Fanconi Anemia Genes Research Articles

Research progress of the Fanconi anemia pathway and premature ovarian insufficiency†.

The Fanconi anemia pathway is a key pathway involved in the repair of deoxyribonucleic acidinterstrand crosslinking damage, which chiefly includes the following four modules: lesion recognition, Fanconi anemia core complex recruitment, FANCD2-FANCI complex monoubiquitination, and downstream events (nucleolytic incision, translesion synthesis, and homologous recombination). Mutations or deletions of multiple Fanconi anemia genes in this pathway can damage the interstrand crosslinking repair pathway and disrupt primordial germ cell development and oocyte meiosis, thereby leading to abnormal follicular development. Premature ovarian insufficiency is a gynecological clinical syndrome characterized by amenorrhea and decreased fertility due to decreased oocyte pool, accelerated follicle atresia, and loss of ovarian function in women <40years old. Furthermore, in recent years, several studies have detected mutations in the Fanconi anemia gene in patients with premature ovarian insufficiency. In addition, some patients with Fanconi anemia exhibit symptoms of premature ovarian insufficiency and infertility. The Fanconi anemia pathway and premature ovarian insufficiency are closely associated.

A comprehensive analysis of Fanconi anemia genes in Chinese patients with high-risk hereditary breast cancer

BackgroundFour Fanconi anemia (FA) genes (BRCA1, BRCA2, PALB2 and RAD51C) are defined as breast cancer (BC) susceptibility genes. Other FA genes have been inconsistently associated with BC. Thus, the role of other FA genes in BC should be explored in specific populations.MethodsMutations in 16 FA genes were screened with a 98-gene panel sequencing assay in a cohort of 1481 Chinese patients with high-risk hereditary BC. The association between mutations and clinicopathological characteristics as well as prognosis was analyzed. The risk of BC in carriers of FA gene mutations was assessed in the Genome Aggregation Database and the Westlake Biobank for Chinese cohort.ResultsA total of 2.57% (38/1481) BC patients were identified who had 12 other FA gene germline mutations. Among them, the most frequently mutated gene was FANCA (8/1481, 0.54%). These 38 patients carried 35 distinct pathogenic/likely pathogenic variants, of which 21 were novel. We found one rare FANCB deleterious variant (c.1327-3dupT) in our cohort. There was a statistically significant difference in lymph node status between FA gene mutation carriers and non-carriers (p = 0.041). We observed a trend that mutation carriers had larger tumor sizes, lower estrogen receptor (ER) and progesterone receptor (PR) positivity rates, and lower 3.5-year invasive disease-free survival (iDFS) and distant recurrence-free survival (DRFS) rates than non-carriers (tumor size > 2 cm: 51.43% vs. 45.63%; ER positivity rates: 51.43% vs. 60.81%; PR positivity rates: 48.57% vs. 55.16%; 3.5-year iDFS rates: 58.8% vs. 66.7%; 3.5-year DRFS rates: 58.8% vs. 68.8%). The frequency of the mutations in FANCD2, FANCM and BRIP1 trended to be higher among BC cases than that in controls (p = 0.055, 0.08 and 0.08, respectively).ConclusionThis study comprehensively estimated the prevalence, clinicopathological characteristics, prognosis and risk of BC associated with deleterious variants in FA genes in Chinese high-risk hereditary BC patients. It enriches our understanding of the role of FA genes with BC.

Fance deficiency inhibits primordial germ cell proliferation associated with transcription–replication conflicts accumulate and DNA repair defects

Fanconi anemia (FA) gene mutations are critical components in the genetic etiology of premature ovarian insufficiency (POI). Fance−/− mice detected meiotic arrest of primordial germ cells (PGCs) as early as embryonic day (E) 13.5 and exhibited decreased ovarian reserve after birth. However, the mechanism of Fance defect leading to dysgenesis of PGCs is unclear. We aimed to explore the effect of Fance defects on mitotic proliferation of PGCs. Combined with transcriptomic sequencing and validation, we examined the effect of Fance defects on cell cycle, transcription–replication conflicts (TRCs), and multiple DNA repair pathways in PGCs during active DNA replication at E11.5 and E12.5. Results showed Fance defects cause decreased numbers of PGCs during rapid mitosis at E11.5 and E12.5. Mitotic cell cycle progression of Fance−/− PGCs was blocked at E11.5 and E12.5, shown by decreased cell proportions in S and G2 phases and increased cell proportions in M phase. RNA-seq suggested the mechanisms involved in DNA replication and repair. We found Fance−/− PGCs accumulate TRCs during active DNA replication at E11.5 and E12.5. Fance−/− PGCs down-regulate multiple DNA repair pathways at E11.5 and E12.5 including the FA pathway, homologous recombination (HR) pathway, and base excision repair (BER) pathway. In conclusion, Fance defect impaired the mitotic proliferation of PGCs leading to rapidly decreased numbers and abnormal cell cycle distribution. Proliferation inhibition of Fance−/− PGCs was associated with accumulated TRCs and down-regulation of FA, HR, BER pathways. These provided a theoretical basis for identifying the inherited etiology and guiding potential fertility management for POI.

