Genetic risk of second malignant neoplasm after childhood cancer treatment: A systematic review.

Second malignant neoplasm (SMN) is one of the most severe long-term risks for childhood cancer survivors (CCS), significantly impacting long-term patient survival. While radiotherapy and chemotherapy are known risk factors, the observed inter-individual variability suggests a genetic component contributing to the risk of SMN. This article aims to conduct a systematic review of genetic factors implicated in the SMN risk among CCS. Searches were performed in PubMed, Scopus, and Web of Sciences. Eighteen studies were included (eleven candidate gene studies, three genome-wide association studies, and four whole exome/genome sequencing studies). The included studies were based on different types of first cancers, investigated any or specific types of SMN, and focused mainly on genes involved in drug metabolism and DNA repair pathways. These differences in study design and methods used to characterize genetic variants limit the scope of the results and highlight the need for further extensive and standardized investigations. However, this review provides a valuable compilation of SMN risk-associated variants and genes, facilitating efficient replication and advancing our understanding of the genetic basis for this major risk for CCS.

FANCA deficiency promotes leukaemic progression by allowing the emergence of cells carrying oncogenic driver mutations.

Leukaemia is caused by the clonal evolution of a cell that accumulates mutations/genomic rearrangements, allowing unrestrained cell growth. However, recent identification of leukaemic mutations in the blood cells of healthy individuals revealed that additional events are required to expand the mutated clones for overt leukaemia. Here, we assessed the functional consequences of deleting the Fanconi anaemia A (Fanca) gene, which encodes a DNA damage response protein, in Spi1 transgenic mice that develop preleukaemic syndrome. FANCA loss increases SPI1-associated disease penetrance and leukaemic progression without increasing the global mutation load of leukaemic clones. However, a high frequency of leukaemic FANCA-depleted cells display heterozygous activating mutations in known oncogenes, such as Kit or Nras, also identified but at low frequency in FANCA-WT mice with preleukaemic syndrome, indicating that FANCA counteracts the emergence of oncogene mutated leukaemic cells. A unique transcriptional signature is associated with the leukaemic status of FANCA-depleted cells, leading to activation of MDM4, NOTCH and Wnt/ß-catenin pathways. We show that NOTCH signalling improves the proliferation capacity of FANCA-deficient leukaemic cells. Collectively, our observations indicate that loss of the FANC pathway, known to control genetic instability, fosters the expansion of leukaemic cells carrying oncogenic mutations rather than mutation formation. FANCA loss may contribute to this leukaemogenic progression by reprogramming transcriptomic landscape of the cells.

Fanca deficiency is associated with alterations in osteoclastogenesis that are rescued by TNFα.

BACKGROUND: Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, which includes bone-forming and bone-resorbing cells, i.e., osteoblasts (OBs) and osteoclasts (OCs). OBs originate from mesenchymal progenitors, while OCs are derived from HSCs. Self-renewal, proliferation and differentiation of HSCs are under the control of regulatory signals generated by OBs and OCs within the BM niche. Consequently, OBs and OCs control both bone physiology and hematopoiesis. Since the human developmental and bone marrow failure genetic syndrome fanconi anemia (FA) presents with skeletal abnormalities, osteoporosis and HSC impairment, we wanted to test the hypothesis that the main pathological abnormalities of FA could be related to a defect in OC physiology and/or in bone homeostasis. RESULTS: We revealed here that the intrinsic differentiation of OCs from a Fanca-/- mouse is impaired in vitro due to overactivation of the p53-p21 axis and defects in NF-kB signaling. The OC differentiation abnormalities observed in vitro were rescued by treating Fanca-/- cells with the p53 inhibitor pifithrin-α, by treatment with the proinflammatory cytokine TNFα or by coculturing them with Fanca-proficient or Fanca-deficient osteoblastic cells. CONCLUSIONS: Overall, our results highlight an unappreciated role of Fanca in OC differentiation that is potentially circumvented in vivo by the presence of OBs and TNFα in the BM niche.

Safeguarding DNA Replication: A Golden Touch of MiDAS and Other Mechanisms.

