Cells often utilize multiple pathways to repair the same DNA lesion, and pathway choice has profound implications for the fidelity of genome maintenance. DNA interstrand cross-links (ICLs) block DNA replication and transcription by covalently linking the two strands of DNA, and the cytotoxicity of ICLs is exploited for chemotherapy. In Xenopus egg extracts, replication fork collision with ICLs initiates two distinct repair pathways. The NEIL3 glycosylase can cleave the cross-link 1 , but if this fails, the Fanconi anemia (FA) proteins incise the phosphodiester backbone surrounding the ICL, generating a double-strand break (DSB) intermediate that is repaired by homologous recombination 2 . How the simpler NEIL3 pathway is prioritized over the FA pathway, which can cause genomic rearrangements, is unknown. Here, we show that the E3 ubiquitin ligase Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Summary Reactive aldehydes arise as by-products of metabolism and are normally cleared by multiple families of enzymes. We find that mice lacking two aldehyde detoxifying enzymes, mitochondrial ALDH2 and cytoplasmic ADH5, have greatly shortened lifespans and develop leukemia. Hematopoiesis is disrupted profoundly, with a reduction of hematopoietic stem cells and common lymphoid progenitors causing a severely depleted acquired immune system. We show that formaldehyde is a common substrate of ALDH2 and ADH5 and establish methods to quantify elevated blood formaldehyde and formaldehyde-DNA adducts in tissues. Bone-marrow-derived progenitors actively engage DNA repair but also imprint a formaldehyde-driven mutation signature similar to aging-associated human cancer mutation signatures. Furthermore, we identify analogous genetic defects in children causing a previously uncharacterized inherited bone marrow failure and pre-leukemic syndrome. Endogenous formaldehyde clearance alone is therefore critical for hematopoiesis and in limiting mutagenesis in somatic tissues.

We recently demonstrated that acute myeloid leukemia (AML) cell lines and patient-derived blasts release exosomes that carry RNA and protein; following an in vitro transfer, AML exosomes produce proangiogenic changes in bystander cells. We reasoned that paracrine exosome trafficking may have a broader role in shaping the leukemic niche. In a series of in vitro studies and murine xenografts, we demonstrate that AML exosomes downregulate critical retention factors (Scf, Cxcl12) in stromal cells, leading to hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow. Exosome trafficking also regulates HSPC directly, and we demonstrate declining clonogenicity, loss of CXCR4 and c-Kit expression, and the consistent repression of several hematopoietic transcription factors, including c-Myb, Cebp-β and Hoxa-9. Additional experiments using a model of extramedullary AML or direct intrafemoral injection of purified exosomes reveal that the erosion of HSPC function can occur independent of direct cell–cell contact with leukemia cells. Finally, using a novel multiplex proteomics technique, we identified candidate pathways involved in the direct exosome-mediated modulation of HSPC function. In aggregate, this work suggests that AML exosomes participate in the suppression of residual hematopoietic function that precedes widespread leukemic invasion of the bone marrow directly and indirectly via stromal components.