Genomic analyses of germline and somatic variation in high-grade serous ovarian cancer

BackgroundHigh-grade serous ovarian cancers (HGSCs) display a high degree of complex genetic alterations. In this study, we identified germline and somatic genetic alterations in HGSC and their association with relapse-free and overall survival. Using a targeted capture of 557 genes involved in DNA damage response and PI3K/AKT/mTOR pathways, we conducted next-generation sequencing of DNA from matched blood and tumor tissue from 71 HGSC participants. In addition, we performed the OncoScan assay on tumor DNA from 61 participants to examine somatic copy number alterations (SCNA).ResultsApproximately one-third of tumors had loss-of-function (LOF) germline (18/71, 25.4%) or somatic (7/71, 9.9%) variants in the DNA homologous recombination repair pathway genes BRCA1, BRCA2, CHEK2, MRE11A, BLM, and PALB2. LOF germline variants also were identified in other Fanconi anemia genes and in MAPK and PI3K/AKT/mTOR pathway genes. Most tumors harbored somatic TP53 variants (65/71, 91.5%). Using the OncoScan assay on tumor DNA from 61 participants, we identified focal homozygous deletions in BRCA1, BRCA2, MAP2K4, PTEN, RB1, SLX4, STK11, CREBBP, and NF1. In total, 38% (27/71) of HGSC patients harbored pathogenic variants in DNA homologous recombination repair genes. For patients with multiple tissues from the primary debulking or from multiple surgeries, the somatic mutations were maintained with few newly acquired point mutations suggesting that tumor evolution was not through somatic mutations. There was a significant association of LOF variants in homologous recombination repair pathway genes and high-amplitude somatic copy number alterations. Using GISTIC analysis, we identified NOTCH3, ZNF536, and PIK3R2 in these regions that were significantly associated with an increase in cancer recurrence and a reduction in overall survival.ConclusionsFrom 71 patients with HGCS, we performed targeted germline and tumor sequencing and provided a comprehensive analysis of these 557 genes. We identified germline and somatic genetic alterations including somatic copy number alterations and analyzed their associations with relapse-free and overall survival. This single-site long-term follow-up study provides additional information on genetic alterations related to occurrence and outcome of HGSC. Our findings suggest that targeted treatments based on both variant and SCNA profile potentially could improve relapse-free and overall survival.

Investigation of cancer predisposition in Fanconi anemia heterozygotes: A DiscovEHR cohort population study.

10589 Background: Fanconi anemia (FA) is a cancer predisposition syndrome caused by biallelic pathogenic variants in one of 22 genes involved in DNA intrastrand crosslink repair. 20 of these genes have an autosomal recessive inheritance pattern. Of the FA genes, 5 ( BRCA1, BRCA2, BRIP1, PALB2, RAD51C) are known cancer predisposition genes (CPG) when inherited in monoallelic autosomal dominant manner, though the heterozygous predisposition status of the remaining 15 remains unclear. Large population-level exome sequencing projects with linked health records such as the DiscovEHR cohort serve as promising tools to establish sufficient power to investigate all FA genes. Quantifying cancer risk is important for the counseling and surveillance of FA heterozygotes, especially with the ever-increasing use of genetic testing identifying these individuals. Methods: 170,503 individuals enrolled in the DiscovEHR Cohort were analyzed for pathogenic/likely pathogenic variants in 22 FA genes and identified 5834 subjects, the case group. ICD10 phenotype classifications for these subjects were curated to Phecodes and ICD-O-3 Site Recodes for two control arms of analysis. The first control arm included all DiscovEHR subjects excluding the case group and those with a variant of uncertain significance in the genes of interest with a deleterious in-silico score. The second control arm included individuals from the Surveillance, Epidemiology, and End Results (SEER) database. Phecode analysis yielded odds ratios adjusted for age, sex, race, smoking status, and BMI. SEER analysis yielded Standardized Incidence Ratio scores adjusted for age, sex, and birth cohort. When both arms of the analysis were completed Phecodes and SEER analyses were matched and positive signals for cancer risk were defined by statistically significant ratios in both. Results: The 5 known CPGs demonstrated multiple signals for cancer predisposition as expected (p &lt; 0.0001). Results for FANCA (p = 0.4231) and FANCC (p = 0.8142) heterozygotes validated recent results from our group showing no increased risk of cancer in relatives of FA patients with these genotypes. Ten other FA genes also showed no increased risk of cancer. 3 genes suggested a possible increased risk of specific cancers in heterozygotes and are currently being evaluated in a second large population exome cohort to validate these findings. Conclusions: This is the largest study to date on the cancer predisposition risk in Fanconi anemia heterozygotes. We have confirmed the known risk in 5 well-described CPGs and the lack of risk amongst FANCA and FANCC heterozygotes, the most common in population. Further analysis via an additional large population cohort is underway to validate any possible novel cancer predispositions in 3 genes. For the remaining FA genes, this population analysis supports previous literature on known CPGs and the lack of cancer predisposition in most FA genes.

