Variations in the TP53 and KRAS genes indicate a particularly adverse prognosis in relapsed pediatric T-ALL. We hypothesized that these variations might be subclonally present at disease onset and contribute to relapse risk. To test this, we examined two cohorts of children diagnosed with T-ALL: one with 81 patients who relapsed and 79 matched non-relapsing controls, and another with 226 consecutive patients, 30 of whom relapsed. In cohort 1, targeted sequencing revealed TP53 clonal and subclonal variants in 6 of 81 relapsing patients but none in the non-relapsing group (p=0.014). KRAS alterations were found in 9 of 81 relapsing patients compared to 2 of 79 non-relapsing patients (p=0.032). Survival analysis showed that none of the relapsed patients with TP53 and/or KRAS alterations survived, whereas 19 of 67 relapsed patients without such variants did with a minimum follow-up time of 3 years (p=0.023). In cohort 2, none of the relapsing patients but 10 of 196 non-relapsing patients carried TP53 or KRAS variants, indicating that mutation status alone does not predict poor prognosis. All ten non-relapsing patients with mutations had a favorable early treatment response. Among the total cohort of 386 patients, 188 showed poor treatment response of whom 69 relapsed. Nine of these poor responders harbored TP53 or KRAS variants. In conclusion, subclonal TP53 and KRAS alterations identified at the time of initial diagnosis, along with a poor treatment response, characterize a subset of children with T-ALL who face a dismal prognosis and may benefit from alternative treatment approaches.

Abstract It has been the hypothesis of this study that clonal or subclonal variants of TP53 and KRAS that signify ultra-high risk in relapsed pediatric T-ALL indicate a poor prognosis at initial disease. We analyzed two cohorts of 160 and 226 BFM-patients, one consisting of 81 patients who later relapsed and 79 matched non-relapsing controls. Cohort-2 consisted of consecutive patients of whom 30 relapsed. In cohort-1 targeted sequencing revealed TP53 clonal and subclonal variants in 6/81 relapsing but in none of the non-relapsing patients (p=0.014). KRAS alterations occurred in 9/81 relapsing and 2/79 non-relapsing patients (p=0.032). No relapsing patient with TP53 and/or KRAS alterations survived, whereas 19/67 relapsing patients without such variants did (p=0.023). In cohort-2, 196 patients without relapse included 10 with TP53 or KRAS variants, all of whom were non-high risk based on treatment response. However, in the entire group of 386 patients the 188 patients with poor treatment response included 9 with such a variant of whom 8 relapsed and died. Of these, 5 had not undergone stem-cell-transplantation. In conclusion, these data show that subclonal and clonal TP53 and KRAS alterations at initial diagnosis indicate a dismal prognosis in children with T-ALL and poor treatment response.

