitation and ubiquitination assays using purified SRSF1 and ID2. DART assay, S9.6 immunofluorescence, and S9.6 slot-blot to measure R loops in cells. Immunoprecipitation of mRNPs used to detect mature mRNA-bound ribonucleoproteins. RNA FISH to measure mRNA export. Results: In this study, we provide strong evidence that the FA pathway coordinates the prevention of R-loop formation and subsequent genomic instability through its interaction with splicing factors via regulation of mRNA export. First, we demonstrate that knockdown of SRSF1, which is known to result in increased R-loops, also is associated with diminished activation of the FA pathway. Second, the central FA complex ID2 binds to SRSF1 in cells and in recombinant protein assays. Further, we demonstrate that SRSF1 activates the FA pathway by binding ID2 and stimulates its monoubiquitination in an RNA-dependent fashion. This activity is associated with co-localization in cells at transcriptional-DNA damage inducible R loops. In turn, FANCD2 monoubiquitination proves crucial for the assembly of the SRSF1-NXF1 nuclear export complex and mRNA export. Importantly, cancer-associated SRSF1 mutants fail to interact with FANCD2, leading to inefficient FANCD2 monoubiquitination, decreased mRNA export and R-loops accumulation. Conclusion: Our findings uncover a novel mechanism of FANCD2 and SRSF1 in the prevention of R-loop formation via coupling of mRNA export to the DNA damage response, thus preventing genomic instability. Citation Format: Anne Olazabal-Herrero, Fengshan Liang, Arijit Dutta, Yuxin Huang, Zhuobin Liang, Abhishek Gupta, Yaqun Teng, Li Lan, Xiaoyong Chen, Huadong Pei, Manoj Pillai, Patrick Sung, Gary Kupfer. The Fanconi Anemia pathway protein complex FANCI/FANCD2 couples the DNA damage response to R-loop regulation through SRSF1-mediated mRNA export [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6100.

Abstract PD14-03: PD14-03 Reappraising the Fanconi Anemia DNA repair pathway in breast cancer risk and precision intervention: Insights and opportunities from the City of Hope INSPIRE study