Mutational landscape of intracranial germ cell tumors.

e14060 Background: Intracranial germ cell tumours (GCTs) are relatively rare tumours of the central nervous system, which account for 1% to 3% of cases. The pathogenesis of intracranial GCTs is unknown, molecular diagnostic methods currently play only a minor role in the diagnosis of CNS germ cell tumours, which generally rest on histopathological and secretory features. The objective of this study was to explore the molecular profile associated with Chinese intracranial GCT patients. Methods: This study retrospectively analyzed the genomic features of 24 intracranial GCT patients. Next-generation sequencing (NGS) of 551-gene panel was performed to detect gene mutations in tumor and blood samples of the patients. Results: The tumors originated most frequently in the pineal region (41.6%, 10/24), followed by the sellar tumor (16.6%, 4/24). The average age of the patients was 14 years (range, 4-55years), There is a male preponderance of 2:1. The most common mutation genes were KIT (4.23%, n=6), KRAS (2.82%, n=4) and PTEN (2.11%, n=3). KIT mutations are mainly missense mutations concentrated in exon 17, exon 11 and exon 13. Exon 2 and exon 3 of KRAS gene were the main mutation hotspots, mainly G12X(2.08%) missense mutation. The co-mutation of KIT and KRAS was only in one patient. Unexpectedly, four patients (16.6%) carried germline deleterious mutations, in FANCA(n=1), FANCD2(n=1), FANCE(n=1), SDHA(n=1). Two of the patients with germline mutations co-mutated with KIT and KRAS, respectively. Conclusions: 16.6% of the intracranial GCT patients carry germline deleterious mutations, and 12.5% in Fanconi anemia (FA) gene. Overall, these findings indicate that the Fanconi anemia (FA) pathway and the PI3K/AKT pathway play a significant role in intracranial GCTs tumorigenesis.

The application of a multigene NGS assay in homologous recombination deficiency tracking.

e15169 Background: Several tumor types have been efficiently treated with PARP inhibitors, a class of proteins involved in DNA repair pathway, which are approved for the treatment of ovarian, breast, prostate, and pancreatic cancer. The BRCA1/2 genes are essential for homologous recombination, and tumors which carry this type of mutations appear to have a defective homologous recombination pathway as a result of the loss of the second non mutated allele in the tumor. However, both epigenetic changes and mutations in many other genes involved in the HR pathway, including Fanconi anemia genes and the ΑRID1A, ATM, ATRX, BAP1, BARD1, BLM, BRIP1, CHEK1/2, MRE11A, NBN, PALB2, RAD50, RAD51, WRN may be responsible for the HRD phenomenon. In addition to mutational status, useful for the detection of the patients that would benefit from PARPi treatment could be the presence of certain genomic scars in the tumor, such as the loss of heterozygosity (LOH). Methods: In this study 406 samples from patients with various tumor types were analyzed using a targeted NGS assay for the analysis of 513 genes associated with targeted and immuno-oncology therapies. This assay detects relevant SNVs, indels, CNVs, gene fusions, splice variants in addition to TMB and MSI simultaneously. It also measures genomic instability using sample-level LOH in addition to the analysis for 31 HR related gene alterations. Moreover, the %gLOH was also calculated by the OncoScan™ CNV Assay in 25 samples in order to compare the results obtained by these two methodologies. Results: An HR gene mutation was detected in 20.93% of the tumors analyzed, with BRCA1/2 genes being the most prevalent HR altered genes detected in 5.17% of the tumors. The majority of the non BRCA1/2 HR mutations were in ARID1A (4.18%), ATRX (2.21%) and ATM (1.97%) genes. The %gLOH was highly correlated with the presence of mutations in the BRCA1/2 genes since 76.19% (16/21) of the tumors harboring such alterations had a high %LOH value (p = 0.007). Additionally, the LOH status was highly correlated to the presence of TP53 mutations, while a negative correlation to KRAS mutations was observed. Furthermore, there was no association of %LOH and TMB value in our cohort. Lin's Concordance Correlation Coefficient was 0.93 for the 25 evaluable samples examined simultaneously by both assays, indicating an almost perfect agreement. Conclusions: A high rate of HR mutations was detected in ovarian, breast and prostate which is consistent with the PARPi approval in these tumor types. The presence of BRCA1/2 mutations was highly associated with increased LOH value whereas it did not correlate to other HR mutations. Additionally, the pancancer presence of HR gene alterations indicates that the use of such genes as biomarkers of platinum and PARPi treatments should be evaluated in a wider range of tumor types. In conclusion the addition of analysis of the gLOH seems to be useful for the detection of additional patients eligible for PARPis.

