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Fanconi anemia (FA) is a DNA repair syndrome generated by mutations in any of the 22 FA genes discovered to date1,2. Mutations in FANCA account for more than 60% of FA cases worldwide3,4. Clinically, FA is associated with congenital abnormalities and cancer predisposition. However, bone marrow failure is the primary pathological feature of FA that becomes evident in 70–80% of patients with FA during the first decade of life5,6. In this clinical study (ClinicalTrials.gov, NCT03157804; European Clinical Trials Database, 2011-006100-12), we demonstrate that lentiviral-mediated hematopoietic gene therapy reproducibly confers engraftment and proliferation advantages of gene-corrected hematopoietic stem cells (HSCs) in non-conditioned patients with FA subtype A. Insertion-site analyses revealed the multipotent nature of corrected HSCs and showed that the repopulation advantage of these cells was not due to genotoxic integrations of the therapeutic provirus. Phenotypic correction of blood and bone marrow cells was shown by the acquired resistance of hematopoietic progenitors and T lymphocytes to DNA cross-linking agents. Additionally, an arrest of bone marrow failure progression was observed in patients with the highest levels of gene marking. The progressive engraftment of corrected HSCs in non-conditioned patients with FA supports that gene therapy should constitute an innovative low-toxicity therapeutic option for this life-threatening disorder.

Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage 1 – 3 . The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes 4 – 7 . Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas 8 (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 papillomavirus 9 (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. Defective DNA interstrand crosslink repair in Fanconi anaemia drives extensive genomic rearrangements, thereby substantially increasing the risk of cancer development.

BRCA1 is a tumor suppressor that regulates DNA repair by homologous recombination. Germline mutations in BRCA1 are associated with increased risk of breast and ovarian cancer and BRCA1 deficient tumors are exquisitely sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. Therefore, uncovering additional components of this DNA repair pathway is of extreme importance for further understanding cancer development and therapeutic vulnerabilities. Here, we identify EDC4, a known component of processing-bodies and regulator of mRNA decapping, as a member of the BRCA1-BRIP1-TOPBP1 complex. EDC4 plays a key role in homologous recombination by stimulating end resection at double-strand breaks. EDC4 deficiency leads to genome instability and hypersensitivity to DNA interstrand cross-linking drugs and PARP inhibitors. Lack-of-function mutations in EDC4 were detected in BRCA1/2 -mutation-negative breast cancer cases, suggesting a role in breast cancer susceptibility. Collectively, this study recognizes EDC4 with a dual role in decapping and DNA repair whose inactivation phenocopies BRCA1 deficiency. Mutations in BRCA1 are associated with an increased risk of breast and ovarian cancer. Here the authors show that EDC4, a component of P-bodies, is a member of the BRCA1 complex with roles in stimulating end resection at breaks.

Allogeneic haematopoietic stem-cell transplantation is the standard treatment for bone marrow failure (BMF) in patients with Fanconi anaemia, but transplantation-associated complications such as an increased incidence of subsequent cancer are frequent. The aim of this study was to evaluate the safety and efficacy of the infusion of autologous gene-corrected haematopoietic stem cells as an alternative therapy for these patients. This was an open-label, investigator-initiated phase 1/2 clinical trial (FANCOLEN-1) and long-term follow-up trial (up to 7 years post-treatment) in Spain. Mobilised peripheral blood (PB) CD34+ cells from nine patients with Fanconi anaemia-A in the early stages of BMF were transduced with a therapeutic FANCA-encoding lentiviral vector and re-infused without any cytotoxic conditioning treatment. The primary efficacy endpoint of FANCOLEN-1 was the engraftment of transduced cells, as defined by the detection of at least 0·1 therapeutic vector copies per nucleated cell of patient bone marrow (BM) or PB at the second year post-infusion, without this percentage having declined substantially over the previous year. The safety coprimary endpoint was adverse events during the 3 years after infusion. The completed open-label phase 1/2 and the ongoing long-term clinical trials are registered with ClinicalTrials.gov, NCT03157804; EudraCT, 2011–006100–12; and NCT04437771, respectively. There were eight evaluable treated patients with Fanconi anaemia-A. Patients were recruited between Jan 7, 2016 and April 3, 2019. The primary endpoint was met in five of the eight evaluable patients (62·50%). The median number of therapeutic vector copies per nucleated cell of patient BM and PB at the second year post-infusion was 0·18 (IQR 0·01–0·20) and 0·06 (0·01–0·19), respectively. No genotoxic events related to the gene therapy were observed. Most treatment-emergent adverse events (TEAEs) were non-serious and assessed as not related to therapeutic FANCA-encoding lentiviral vector. Nine serious adverse events (grade 3–4) were reported in six patients, one was considered related to medicinal product infusion, and all resolved without sequelae. Cytopenias and viral infections (common childhood illnesses) were the most frequently reported TEAEs. These results show for the first time that haematopoietic gene therapy without genotoxic conditioning enables sustained engraftment and reversal of BMF progression in patients with Fanconi anaemia. European Commission, Instituto de Salud Carlos III, and Rocket Pharmaceuticals.