Abstract Background: Fanconi Anemia (FA) proteins facilitate homologous recombination (HR)-mediated repair of DNA interstrand cross-links. Germline monoallelic, pathogenic/likely pathogenic (P/LP) variants in the highly-penetrant (HP) breast cancer (BC) FA genes, BRCA1 (FANCS), BRCA2 (FANCD1) and PALB2 (FANCN)), compromise HR and predispose to hereditary BC. The effects of monoallelic, pathogenic variants in other non-HP BC FA genes upon HR and BC predisposition remain less understood. In this investigation we report the germline mutational landscape of FA gene P/LP variants and somatic molecular consequences of patients with BC diagnoses from City of Hope’s (COH) INSPIRE (Implementing Next-generation Sequencing for Precision Intervention and Risk Evaluation) study. Methods: COH-INSPIRE is a universal access study open to all patients at COH with a personal and/or family history of cancer. Patients undergo custom panel-based germline genetic testing to detect P/LP single nucleotide variants (SNVs), short insertions/deletions (indels) and exon-level deletions/duplications in 155 cancer-predisposition genes including the HP BC FA genes and 15 non-HP BC FA genes [FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ (BRIP1), FANCL, FANCM, FANCO (RAD51C), FANCP (SLX4), FANCQ (ERCC4) and FANCU (XRCC2)]. Patients’ tumor specimens undergo somatic tumor (>400X)-normal (>180X) whole exome and transcriptome sequencing (>50 million reads). Somatic sequencing identifies P/LP SNVs, indels, copy number events, and fusions. Secondary analyses assessed somatic homologous recombination deficiency (HRD) by examining tumor mutational signatures, as well as an ensemble HRD score derived by combining individual genomic loss of heterozygosity, telomeric allelic imbalance and large-scale molecular transition scores. Reference comparison of germline and somatic features to current FDA therapeutic guidelines and NIH clinical trials registrations determined eligibility for precision therapeutic intervention and clinical trial enrollment. Results: Of 7,584 patients enrolled in COH-INSPIRE, 1,651 (21.8%) patients had a BC diagnosis. Germline panel testing of BC patients identified 204 (12.4%) with germline P/LP variant in a FA gene. Greater than one third of FA gene-altered BC patients (37.7%) carried a P/LP variant in a non-HP BC FA gene. We observed that BC patients with a non-HP BC FA gene variant may demonstrate HR compromise as evidenced by presence of a Signature 3 mutational profile or an elevated combined HRD score (> 33 and/or > 42). (Table 1) Further, we identified ostensible segregation of triple negative BC in a family harboring a germline pathogenic variant in FANCG. With regard to precision clinical actionability (i.e. qualification for targeted therapeutic intervention [PARP inhibitor (PARPi)] and/or clinical trial) for patients with advanced stage BC: All patients with germline P/LP HP BC FA gene variant and 20.7% (N=16) of patients with a P/LP FA non-HPBC FA gene variant met criteria for treatment with on/off-label PARPi. 100% of patients with advanced BC with germline P/LP HP BC or non-HPBC FA gene variant qualified for a clinical trial. Conclusions: Patients with BC often carry a germline monoallelic, P/LP FA gene variant; in more than one third, the FA gene alteration occurs in a non-HP BC FA gene. BC patients harboring a monoallelic germline non-HP BC P/LP FA gene may exhibit somatic mutational signatures and HRD scoring consistent with compromise of HR. Somatic tumor evaluation of BC patients with germline P/LP non-HP BC FA gene variants expands opportunities for precision therapeutic intervention and clinical trial enrollment. Continued appraisal will clarify emerging questions of germline non-HP P/LP FA gene-associated autosomal dominant BC risk and management as well as facilitate optimization of precision BC care. Table 1 Summary Molecular Features of BC patients with P/LP Variants in FA gene from COH-INSPIRE Citation Format: Laura Kruper, Kevin McDonnell, Joseph Bonner, Kevin K. Tsang, Veronica Jones, Joanne Mortimer, Sidney S. Lindsey, Ilana Solomon, Heather Hampel, Wai Park, Gregory E. Idos, Stacy Gray, Stephen Gruber. PD14-03 Reappraising the Fanconi Anemia DNA repair pathway in breast cancer risk and precision intervention: Insights and opportunities from the City of Hope INSPIRE study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD14-03.

Genome-wide siRNA screens identify RBBP9 function as a potential target in Fanconi anaemia-deficient head-and-neck squamous cell carcinoma

Fanconi anaemia (FA) is a rare chromosomal-instability syndrome caused by mutations of any of the 22 known FA DNA-repair genes. FA individuals have an increased risk of head-and-neck squamous-cell-carcinomas (HNSCC), often fatal. Systemic intolerance to standard cisplatin-based protocols due to somatic-cell hypersensitivity underscores the urgent need to develop novel therapies. Here, we performed unbiased siRNA screens to unveil genetic interactions synthetic-lethal with FA-pathway deficiency in FA-patient HNSCC cell lines. We identified based on differential-lethality scores between FA-deficient and FA-proficient cells, next to common-essential genes such as PSMC1, PSMB2, and LAMTOR2, the otherwise non-essential RBBP9 gene. Accordingly, low dose of the FDA-approved RBBP9-targeting drug Emetine kills FA-HNSCC. Importantly both RBBP9-silencing as well as Emetine spared non-tumour FA cells. This study provides a minable genome-wide analyses of vulnerabilities to address treatment challenges in FA-HNSCC. Our investigation divulges a DNA-cross-link-repair independent lead, RBBP9, for targeted treatment of FA-HNSCCs without systemic toxicity.

Genomic signature of Fanconi anaemia DNA repair pathway deficiency in cancer.

Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage1-3. The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes4-7. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas8 (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus9 (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.

