Letters to the Editor to etoposide, which did cause significant cell death at The insecticides permethrin and chlorpyrifos show doses as low as 1 µM, neither permethrin nor chlorpyri- limited genotoxicity and no leukemogenic poten- fos induced cell death/apoptosis at doses 50-fold higher tial in human and murine hematopoietic stem pro- (Online Supplementary Figure S1A), supporting the con- genitor cells cept that the absence of high-grade DNA fragmentation may represent a chromatin physical impediment for Epidemiological and clinical studies have revealed that MLL DSB to fuse in-frame with a partner gene and maternal exposure to pesticides-insecticides during preg- encode an oncogenic fusion protein. Inverse polymerase nancy is associated with an increased risk of infant or chain reaction assays confirmed the absence of in-frame childhood acute leukemia.1-3 The household insecticides MLL fusions (data not shown). Overall, these data indicate permethrin and chlorpyrifos, which are members of the that chronic exposure to insecticides neither enriches for pyrethroid and organophosphate families of pesticides, MLL breaks nor generates MLL fusion oncogenes or respectively, have been associated with both the induc- gross genomic instability. tion of MLL rearrangements (MLLr) and the develop- To test the bona fide ability of permethrin and chlor- ment of infant acute leukemia.4-9 Despite the epidemio- pyrifos to function as topoisomerase II poisons, and their logical association between insecticides and increased ability to generate DNA-DSB we performed an in vivo risk of leukemia, whether such insecticides act as topo complex of enzyme assays to analyze covalent genomic isomerase II DNA-damaging poisons remains unknown DNA/topoisomerase II complexes in live cells.12 and studies testing the biological plausibility of such an Treatment of hESC with permethrin and chlorpyrifos at association are lacking. Here, we assessed the genotoxi- 10–500 µM induced only minimal poisoning of the city, induction of MLLr and leukemogenic potential of topoisomerase II isoforms (a and b), whereas as little as permethrin and chlorpyrifos by taking advantage of cut- 1 µM etoposide induced significant poisoning of both ting-edge in vitro and in vivo models using prenatal, isoforms (Online Supplementary Figure S1B). Similarly, neonatal and adult hematopoietic stem and progenitor monitoring of g-H2AX by western blotting (Online cells (HSPC). Our data suggest that the insecticides test- Supplementary Figure S1C) or fluorescent activated cell ed show no cytotoxicity, limited genotoxicity and no sorting (FACS) analysis (Online Supplementary Figure leukemogenic potential in human and murine HSPC in S1D-F) revealed that the pesticides were unable to vitro and in vivo. induce DSB in hESC, neonatal or adult CD34+ cells at We initially assessed whether acute exposure to etopo- relatively high concentrations, whereas etoposide side (1 µM), permethrin or chlorpyrifos (10 µM or 50 potently induced DSB that were slightly repaired to µM) induces MLL breaks in undifferentiated human some extent over time in somatic CD34+ cells but not in embryonic stem cells (hESC) and CD34+ HSPC derived hESC (Online Supplementary Figure S1F). Overall, the data from hESC, cord blood or adult peripheral blood (Figure indicate that permethrin and chlorpyrifos are not topoi- 1A). Acute exposure (24 h) to either permethrin or chlor- somerase II poisons and do not generate DNA-DSB in pyrifos consistently induced MLL breaks in 3–7% of embryonic or somatic CD34+ cells. embryonic, neonatal and adult CD34+ cells in a dose- We next sought to determine whether prolonged in independent manner (Figure 1B, C). Embryonic and vivo exposure of human CD34+ HSPC to pesticides somatic CD34+ cells were sensitive to the three treat- induces MLL breaks or initiates leukemia (Figure 2A). ments (Figure 1C). Of note, whereas embryonic and Immunodeficient (NSG) mice xenotransplanted with neonatal CD34+ cells were slightly more sensitive to cord blood-derived human CD34+ HSPC were exposed etoposide than to insecticides (Figure 1C), no differences for 12 weeks to permethrin or chlorpyrifos in the drink- were found for adult CD34+ cells, suggesting that the ing water to mimic the environmental exposure in potential genotoxicity of the insecticides