DNA replication is a tightly regulated fundamental process allowing the correct duplication and transfer of the genetic information from the parental cell to the progeny. It involves the coordinated assembly of several proteins and protein complexes resulting in replication fork licensing, firing and progression. However, the DNA replication pathway is strewn with hurdles that affect replication fork progression during S phase. As a result, cells have adapted several mechanisms ensuring replication completion before entry into mitosis and segregating chromosomes with minimal, if any, abnormalities. In this review, we describe the possible obstacles that a replication fork might encounter and how the cell manages to protect DNA replication from S to the next G1.

A new frontier in Fanconi anemia: From DNA repair to ribosome biogenesis.

Described by Guido Fanconi almost 100 years ago, Fanconi anemia (FA) is a rare genetic disease characterized by developmental abnormalities, bone marrow failure (BMF) and cancer predisposition. The proteins encoded by FA-mutated genes (FANC proteins) and assembled in the so-called FANC/BRCA pathway have key functions in DNA repair and replication safeguarding, which loss leads to chromosome structural aberrancies. Therefore, since the 1980s, FA has been considered a genomic instability and chromosome fragility syndrome. However, recent findings have demonstrated new and unexpected roles of FANC proteins in nucleolar homeostasis and ribosome biogenesis, the alteration of which impacts cellular proteostasis. Here, we review the different cellular, biochemical and molecular anomalies associated with the loss of function of FANC proteins and discuss how these anomalies contribute to BMF by comparing FA to other major inherited BMF syndromes. Our aim is to determine the extent to which alterations in the DNA damage response in FA contribute to BMF compared to the consequences of the loss of function of the FANC/BRCA pathway on the other roles of the pathway.

Molecular Landscape of Therapy-related Myeloid Neoplasms in Patients Previously Treated for Gynecologic and Breast Cancers.

Definition of therapy-related myeloid neoplasms (TRMN) is only based on clinical history of exposure to leukemogenic therapy. No specific molecular classification combining therapy-related acute myeloid leukemia and therapy-related myelodysplastic syndromes has been proposed. We aimed to describe the molecular landscape of TRMN at diagnosis, among 77 patients with previous gynecologic and breast cancer with a dedicated next-generation sequencing panel covering 74 genes. We investigated the impact of clonal hematopoiesis of indeterminate potential-associated mutations (CHIP-AMs defined as presence at TRMN stage of mutations described in CHIP with a frequency >1%) on overall survival (OS) and the clinical relevance of a modified genetic ontogeny-based classifier that categorized patients in 3 subgroups. The most frequently mutated genes were TP53 (31%), DNMT3A (19%), IDH1/2 (13%), NRAS (13%), TET2 (12%), NPM1 (10%), PPM1D (9%), and PTPN11 (9%). CHIP-AMs were detected in 66% of TRMN patients, with no impact on OS. Yet, patients with CHIP-AM were older and had a longer time interval between solid tumor diagnosis and TRMN. According to our modified ontogeny-based classifier, we observed that the patients with TP53 or PPM1D mutations had more treatment lines and complex karyotypes, the "MDS-like" patients were older with more gene mutations, while patients with "De novo/pan-AML" mutations were younger with more balanced chromosomal translocations. Median OS within each subgroup was 7.5, 14.5, and 25.2 months, respectively, with statistically significant difference in multivariate analysis. These results support the integration of cytogenetic and molecular markers into the future TRMN classification to reflect the biological diversity of TRMN and its impact on outcomes.

Abnormal migration behavior linked to Rac1 signaling contributes to primordial germ cell exhaustion in Fanconi anemia pathway-deficient Fancg-/- embryos.

Fanconi anemia (FA) is a rare human genetic disorder characterized by bone marrow failure, predisposition to cancer and developmental defects including hypogonadism. Reproductive defects leading to germ cell aplasia are the most consistent phenotypes seen in FA mouse models. We examined the role of the nuclear FA core complex gene Fancg in the development of primordial germ cells (PGCs), the embryonic precursors of adult gametes, during fetal development. PGC maintenance was severely impaired in Fancg-/- embryos. We observed a defect in the number of PGCs starting at E9.5 and a strong attrition at E11.5 and E13.5. Remarkably, we observed a mosaic pattern reflecting a portion of testicular cords devoid of PGCs in E13.5 fetal gonads. Our in vitro and in vivo data highlight a potential role of Fancg in the proliferation and in the intrinsic cell motility abilities of PGCs. The random migratory process is abnormally activated in Fancg-/- PGCs, altering the migration of cells. Increased cell death and PGC attrition observed in E11.5 Fancg-/- embryos are features consistent with delayed migration of PGCs along the migratory pathway to the genital ridges. Moreover, we show that an inhibitor of RAC1 mitigates the abnormal migratory pattern observed in Fancg-/- PGCs.

