Two Aldehyde Clearance Systems Are Essential to Prevent Lethal Formaldehyde Accumulation in Mice and Humans.

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.

The ribonuclease domain function is dispensable for SLFN11 to mediate cell fate decision during replication stress response.

The SLFN11 gene participates in cell fate decision following cancer chemotherapy and encodes the N-terminal ribonuclease (RNase) domain and the C-terminal helicase/ATPase domain. How these domains contribute to the chemotherapeutic response remains controversial. Here, we expressed SLFN11 containing mutations in two critical residues required for RNase activity in SLFN11-/- cells. We found that this mutant was still able to suppress DNA damage tolerance, destabilized the stalled replication forks, and perturbed recruitment of the fork protector RAD51. In contrast, we confirmed that the helicase domain was essential to accelerate fork degradation. The fork degradation by the RNase mutant was dependent on both DNA2 and MRE11 nuclease, but not on MRE11's novel interactor FXR1. Collectively, these results supported the view that the RNase domain function is dispensable for SLFN11 to mediate cell fate decision during replication stress response.

SLFN11 promotes stalled fork degradation that underlies the phenotype in Fanconi anemia cells.

Fanconi anemia (FA) is a hereditary disorder caused by mutations in any 1 of 22 FA genes. The disease is characterized by hypersensitivity to interstrand crosslink (ICL) inducers such as mitomycin C (MMC). In addition to promoting ICL repair, FA proteins such as RAD51, BRCA2, or FANCD2 protect stalled replication forks from nucleolytic degradation during replication stress, which may have a profound impact on FA pathophysiology. Recent studies showed that expression of the putative DNA/RNA helicase SLFN11 in cancer cells correlates with cell death on chemotherapeutic treatment. However, the underlying mechanisms of SLFN11-mediated DNA damage sensitivity remain unclear. Because SLFN11 expression is high in hematopoietic stem cells, we hypothesized that SLFN11 depletion might ameliorate the phenotypes of FA cells. Here we report that SLFN11 knockdown in the FA patient-derived FANCD2-deficient PD20 cell line improved cell survival on treatment with ICL inducers. FANCD2-/-SLFN11-/- HAP1 cells also displayed phenotypic rescue, including reduced levels of MMC-induced chromosome breakage compared with FANCD2-/- cells. Importantly, we found that SLFN11 promotes extensive fork degradation in FANCD2-/- cells. The degradation process is mediated by the nucleases MRE11 or DNA2 and depends on the SLFN11 ATPase activity. This observation was accompanied by an increased RAD51 binding at stalled forks, consistent with the role of RAD51 antagonizing nuclease recruitment and subsequent fork degradation. Suppression of SLFN11 protects nascent DNA tracts even in wild-type cells. We conclude that SLFN11 destabilizes stalled replication forks, and this function may contribute to the attrition of hematopoietic stem cells in FA.

Analysis of disease model iPSCs derived from patients with a novel Fanconi anemia-like IBMFS ADH5/ALDH2 deficiency.

We have recently discovered Japanese children with a novel Fanconi anemia-like inherited bone marrow failure syndrome (IBMFS). This disorder is likely caused by the loss of a catabolic system directed toward endogenous formaldehyde due to biallelic variants in ADH5 combined with a heterozygous ALDH2*2 dominant-negative allele (rs671), which is associated with alcohol-induced Asian flushing. Phytohemagglutinin-stimulated lymphocytes from these patients displayed highly increased numbers of spontaneous sister chromatid exchanges (SCEs), reflecting homologous recombination repair of formaldehyde damage. Here, we report that, in contrast, patient-derived fibroblasts showed normal levels of SCEs, suggesting that different cell types or conditions generate various amounts of formaldehyde. To obtain insights about endogenous formaldehyde production and how defects in ADH5/ALDH2 affect human hematopoiesis, we constructed disease model cell lines, including induced pluripotent stem cells (iPSCs). We found that ADH5 is the primary defense against formaldehyde, and ALDH2 provides a backup. DNA repair capacity in the ADH5/ALDH2-deficient cell lines can be overwhelmed by exogenous low-dose formaldehyde, as indicated by higher levels of DNA damage than in FANCD2-deficient cells. Although ADH5/ALDH2-deficient cell lines were healthy and showed stable growth, disease model iPSCs displayed drastically defective cell expansion when stimulated into hematopoietic differentiation in vitro, displaying increased levels of DNA damage. The expansion defect was partially reversed by treatment with a new small molecule termed C1, which is an agonist of ALDH2, thus identifying a potential therapeutic strategy for the patients. We propose that hematopoiesis or lymphocyte blastogenesis may entail formaldehyde generation that necessitates elimination by ADH5/ALDH2 enzymes.