may have a humans. Our target concentration for pesticide exposure more relevant etiological impact in adult MLLr acute was 10 mg/kg/day, and 5 mg/kg/day for etoposide. As leukemia. the actual concentration to which mice are exposed will Chronic exposure to low doses of etoposide has been depend on their weight and their consumption of drink- reported to induce apoptosis, MLL breaks and major ing water over the experimental period, we measured chromosomal abnormalities in hESC.10 To test whether both parameters weekly. Results revealed an actual this occurred after exposure to insecticides, we treated intake of permethrin, chlorpyrifos and etoposide only hESC for 24 h with 10 µM of either permethrin or chlor- 10–20% lower than the theoretical concentrations (8.5, pyrifos followed by a daily “booster” dose (2 µM) for 40 9 and 4.2 mg/kg/day, respectively) (Online Supplementary days (Figure 1D). After 5 days of recovery (without treat- Figure S2A-C). Importantly, the permethrin and chlor- ments), MLLr breaks and gross genomic abnormalities pyrifos metabolites 3-BPA and TCPy were readily were assayed by interphase fluorescence in situ detected by gas chromatography-mass spectrometry in hybridization (iFISH), G-banding and comparative serum and urine as soon as 48 h after pesticide exposure, genomic hybridization (Figure 1E-G). In contrast to the confirming consistent exposure through administration frequency of MLL breaks (3–7%) observed after acute of drinking water (Online Supplementary Figure S2D). exposure (Figure 1C), MLL breaks were scarcely iFISH analysis at sacrifice revealed a small but significant detectable upon chronic exposure to permethrin or increase in the frequency of bone marrow-engrafted chlorpyrifos, suggesting a legitimate repair of the DNA human CD45+ cells harboring MLL breaks in etoposide- damage/double strand breaks (DSB) at the MLL locus treated mice, but not in permethrin- or chlorpyrifos- (Figure 1E). Likewise, karyotyping and comparative treated mice (Figure 2B). FACS analysis at sacrifice genomic hybridization analysis revealed no numerical or revealed similar levels of human graft and normal multi- structural chromosomal alterations (Figure 1F) or DNA lineage (immature, myeloid and B-cell lymphoid) gains or losses after chronic exposure (Figure 1G). It has engraftment in the bone marriow and peripheral blood been previously suggested that the chromosomal topol- of etoposide-, permethrin- and chlorpyrifos-treated mice ogy and chromatin structure resulting from early apop- (Figure 2C, Online Supplementary Figure S2E), with no tosis may represent the underlying substrate for MLL evidence of splenomegaly (Figure 2D). These findings chromosomal translocations to occur.11 Here, in contrast indicate that chronic in vivo exposure to the indicated 544 haematologica | 2022; 107(2) Letters to the Editor A B C D E F G Figure 1. In vitro induction of MLL rearrangements in embryonic, neonatal and adult human CD34+ hematopoietic stem and progenitor cells following acute and chronic exposure to etoposide, permethrin and chlorpyrifos. (A) Experimental design to assess the induction of MLL breaks in human undifferentiated embryonic stem cells (hESC) and CD34+ hematopoietic stem and progenitor cells (HSPC) after 24 h single-pulse exposure to the indicated treatments (etoposide, ETO; permethrin, PER; and chlorpyrifos, CPF). (B) Representative interphase fluorescence in situ hybridization (iFISH) images showing MLL germline and rearranged (MLLr) human CD34+ cells. (C) Frequency of MLL breaks in undifferentiated hESC and embryonic, neonatal and adult CD34+ HSPC after 24 h sin- gle-pulse exposure to the indicated treatments (n=3 independent experiments for each cell type). Asterisks indicate statistically significant differences of a given treatment as compared with dimethylsulfoxide (DMSO, vehicle treatment) *P<0.05, **P<0.01. Dotted lines in the graphs show the percentage of MLL breaks in the DMSO-treated control groups. A minimum of 500 nuclei were analyzed except in some samples for which 80-400 nuclei were analyzed. (D) Experimental design to analyze the frequency of MLL breaks and gross chromosomal damage in hESC after continuous exposure to PER and CPF. (E) Frequency of MLL breaks detected by iFISH 45 days after chronic treatment with either PER