The WD40-containing E3 ubiquitin ligase RFWD3 has been recently linked to the repair of DNA damage by homologous recombination (HR). Here we have shown that an RFWD3 mutation within the WD40 domain is connected to the genetic disease Fanconi anemia (FA). An individual presented with congenital abnormalities characteristic of FA. Cells from the patient carrying the compound heterozygous mutations c.205_206dupCC and c.1916T>A in RFWD3 showed increased sensitivity to DNA interstrand cross-linking agents in terms of increased chromosomal breakage, reduced survival, and cell cycle arrest in G2 phase. The cellular phenotype was mirrored in genetically engineered human and avian cells by inactivation of RFWD3 or introduction of the patient-derived missense mutation, and the phenotype was rescued by expression of wild-type RFWD3 protein. HR was disrupted in RFWD3-mutant cells as a result of impaired relocation of mutant RFWD3 to chromatin and defective physical interaction with replication protein A. Rfwd3 knockout mice appear to have increased embryonic lethality, are subfertile, show ovarian and testicular atrophy, and have a reduced lifespan resembling that of other FA mouse models. Although RFWD3 mutations have thus far been detected in a single child with FA, we propose RFWD3 as an FA gene, FANCW, supported by cellular paradigm systems and an animal model.

The tumor suppressor FANCD1/BRCA2 is crucial for DNA homologous recombination repair (HRR). BRCA2 biallelic pathogenic variants result in a severe form of Fanconi anemia (FA) syndrome, whereas monoallelic pathogenic variants cause mainly hereditary breast and ovarian cancer predisposition. For decades, the co-occurrence in trans with a clearly pathogenic variant led to assume that the other allele was benign. However, here we show a patient with biallelic BRCA2 (c.1813dup and c.7796 A > G) diagnosed at age 33 with FA after a hypertoxic reaction to chemotherapy during breast cancer treatment. After DNA damage, patient cells displayed intermediate chromosome fragility, reduced survival, cell cycle defects, and significantly decreased RAD51 foci formation. With a newly developed cell-based flow cytometric assay, we measured single BRCA2 allele contributions to HRR, and found that expression of the missense allele in a BRCA2 KO cellular background partially recovered HRR activity. Our data suggest that a hypomorphic BRCA2 allele retaining 37–54% of normal HRR function can prevent FA clinical phenotype, but not the early onset of breast cancer and severe hypersensitivity to chemotherapy.

Background Individuals diagnosed with Fanconi anemia (FA) present an incidence of 500- to 700-fold higher to develop head and neck squamous carcinomas (HNSCCs) compared to the general population. Effective anticancer treatments for FA-HNSCCs are missing. Several studies demonstrated that FA-HNSCCs overexpress the epithelial growth factor receptor (EGFR) and their viability is highly dependent on this pathway, as FA-HNSCCs cells are highly sensitive to EGFR inhibitors such as afatinib in preclinical models, which led to an orphan drug designation by EMA in 2018. Methods The AFAN trial is a phase Ib/II, single arm, non-randomized, open-label, multicenter study to determine whether afatinib is effective and safe in patients with FA and advanced / metastatic HNSCC. Patients could be treatment-naïve or progressed to a previous systemic treatment with immunotherapy, chemotherapy or cetuximab. Afatinib will be administered orally at a starting dose of 20 mg /day (weeks 1–2), escalating to 30 mg / day at weeks 3–4 and to 40 mg / day thereafter provided no adverse events occur. Treatment will be maintained until disease progression / secondary primary tumor, loss to follow-up, unacceptable toxicity, patient withdrawal or death. Dose reductions and delays will be allowed. All patients will undergo periodic tumor assessments by CT or MRI scan every 12 weeks (3 months) from the start of the study treatment until progression / SPT or patient withdrawal. The primary endpoint is objective response rate (ORR) after 9 months of study treatment initiation according to RECIST V1.1. Secondary endpoints include disease control rate, duration of response, disease-free survival, overall survival, safety, patient reported outcomes and ancillary studies. The expected sample size is 25 patients calculated using a Simon II stage design (α = 0.1; β = 80%), taking as null hypothesis a 9-month ORR of 20% and an alternative ORR of 40%. Discussion The AFAN trial will investigate if afatinib is an effective treatment in FA patients with unresectable and / or metastatic locoregionally advanced squamous cell carcinoma of the oral cavity, oropharynx or hypopharynx or larynx. Trial registration EU CT 20,245,114,772,900 / www.clinicaltrials.gov NCT06648096 (August, 1st 2024).