Genome-Wide CRISPR Screen Reveals Dependency of PPM1D-mutant Cells on DNA Repair

PPM1D (Protein Phosphatase Mg2+/Mn2+ Dependent 1D) is a protein phosphatase frequently mutated in clonal hematopoiesis (CH), therapy-related myeloid neoplasms (t-MN), and solid tumors. PPM1D is best known for its role as a negative regulator of the DNA damage response. Mutations in the terminal exon of PPM1D are gain-of-function and lead to a truncated, dominant form of PPM1D with preserved phosphatase activity. This stabilized, mutant protein leads to persistent dephosphorylation of PPM1D-targets including p53 and other key DNA damage response proteins. Our lab previously demonstrated that mutant Ppm1d hematopoietic stem and progenitor cells (HSPCs) outcompete their wild type (WT) counterparts in vivo following bone marrow transplantation and repeated cisplatin exposure. Given the prevalence of PPM1D mutations in CH and cancer, PPM1D is an attractive therapeutic target and ongoing efforts are underway to develop a small molecular inhibitor. We have sought to identify genetic vulnerabilities of PPM1D mutant cells by performing a whole-genome CRISPR/Cas9 dropout screen in PPM1D WT and mutant leukemia cell lines. Analysis of gene candidates that were essential for mutant cell survival, but not WT, showed an enrichment for genes involved in DNA repair including the Fanconi Anemia (FA) pathway genes. Given the role of PPM1D in the inhibition of p53 and the DNA damage response, we hypothesized that mutant PPM1D cells have increased genomic instability and therefore, rely on the non-canonical FA DNA repair pathway for survival. Indeed, we have found that PPM1D mutant cells harbored more single- and double-stranded DNA breaks as well as chromosomal aberrations both without external stressors and after cisplatin treatment compared to their WT counterparts. We have also found that PPM1D mutant cells exhibit significantly more FANCD2 foci at baseline compared to WT cells, suggesting increased activation of the FA pathway. In addition to interstrand crosslink repair, the FA pathway also plays a critical role in replication fork protection during replication stress to prevent the accumulation of toxic double-stranded breaks. To assess replication stress and kinetics in WT and PPM1D mutant cells, we performed DNA fiber assays to visualize nascent DNA synthesis at the single-molecule resolution. Surprisingly, in comparison to WT cells, PPM1D mutant cells had increased replication fork speed. These results demonstrate that PPM1D mutant cells have more genome instability and activate the FA pathway for DNA repair and replication progression. Increased DNA damage often results in increased mutagenesis and accumulation of single-base substitutions. To validate our cell line findings and determine if Ppm1d-mutant HSPCs harbor more mutations, we performed whole-genome sequencing on WT and Ppm1d HSPCs from single-cell derived methocult colonies. Surprisingly, we did not find a significant increase in the overall mutation burden in the Ppm1d-mutant colonies compared to WT. This suggests that while Ppm1d-mutant cells have more genomic instability, the fidelity of repair is maintained. This may be due to a delay in the recruitment of DNA repair factors. Overall, our work identifies genetic candidates that give rise to opportunities for synthetic lethal approaches to target PPM1D-mutant clonal expansion in patients with PPM1D-mutated malignancies and those at high-risk for developing t-MN.

A phase II study evaluating the efficacy of niraparib and dostarlimab (TSR-042) in recurrent/metastatic head and neck squamous cell carcinoma.