Effects of the major formaldehyde catalyzer ADH5 on phenotypes of fanconi anemia zebrafish model.

BACKGROUND: Fanconi anemia (FA) is a devastating hereditary disorder for which we desperately need a novel therapeutic strategy. It is caused by mutations in one of at least 22 genes in the FA pathway and is characterized by developmental abnormalities, bone marrow failure, and cancer predisposition. The FA pathway is required for the efficient repair of damaged DNA, including interstrand cross-links (ICL). Recent studies indicate formaldehyde as an ultimate endogenous cause of DNA damage in FA pathophysiology. Formaldehyde can form DNA adducts as well as ICLs by inducing covalent linkages between opposite strands of double-stranded DNA. METHODS AND RESULTS: In this study, we generated a disease model of FA in zebrafish by disrupting the ube2t or fancd2 gene, which resulted in a striking phenotype of female-to-male sex reversal. Since formaldehyde is detoxified from the body by alcohol dehydrogenase 5 (ADH5), we generated fancd2-/-/adh5-/- zebrafish. We observed a body size reduction and a lower number of mature spermatozoa than wild-type or single knockout zebrafish. To evaluate if increased activity in ADH5 can affect the FA phenotype, we overexpressed human ADH5 in fancd2-/- zebrafish. The progress of spermatogenesis seemed to be partially recovered due to ADH5 overexpression. CONCLUSIONS: Our results suggest potential utility of an ADH5 enzyme activator as a therapeutic measure for the clearance of formaldehyde and treatment of FA.

[Aldehyde degradation deficiency (ADD) syndrome: discovery of a novel fanconi anemia-like inherited BMF syndrome due to combined ADH5/ALDH2 deficiency].

We have recently described the identification of a novel inherited bone marrow failure syndrome. The first set of patients was diagnosed through the exome analysis of cells from Japanese patients with hypoplastic anemia, which have been deposited to the JCRB cell bank for quite some time previously. Originally, these cases were diagnosed with a novel disorder based on increased levels of sister chromatid exchanges in lymphocytes; however, causative genes were clarified only after applying the recently developed next-generation sequencing technology. Aldehyde degradation deficiency syndrome (ADDS) is caused by combined defects in two genes, ADH5 and ALDH2, which are both critical for degrading endogenously generated formaldehyde. Formaldehyde is highly reactive and toxic to biological molecules including DNA, and its endogenous generation in the absence of the degradation system results in DNA damage that overwhelms the DNA repair capacity, leading to the development of BMF with loss of hematopoietic stem cells and progression to MDS/leukemia. In this short review, we would like to summarize what is known today about ADDS for a wide readership of hematology clinicians in Japan.

A simple and highly sensitive method of measuring heme oxygenase activity.

Heme oxygenase (HO) is a rate-limiting step of heme degradation, which catalyzes the conversion of heme into biliverdin, iron, and CO. HO has been characterized in microorganisms, insects, plants, and mammals. Previously used assays of HO activity were complicated and had low sensitivity. We found that the use of an eel bilirubin-bound fluorescent protein, UnaG, can achieve a highly sensitive and simple assay of HO activity. Using several enzyme sources including human culture cells, homogenates of plant tissues, and recombinant yeast HO, data were successfully obtained. The present method can facilitate the examination of HO in various organisms.

Fanconi anemia and Aldehyde Degradation Deficiency Syndrome: Metabolism and DNA repair protect the genome and hematopoiesis from endogenous DNA damage.

We have identified a set of Japanese children with hypoplastic anemia caused by combined defects in aldehyde degrading enzymes ADH5 and ALDH2. Their clinical characteristics overlap with a hereditary DNA repair disorder, Fanconi anemia. Our discovery of this disorder, termed Aldehyde Degradation Deficiency Syndrome (ADDS), reinforces the notion that endogenously generated aldehydes exert genotoxic effects; thus, the coupled actions of metabolism and DNA repair are required to maintain proper hematopoiesis and health.