Background: Relapse of pediatric T-cell acute lymphoblastic leukemia (T-ALL) remains a substantial challenge in pediatric oncology. Patients who suffer relapse face a dismal prognosis caused by therapy resistance. There are no molecular biomarkers reliably predicting risk. In T-ALL relapse, inactivating variants in TP53 were previously described to identify approximately 10% patients who all experienced fatal relapse (Hof et al. 2011; Richter-Pechanska et al. 2017). Variants in other genes were described to be either associated with a poor prognosis in relapse (MSH6, USP7, IL7R, CNOT3, KRAS and NRAS (Richter-Pechanska et al. 2017)) or to be characteristic for relapse (CCDC88A (Richter-Pechanska et al. 2017), NT5C2 (Kunz et al. 2015; Meyer et al. 2013)). Aims: Considering that clonal evolution is a known mechanism that drives therapy resistance and relapse in cancer (Ding et al. 2012; O’Leary et al. 2018), we tested the hypothesis that subclonal or clonal variants that signify ultra-high-risk at the time of relapse may also be prognostic at the time of initial disease. Methods: Two cohorts of pediatric patients at initial diagnosis of T-ALL who were treated on ALL-BFM protocols were analyzed. Cohort-1 comprised a total of 160 samples that were selected based on a case-control design: 81 patients who later relapsed and 79 who reained in first complete remission for at least three years matched for treatment response/intensity, age and sex. In cohort-2, 226 samples of unselected consecutive T-ALL patients were analyzed. We performed targeted deep sequencing of 9 genes (TP53, KRAS, NRAS, MSH6, USP7, IL7R, CNOT3, CCDC88A, NT5C2) using the Agilent Haloplex High Sensitivity kit with unique molecular identifiers to enable the reliable detection of variants with very low allele frequencies (average read depth: 1 012 +/- 642 and 723 +/- 491 reads). Results: Overall, we identified 75 variants in 7/9 targeted genes in 54/160 (34%) T-ALL patients of cohort-1. The variants tended to be more common in relapsing than in non-relapsing patients (33 vs. 21; Chi2, p=0.058). The mean allele frequency of the detected variants was 24.7% (SD±18; range 0.8-83). More than half of the variants (43/75) were found at allele frequencies <30% and were thus considered subclonal. Interestingly, TP53 variants were identified exclusively in patients later developing a relapse (6/81; Chi2, p=0.014). Variants of KRAS were also significantly more frequent in relapsing patients (9/81 vs. 2/79; Chi2, p=0.032). All of these 14 patients with TP53 and/or KRAS variants died in relapse, whereas 19/67 without such variants survived the relapse (p=0.023). In the unselected patients of cohort-2, a total of 66 variants (subclonal: n=32, clonal: n=34) were found in 8/30 (27%) relapsing and 48/196 (25%) non-relapsing patients. TP53 and KRAS variants were not enriched in patients who later relapsed, indicating that the presence of these variants per se does not predict the risk of relapse in unselected patients. We thus extended our analysis by also considering treatment response. Of all the 386 analyzed patients, 188 were stratified into the high-risk arm because of poor treatment response. Of these 188 patients, 9 carried subclonal or clonal TP53 and/or KRAS variants, of whom 8 suffered a relapse and subsequently died. Of these, 5 had not received stem cell transplantation. Summary/Conclusion: In conclusion, our findings indicate that the combination of TP53 and/or KRAS variants with poor treatment response identifies a subgroup of patients with a dismal prognosis who might benefit from treatment intensification or experimental treatment approaches such as CD1a or CD7 CAR-T cell therapy.Keywords: T-ALL, Acute lymphoblastic leukemia, High risk, Risk factor

Background: Anemia affects ~25% of the world population and is frequently caused by iron deficiency as a consequence of malnutrition or inflammation. Only in rare cases anemia is due to mutations in genes responsible for balancing iron homeostasis. Among these, loss of function variants of MT2 cause iron-refractory iron-deficiency anemia (IRIDA); a disease characterized by inappropriately high levels of hepcidin. Hepcidin is a hepatic hormone that limits dietary iron uptake and iron release from intracellular stores and thus renders IRIDA patients resistant to iron therapies. In mice, a form of anemia resistant to iron supplementation is further caused by mutations in Transferrin Receptor 1 (TfR1; Tfrc gene). TfR1 internalizes iron-bound transferrin, a process fundamental for erythropoiesis. However, up to date, the sole human report describing a TfR1 mutation (p.Tyr20His) showed that it impairs the immunological compartment rather than erythroblast maturation. Here, we report pediatric patients with previously undescribed mutations in TFRC, diagnosed with microcytic hypochromic anemia, partially resistant to iron therapies. Aims: Identification of novel disease alleles that cause anemia. Methods: Whole exome sequencing (WES) of DNA of patients with anemia. Functional characterization of novel mutations identified in TFRC gene was assessed in HeLa cells overexpressing TfR1 wild-type and mutated by western blotting and wide-field microscopy. Results: A 4-year old boy manifested symptoms of anemia and the analysis of hematological parameters revealed low levels of hemoglobin, MCV and MCH. Plasma iron, transferrin saturation and hepcidin were within physiological ranges. Unexpectedly, soluble TfR1 (sTfR1) levels were almost undetectable and the bone marrow smear revealed increased erythropoiesis and iron deficiency in this compartment. The condition of hypochromic microcytic anemia was persistent over years and could not be corrected by oral iron therapy. WES analysis identified a TFRC_c.941C>T homozygous mutation, encoding for TfR1P314L. Relatives carrying the same mutation in heterozygosity were not anemic. This indicates that TFRC_c.941C>T mutation causes a recessive form of anemia. Overexpression of a construct expressing the TfR1P314L mutant in HeLa cells showed a reduction in total and secreted TfR1, thus reflecting upon the observation of very low sTfR1 levels in the blood of the patient. We further show that lower TfR1 levels are likely due to a faster degradation rate of TfR1P314L compared to TfR1WT. In addition, the TfR1P314L mutated protein showed impaired internalization of fluorescently labeled transferrin, most likely explaining the reduced iron levels observed in the bone marrow. In addition to this index patient, 3 additional children of 2 independent families were identified with TFRC mutations and hypochromic microcytic anemia. The genetic analysis detected a TFRC_c.967C>G heterozygous mutation encoding for TfR1P323A and a TFRC_c.934G>A homozygous mutation, encoding for TfR1G312R. All three TFRC mutations are highly conserved across species and are located in the same extracellular loop of TfR1. Summary/Conclusion: We identified a novel subtype of juvenile hereditary hypochromic microcytic anemia that is partially resistant to iron therapies and caused by missense mutations in the TFRC gene. Reduced endocytosis of iron-bound transferrin may contribute to insufficient iron supply for erythropoiesis and resistance to iron supplementation. In the clinical practice, our findings suggest to include TFRC sequencing analysis for patients with unexplained anemia.