or CPF (n=3 independent experiments). A minimum of 400 nuclei was analyzed per experi- ment. (F) Representative image of a G-banding karyotype 45 days after chronic treatment with either PER, CPF or DMSO (n=3). (G) Representative image of DNA copy number variation profiling by comparative genomic hybridization array analysis 45 days after chronic treatment with either PER or CPF. CGH: comparative genomic hybridization; AMP: amplification; DEL: deletion; LOH: loss of heterozygosity. haematologica | 2022; 107(2) 545 Letters to the Editor doses of etoposide or the pesticides fails to induce MLL cides (20 mg/kg/day) in drinking water from gestational breaks or to initiate leukemia in NSG-reconstituting day 0.5 to day 21 (Figure 3A). No significant differences human CD34+ HSPC. were found between treatment groups for the number of We next assessed whether chronic exposure to pesti- pups per litter at birth (range, 11–18) or sex distribution cides during embryonic development induces Mll breaks (Online Supplementary Figure S3A, B). The mothers and in developing murine HSPC. To do this, CD1 male and one-half of the litter were euthanized at weaning to ana- female mice were mated and pregnant CD1 females lyze the impact of etoposide and pesticides on the Mll were exposed to etoposide (10 mg/kg/day) or to pesti- locus and on hematopoietic homeostasis, and the A B C D Figure 2. Continuous exposure to permethrin or chlorpyrifos fails to induce MLL breaks or leukemia in NSG-reconstituting human CD34+ hematopoietic stem and progenitor cells. (A) Experimental design to determine whether prolonged in vivo exposure of human CD34+ hematopoietic stem and progenitor cells (HSPC) to insecticides induces MLL breaks or can initiate leukemia. In brief, 32 NSG mice were irradiated and cord blood-derived human CD34+ cells were transplanted into the bone marrow 6–8 h later. Four days later, mice were divided into four groups (8 mice/group) to initiate exposure in drinking water to etoposide (ETO, 5 mg/kg/day), permethrin (PER, 10 mg/kg/day), and chlorpyrifos (CPF, 10 mg/kg/day) or 0.1% dimethylsulfoxide (DMSO). The presence of the PER and CPF metabolites 3-BPA and TCPy in urine and serum was analyzed 48 h later by gas chromatography-mass spectometry. Mice were sacrificed for interphase fluores- cence in situ hybridization (iFISH) and fluorescence activated cell sorting (FACS) analysis after 12 weeks of continuous treatment. (B) Left, scheme depicting the human chromosome 11 and the 11q23 region where the MLL probe hybridizes. Middle, representative iFISH image showing human cells with germline MLL or MLL rearrangement (MLLr). Right, percentage of human CD45+ cells harboring MLL breaks detected by iFISH at sacrifice. A minimum of 500 nuclei per sam- ple was analyzed, except in one mouse from the DMSO group, for which only 216 nuclei could be analyzed. (C) Upper panels show the percentage of human engraftment (CD45+HLA-ABC+ cells) in bone marrow and peripheral blood. Lower panels show the relative proportion of immature (CD34+), myeloid (CD33+) and B-cell (CD19+) populations within human engraftment in bone marrow and peripheral blood. (D) Representative macroscopic images of spleens at sacrifice for each experimental group. GC-MS: gas chromatography-mass spectrometry. 546 haematologica | 2022; 107(2) Letters to the Editor remaining half of the litter was maintained for 32 weeks progenitors in the mothers and adult offspring, but not for analysis at adulthood (Figure 3A). iFISH analysis in the pups (Figure 3B, C, Online Supplementary Figure revealed that exposure to permethrin or chlorpyrifos S3C). during pregnancy failed to induce Mll breaks in bone Finally, we analyzed whether prenatal exposure to marrow Lin-Kit+ progenitors from mothers, pups or adult etoposide or pesticides affects the hematopoietic home- offspring, whereas etoposide exposure induced Mll ostasis of mothers and pups (at weaning) and adult off- breaks in a small but significant proportion of Lin-Kit+ spring. Analysis at sacrifice revealed no significant differ- A B C D E Figure 3. Legend on following page haematologica | 2022; 107(2) 547 Letters to the Editor Figure 3. Continuous exposure to permethrin or chlorpyrifos during pregnancy fails to induce Mll breaks in bone marrow progenitors or alterations in the hematopoietic