Mouse Slfn8 and Slfn9 genes complement human cells lacking SLFN11 during the replication stress response.

The Schlafen (SLFN)11 gene has been implicated in various biological processes such as suppression of HIV replication, replication stress response, and sensitization of cancer cells to chemotherapy. Due to the rapid diversification of the SLFN family members, it remains uncertain whether a direct ortholog of human SLFN11 exists in mice. Here we show that mSLFN8/9 and hSLFN11 were rapidly recruited to microlaser-irradiated DNA damage tracks. Furthermore, Slfn8/9 expression could complement SLFN11 loss in human SLFN11-/- cells, and as a result, reduced the growth rate to wild-type levels and partially restored sensitivity to DNA-damaging agents. In addition, both Slfn8/9 and SLFN11 expression accelerated stalled fork degradation and decreased RPA and RAD51 foci numbers after DNA damage. Based on these results, we propose that mouse Slfn8 and Slfn9 genes may share an orthologous function with human SLFN11. This notion may facilitate understanding of SLFN11's biological role through in vivo studies via mouse modeling.

Lack of impact of the ALDH2 rs671 variant on breast cancer development in Japanese BRCA1/2-mutation carriers.

The aldehyde degrading function of the ALDH2 enzyme is impaired by Glu504Lys polymorphisms (rs671, termed A allele), which causes alcohol flushing in east Asians, and elevates the risk of esophageal cancer among habitual drinkers. Recent studies suggested that the ALDH2 variant may lead to higher levels of DNA damage caused by endogenously generated aldehydes. This can be a threat to genome stability and/or cell viability in a synthetic manner in DNA repair-defective settings such as Fanconi anemia (FA). FA is an inherited bone marrow failure syndrome caused by defects in any one of so far identified 22 FANC genes including hereditary breast and ovarian cancer (HBOC) genes BRCA1 and BRCA2. We have previously reported that the progression of FA phenotypes is accelerated with the ALDH2 rs671 genotype. Individuals with HBOC are heterozygously mutated in either BRCA1 or BRCA2, and the cancer-initiating cells in these patients usually undergo loss of the wild-type BRCA1/2 allele, leading to homologous recombination defects. Therefore, we hypothesized that the ALDH2 genotypes may impact breast cancer development in BRCA1/2 mutant carriers. We genotyped ALDH2 in 103 HBOC patients recruited from multiple cancer centers in Japan. However, we were not able to detect any significant differences in clinical stages, histopathological classification, or age at clinical diagnosis across the ALDH2 genotypes. Unlike the effects in hematopoietic cells of FA, our current data suggest that there is no impact of the loss of ALDH2 function in cancer initiation and development in breast epithelium of HBOC patients.

Enhancements of the production of bilirubin and the expression of ß-globin by carbon monoxide during erythroid differentiation.

Heme is degraded by heme oxygenase to form iron, carbon monoxide (CO), and biliverdin. However, information about the catabolism of heme in erythroid cells is limited. In this study, we showed the production and export of bilirubin in murine erythroleukemia (MEL) cells. The production of bilirubin by MEL cells was enhanced when heme synthesis was induced. When mouse bone marrow cells were induced with erythropoietin to differentiate into erythroid cells, the synthesis of bilirubin increased. The expression of ß-globin was enhanced by CO at the transcriptional level. These results indicate that constant production of CO from heme regulates erythropoiesis.

Continuous de novo biosynthesis of haem and its rapid turnover to bilirubin are necessary for cytoprotection against cell damage.

It is well known that haem serves as the prosthetic group of various haemoproteins that function in oxygen transport, respiratory chain, and drug metabolism. However, much less is known about the functions of the catabolites of haem in mammalian cells. Haem is enzymatically degraded to iron, carbon monoxide (CO), and biliverdin, which is then converted to bilirubin. Owing to difficulties in measuring bilirubin, however, the generation and transport of this end product remain unclear despite its clinical importance. Here, we used UnaG, the recently identified bilirubin-binding fluorescent protein, to analyse bilirubin production in a variety of human cell lines. We detected a significant amount of bilirubin with many non-blood cell types, which was sensitive to inhibitors of haem metabolism. These results suggest that there is a basal level of haem synthesis and its conversion into bilirubin. Remarkably, substantial changes were observed in the bilirubin generation when cells were exposed to stress insults. Since the stress-induced cell damage was exacerbated by the pharmacological blockade of haem metabolism but was ameliorated by the addition of biliverdin and bilirubin, it is likely that the de novo synthesis of haem and subsequent conversion to bilirubin play indispensable cytoprotective roles against cell damage.