The iron chelator deferasirox (DFX) can improve anemia, as observed in patients with transfusional iron overload, such as patients with myelodysplastic syndrome (MDS),(1-4) myeloproliferative neoplasms,(5) aplastic anemia,(2,6) pure red cell aplasia,(7) and the iron metabolism disorder aceruloplasminemia.(8) How DFX improves anemia is unclear. It may increase erythropoietin production, improve iron availability for hematopoietic tissue,(9) and modify the bone marrow (BM) microenvironment by reducing oxidative stress(10,11) and inflammation.(12) Here, we describe the sustained and dose-dependent erythroid improvement in a patient with constitutional variants in the TRIB2 and ABCB6 genes. A 55-year-old woman presented with chest pain and a 2-week history of dyspnea on exertion. A diagnosis of macrocytic anemia was made (hemoglobin [Hb], 10.8 g/dL; mean corpuscular volume, 103 fL) and, 5 weeks later, she was referred to our hospital because of hyporegenerative, transfusion-dependent anemia (Hb, 7.8 g/dL). Laboratory evaluation revealed moderate thrombocytosis and leukopenia, hyperferritinemia, and elevated inflammatory parameters (C-reactive protein [CRP], alpha-1-globulin; Table 1). Her last documented Hb level from 2013 had been normal. She had received 1 red blood cell (RBC) transfusion a few days before the referral but no other lifetime transfusions. Further workup was notable for mild splenomegaly, type C gastritis, and IgM-kappa monoclonal gammopathy of undetermined significance. RBC morphology on the peripheral blood smear was overall normal. BM examination showed mild hypercellularity, megakaryocytic and erythroid hyperplasia, impaired erythroid maturation, and interstitial lymphocytosis (Figure 1A). Plasma cell percentage was normal. Ferritin and hemosiderin were considerably increased and ring sideroblasts were absent on examination of iron staining on BM smears. The karyotype was normal, no mutations were detected with a panel of 54 genes associated with myeloid disorders, and the cause of anemia remained unclear. The patient subsequently required about 4 RBC units per month (Figure 1B) because of symptomatic anemia. After 4 months and a total of 15 transfusions, iron chelation therapy (DFX 12 mg/kg daily) was initiated because of iron overload indicated by serum ferritin levels (Figure 1B). Four months after starting DFX, Hb levels increased, the patient became transfusion-independent, and DFX was stopped. However, 2 months later, erythroid normalization was lost. Findings of a BM reexamination were unchanged from initial consultation. Three weeks after reinitiated transfusions plus standard-dose DFX (17 mg/kg daily), Hb levels increased and the patient became transfusion-independent again, this time within several weeks (Figure 1C). DFX remained the only plausible cause of this second remission of anemia and the regained erythroid response was maintained (median Hb, 14.1 g/dL; Figure 1B,D) with DFX. Six months later, treatment was interrupted because of cholestatic liver disease, Coombs-negative hemolysis, abdominal discomfort, and cholecystolithiasis. Both the cholestatic disease and the hemolysis were pre-sumed to be due to DFX because no other cause could be identified, and liver and hemolysis parame-ters normalized after stopping DFX. The patient subsequently maintained Hb levels $12 g/dL for 6 months without DFX (Figure 1B).