TPS6105 Background: Despite improvement in outcomes with checkpoint (PD-1) inhibitors in some patients (pts) with recurrent and metastatic head and neck squamous cell carcinoma (RM HNSCC), overall survival (OS) remains poor, necessitating novel therapy combinations. Fanconi Anemia (FA) and FA-related DNA repair pathways alterations have been associated with cumulative tumor mutational burden (TMB) due to genomic instability. High TMB is associated with improved response to PD-1 inhibitors. DNA pathway repair mutations have been reported in 17% of sporadic HNSCC. Ataxia-Telangiesctasia Mutated (ATM) loss has been reported in 60% of human HNSCC biopsies and FA-related defects were reported in 15-21% of human HNSCC biopsies and cell lines. Poly (ADP-ribose) polymerase (PARP) inhibition has already demonstrated efficacy as a single agent in multiple cancers which harbor a DNA repair defect. Preclinical data also showed PARP inhibitor (PARPi) upregulates PD-L1 expression in cancer lines and animal models via inactivation of glycogen synthase kinase 3 (GSK3β). Importantly, blockade of PD-1 re-sensitized PARPi treated cells to induce T-cell mediated cytotoxicity. Combination of PARPi and anti-PD-1 therapy significantly increased therapeutic efficacy in vivo compared to single agent. Therefore, we hypothesized that the combination of a PARPi (niraparib) and PD-1 blockade (dostarlimab) in our ongoing phase II study would enhance overall response (OR) and survival in R/M HNSCC pts (NCT04313504). Methods: Eligible pts must have a confirmed histopathology of non-cutaneous RM HNSCC not amenable to local therapy, progressive disease (PD) after at least one line of platinum-based chemotherapy and/or immunotherapy, age>18, ECOG <2, no prior therapy with PARPi and adequate bone marrow, liver, and renal function. Seven of 14 pts have been enrolled in the first stage of the study. If 8 or more pts develop either stable disease (SD), partial (PR) or complete response (CR), 9 additional patients will be accrued for a total of 23 patients. Pts will receive 200 mg PO niraparib daily in 28-day cycles along with dostarlimab 500mg (Q3W) for 4 doses followed by 1000mg (Q6W) until PD or unacceptable toxicity. An interim analysis will be performed to assess efficacy and safety after 14 patients become evaluable. Tumor response will be assessed per RECIST v1.1. Primary endpoint is best OR including SD, PR, and CR; Secondary endpoints include adverse events determined by CTCAE v5.0, progression free survival and OS. Integrated tissue analysis includes DNA pathway repair defects and TMB. Additional exploratory analyses including peripheral and immune cell activation will be performed on tissue and plasma samples. Clinical trial information: NCT04313504.

Fanconi anemia: current insights regarding epidemiology, cancer, and DNA repair.

Fanconi anemia is a genetic disorder that is characterized by bone marrow failure, as well as a predisposition to malignancies including leukemia and squamous cell carcinoma (SCC). At least 22 genes are associated with Fanconi anemia, constituting the Fanconi anemia DNA repair pathway. This pathway coordinates multiple processes and proteins to facilitate the repair of DNA adducts including interstrand crosslinks (ICLs) that are generated by environmental carcinogens, chemotherapeutic crosslinkers, and metabolic products of alcohol. ICLs can interfere with DNA transactions, including replication and transcription. If not properly removed and repaired, ICLs cause DNA breaks and lead to genomic instability, a hallmark of cancer. In this review, we will discuss the genetic and phenotypic characteristics of Fanconi anemia, the epidemiology of the disease, and associated cancer risk. The sources of ICLs and the role of ICL-inducing chemotherapeutic agents will also be discussed. Finally, we will review the detailed mechanisms of ICL repair via the Fanconi anemia DNA repair pathway, highlighting critical regulatory processes. Together, the information in this review will underscore important contributions to Fanconi anemia research in the past twodecades.