Fanconi anemia (FA) is a DNA repair disorder resulting from mutations in genes encoding for FA DNA repair complex components and is characterized by variable congenital abnormalities, bone marrow failure (BMF), and high incidences of malignancies. FA mosaicism arises from reversion or other compensatory mutations in hematopoietic cells and may be associated with BMF reversal and decreased blood cell sensitivity to DNA-damaging agents (clastogens); this sensitivity is a phenotypic and diagnostic hallmark of FA. Uncertainty regarding the clinical significance of FA mosaicism persists; in some cases, patients have survived multiple decades without BMF or hematologic malignancy, and in others hematologic failure occurred despite the presence of clastogen-resistant cell populations. Assessment of mosaicism is further complicated because clinical evaluation is frequently based on clastogen resistance in lymphocytes, which may arise from reversion events both in lymphoid-specific lineages and in more pluripotent hematopoietic stem/progenitor cells (HSPCs). In this review, we describe diagnostic methods and outcomes in published mosaicism series, including the substantial intervals (1–6 years) over which blood counts normalized, and the relatively favorable clinical course in cases where clastogen resistance was demonstrated in bone marrow progenitors. We also analyzed published FA mosaic cases with emphasis on long-term clinical outcomes when blood count normalization was identified. Blood count normalization in FA mosaicism likely arises from reversion events in long-term primitive HSPCs and is associated with low incidences of BMF or hematologic malignancy. These observations have ramifications for current investigational therapeutic programs in FA intended to enable gene correction in long-term repopulating HSPCs.

In about 10% of patients affected by Fanconi anemia (FA) the diagnosis is delayed until adulthood, and the presenting symptom in these “occult” FA cases is often a solid cancer and cancer treatment–related toxicity. Highly predictive clinical parameter(s) for diagnosing such an adult-onset cases are missing. (1) Exome sequencing (ES), (2) Sanger sequencing of FANCA, (3) diepoxybutane (DEB)-induced chromosome breakage test. ES identified a pathogenic homozygous FANCA variant in a patient affected by Sertoli cell–only syndrome (SCOS) and in his azoospermic brother. Although they had no overt anemia, chromosomal breakage test revealed a reverse somatic mosaicism in the former and a typical FA picture in the latter. In 27 selected SCOS cases, 1 additional patient showing compound heterozygous pathogenic FANCA variants was identified with positive chromosomal breakage test. We report an extraordinarily high frequency of FA in a specific subgroup of azoospermic patients (7.1%). The screening for FANCA pathogenic variants in such patients has the potential to identify undiagnosed FA before the appearance of other severe clinical manifestations of the disease. The definition of this high-risk group for “occult” FA, based on specific testis phenotype with mild/borderline hematological alterations, is of unforeseen clinical relevance.

Cancer is a complex disease resulting from the accumulation of genetic dysfunctions. Tumor heterogeneity causes the molecular variety that divergently controls responses to chemotherapy, leading to the recurrent problem of cancer reappearance. For many decades, efforts have focused on identifying essential tumoral genes and cancer driver mutations. More recently, prompted by the clinical success of the synthetic lethality (SL)-based therapy of the PARP inhibitors in homologous recombinant deficient tumors, scientists have centered their novel research on SL interactions (SLI). The state of the art to find new genetic interactions are currently large-scale forward genetic CRISPR screens. CRISPR technology has rapidly evolved to be a common tool in the vast majority of laboratories, as tools to implement CRISPR screen protocols are available to all researchers. Taking advantage of SLI, combinatorial therapies have become the ultimate model to treat cancer with lower toxicity, and therefore better efficiency. This review explores the CRISPR screen methodology, integrates the up-to-date published findings on CRISPR screens in the cancer field and proposes future directions to uncover cancer regulation and individual responses to chemotherapy.