The mechanisms underlying T-ALL relapse remain essentially unknown. Multilevel-omics in 38 matched pairs of initial and relapsed T-ALL revealed 18 (47%) type-1 (defined by being derived from the major ancestral clone) and 20 (53%) type-2 relapses (derived from a minor ancestral clone). In both types of relapse, we observed known and novel drivers of multidrug resistance including MDR1 and MVP, NT5C2 and JAK-STAT activators. Patients with type-1 relapses were specifically characterized by IL7R upregulation. In remarkable contrast, type-2 relapses demonstrated (1) enrichment of constitutional cancer predisposition gene mutations, (2) divergent genetic and epigenetic remodeling, and (3) enrichment of somatic hypermutator phenotypes, related to BLM, BUB1B/PMS2 and TP53 mutations. T-ALLs that later progressed to type-2 relapses exhibited a complex subclonal architecture, unexpectedly, already at the time of initial diagnosis. Deconvolution analysis of ATAC-Seq profiles showed that T-ALLs later developing into type-1 relapses resembled a predominant immature thymic T-cell population, whereas T-ALLs developing into type-2 relapses resembled a mixture of normal T-cell precursors. In sum, our analyses revealed fundamentally different mechanisms driving either type-1 or type-2 T-ALL relapse and indicate that differential capacities of disease evolution are already inherent to the molecular setup of the initial leukemia.

Muckenthaler et al describe a novel form of hemochromatosis caused by a constitutional PIGA mutation in 3 children with associated neurologic dysfunction. Hemochromatosis results from decreased hepcidin, which is regulated by HFE, hemojuvelin (HJV), and transferrin receptor 2. HJV is a glycosylphosphatidylinositol-linked protein, so PIGA mutation leads to decreased HJV expression. Interestingly, none of the children had evidence of paroxysmal nocturnal hemoglobinuria. The cause of the novel association with central nervous system manifestations remains to be elucidated.

Background: Relapse is the main cause of death from pediatric acute precursor T-cell leukemia (T-ALL), but the underlying mechanisms of disease evolution from initial disease to relapse remain incompletely understood and show remarkable interpatient heterogeneity. Aims: As cross-sectional studies failed to identify unifying determinants of relapse, we adopted a longitudinal strategy and performed multi-omic analyses in 13 matched pairs of initial diagnosis and relapse samples and their matched PDXs. We extended this set by WES and methylome analyses in an additional cohort of 25 matched DNA samples from patient cells collected at initial diagnosis, remission, and relapse. Methods: Thirty-eight patients were recruited from the ALL‐BFM 2000/2009, CoALL97/03/09, and ALL‐REZ BFM 2002 trials or from Schneider Children’s Medical Center of Israel at time points of initial diagnosis, remission, and relapse. Material from 13 matched pairs of PDXs (RNA, cells) was used for multi-omic analyses, including DNA-Seq (WES), RNA-Seq, ATAC-Seq and methylation analysis with EPIC arrays. Results: Based on the profile of SNVs and InDels we distinguished 18 (47%) type-1 (derived from the major ancestral clone) and 20 (53%) type-2 relapses (derived from a minor ancestral clone). We observed stronger remodeling on the way to type 2 than to type 1 relapses reflected by more evident changes in methylation, chromatin accessibility and gene expression. At the time of relapse, 3/20 type 2 patients exhibited a hypermutator phenotype, probably caused by gains of mutations in TP53, BLM and BUB1B combined with PMS2. Moreover, type 2 T-ALLs were predominantly TAL1-driven (4/8) in contrary to type 1 (0/5). T-ALLs that later progressed to type-2 relapses exhibited a complex subclonal architecture, unexpectedly, already at the time of initial diagnosis. The fraction of subclonal mutations of those T-ALLs that later developed into a type-2 relapse was significantly higher already at the time of initial diagnosis than in those T-ALLs that later developed into a type-1 relapse (p=0.0387; Fisher’s exact), a difference that became even more pronounced at the time of relapse (p<0.0001). On the other hand, relapse type 1 T-ALLs exhibited overexpression of IL7R, its ligand HGF, and repressors of cytokine signaling (SOCS1, SOCS2, SOCS3) which regulates the IL7R pathway via negative feedback loop. Deconvolution analysis of ATAC-Seq profiles showed that T-ALLs later developing into type-1 relapses resembled a predominant immature thymic T-cell population, whereas T-ALLs developing into type-2 relapses resembled a mixture of normal T-cell precursors. Moreover, an analysis of remission samples revealed a significant enrichment of mutations in constitutional cancer predisposition genes (CPG) in type 2 patients, thus indicating fundamental differences between these two groups of patients. In both types of relapse, we observed known and novel drivers of drug resistance including MDR1 and MVP and NT5C2. Image:Summary/Conclusion: In sum, our comprehensive analyses revealed fundamentally different mechanisms driving either type-1 or type-2 T-ALL relapse and indicate that differential capacities of disease evolution are already inherent to the molecular setup of the initial leukemia. Leukemias of patients with type-1 relapses were often characterized by upregulation of the IL7R pathway, whereas type-2 relapses were characterized by (i) an enrichment of TAL-1 fusion, (ii) and of constitutional mutations in CPG, (iii) divergent genetic and epigenetic remodeling, and (iv) an enrichment of somatic hypermutator phenotypes.