Abstract 1371: Tumor treating fields exposure causes an imbalance of reactive oxygen homeostasis likely through the cytosolic function of the fanconi′s anemia genes

Abstract The use of Tumor Treating Fields (TTFields) has been shown to significantly benefit patients with non-resectable locally advanced or metastatic glioblastoma and malignant pleural mesothelioma. Although the disruption of mitosis that was initially attributed as the mechanism of tumor cell killing, in subsequent studies, it has been shown that TTFields inhibits DNA damage repair by downregulating DNA repair genes and proteins, including those associated with Fanconi's Anemia (FA). As a result, radiation-induced DNA damage repair is reduced and DNA replication fork protection is impaired, leading to replication stress and replication fork collapse. As the FA genes play a critical role in multiple repair pathways, TTFields is likely to increase susceptibility to other chemo agents besides radiation that cause DNA damage which is repaired by FA-dependent DNA repair and replication fork maintenance pathways in nuclei. In addition to their nuclear role, the FA proteins also regulate mitophagy in the cytosol. Using a panel of non-small cell lung cancer cell lines, it was found that TTFields exposure disrupted the clearance of damaged mitochondria despite the presence of a well-defined membrane markers for autophagosomal destruction of damaged mitochondria, that being PINK1 and parkin. Analysis of electron micrographs revealed disrupted mitochondrial cristae, while radical oxygen metabolism studies identified an imbalance leading to the production of reactive oxygen species (ROS) and ultimately, cell death. The downregulation of key mitochondrial genes including DAPIT, OSCP1, ATP5S, ATP5B and COXIV that are related to electron transport and mitochondrial integrity was confirmed by gene expression analysis. The small molecule dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, changes the metabolic state of cancer cells from cytoplasmic glycolysis to mitochondrial glucose oxidation, resulting in a decrease in mitochondrial membrane potential and an increase in ROS production in cancer cells. A combination of DCA with TTFields is therefore being investigated as a means of exploiting mitochondrial dysfunction caused by TTFields. The inhibition of mitophagy suggests a new mechanism of action for TTFields exposure tied to the downregulation of the FA pathway genes, the result being both nuclear and cytosolic perturbations in cell homeostasis. This study offers an evidence-based rationale for combining TTFields with various chemotherapy agents that may cause DNA damage, induce replication stress, and expose cells to radical oxygen byproducts of disrupting electron transport chains and the disruption of mitochondrial integrity. Citation Format: Narasimha Kumar Karanam, Michael Story. Tumor treating fields exposure causes an imbalance of reactive oxygen homeostasis likely through the cytosolic function of the fanconi′s anemia genes [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 1371.

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.