The RPA inhibitor HAMNO sensitizes Fanconi anemia pathway-deficient cells

ABSTRACT The Fanconi anemia (FA) DNA repair pathway is required for DNA inter-strand crosslink (ICL) repair. Besides its role in ICL repair, FA proteins play a central role in stabilizing stalled replication forks, thereby ensuring genome integrity. We previously demonstrated that depletion of replication protein A (RPA) induces the activation of FA pathway leading to FANCD2 monoubiquitination and FANCD2 foci formation. Thus, we speculated that FA-deficient cells would be more sensitive to RPA inhibition compared to FA-proficient cells. Following treatment with RPA inhibitor HAMNO, we observed significant induction in FANCD2 monoubiquitination and foci formation as observed in RPA depletion. In addition, HAMNO treatment caused increased levels of γ-H2AX and S-phase accumulation in FA-deficient cells. Importantly, FA-deficient cells showed more increased sensitivity to HAMNO than FA-proficient cells. Moreover, in combination with cisplatin, HAMNO further enhanced the cytotoxicity of cisplatin in FA-deficient cells, while being less toxic against FA-proficient cells. This result suggests that RPA inhibition might be a potential therapeutic candidate for the treatment of FA pathway-deficient tumors.

HIV-1 exploits the Fanconi anemia pathway for viral DNA integration.

SUMMARYThe integration of HIV-1 DNA into the host genome results in single-strand gaps and 2-nt overhangs at the ends of viral DNA, which must be repaired by cellular enzymes. The cellular factors responsible for the DNA damage repair in HIV-1 DNA integration have not yet been well defined. We report here that HIV-1 infection potently activates the Fanconi anemia (FA) DNA repair pathway, and the FA effector proteins FANCI-D2 bind to the C-terminal domain of HIV-1 integrase. Knockout of FANCI blocks productive viral DNA integration and inhibits the replication of HIV-1. Finally, we show that the knockout of DNA polymerases or flap nuclease downstream of FANCI-D2 reduces the levels of integrated HIV-1 DNA, suggesting these enzymes may be responsible for the repair of DNA damages induced by viral DNA integration. These experiments reveal that HIV-1 exploits the FA pathway for the stable integration of viral DNA into host genome.

A functionally impaired missense variant identified in French Canadian families implicates FANCI as a candidate ovarian cancer-predisposing gene

BackgroundFamilial ovarian cancer (OC) cases not harbouring pathogenic variants in either of the BRCA1 and BRCA2 OC-predisposing genes, which function in homologous recombination (HR) of DNA, could involve pathogenic variants in other DNA repair pathway genes.MethodsWhole exome sequencing was used to identify rare variants in HR genes in a BRCA1 and BRCA2 pathogenic variant negative OC family of French Canadian (FC) ancestry, a population exhibiting genetic drift. OC cases and cancer-free individuals from FC and non-FC populations were investigated for carrier frequency of FANCI c.1813C>T; p.L605F, the top-ranking candidate. Gene and protein expression were investigated in cancer cell lines and tissue microarrays, respectively.ResultsIn FC subjects, c.1813C>T was more common in familial (7.1%, 3/42) than sporadic (1.6%, 7/439) OC cases (P = 0.048). Carriers were detected in 2.5% (74/2950) of cancer-free females though female/male carriers were more likely to have a first-degree relative with OC (121/5249, 2.3%; Spearman correlation = 0.037; P = 0.011), suggesting a role in risk. Many of the cancer-free females had host factors known to reduce risk to OC which could influence cancer risk in this population. There was an increased carrier frequency of FANCI c.1813C>T in BRCA1 and BRCA2 pathogenic variant negative OC families, when including the discovery family, compared to cancer-free females (3/23, 13%; OR = 5.8; 95%CI = 1.7–19; P = 0.005). In non-FC subjects, 10 candidate FANCI variants were identified in 4.1% (21/516) of Australian OC cases negative for pathogenic variants in BRCA1 and BRCA2, including 10 carriers of FANCI c.1813C>T. Candidate variants were significantly more common in familial OC than in sporadic OC (P = 0.04). Localization of FANCD2, part of the FANCI-FANCD2 (ID2) binding complex in the Fanconi anaemia (FA) pathway, to sites of induced DNA damage was severely impeded in cells expressing the p.L605F isoform. This isoform was expressed at a reduced level, destabilized by DNA damaging agent treatment in both HeLa and OC cell lines, and exhibited sensitivity to cisplatin but not to a poly (ADP-ribose) polymerase inhibitor. By tissue microarray analyses, FANCI protein was consistently expressed in fallopian tube epithelial cells and only expressed at low-to-moderate levels in 88% (83/94) of OC samples.ConclusionsThis is the first study to describe candidate OC variants in FANCI, a member of the ID2 complex of the FA DNA repair pathway. Our data suggest that pathogenic FANCI variants may modify OC risk in cancer families.