Aberrant B-cell receptor (BCR)/NF-kB signaling is a hallmark feature of B-cell non-Hodgkin lymphomas (B-NHL), especially in diffuse large B-cell lymphoma (DLBCL). Recurrent mutations in this cascade, e.g. in CD79B, CARD11, or NFKBIZ, and also in the Toll-like receptor pathway transducer MyD88, all deregulate NF-kB, but their differential impact on lymphoma development and biology remains to be determined. We functionally investigate here primary mouse lymphomas that formed in recipient mice of Eµ-myc transgenic hematopoietic stem cells (HSC) stably transduced with naturally occurring NF-kB mutants. While most mutants supported Myc-driven lymphoma formation through repressed apoptosis, CARD11- or MyD88-mutant lymphoma cells selectively presented with a macrophage-activating secretion profile, which, in turn, strongly enforced TGF-b-mediated senescence in the lymphoma cell compartment. However, MyD88- or CARD11-mutant Eµ-myc lymphomas exhibited high-level expression of the immune checkpoint mediator PD-L1, thus preventing their efficient clearance by adaptive host immunity. Conversely, these mutant-specific dependencies were therapeutically exploitable by anti-PD1 checkpoint blockade, leading to direct T-cell-mediated lysis of predominantly but not exclusively senescent lymphoma cells. Importantly, mouse-based mutant MyD88- and CARD11-derived signatures marked DLBCL subgroups exhibiting mirroring phenotypes with respect to the triad of senescence induction, macrophage attraction, and evasion of cytotoxic T-cell immunity. Complementing genomic subclassification approaches, our functional, cross-species investigation unveils pathogenic principles and therapeutic vulnerabilities applicable to and testable in human DLBCL subsets that may inform future personalized treatment strategies.

Derailed cytokine and immune cell networks account for the organ damage and the clinical severity of COVID-19 (refs. 1-4). Here we show that SARS-CoV-2, like other viruses, evokes cellular senescence as a primary stress response in infected cells. Virus-induced senescence (VIS) is indistinguishable from other forms of cellular senescence and is accompanied by a senescence-associated secretory phenotype (SASP), which comprises pro-inflammatory cytokines, extracellular-matrix-active factors and pro-coagulatory mediators5-7. Patients with COVID-19 displayed markers of senescence in their airway mucosa in situ and increased serum levels of SASP factors. In vitro assays demonstrated macrophage activation with SASP-reminiscent secretion, complement lysis and SASP-amplifying secondary senescence of endothelial cells, which mirrored hallmark features of COVID-19 such as macrophage and neutrophil infiltration, endothelial damage and widespread thrombosis in affected lung tissue1,8,9. Moreover, supernatant from VIS cells, including SARS-CoV-2-induced senescence, induced neutrophil extracellular trap formation and activation of platelets and the clotting cascade. Senolytics such as navitoclax and a combination of dasatinib plus quercetin selectively eliminated VIS cells, mitigated COVID-19-reminiscent lung disease and reduced inflammation in SARS-CoV-2-infected hamsters and mice. Our findings mark VIS as a pathogenic trigger of COVID-19-related cytokine escalation and organ damage, and suggest that senolytic targeting of virus-infected cells is a treatment option against SARS-CoV-2 and perhaps other viral infections.