homeostasis of mothers, pups or adult offspring in mice. (A) Experimental design to determine whether prenatal exposure to etoposide (ETO), permethrin (PER) or chlorpyrifos (CPF) induces Mll breaks or hematopoietic alterations in mothers, pups or adult offspring in mice. In brief, pregnant CD1 female mice were exposed to PER (20 mg/kg/day), CPF (20 mg/kg/day), ETO (10mg/kg/day) or 0.1 % dimethylsulfoxide (DMSO) from day 0 to day 21 of gestation. The number of pups per litter and their sex distribution were analyzed at birth. The mothers and one-half of the litter were analyzed at weaning while the remaining one-half of the offspring were maintained for 32 weeks for analysis in adulthood. (B) Upper panel, scheme depicting mouse chromosome 9 and the region where mouse Mll probes hybridize (UCSC, GRCm38/mm10). Lower left panel, a representative fluorescence in situ hybridization (FISH) image of a mouse metaphase and presence of fluorescence signals in both Mll alleles. Lower right panel, a zoom image of mouse chromosome 9 by DAPI banding, with and without the BAC fluorescence signal revealing the Mll gene localization. (C) Percentage of mouse bone marrow (BM) LK cells with Mll breaks detected by interphase FISH at sac- rifice. DMSO and ETO were used as negative and positive controls, respectively. The numbers in bars indicate the number of mice analyzed. (D) Fluorescence activated cells sorting (FACS) BM analysis of the Lin- Sca-1+ Kit+ (LSK) subpopulation (upper panels), hematopoietic stem and progenitor cell subsets (middle panels), and mature cells (lower panels) in mothers, pups and adult offspring. (E) Representative macroscopic images of spleens (upper panel) and livers (lower panel) at sacrifice of mothers, pups and adult offspring exposed to the indicated treatments. HSC: hematopoietic stem cells; MPP: multipotent progenitors; HPC: hematopoietic progenitor cells. ences in the proportions of mature cells (myeloid, T and mal translocations encoding MLL fusions. This explains B) in peripheral blood or total Lin-Sca+Kit+ progenitors, their limited genotoxicity and no leukemogenic potential hematopoietic stem cells, multipotent progenitors or and reinforces why these compounds are still considered hematopoietic progenitor cells in the bone marrow as non-classifiable carcinogens. Linking environmental between treatments in any group (Figure 3D, Online or genotoxic exposure to causal and/or functional MLL Supplementary Figure S3D). Similarly, prenatal exposure chromosomal translocations has long been to etoposide or pesticides did not affect the hematopoi- controversial,11 and the cutting-edge in vitro and in vivo etic homeostasis in peripheral blood, as determined by cellular models employed in the present study also have absolute numbers of white blood cells, red blood cells obvious limitations. For instance, site-specific cleavage and platelets (Online Supplementary Figure S3E). Lastly, within the MLL break cluster region (bcr) has been no evidence of splenomegaly or hepatomegaly was shown to be induced by either topoisomerase II poisons observed in mothers, pups or adult offspring (Figure 3E). but also genotoxic chemotherapeutic agents which do Our results thus suggest that chronic exposure to perme- not target topoisomerase II and even by non-genotoxic thrin or chlorpyrifos during pregnancy does not induce stimuli of apoptotic cell death. In addition, MLL chromo- Mll breaks in bone marrow progenitors or alterations in somal translocations have been linked to higher-order the hematopoietic homeostasis of mothers, pups or adult chromatin fragmentation that occurs during the initial offspring. stages of apoptosis, suggesting that the generation of A unique strength of the present study is the cutting- MLL chromosomal translocations (and likely others) are edge in vivo models employed to assess the genotoxicity part of a generalized acute apoptotic response-mediated and leukemogenesis potential of etoposide, permethrin higher-order chromatin fragmentation which ultimately or chlorpyrifos. The NSG mice model was established to renders a chromosome topology and chromatin struc- mimic the adult exposure associated with occupational ture prone to chromosomal DNA exchanges.14 This is risk, whereas the CD1 mice model attempted to mimic further supported by