Regulation of the Fanconi Anemia DNA Repair Pathway by Phosphorylation and Monoubiquitination.

The Fanconi anemia (FA) DNA repair pathway coordinates a faithful repair mechanism for stalled DNA replication forks caused by factors such as DNA interstrand crosslinks (ICLs) or replication stress. An important role of FA pathway activation is initiated by monoubiquitination of FANCD2 and its binding partner of FANCI, which is regulated by the ATM-related kinase, ATR. Therefore, regulation of the FA pathway is a good example of the contribution of ATR to genome stability. In this short review, we summarize the knowledge accumulated over the years regarding how the FA pathway is activated via phosphorylation and monoubiquitination.

Novel Ubiquitinated Proteins Downstream of the Fanconi Anemia Core Complex

The Fanconi anemia (FA) pathway is a major player in the control of DNA replication integrity in response to replication stress. Germline defect in the pathway results in the FA syndrome characterized by developmental abnormalities, bone marrow (BM) failure, and genome instability which greatly elevates the incidence of cancers. A pivotal step in the activation of the FA DNA repair pathway is the monoubiquitination of the FANCD2 and FANCI proteins (ID2) by the FA core complex, a unique ubiquitin ligase complex which includes eight proteins (FANCA-FANCG, FANCL, and FAAP100) and UBE2T/FANCT. This monoubiquitination event enables the recruitment of the ID2 complex to chromatin and nuclear foci at sites of DNA damage. Cells with mutations in any of the FA core complex proteins lack the ability to monoubiquitinated ID2, making ID2 ubiquitination a convergence point in the pathway, with an estimation of>90% FA patients defective in this step. Additionally, somatic mutations In FA genes render tumor cells sensitive to DNA crosslinking agents, so identification of FA pathway defects provides an opportunity for therapeutic targeting. In search for additional potential target/substrate of this unique FA core ubiquitin ligase complex, we performed a high throughput genome-wide ubiquitin-specific proteomics (UbiScan) screen and found, in addition to the ID2 complex, many ubiquitinated proteins are dysregulated (mostly downregulated) in FA deficient cells compared with that of FA proficient cells.We used a Ubiquitin Remnant Motif (K- ∑-GG) Antibody Bead Conjugate (Cell Signaling Technology), a proprietary ubiquitin branch (“K- ∑-GG”) antibody with specificity for a di-glycine tag that is the remnant of ubiquitin left on protein substrates after trypsin digestion, to enrich ubiquitinated peptides from trypsin digested cell samples (shNT vs shFANCA). This enrichment is followed by LC-MS/MS analysis for quantitative profiles of hundreds to over a thousand nonredundant ubiquitinated sequences. We were successful in demonstrating that under steady-state conditions (without proteasome inhibitor treatment), the ubiquitinated forms of both FANCD2 and FANCI proteins are much higher in control (shNT) HeLa cells compared with that of the cells depleted of FANCA (shFANCA).We then collaborated with the Cell signaling technology to perform a high throughput UbiScan® analysis of total ubiquitinated proteins both in total nuclei and chromatin fractions under replicative stress conditions. UbiScan® enables researchers to isolate, identify and quantitate large numbers of ubiquitin-modified cellular peptides with a high degree of specificity and sensitivity, providing a global overview of the ubiquitination sites in cellular proteins in cell and tissue samples without preconceived biases about where these modified sites occur. A total of 16,249 redundant modified peptide assignments to 7,856 modified sites for the Ubiquitin K-GG Remnant Motif Antibody were obtained. As expected, the amount of monoubiquitinated FANCD2 (at K651) and FANCI (at K523) were highly reduced in both the nuclear and chromatin fractions of Hela cells depleted of FANCA (shA). Consistent with the earlier findings, the amount of ubiquitinated ID2 proteins were extremely low in the chromatin fraction of the Hela cells depleted of FANCA.Since there are numerous ubiquitinated proteins found to be dysregulated in our UbiScan analyses, we used the following criteria to select the target proteins based on; a) -fold changes, and b) proteins that are known to participate in the DNA repair signaling pathways. We validated our UbiScan results by using an assay system to detect endogenous protein ubiquitination. We also found a significant reduction in the ubiquitination of several DNA repair-related proteins (found in our UbiScan analysis) in FANCA deficient cells. To assess FA pathway functions, we generated HAP1 and appropriate cells knock out of these select ubiquitinated target proteins by using CRISPR-Cas9 system. Then, the KO cells were examined for FA pathway functions. These results will be discussed.In conclusion, our findings reveal that the FA core ubiquitin ligase complex regulates (directly or indirectly) the ubiquitinated levels of many novel proteins outside of the ID2 complex, and these novel target proteins may provide important additional mechanistic insights into the FA DNA repair pathway. DisclosuresNo relevant conflicts of interest to declare.