Beyond DNA repair and chromosome instability-Fanconi anaemia as a cellular senescence-associated syndrome.

Fanconi anaemia (FA) is the most frequent inherited bone marrow failure syndrome, due to mutations in genes encoding proteins involved in replication fork protection, DNA interstrand crosslink repair and replication rescue through inducing double-strand break repair and homologous recombination. Clinically, FA is characterised by aplastic anaemia, congenital defects and cancer predisposition. In in vitro studies, FA cells presented hallmarks defining senescent cells, including p53-p21 axis activation, altered telomere length, mitochondrial dysfunction, chromatin alterations, and a pro-inflammatory status. Senescence is a programme leading to proliferation arrest that is involved in different physiological contexts, such as embryogenesis, tissue remodelling and repair and guarantees tumour suppression activity. However, senescence can become a driving force for developmental abnormalities, aging and cancer. Herein, we summarise the current knowledge in the field to highlight the mutual relationships between FA and senescence that lead us to consider FA not only as a DNA repair and chromosome fragility syndrome but also as a "senescence syndrome".

Fanconi anemia A protein participates in nucleolar homeostasis maintenance and ribosome biogenesis.

Fanconi anemia (FA), the most common inherited bone marrow failure and leukemia predisposition syndrome, is generally attributed to alterations in DNA damage responses due to the loss of function of the DNA repair and replication rescue activities of the FANC pathway. Here, we report that FANCA deficiency, whose inactivation has been identified in two-thirds of FA patients, is associated with nucleolar homeostasis loss, mislocalization of key nucleolar proteins, including nucleolin (NCL) and nucleophosmin 1 (NPM1), as well as alterations in ribosome biogenesis and protein synthesis. FANCA coimmunoprecipitates with NCL and NPM1 in a FANCcore complex-independent manner and, unique among the FANCcore complex proteins, associates with ribosomal subunits, influencing the stoichiometry of the translational machineries. In conclusion, we have identified unexpected nucleolar and translational consequences specifically associated with FANCA deficiency that appears to be involved in both DNA damage and nucleolar stress responses, challenging current hypothesis on FA physiopathology.

Tipping the Scale: MYC Gains Weight in Fanconi Anemia Bone Marrow Failure Progression.

Fanconi anemia (FA) is an inherited syndrome of bone marrow failure (BMF) due to disrupted DNA repair. In this issue of Cell Stem Cell, Rodríguez et al. (2021) show that blood stem cells from FA patients have abnormal and inflammation-induced MYC expression, which promotes their proliferation in the face of increasing DNA damage.

The underestimated role of the microphthalmia-associated transcription factor (MiTF) in normal and pathological haematopoiesis.

Haematopoiesis, the process by which a restrained population of stem cells terminally differentiates into specific types of blood cells, depends on the tightly regulated temporospatial activity of several transcription factors (TFs). The deregulation of their activity or expression is a main cause of pathological haematopoiesis, leading to bone marrow failure (BMF), anaemia and leukaemia. TFs can be induced and/or activated by different stimuli, to which they respond by regulating the expression of genes and gene networks. Most TFs are highly pleiotropic; i.e., they are capable of influencing two or more apparently unrelated phenotypic traits, and the action of a single TF in a specific setting often depends on its interaction with other TFs and signalling pathway components. The microphthalmia-associated TF (MiTF) is a prototype TF in multiple situations. MiTF has been described extensively as a key regulator of melanocyte and melanoma development because it acts mainly as an oncogene. Mitf-mutated mice show a plethora of pleiotropic phenotypes, such as microphthalmia, deafness, abnormal pigmentation, retinal degeneration, reduced mast cell numbers and osteopetrosis, revealing a greater requirement for MiTF activity in cells and tissue. A growing amount of evidence has led to the delineation of key roles for MiTF in haematopoiesis and/or in cells of haematopoietic origin, including haematopoietic stem cells, mast cells, NK cells, basophiles, B cells and osteoclasts. This review summarizes several roles of MiTF in cells of the haematopoietic system and how MiTFs can impact BM development.