Genome-Scale Analysis of Cellular Restriction Factors That Inhibit Transgene Expression from Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) vectors are one of the leading platforms for gene delivery for the treatment of human genetic diseases, but the antiviral cellular mechanisms that interfere with optimal transgene expression are incompletely understood. Here, we performed two genome-scale CRISPR screens to identify cellular factors that restrict transgene expression from recombinant AAV vectors. Our screens revealed several components linked to DNA damage response, chromatin remodeling, and transcriptional regulation. Inactivation of the Fanconi anemia gene FANCA; the human silencing hub (HUSH)-associated methyltransferase SETDB1; and the gyrase, Hsp90, histidine kinase, and MutL (GHKL)-type ATPase MORC3 led to increased transgene expression. Moreover, SETDB1 and MORC3 knockout improved transgene levels of several AAV serotypes as well as other viral vectors, such as lentivirus and adenovirus. Finally, we demonstrated that the inhibition of FANCA, SETDB1, or MORC3 also enhanced transgene expression in human primary cells, suggesting that they could be physiologically relevant pathways that restrict AAV transgene levels in therapeutic settings. IMPORTANCE Recombinant AAV (rAAV) vectors have been successfully developed for the treatment of genetic diseases. The therapeutic strategy often involves the replacement of a defective gene by the expression of a functional copy from the rAAV vector genome. However, cells possess antiviral mechanisms that recognize and silence foreign DNA elements thereby limiting transgene expression and its therapeutic effect. Here, we utilize a functional genomics approach to uncover a comprehensive set of cellular restriction factors that inhibit rAAV-based transgene expression. Genetic inactivation of selected restriction factors increased rAAV transgene expression. Hence, modulation of identified restriction factors has the potential to enhance AAV gene replacement therapies.

Replication stress increases mitochondrial metabolism and mitophagy in FANCD2 deficient fetal liver hematopoietic stem cells.

Fanconi Anemia (FA) is an inherited bone marrow (BM) failure disorder commonly diagnosed during school age. However, in murine models, disrupted function of FA genes leads to a much earlier decline in fetal liver hematopoietic stem cell (FL HSC) number that is associated with increased replication stress (RS). Recent reports have shown mitochondrial metabolism and clearance are essential for long-term BM HSC function. Intriguingly, impaired mitophagy has been reported in FA cells. We hypothesized that RS in FL HSC impacts mitochondrial metabolism to investigate fetal FA pathophysiology. Results show that experimentally induced RS in adult murine BM HSCs evoked a significant increase in mitochondrial metabolism and mitophagy. Reflecting the physiological RS during development in FA, increase mitochondria metabolism and mitophagy were observed in FANCD2-deficient FL HSCs, whereas BM HSCs from adult FANCD2-deficient mice exhibited a significant decrease in mitophagy. These data suggest that RS activates mitochondrial metabolism and mitophagy in HSC.

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 (&amp;gt;400X)-normal (&amp;gt;180X) whole exome and transcriptome sequencing (&amp;gt;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 (&amp;gt; 33 and/or &amp;gt; 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.

Pan-cancer analysis of the prognostic and immunological role of Fanconi anemia complementation group E.

Fanconi anemia (FA) genes contribute to tumorigenesis by regulating DNA repair. Despite its importance for assembly and functionality of the FA core complex, no pan-cancer analysis of FANCE was performed. We aimed to provide a comprehensive understanding of the role of FANCE in cancers. Based on The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE), Genotype Tissue-Expression (GTEx), Human Protein Atlas (HPA), Gene Expression Omnibus (GEO), and Cancer Single-cell Atlas (CancerSEA) databases, we investigated the carcinogenicity of FANCE using various bioinformatics methods, including FANCE expression and prognosis, immune invasion, tumor mutation burden, microsatellite instability, and neoantigens. We monitored Fance mutations in mice that caused tumorigenesis. FANCE expression and activity scores were upregulated in 15 and 21 cancers. High expression of FANCE affected shorter overall survival (OS) in seven cancers and longer overall survival in three cancers. It was correlated with shorter overall survival and progression-free interval (PFI) in endometrial cancer and longer overall survival and PFI in cervical cancer. FANCE expression negatively correlated with stromal/immune scores in 21 cancers including cervical cancer, endometrial cancer, and ovarian cancer. FANCE expression negatively correlated with CD8 T cells in endometrial cancer and positively correlated with M1 macrophages in cervical cancer, possibly related to cancer prognosis. FANCE positively correlated with immune checkpoint inhibitors PD-1, PD-L1, and CTLA4 in endometrial cancer and ovarian cancer. FANCE expression positively correlated with microsatellite instability, tumor mutational burden, and neoantigens in 7, 22, and five cancers, especially in endometrial cancer, potentially increasing the effectiveness of immunotherapy. Single-cell sequencing data showed FANCE was primarily expressed in cancer cells in cervical and ovarian cancer, and in fibroblasts in endometrial cancer. Fance heterozygous mutant mice had increased tumor incidences and shorter overall survival and tumor-free survival (TFS) than Fance homozygous mutant mice and wild-type mice. Conclusively, FANCE potential to serve as a biomarker for cancer prognosis and may predict cancer immunotherapy responses. Fance heterozygous mutant resulted in increased tumorigenesis and poor prognosis in mice.