the ambiguity of MLL transloca- prenatal exposure to topoisomerase II poisons and insec- tions partnering with a large number of different chro- ticides suggested to be involved in the etiology of infant mosomal loci. leukemia. Continuous exposure to permethrin or chlor- pyrifos in both models failed to induce MLL breaks or Virginia C. Rodriguez-Cortez,1 María Pilar Navarrete- alterations in hematopoietic homeostasis, confirming Meneses,2* Oscar Molina,1* Talia Velasco-Hernandez,1 the in vitro results of limited genotoxicity and no leuke- Jessica Gonzalez,3 Paola Romecin,1 Francisco Gutierrez- mogenic potential of permethrin or chlorpyrifos in Agüera,1 Heleia Roca-Ho,1 Meritxell Vinyoles,1 Eric human and murine HSPC after chronic exposure. The Kowarz,4 Pedro Marin,5 Sandra Rodriguez-Perales,6 Carlos fact that MLL breaks are acutely induced by permethrin Gomez-Marin,7 Patricia Perez-Vera,2 Felipe Cortes- or chlorpyrifos but are not sustained upon long-term Ledesma,7 Anna Bigas,1,3,8 Andrea Terron,9 Clara Bueno1,8 chronic exposure in vitro or in vivo indicates a legitimate and Pablo Menendez1,8,10 repair of the DNA damage/DSB in the MLL locus. Of 1 note, although long-term in vivo exposure to etoposide Josep Carreras Leukemia Research Institute. Department of did induce MLLr in some hematopoietic progenitors, it Biomedicine. School of Medicine, University of Barcelona. Barcelona, failed to initiate leukemia in either in vivo models, in line Spain; 2Laboratorio de Genética y Cáncer, Departamento de Genética with a previous study confirming that in utero exposure Humana, Instituto Nacional de Pediatría, Ciudad de México, México; 3 to etoposide did not trigger the development of leukemia Cancer Research Program, Institut Hospital del Mar d'Investigacions in either Atm+/+ or Atm-/- mice.13 The eventual develop- Mèdiques, Hospital del Mar, Barcelona, Spain; 4Institute of ment of overt leukemia might depend on the survival Pharmaceutical Biology/DCAL, Goethe-University of Frankfurt, and proliferative advantage of minor MLLr pre-leukemic Frankfurt/Main, Germany; 5Hematology Department. Hospital Clínic clones, targeting the right cell-of-origin, on stromal bone de Barcelona, Barcelona, Spain; 6Molecular Cytogenetics Group, marrow interactions and also on the acquisition of sec- Human Cancer Genetics Program, Spanish National Cancer Research ondary cooperating oncogenic alterations. The clearance Center (CNIO), Madrid, Spain; 7Topology and DNA Breaks Group, and lack of selection of MLLr clones is consistent with Spanish National Cancer Center (CNIO), Madrid, Spain; 8Centro de the development of MLLr treatment-related acute Investigación Biomedica en Red-Oncología (CIBERONC), Madrid, leukemia in adults or infant leukemia in only a rare Spain; 9European Food and Safety Authority. Parma. Italy and 10 subset of patients exposed to topoisomerase II poisons. Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Our results clearly suggest that permethrin and chlor- Barcelona, Spain. pyrifos induce MLL breaks in human HSPC across *MPNM and OM contributed equally. ontogeny. However, such insecticide-induced DNA-DSB Correspondence: are successfully repaired, and do not involve chromoso- PABLO MENÉNDEZ - pmenendez@carrerasresearch.org 548 haematologica | 2022; 107(2) Letters to the Editor VIRGINIA RODRÍGUEZ-CORTEZ - vrodriguez@carrerasre- EXPLORA Project BIO2017-91272-EXP and AECC_Lab_2020 search.org Project. PM is an investigator of the Spanish Cell Therapy cooper- doi:10.3324/haematol.2021.279047 ative network (TERCEL). Received: April 21, 2021. Accepted: October 19, 2021. References Pre-published: October 28, 2021. 1. Infante-Rivard C, Labuda D, Krajinovic M, Sinnett D. Risk of child- Disclosures: no conflicts of interest to disclose. hood leukemia associated with exposure to pesticides and with gene polymorphisms. Epidemiology. 1999;10(5):481-487. Contributions:VR-C and MPN-M: conceptualization, methodol- 2. Ma X, Buffler PA, Gunier RB, et al. Critical windows of exposure to ogy, formal analysis and investigation, writing and preparation of household pesticides and risk of childhood leukemia. Environ the original draft; OM and TV-H: methodology, formal analysis Health Perspect. 