Hypoxia increases mutational load of breast cancer cells through frameshift mutations.

Tumor hypoxia-induced downregulation of DNA repair pathways and enhanced replication stress are potential sources of genomic instability. A plethora of genetic changes such as point mutations, large deletions and duplications, microsatellite and chromosomal instability have been discovered in cells under hypoxic stress. However, the influence of hypoxia on the mutational burden of the genome is not fully understood. Here, we attempted to elucidate the DNA damage response and repair patterns under different types of hypoxic stress. In addition, we examined the pattern of mutations exclusively induced under chronic and intermittent hypoxic conditions in two breast cancer cell lines using exome sequencing. Our data indicated that hypoxic stress resulted in transcriptional downregulation of DNA repair genes which can impact the DNA repair induced during anoxic as well as reoxygenated conditions. In addition, our findings demonstrate that hypoxic conditions increased the mutational burden, characterized by an increase in frameshift insertions and deletions. The somatic mutations were random and non-recurring, as huge variations within the technical duplicates were recognized. Hypoxia also resulted in an increase in the formation of potential neoantigens in both cell lines. More importantly, these data indicate that hypoxic stress mitigates DNA damage repair pathways and causes an increase in the mutational burden of tumor cells, thereby interfering with hypoxic cancer cell immunogenicity.

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.

Large deletions in immunoglobulin genes are associated with a sustained absence of DNA Polymerase η.

Somatic hypermutation of immunoglobulin genes is a highly mutagenic process that is B cell-specific and occurs during antigen-driven responses leading to antigen specificity and antibody affinity maturation. Mutations at the Ig locus are initiated by Activation-Induced cytidine Deaminase and are equally distributed at G/C and A/T bases. This requires the establishment of error-prone repair pathways involving the activity of several low fidelity DNA polymerases. In the physiological context, the G/C base pair mutations involve multiple error-prone DNA polymerases, while the generation of mutations at A/T base pairs depends exclusively on the activity of DNA polymerase η. Using two large cohorts of individuals with xeroderma pigmentosum variant (XP-V), we report that the pattern of mutations at Ig genes becomes highly enriched with large deletions. This observation is more striking for patients older than 50 years. We propose that the absence of Pol η allows the recruitment of other DNA polymerases that profoundly affect the Ig genomic landscape.

SMC5/6 acts jointly with Fanconi anemia factors to support DNA repair and genome stability.

SMC5/6 function in genome integrity remains elusive. Here, we show that SMC5 dysfunction in avian DT40 B cells causes mitotic delay and hypersensitivity toward DNA intra- and inter-strand crosslinkers (ICLs), with smc5 mutants being epistatic to FANCC and FANCM mutations affecting the Fanconi anemia (FA) pathway. Mutations in the checkpoint clamp loader RAD17 and the DNA helicase DDX11, acting in an FA-like pathway, do not aggravate the damage sensitivity caused by SMC5 dysfunction in DT40 cells. SMC5/6 knockdown in HeLa cells causes MMC sensitivity, increases nuclear bridges, micronuclei, and mitotic catastrophes in a manner similar and non-additive to FANCD2 knockdown. In both DT40 and HeLa systems, SMC5/6 deficiency does not affect FANCD2 ubiquitylation and, unlike FANCD2 depletion, RAD51 focus formation. SMC5/6 components further physically interact with FANCD2-I in human cells. Altogether, our data suggest that SMC5/6 functions jointly with the FA pathway to support genome integrity and DNA repair and may be implicated in FA or FA-related human disorders.

Microphthalmia transcription factor expression contributes to bone marrow failure in Fanconi anemia.

Hematopoietic stem cell (HSC) attrition is considered the key event underlying progressive BM failure (BMF) in Fanconi anemia (FA), the most frequent inherited BMF disorder in humans. However, despite major advances, how the cellular, biochemical, and molecular alterations reported in FA lead to HSC exhaustion remains poorly understood. Here, we demonstrated in human and mouse cells that loss-of-function of FANCA or FANCC, products of 2 genes affecting more than 80% of FA patients worldwide, is associated with constitutive expression of the transcription factor microphthalmia (MiTF) through the cooperative, unscheduled activation of several stress-signaling pathways, including the SMAD2/3, p38 MAPK, NF-κB, and AKT cascades. We validated the unrestrained Mitf expression downstream of p38 in Fanca-/- mice, which display hallmarks of hematopoietic stress, including loss of HSC quiescence, DNA damage accumulation in HSCs, and reduced HSC repopulation capacity. Importantly, we demonstrated that shRNA-mediated downregulation of Mitf expression or inhibition of p38 signaling rescued HSC quiescence and prevented DNA damage accumulation. Our data support the hypothesis that HSC attrition in FA is the consequence of defects in the DNA-damage response combined with chronic activation of otherwise transiently activated signaling pathways, which jointly prevent the recovery of HSC quiescence.

