A CRISPR-Cas9 screen identifies EXO1 as a formaldehyde resistance gene.

Fanconi Anemia (FA) is a rare, genome instability-associated disease characterized by a deficiency in repairing DNA crosslinks, which are known to perturb several cellular processes, including DNA transcription, replication, and repair. Formaldehyde, a by-product of metabolism, is thought to drive FA by generating DNA interstrand crosslinks (ICLs) and DNA-protein crosslinks (DPCs). However, the impact of formaldehyde on global cellular pathways has not been investigated thoroughly. Herein, using a pangenomic CRISPR-Cas9 screen, we identify EXO1 as a critical regulator of formaldehyde-induced DNA lesions. We show that EXO1 knockout cell lines exhibit formaldehyde sensitivity leading to the accumulation of replicative stress, DNA double-strand breaks, and quadriradial chromosomes, a typical feature of FA. After formaldehyde exposure, EXO1 is recruited to chromatin, protects DNA replication forks from degradation, and functions in parallel with the FA pathway to promote cell survival. In vitro, EXO1-mediated exonuclease activity is proficient in removing DPCs. Collectively, we show that EXO1 limits replication stress and DNA damage to counteract formaldehyde-induced genome instability.

A new step in understanding stem cell mobilization in patients with Fanconi anemia: A bridge to gene therapy.

BACKGROUND: Fanconi anemia (FA) is an inherited disorder characterized clinically by congenital abnormalities, progressive bone marrow failure (BMF), and a predisposition to malignancy. Gene therapy (GT) of FA, via the infusion of gene-corrected peripheral blood (PB) autologous hematopoietic stem cells (HSCs), may constitute a cure for BMF. GT bypasses the donor restrictions and adverse events associated with allogenic HSC transplantation. However, adequate harvesting of PB-HSCs is a crucial determinant of successful engraftment in gene therapy. Harvesting the low numbers of HSCs in patients with FA is particularly challenging. STUDY DESIGN AND METHODS: This open-label phase I/II trial evaluates the feasibility and safety of co-administration of G-CSF and plerixafor in patients with FA for the mobilization and harvesting of peripheral HSCs, intending to use them in a gene therapy trial. Patients with mutations in the FANCA gene received two subcutaneous injections of G-CSF (6 µg/kg × 2/d from D1 to D8. Plerixafor (0.24 mg/kg/d) was administered 2 h before apheresis (from D5 onward). RESULTS: CD34+ cells were mobilized for four patients quickly but transiently after the plerixafor injection. One patient had a CD34+ cell count of over 100/µl; the mobilization peaked 2 h after the injection and lasted for more than 9 h. There were no short-term adverse events associated with the mobilization or harvesting procedures. CONCLUSION: Our data in patients with FA show that the mobilization of HSCs with G-CSF and plerixafor is safe and more efficient in younger individuals without BMF.

The FANC/BRCA Pathway Releases Replication Blockades by Eliminating DNA Interstrand Cross-Links.

DNA interstrand cross-links (ICLs) represent a major barrier blocking DNA replication fork progression. ICL accumulation results in growth arrest and cell death-particularly in cell populations undergoing high replicative activity, such as cancer and leukemic cells. For this reason, agents able to induce DNA ICLs are widely used as chemotherapeutic drugs. However, ICLs are also generated in cells as byproducts of normal metabolic activities. Therefore, every cell must be capable of rescuing lCL-stalled replication forks while maintaining the genetic stability of the daughter cells in order to survive, replicate DNA and segregate chromosomes at mitosis. Inactivation of the Fanconi anemia/breast cancer-associated (FANC/BRCA) pathway by inherited mutations leads to Fanconi anemia (FA), a rare developmental, cancer-predisposing and chromosome-fragility syndrome. FANC/BRCA is the key hub for a complex and wide network of proteins that-upon rescuing ICL-stalled DNA replication forks-allows cell survival. Understanding how cells cope with ICLs is mandatory to ameliorate ICL-based anticancer therapies and provide the molecular basis to prevent or bypass cancer drug resistance. Here, we review our state-of-the-art understanding of the mechanisms involved in ICL resolution during DNA synthesis, with a major focus on how the FANC/BRCA pathway ensures DNA strand opening and prevents genomic instability.

Microcephalus, medulloblastoma and excessive toxicity from chemotherapy: an unusual presentation of Fanconi anaemia.

UNLABELLED: Fanconi anaemia is a genetically and phenotypically heterogeneous disorder with different forms of clinical presentation. In this case the patient had suffered from microcephalus and delayed motor development from birth, but extensive investigation did not disclose any aetiology. At 3.5 y she developed a cerebellar medulloblastoma which was treated with surgery and chemotherapy. Following chemotherapy with alkylating agents she suffered from severe bone marrow aplasia which caused life-threatening infections, feeding problems and impaired kidney function. Fanconi anaemia was suspected, but it took 2 mo before the chromosome fragility test came out positive. From the moment diagnosis of Fanconi anaemia was made, no further active treatment was given. The patient's condition improved for some time, but she relapsed and died exactly 1 y after the first diagnosis of brain tumour. CONCLUSION: Fanconi anaemia must always be suspected in patients who experience excessive toxicity from chemotherapy regardless of the type of malignancy and congenital malformations.