Fanci-FANCD2 Promotes Genome Stability and DNA Repair By Down-Regulating BLM Helicase Activity

Background: Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure, developmental defects, and higher risk of cancer. Mutations in FA genes have been detected commonly in a large swath of cancers. In the FA DNA repair pathway, DNA damage induces the mono-ubiquitination of the FANCI-FANCD2 (ID2) heterodimer and this regulation licenses the execution of downstream DNA damage signaling and repair steps. In response to replication stress, FANCD2 also prevents replication fork collapse during S phase. Bloom syndrome (BS) is also a genomic instability disease, characterized by growth abnormalities and cancer predisposition. The single BS protein, BLM helicase, participates in DNA repair by promoting DNA end resection and double Holliday junction dissolution. It has been shown that BLM is involved in restart of stalled replication fork. FA and BS have functional interactions. In tumor DNA sequencing of the Yale Precision Tumor board, we identified a somatic 6 amino acid deletion in FANCD2 in a head and neck tumor, while a germline point mutation was found on the other allele. We have identified a FANCD2-L822A mutant with defective BLM binding, which was used to further investigate the role of FANCD2-BLM interaction in genome stability and DNA repair.Methods: Highly purified proteins were used to investigate how ID2 affects helicase and DNA end resection activity of the BLM complex. HeLa, FANCD2-deficient, and FANCD2 corrected fibroblast cell lines were used to examine pRPA2 and RAD51 foci formation. We also used DNA fiber assay to detect end resection and isolation of proteins on nascent DNA (iPOND) assay to examine the RAD51 recruitment on replication fork.Results: A somatic 6 amino acid deletion (p819-824) in FANCD2 was identified in a head and neck tumor. FA-D2 mutant cells expressing the mutant cDNA demonstrated defects in FANCD2 mono-ubiquitination and DNA damage hypersensitivity. A FANCD2-L822A mutant with defective BLM binding was identified (Figure A, B). We found that Bloom helicase and its DNA end resection activity within BLM-DNA2-RPA were negatively regulated by the heterodimer ID2 (Figure C, D). Both DNA and BLM binding of the ID2 are required for the inhibitory function. The premature DNA end resection and HU sensitivity in FANCD2 deficient and mutant cells are rescued by BLM knockdown. By iPOND assay, we discovered that FANCD2 antagonizes BLM to promote RAD51 recruitment on HU-stalled replication fork.Conclusions: Our study suggests that the DNA end resection activity of BLM-DNA2 is tightly regulated by FANCD2 to ensure that the nuclease DNA2 normally resects the DNA intermediate needed for efficient DNA repair and RAD51 recruitment to protect replication forks. Our findings highlight that ID2-BLM interaction functions in DNA damage repair to maintain genome stability. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

FAN1's protection against CGG repeat expansion requires its nuclease activity and is FANCD2-independent.