Neurotransmitter transporter family including SLC6A6 and SLC6A13 contributes to the 5-aminolevulinic acid (ALA)-induced accumulation of protoporphyrin IX and photodamage, through uptake of ALA by cancerous cells.

δ-Aminolevulinic acid (ALA)-induced protoporphyrin accumulation is widely used in the treatment of cancer, as photodynamic therapy (PDT). To clarify the mechanisms of ALA uptake by tumor cells, we have examined the ALA-induced accumulation of protoporphyrin by the treatment of colon cancer DLD-1 and epithelial cancer HeLa cells with γ-aminobutyric acid (GABA)-related compounds. When the cells were treated with GABA, taurine and ß-alanine, the level of protoporphyrin was decreased, suggesting that plasma membrane transporters involved in the transport of neurotransmitters contribute to the uptake of ALA. By transfection with neurotransmitter transporters SLC6A6, SLC6A8 and SLC6A13 cDNA, the ALA- and ALA methylester-dependent accumulation of protoporphyrin markedly increased in HEK293T cells, dependent on an increase in the uptake of ALA. When ALA-treated cells were exposed to white light, the extent of photodamage increased in SLC6A6- and SLC6A13-expressing cells. Conversely, knockdown of SLC6A6 or SLC6A13 with siRNAs in DLD-1 and HeLa cells decreased the ALA-induced accumulation. The expression of SLC6A6 and SLC6A13 was found in some cancer cell lines. Immunohistochemical studies revealed that the presence of these transporters was elevated in colon cancerous cells. These results indicated that neurotransmitter transporters including SLC6A6 and SLC6A13 mediate the uptake of ALA and can play roles in the enhancement of ALA-induced accumulation of protoporphyrin in cancerous cells.

p53 directly regulates the transcription of the human frataxin gene and its lack of regulation in tumor cells decreases the utilization of mitochondrial iron.

Mitochondrial frataxin functions in iron homeostasis, biogenesis of iron-sulfur clusters, protection from oxidative stress and apoptosis, and as a tumor suppressor protein. We examined regulation of the expression of the human frataxin by p53. Pifithrin-α, an inhibitor of p53 function, and knockdown of p53 decreased the level of frataxin mRNA in human kidney HEK 293T cells. The transcriptional activity of the human frataxin gene is enhanced by the proximal promoter containing the p53-responsive element (p53RE) on the gene. Chromatin immunoprecipitation assay and electrophoretic mobility shift assay confirmed the binding of p53 to the human frataxin p53RE. The expression of wild-type p53 in human cancer HeLa cells increased the reporter activity carrying p53RE at the region of -209 to -200bp of the frataxin promoter. Finally, when the HeLa cells overexpressing frataxin were treated with 5-aminolevulinic acid (ALA), there was less accumulation of protoporphyrin than HeLa control cells, and it was sharply decreased by the addition of iron citrate, suggesting that the utilization of mitochondrial iron for heme biosynthesis can be dependent on the level of frataxin. Alternatively, the low expression of frataxin not regulated by p53 in tumor cells lowers the utilization of iron in mitochondria, causing the tumor-specific ALA-induced accumulation of protoporphyrin.

Imaging of heme/hemeproteins in nucleus of the living cells expressing heme-binding nuclear receptors.

Several factors involved in the core circadian rhythm are PAS domain proteins, one of which, neuronal PAS2 (NPAS2), contains a heme-binding motif. It is thought that heme controls the transcriptional activity of core circadian factors BMAL1-NPAS2, and that the heme-binding nuclear receptor REV-erbα negatively regulates the expression of BMAL1. To examine the role of heme in the nucleus, we expressed nuclear hemeproteins including the nuclear localization signal-added cytoglobin, NPAS2 and REV-erbα. Then, the living cells expressing these proteins were treated with 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA). The fluorescent signal derived from DCFH-DA was observed in the nucleus. When the cells were cultured with hemin, the signal of heme in the nucleus increased. Considering that DCFH-DA reacted with heme, we propose that the use of DCFH-DA could be useful in detection of the heme moiety of hemeprotein in vivo.