Acetaldehyde is a highly reactive, DNA damaging metabolite, produced upon alcohol consumption 1. Impaired acetaldehyde detoxification is common in the Asian population, and is associated with alcohol related cancers 1,2. Cellular protection against acetaldehyde-induced damage is provided by DNA crosslink repair; when impaired this causes Fanconi anaemia (FA), a disease resulting in failed blood production and cancer predisposition 3,4. Strikingly, combined inactivation of acetaldehyde detoxification and the FA pathway induces mutation, accelerates malignancies and causes the rapid attrition of blood stem cells 5-7. A key question concerns the nature of DNA damage caused by acetaldehyde, and how this is repaired. Here we generate acetaldehyde-induced DNA interstrand crosslinks (AA-ICLs) and determine their repair mechanism in Xenopus egg extract. We discover that two replication-coupled pathways repair these lesions. The first is the FA pathway, that operates using excision, analogous to the mechanism used for chemotherapeutic crosslinks caused by cisplatin. Yet, this AA-ICL repair results in elevated mutation frequency and altered mutational spectrum. The second repair modality requires replication fork convergence but unexpectedly does not involve DNA incisions, instead the acetaldehyde-crosslink itself is broken. The Y-family DNA polymerase REV1 completes repair, culminating in a distinct mutation spectrum. This work defines how DNA interstrand crosslinks caused by an endogenous and alcohol-derived metabolite are repaired, identifying an excision-independent mechanism. To study the repair of alcohol-induced DNA damage, we generated an acetaldehyde-crosslinked DNA substrate. Acetaldehyde reacts with guanine creating a crosslink precursor, N2-propanoguanine (PdG) (Fig. 1a) 8. In a 5'-CpG sequence, PdG reacts with the N2-amine of guanine on the opposite strand to create an interstrand acetaldehyde crosslink (AA-ICL). The crosslink exists in equilibrium between three states 9. We synthesized a site-specific native AANAT-ICL within an oligonucleotide duplex (Extended Data Fig. 1a, b, d, Supplementary Information Fig. 1). A control reaction of PdG with deoxyinosine (dIno), lacking an N2-amine, did not crosslink, confirming AANAT-ICL site-specificity (Extended Data Fig. 1c, for gel source data see Supplementary Information Fig. 2). AANAT-ICLs were stable at physiological pH and temperature (< 10% reversal after 72 h at 37 C) (Extended Data Fig. 1e). Elevated temperature (55 C) or acid did however reverse AANAT-ICL, consistent with Schiff base Top strand Unhooked Bottom strand Unhooked

ObjectiveRecent evidence indicates that the adult hematopoietic system is susceptible to diet-induced lineage skewing. It is not known whether the developing hematopoietic system is subject to metabolic programming via in utero high-fat diet (HFD) exposure, an established mechanism of adult disease in several organ systems. We previously reported substantial losses in offspring liver size with prenatal HFD. As the liver is the main hematopoietic organ in the fetus, we asked whether the developmental expansion of the hematopoietic stem and progenitor cell (HSPC) pool is compromised by prenatal HFD and/or maternal obesity.MethodsWe used quantitative assays, progenitor colony formation, flow cytometry, transplantation, and gene expression assays with a series of dietary manipulations to test the effects of gestational high-fat diet and maternal obesity on the day 14.5 fetal liver hematopoietic system.ResultsMaternal obesity, particularly when paired with gestational HFD, restricts physiological expansion of fetal HSPCs while promoting the opposing cell fate of differentiation. Importantly, these effects are only partially ameliorated by gestational dietary adjustments for obese dams. Competitive transplantation reveals compromised repopulation and myeloid-biased differentiation of HFD-programmed HSPCs to be a niche-dependent defect, apparent in HFD-conditioned male recipients. Fetal HSPC deficiencies coincide with perturbations in genes regulating metabolism, immune and inflammatory processes, and stress response, along with downregulation of genes critical for hematopoietic stem cell self-renewal and activation of pathways regulating cell migration.ConclusionsOur data reveal a previously unrecognized susceptibility to nutritional and metabolic developmental programming in the fetal HSPC compartment, which is a partially reversible and microenvironment-dependent defect perturbing stem and progenitor cell expansion and hematopoietic lineage commitment.

Key PointsFancc −/− mice experience previously unrecognized late gestational lethality. Fancc −/− fetal mouse hematopoiesis is quantitatively and qualitatively deficient.

SummaryOur mechanistic understanding of Fanconi anemia (FA) pathway function in hematopoietic stem and progenitor cells (HSPCs) owes much to their role in experimentally induced DNA crosslink lesion repair. In bone marrow HSPCs, unresolved stress confers p53-dependent apoptosis and progressive cell attrition. The role of FA proteins during hematopoietic development, in the face of physiological replicative demand, remains elusive. Here, we reveal a fetal HSPC pool in Fancd2−/− mice with compromised clonogenicity and repopulation. Without experimental manipulation, fetal Fancd2−/− HSPCs spontaneously accumulate DNA strand breaks and RAD51 foci, associated with a broad transcriptional DNA-damage response, and constitutive activation of ATM as well as p38 stress kinase. Remarkably, the unresolved stress during rapid HSPC pool expansion does not trigger p53 activation and apoptosis; rather, it constrains proliferation. Collectively our studies point to a role for the FA pathway during hematopoietic development and provide a new model for studying the physiological function of FA proteins.

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