The Repeat Expansion Diseases, a large group of human diseases that includes the fragile X-related disorders (FXDs) and Huntington's disease (HD), all result from expansion of a disease-specific microsatellite via a mechanism that is not fully understood. We have previously shown that mismatch repair (MMR) proteins are required for expansion in a mouse model of the FXDs, but that the FANCD2 and FANCI associated nuclease 1 (FAN1), a component of the Fanconi anemia (FA) DNA repair pathway, is protective. FAN1's nuclease activity has been reported to be dispensable for protection against expansion in an HD cell model. However, we show here that in a FXD mouse model a point mutation in the nuclease domain of FAN1 has the same effect on expansion as a null mutation. Furthermore, we show that FAN1 and another nuclease, EXO1, have an additive effect in protecting against MSH3-dependent expansions. Lastly, we show that the loss of FANCD2, a vital component of the Fanconi anemia DNA repair pathway, has no effect on expansions. Thus, FAN1 protects against MSH3-dependent expansions without diverting the expansion intermediates into the canonical FA pathway and this protection depends on FAN1 having an intact nuclease domain.

Impact of Tumor Treating Fields (TTFields) on DNA Damage Repair in Mesothelioma

Tumor Treating Fields (TTFields) are low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields delivered to solid tumors noninvasively and locoregionally. TTFields have demonstrated a promising median overall survival in patients with malignant pleural mesothelioma (MPM), an aggressive thoracic cancer with poor prognosis, without increases in systemic toxicity (STELLAR clinical trial). Consequently, TTFields therapy combined with pemetrexed and a platinum-based chemotherapy agent are approved as first line treatment for unresectable MPM in the US and Europe. While efficacy of TTFields for MPM treatment is well-established, the underlying mechanism of action needs further elucidation. This study investigated the TTFields effect on DNA damage and repair in MPM and tested the combination of TTFields with DNA damaging agents such as cisplatin and pemetrexed, in vitro and in vivo.TTFields frequencies ranging from 100-400 kHz were applied to human MPM cell lines (NCI-H2052 and MSTO-211H) to identify the most effective frequency. To detect DNA double strand breaks (DSB), the effect of optimal TTFields frequency on the formation of ɣH2AX foci was examined by fluorescent microscopy. Levels of DNA damage repair related proteins were evaluated via immunoblotting of cell lysates. The combined cytotoxic effect of TTFields with cisplatin or pemetrexed was tested in vitro. Efficacy of TTFields concomitant with both cisplatin and pemetrexed was examined in C57BL/6 mice inoculated subcutaneously with RN-5 cells by measuring tumor volume and DNA damage within the tumor.The optimal TTFields frequency was identified as 150 kHz in both MPM cell lines, displaying significant cytotoxicity and formation of DNA DSB. These effects were accompanied by increased expression of p21 and p27, proteins involved in DNA damage-induced cell cycle arrest, and reduced expression of proteins from the Fanconi Anemia (FA) DNA repair pathway - FANCA, FANCD2, FANCJ, and BRCA1. Co-treatment of TTFields with cisplatin or pemetrexed augmented efficacy versus each treatment alone, with the TTFields-pemetrexed combination displaying an additive effect, and the co-administration of TTFields with cisplatin demonstrating synergistic interaction. In vivo, tumor volume fold change was significantly decreased for the combination of TTFields with chemotherapy (cisplatin + pemetrexed) versus control, while levels of DNA damage within the tumor were increased.Efficacy of TTFields for the treatment of MPM is associated with increased DNA damage, elevated levels of DNA-damage related cell cycle arrest proteins, and reduced expression of FA pathway proteins. This latter effect may account for the synergistic interaction displayed for the TTFields-cisplatin combination, as cisplatin is known to cause DNA damage that requires the FA pathway for repair.