2002;110(9):955-960. and investigation; JG, PR, FG-A, HR-H, EK, MV, SR-P and 3. Turner MC, Wigle DT, Krewski D. Residential pesticides and child- CG-M: methodology; PMa, AB, AT, PP-V and FC-L: resourses; hood leukemia: a systematic review and meta-analysis. Environ Health Perspect. 2010;118(1):33-41. CB and Pme: conceptualization, writing, review and editing, super- 4. Navarrete-Meneses MP, Pedraza-Meléndez AI, Salas-Labadía C, vision. Moreno-Lorenzana D, Pérez-Vera P. Low concentrations of perme- Acknowledgments:the authors would like to thank the staff of the thrin and malathion induce numerical and structural abnormalities mass spectrometry unit of the scientific and technological services of in KMT2A and IGH genes in vitro. J Appl Toxicol. 2018;38(9):1262- the University of Barcelona for pesticide metabolite analysis. We are 1270. 5. Navarrete-Meneses MDP, Pérez-Vera P. Pyrethroid pesticide expo- also grateful to the staff at the animal facility of the scientific park of sure and hematological cancer: epidemiological, biological and Barcelona for urine collection in metabolic cages. molecular evidence. Rev Environ Health. 2019;34(2):197-210. Funding: this work was supported by CERCA/Generalitat de 6. Lu C, Liu X, Liu C, et al. Chlorpyrifos induces MLL translocations Catalunya and Fundació Josep Carreras-Obra Social la Caixa through caspase 3-dependent genomic instability and topoiso- merase II inhibition in human fetal liver hematopoietic stem cells. through institutional support. Pablo Menendez’s laboratory was Toxicol Sci. 2015;147(2):588-606. supported by the Spanish Ministry of Science and Innovation 7. Ferreira JD, Couto AC, Pombo-de-Oliveira MS, Koifman S. In utero (PID2019-108160RB-I00 / AEI / 10.13039/501100011033), pesticide exposure and leukemia in Brazilian children < 2 years of Retos Colaboración with Banc de Sang i Teixits (RTC-2017-6367- age. Environ Health Perspect. 2013;121(2):269-275. 1), the European Food and Safety Authority 8. Ding G, Shi R, Gao Y, et al. Pyrethroid pesticide exposure and risk (EFSA.PRAS.2018.04-CT1), and the European Research Council. of childhood acute lymphocytic leukemia in Shanghai. Environ Sci VR-C and MV were supported by Juan de la Cierva fellowships Technol. 2012;46(24):13480-13487. 9. Borkhardt A, Wilda M, Fuchs U, Gortner L, Reiss I. Congenital from the Spanish Ministry of Science and Innovation (FJCI-2015- leukaemia after heavy abuse of permethrin during pregnancy. Arch 24303 and IJCI-2017-33172). CB was supported by the Health Dis Child Fetal Neonatal Ed. 2003;88(5):436-437. Institute Carlos III (ISCIII/FEDER PI17/01028 and PI20/00822). 10. Bueno C, Catalina P, Melen GJ, et al. Etoposide induces MLL PP-V and PN-M were supported by Consejo Nacional de Ciencia rearrangements and other chromosomal abnormalities in human y Tecnología (CB-2012-01-183467) and by Fondos del embryonic stem cells. Carcinogenesis. 2009;30(9):1628-1637. Presupuesto Federal para la Investigación (project 001/2013, 11. Stanulla M, Wang J, Chervinsky DS, Thandla S, Aplan PD. DNA Instituto Nacional de Pediatría). JG was supported by Centro de cleavage within the MLL breakpoint cluster region is a specific event which occurs as part of higher-order chromatin fragmentation dur- Investigación Biomédica en Red en Cáncer (PID2019-104695RB- ing the initial stages of apoptosis. Mol Cell Biol. 1997;17(7):4070. I00). TV-H was supported by a Maria Sklodowska-Marie Curie 12. Schellenberg MJ, Lieberman JA, Herrero-Ruiz A, et al. ZATT fellowship (792923). OM was supported by a Beatriu de Pinós (ZNF451)–mediated resolution of topoisomerase 2 DNA-protein postdoctoral fellowship (BP2016-00048) from the Generalitat de cross-links. Science. 2017;357(6358):1412-1416. Catalunya, a Lady Tata award from the Lady Tata Memorial 13. Nanya M, Sato M, Tanimoto K, Tozuka M, Mizutani S, Takagi M. Trust, and an investigator fellowship from the Spanish Cancer Dysregulation of the DNA damage response and KMT2A rearrangement in fetal liver hematopoietic cells. PLoS One. Research Association (AECC INVES211226MOLI). SR-P is sup- 2015;10(12):1-18. ported by grants from the Spanish National Research and 14. Aplan PD. Chromosomal translocations involving the MLL gene: Development Plan, Instituto de Salud Carlos III, and FEDER molecular mechanisms. DNA Repair (Amst). 2006;5(9-10):1265- (PI17/02303, PI20/01837 and DTS19/00111); AEI/MICIU 1272. haematologica | 2022; 107(2) 549