Targeting PLK1 As a Novel Strategy for Acute Myeloid Leukemias with Fanconi Anemia Pathway Mutations

The Fanconi anemia (FA) signaling network is critical to maintaining genomic integrity during interphase and mitosis. Biallelic germline mutation of any of the 22 genes that constitute this pathway (FANCA-FANCW) results in the disorder FA, which is characterized by congenital malformations, a predisposition to bone marrow failure, and pediatric acute myeloid leukemias (AMLs). Among the general population, acquired genetic disruptions of the FA pathway are present in 18% of sporadic pediatric AMLs, underscoring the importance of this pathway in the prevention of hematological malignancy. FANCA is the gene most commonly mutated in FA and is one of the most frequently disrupted FA pathway genes in sporadic AML. Therefore, the identification of precision therapies for FANCA-deficient AML is a high clinical priority. To identify synthetic lethal targets of FANCA, we previously identified mitotic phosphosignaling pathways required for the survival of FANCA-/- patient-derived fibroblasts through a kinome-wide shRNA screen. This screen identified the mitotic kinases CHEK1, PLK1, and BUB1 as hits. These findings corroborate work by others who have identified synthetic lethal interactions between PLK1 and the FA pathway components FANCG and BRCA1, suggesting inactivation of the FA pathway may sensitize cancers to PLK1 inhibition. The clinical success of PLK1 inhibitors, such as volasertib and onvansertib, has been limited by the absence of predictive biomarkers of response and adverse side effects. To test our hypothesis that FA gene mutations may predict sensitivity to PLK1 inhibition we generated an isogenic panel of human AML cells with a stable knockout of the FANCA gene. Utilizing in vitro cell viability assays, we have demonstrated the selective killing of FA-deficient AMLs by pharmacological PLK1 inhibition through low doses of volasertib and onvansertib. We also found that PLK1-inhibition impairs the hematopoietic potential of Fanca-/- murine hematopoietic stem and progenitor cells by performing in vitro bone marrow colony formation assays. Similarly, we demonstrated that primary human AMLs with multiple disruptions within the FA-pathway were hypersensitive to low doses of volasertib. To investigate the mechanism of synthetic lethality, we performed quantitative proteomic analysis of FANCA immunoprecipitates, which identified PLK1 as a novel FANCA-binding partner during mitosis. We validated this finding through high-resolution immunofluorescence microscopy and proximity ligation assays, which revealed a direct interaction between FANCA and PLK1 at kinetochores. Additionally, we demonstrated that volasertib treatment induces monoubiquitination of FANCD2 and colocalization of FANCA with FANCD2 at gamma-H2AX foci. Together, these findings indicate that PLK1 inhibition causes damage to mitotic chromosomes and subsequent activation of the FA pathway. Our work identifies a mitosis-specific vulnerability of FA-deficient cells and suggests that genetic disruptions of the FA pathway may be predictive of sensitivity to PLK1 inhibition, providing a preclinical rationale for the development of precision therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Adenine base editing efficiently restores the function of Fanconi anemia hematopoietic stem and progenitor cells

Fanconi Anemia (FA) is a debilitating genetic disorder with a wide range of severe symptoms including bone marrow failure and predisposition to cancer. CRISPR-Cas genome editing manipulates genotypes by harnessing DNA repair and has been proposed as a potential cure for FA. But FA is caused by deficiencies in DNA repair itself, preventing the use of editing strategies such as homology directed repair. Recently developed base editing (BE) systems do not rely on double stranded DNA breaks and might be used to target mutations in FA genes, but this remains to be tested. Here we develop a proof of concept therapeutic base editing strategy to address two of the most prevalent FANCA mutations in patient hematopoietic stem and progenitor cells. We find that optimizing adenine base editor construct, vector type, guide RNA format, and delivery conditions leads to very effective genetic modification in multiple FA patient backgrounds. Optimized base editing restored FANCA expression, molecular function of the FA pathway, and phenotypic resistance to crosslinking agents. ABE8e mediated editing in primary hematopoietic stem and progenitor cells from FA patients was both genotypically effective and restored FA pathway function, indicating the potential of base editing strategies for future clinical application in FA.

Investigation of mutations in Fanconi anemia genes and malignancy predisposition in Brazilian patients.