Abstract Introduction Patients who suffer a relapse of pediatric T-cell acute lymphoblastic leukemia (T-ALL) face a dismal prognosis. Prognostic molecular biomarkers that reliably predict the risk of relapse at the time of first diagnosis are not available. Inactivating mutations in TP53 were previously detected in approximately 10% of relapsed patients (Hof et al. J Clin Oncol. 2011) and are invariably associated with fatal outcome (Richter-Pechanska et al. Blood Cancer J. 2017). Mutations in other genes were identified to be either specific for relapse (NT5C2 and CCDC88A) or to be associated with a poor prognosis in relapse (IL7R, KRAS, NRAS, USP7, CNOT3 and MSH6) (Meyer et al. Nat Genet. 2013; Richter-Pechanska et al. Blood Cancer J. 2017). We hypothesized that subclones bearing such mutations can give rise to relapse and analyzed these 9 genes at initial diagnosis of T-ALL with targeted ultra-deep sequencing. Methods Leukemia samples collected at initial diagnosis of 81 children with T-ALL who later relapsed were analyzed. As a control group, we selected 79 children with T-ALL who remained in first remission for at least three years and were matched with regard to treatment response, treatment, age and sex. Targeted deep sequencing was performed by using the Agilent Haloplex High Sensitivity kit with unique molecular identifiers for reliable detection of mutations with very low allele frequencies (average read depth: 1,012x). Results Overall, we detected 75 mutations among 7 targeted genes in 33 / 81 relapsing and 21 / 79 non-relapsing patients. The average allele frequency (AF) of the identified mutations was 25% (0.8% - 83%; SD ± 18%). More than half of the variants (43/75) showed AFs below 30% and were thus classified as subclonal. Interestingly, 7 pathogenic TP53 mutations (subclonal: n=5, clonal: n=2) with AFs of 4.4% - 49.4% were exclusively discovered in 6 patients who experienced a relapse. While 2 of these patients received an allogeneic stem cell transplantation in first remission because of poor treatment response, the remaining 4 patients were treated by chemotherapy in the high-risk (n=1) or medium-risk (n=3) arm. None of the 79 non-relapsing control patients carried TP53 mutations. Consistent with the hypothesis of clonal evolution as a mechanism of relapse in T-ALL, Sanger Sequencing of the relapse sample of one TP53-positive patient confirmed that the subclone harboring the TP53 mutation A159D at initial diagnosis (AF 5.4%) expanded to a major clone (AF 42%) in relapse. The presence of TP53 mutations in two further TP53-positive patients in at least one available post-remission sample is also compatible with clonal selection. However, in a fourth patient the low allele frequency of the TP53 mutation at relapse indicates that the TP53 subclone persisted but did not expand during the development of relapse. In addition to TP53, we identified pathogenic KRAS mutations to be significantly enriched in relapsing patients (9 / 81) compared to non-relapsing patients (2 / 79) at the time of initial diagnosis (chi-squared test, p= 0.032; Table 1). Conclusion Subclonal and clonal mutations in TP53 and KRAS at initial diagnosis were enriched in T-ALL patients who later relapsed and identified approximately 17% of patients suffering a relapse. We thus propose that (subclonal) mutations of TP53 and KRAS may define a subgroup of high-risk T-ALL patients already at the time of first diagnosis. The identification of such mutations may complement the current risk stratification which depends on treatment response and may determine a new molecularly defined subgroup of T-ALLs that may benefit from intensified treatment strategies. Figure 1 Figure 1. Disclosures Schrappe: SigmaTau: Other: research support; Amgen: Other: research support; Servier: Honoraria; Novartis: Honoraria; JazzPharma: Honoraria; Servier: Honoraria, Other: research support; JazzPharma: Honoraria, Other: research support; SHIRE: Other: research support; Novartis: Honoraria, Other: research support. Cario: Novartis: Other: Lecture Fee. Muckenthaler: Silence Therapeutics: Research Funding. Kulozik: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BioMedX: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria.

We aimed at identifying the developmental stage at which leukemic cells of pediatric T-ALLs are arrested and at defining leukemogenic mechanisms based onATAC-Seq. Chromatin accessibility maps of seven developmental stages of human healthy T cells revealed progressive chromatin condensation during T-cell maturation. Developmental stages were distinguished by 2,823 signature chromatin regions with 95% accuracy. Open chromatin surroundingSAE1was identified to best distinguish thymic developmental stages suggesting a potential role ofSUMOylation in T-cell development. Deconvolution using signature regions revealed that T-ALLs, including those with mature immunophenotypes, resemble the most immature populations, which was confirmed byTF-binding motif profiles. We integratedATAC-Seq andRNA-Seq and foundDAB1, a gene not related to leukemia previously, to be overexpressed, abnormally spliced and hyper-accessible in T-ALLs.DAB1-negative patients formed a distinct subgroup with particularly immature chromatin profiles and hyper-accessible binding sites forSPI1(PU.1), aTFcrucial for normal T-cell maturation. In conclusion, our analyses of chromatin accessibility andTF-binding motifs showed that pediatric T-ALLcells are most similar to immature thymic precursors, indicating an early developmental arrest.