Fanconi anemia proteins counteract the implementation of the oncogene-induced senescence program.

Fanconi Anemia (FA), due to the loss-of-function of the proteins that constitute the FANC pathway involved in DNA replication and genetic stability maintainance, is a rare genetic disease featuring bone marrow failure, developmental abnormalities and cancer predisposition. Similar clinical stigmas have also been associated with alterations in the senescence program, which is activated in physiological or stress situations, including the unscheduled, chronic, activation of an oncogene (oncogene induced senescence, OIS). Here, we wanted to determine the crosstalk, if any, between the FANC pathway and the OIS process. OIS was analyzed in two known cellular models, IMR90-hTERT/ER:RASG12V and WI38-hTERT/ER:GFP:RAF1, harboring 4-hydroxytamoxifen-inducible oncogenes. We observed that oncogene activation induces a transitory increase of both FANCA and FANCD2 as well as FANCD2 monoubiquitination, readout of FANC pathway activation, followed by their degradation. FANCD2 depletion, which leads to a pre-senescent phenotype, anticipates OIS progression. Coherently, FANCD2 overexpression or inhibition of its proteosomal-dependent degradation slightly delays OIS progression. The pro-senescence protease cathepsin L, which activation is anticipated during OIS in FANCD2-depleted cells, also participates to FANCD2 degradation. Our results demonstrate that oncogene activation is first associated with FANCD2 induction and activation, which may support initial cell proliferation, followed by its degradation/downregulation when OIS proceeds.

Whole exome sequencing identifies a new mutation in the SLC19A2 gene leading to thiamine-responsive megaloblastic anemia in an Egyptian family.

BACKGROUND: The Solute Carrier Family 19 Member 2 (SLC19A2, OMIM *603941) encodes the thiamine transporter 1 (THTR-1) that brings thiamine (Vitamin B1) into cells. THTR-1 is the only thiamine transporter expressed in bone marrow, cochlear, and pancreatic beta cells. THTR-1 loss-of-function leads to the rare recessive genetic disease Thiamine-Responsive Megaloblastic Anemia (TRMA, OMIM #249270). METHODS: In vitro stimulated blood lymphocytes were used for cytogenetics and the isolation of genomic DNA used to perform whole exome sequencing (WES). To validate identified mutations, direct Sanger sequencing was performed following PCR amplification. RESULTS: A 6-year-old male born from a consanguineous couple presenting bone marrow failure and microcephaly was referred to our clinic for disease diagnosis. The patient presented a normal karyotype and no chromosomal fragility in response to DNA damage. WES analysis led to the identification of a new pathogenic variant in the SLC19A2 gene (c.596C>G, pSer199Ter) allowing to identify the young boy as a TRMA patient. CONCLUSION: Our analysis extend the number of inactivating mutations in SLC19A2 leading to TRMA that could guide future prenatal diagnosis for the family and follow-up for patients.

A journey with common fragile sites: From S phase to telophase.

Some regions of the genome, notably common fragile sites (CFSs), are hypersensitive to replication stress and often involved in the generation of gross chromosome rearrangements in cancer cells. CFSs nest within very large genes and display cell-type-dependent instability. Fragile or not, large genes tend to replicate late in S-phase. A number of data now show that transcription perturbs replication completion across the body of large genes, particularly upon replication stress. However, the molecular mechanisms by which transcription elicits such under-replication and subsequent instability remain unclear. We present here our view of the mechanisms responsible for CFS under-replication and those allowing the cells to cope with this problem in G2 and mitosis. We notably focus on the major role played by the FANC proteins in the protection of CFSs from S phase up to late mitosis. We finally discuss a possible rationale for the conservation of large genes across vertebrate evolution.