This study proposed to identify Fanconi anemia (FA) mutations in Brazilian patients and to investigate their impact on clinical manifestations and malignancies onset. A total of 116 patients were screened for nine mutations in FANCA, FANCC, FANCG. Those with no mutations were investigated by multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing for FANCA, FANCC, FANCE, FANCF, FANCG, FANCD1/BRCA2. Genetic subtype was identified in 107/116 (78 FA-A, 8 FA-C, 13 FA-G, 8 FA-E), with only one mutation in 1/116, and no mutations in 9/116 patients. Before hematopoietic cell transplantation (HCT), malignancies were detected in 16/116 patients (14/78 FA-A, 01/08 FA-C, 01/08 FA-E), and 12 of them were hematological. Observed to expected ratio (O/E) of hematologic malignancy was 303.7 (95% CI=148.6-458.7). This study allowed the identification of biallelic mutations in 91.4% of patients. FANCG and FANCC mutations had significantly earlier bone marrow failure onset, and FANCG severe cytopenia at diagnosis. Despite the inherent limitations of the small number of malignancy events in each genetic subtype, the hematologic malignancies O/E ratio was very high. Cumulative incidence of malignancy before HCT was higher in the third and fourth decades of life, considering HCT and death as competing risks. The cumulative incidence of HCT increased during the first decade, competing with malignancy development.

Significance of hereditary gene alterations for the pathogenesis of adult bone marrow failure versus myeloid neoplasia.

Broader genetic screening has led to the growing recognition of the role of germline variants associated with adult bone marrow failure (BMF) and myeloid neoplasia (MN) not exclusively in children and young adults. In this study, we applied a germline variant panel to 3008 adult BMF and MN cases to assess the importance of germline genetics and its impact on disease phenotype and prognosis. In our cohort, up to 9.7% of BMF and 5.3% of MN cases carried germline variants. Our cohort also included heterozygous carriers of recessive traits, suggesting they contribute to the risk of BMF and MN. By gene category, variants of Fanconi anemia gene family represented the highest-frequency category for both BMF and MN cases, found in 4.9% and 1.7% cases, respectively. In addition, about 1.4% of BMF and 0.19% of MN cases harbored multiple germline variants affecting often functionally related genes as compound heterozygous. The burden of germline variants in BMF and MN was clearly associated with acquisition of monosomy 7. While BMF cases carrying germline variants showed similar overall survival as compared to the wild-type (WT) cases, MN cases with germline variants experienced a significantly shorter overall survival as compared to WT cases.

Correlation of CCNE1 copy number levels with molecular profiles and outcomes for high grade serous ovarian and endometrial cancer (121)

<b>Objectives:</b> This study aimed to characterize <i>CCNE1</i> copy number (CN) in relation to other molecular characteristics of tumors, along with patient outcomes, to help guide future clinical trial design and potential novel targeted therapeutics development for these aggressive molecular subtypes of high-grade serous ovarian (HGSOC) and endometrial cancers (EMCA). <b>Methods:</b> Tumor samples were analyzed using Next-Gen sequencing (NGS) of the DNA (NextSeq, 592 genes or NovaSeq, whole-exome sequencing) and RNA (NovaSeq, whole-transcriptome sequencing) and immunohistochemistry (IHC) (Caris Life Sciences, Phoenix, AZ). PD-L1 IHC used SP-142 (cut-off ≥1%). Microsatellite instability (MSI) was tested by fragment analysis, IHC, and NGS. Tumor mutation burden (TMB) was measured by totaling somatic mutations per tumor (high ≥10 mutations/MB). Relative abundance of immune-cell infiltrates was calculated by MCPCounter. <i>CCNE1</i> amplification (<i>CCNE1</i>^Amp) was defined as CN ≥6 and calculated by NGS/WES. Real-world overall survival (OS) was obtained from insurance claims data, and Kaplan-Meier estimates were calculated for molecularly defined patient cohorts. Statistical significance was determined using Chi-square and Wilcoxon rank-sum test, and p-values adjusted for multiple comparisons (q) to be <0.05. <b>Results:</b> <i>CCNE1</i> was amplified in 608/6306 (9.6%) of HGSOC, 214/2098 (10.2%) of serous EMCA, 191/749 (15.3%) of uterine carcinosarcomas (UCS), and 17/2053 (0.80%) of endometrioid EMCA. <i>TP53</i> mutations positively correlated with <i>CCNE1</i>^Amp. <i>CCNE1</i>^Amp HGSOC had fewer mutations in <i>BRCA1/2</i>, <i>ARID1A</i>, and <i>NF1</i> but increased CN amplification in other genes (Table 1). <i>PTEN</i> mutations were negatively correlated with <i>CCNE1</i>^Amp in UCS and endometrioid EMCA. There was no difference in immuno-oncology biomarkers (TMB, dMMR/MSI, PD-L1). However, increased <i>CCNE1</i> levels were associated with decreased fibroblasts in HGSOC and serous EMCA, decreased B cells in serous EMCA, and decreased CD3+ T cells in UCS. In HGSOC, <i>CCNE1</i>^Amp was associated with increased expression of cell-cycle checkpoint regulators, Fanconi anemia, homologous recombination, mismatch repair, and single-strand break repair pathway genes, but decreased expression of these genes in endometrioid EMCA. In tumors with >1 specimen serially submitted, there was a significant increase in <i>CCNE1</i> CN over time in 29% (49/169) of HGSOC and 23% of EMCA (36/158). There was no difference in <i>CCNE1</i> CN in tumors harboring <i>BRCA</i> reversion mutations (<i>n</i>=8) compared to wild-type (<i>n</i>=5110). <i>CCNE1</i>^Amp correlated with mRNA expression in HGSOC and EMCA. <i>CCNE1</i>^Amp was associated with worse OS after carboplatin-based treatment in HGSOC as well as in serous EMCA and UCS. <b>Conclusions:</b> <i>CCNE1</i>^Amp in HGSOC and EMCA is less common than previously identified. Both HGSOC and endometrioid EMCAs have distinct molecular characteristics of <i>CCNE1</i>^Amp tumors. <i>CCNE1</i> CN may increase with the emergence of treatment resistance. Finally, <i>CCNE1</i>^Amp tumors appear overall non-immunogenic.

Mutated FANCA Gene Role in the Modulation of Energy Metabolism and Mitochondrial Dynamics in Head and Neck Squamous Cell Carcinoma.

Fanconi Anaemia (FA) is a rare recessive genetic disorder characterized by a defective DNA repair mechanism. Although aplastic anaemia is the principal clinical sign in FA, patients develop a head and neck squamous cell carcinoma (HNSCC) with a frequency 500–700 folds higher than the general population, which appears more aggressive, with survival of under two years. Since FA gene mutations are also associated with a defect in the aerobic metabolism and an increased oxidative stress accumulation, this work aims to evaluate the effect of FANCA mutation on the energy metabolism and the relative mitochondrial quality control pathways in an HNSCC cellular model. Energy metabolism and cellular antioxidant capacities were evaluated by oximetric, luminometric, and spectrophotometric assays. The dynamics of the mitochondrial network, the quality of mitophagy and autophagy, and DNA double-strand damage were analysed by Western blot analysis. Data show that the HNSCC cellular model carrying the FANCA gene mutation displays an altered electron transport between respiratory Complexes I and III that does not depend on the OxPhos protein expression. Moreover, FANCA HNSCC cells show an imbalance between fusion and fission processes and alterations in autophagy and mitophagy pathways. Together, all these alterations associated with the FANCA gene mutation cause cellular energy depletion and a metabolic switch to glycolysis, exacerbating the Warburg effect in HNSCC cells and increasing the growth rate. In addition, the altered DNA repair due to the FANCA mutation causes a higher accumulation of DNA damage in the HNSCC cellular model. In conclusion, changes in energy metabolism and mitochondrial dynamics could explain the strict correlation between HNSCC and FA genes, helping to identify new therapeutic targets.

Hematopoietic stem cell regeneration through paracrine regulation of the Wnt5a/Prox1 signaling axis.

The crosstalk between the BM microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration. Here, we show that in mice, deletion of the Fanconi anemia (FA) genes Fanca and Fancc dampened HSC regeneration through direct effects on HSCs and indirect effects on BM niche cells. FA HSCs showed persistent upregulation of the Wnt target Prox1 in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improved long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in WT HSCs mimicked the defective repopulation phenotype of FA HSCs. WT mice but not FA mice showed significant induction by TBI of BM stromal Wnt5a protein. Mechanistically, FA proteins regulated stromal Wnt5a expression, possibly through modulating the Wnt5a transcription activator Pax2. Wnt5a treatment of irradiated FA mice enhanced HSC regeneration. Conversely, Wnt5a neutralization inhibited HSC regeneration after TBI. Wnt5a secreted by LepR+CXCL12+ BM stromal cells inhibited β-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSCs. The detrimental effect of deregulated Wnt5a/Prox1 signaling on HSC regeneration was also observed in patients with FA and aged mice. Irradiation induced upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improved HSC regeneration. Treatment of aged mice with Wnt5a enhanced hematopoietic repopulation. Collectively, these findings identified the paracrine Wnt5a/Prox1 signaling axis as a regulator of HSC regeneration under conditions of injury and aging.