The critical role of leukemia-initiating cells as a therapy-resistant population in myeloid leukemia is well established. However, the molecular signatures of such cells in acute lymphoblastic leukemia remain underexplored. Moreover, their role in therapy response and patient prognosis is yet to be systematically investigated across various types of acute leukemia. We used single-cell multiomics to analyze diagnostic specimens from 96 pediatric patients with acute lymphoblastic, myeloid, and lineage-ambiguous leukemias. Through the integration of single-cell multiomics with extensive bulk RNA sequencing and clinical data sets, we uncovered a prevalent, chemotherapy-resistant subpopulation that resembles hematopoietic stem and progenitor cells (HSPC-like) and is associated with poor clinical outcomes across all subtypes investigated. We identified a core transcriptional regulatory network (TRN) in HSPC-like blasts that is combinatorially controlled by HOXA/AP1/CEBPA. This TRN signature can predict chemotherapy response and long-term clinical outcomes. We identified shared potential therapeutic targets against HSPC-like blasts, including FLT3, BCL2, and the PI3K pathway. Our study provides a framework for linking intratumoral heterogeneity with therapy response, patient outcomes, and the discovery of new therapeutic targets for pediatric acute leukemias.

Donor blood saves lives, yet the potential impact of recurrent large-volume phlebotomy on donor health and hematopoietic stem cells (HSCs) remains largely unexplored. In our study, we conducted a comprehensive screening of 217 older male volunteer donors with a history of extensive blood donation (>100 lifetime donations) to investigate the phenomenon of clonal hematopoiesis (CH). No significant difference in the overall incidence of CH was found in frequent donors (FDs) compared with sporadic donors (<10 lifetime donations; 212 donors). However, upon deeper analysis of mutations in DNMT3A, the most commonly affected gene in CH, we observed distinct mutational patterns between the FD and age/sex-matched control donor cohorts. Functional analysis of FD-enriched DNMT3A variants examined in CRISPR-edited human HSCs demonstrated their competitive outgrowth potential upon stimulation with erythropoietin (EPO), a hormone that increases in response to blood loss. In contrast, clones harboring leukemogenic DNMT3A R882 mutations increase upon stimulation with interferon gamma. Through concurrent mutational and immunophenotypic profiling of primary samples at single-cell resolution, a myeloid bias of premalignant R882 mutant HSCs was found, whereas no significant lineage bias was observed in HSCs harboring EPO-responsive DNMT3A variants. The latter exhibited preferential erythroid differentiation when persistent erythropoietic stress was applied to CRISPR-edited human HSC xenografts. Our data demonstrate a nuanced, ongoing Darwinian evolution at the somatic stem cell level, with EPO identified as a novel environmental factor that favors HSCs carrying certain DNMT3A mutations.

Although the bone marrow is the main site of blood cell production in adults, rare pools of hematopoietic stem and progenitor cells have been found in extramedullary organs. In mice, we have previously shown that the lung contains hematopoietic progenitor cells and is a site of platelet production. Here, in the adult human lung, we show that functional hematopoietic precursors reside in the extravascular spaces with a frequency similar to the bone marrow and are capable of proliferation and engraftment in mice. The gene signature of pulmonary and medullary CD34+ hematopoietic progenitors indicates greater baseline activation of immune-, megakaryocyte/platelet-, and erythroid-related pathways in lung progenitors. Spatial transcriptomics mapped blood progenitors in the lung to an alveolar interstitium niche with only a few cells identified in an intravascular location. In human blood samples collected for stem cell transplantation, CD34+ cells with a lung signature enriched the mobilized pool of hematopoietic stem cells. These results identify the lung as a pool for uniquely programmed blood stem and progenitor cells with the potential to support hematopoiesis in humans.

Multiple Myeloma is driven by clonal plasma cell (cPC)-intrinsic factors and changes in the tumorigenic microenvironment (TME). To investigate if residual polyclonal PCs (pPCs) are disrupted, single-cell (sc) RNAseq and sc B-cell receptor analysis were applied in a cohort of 46 samples with PC dyscrasias and 21 healthy donors (HDs). Out of n=234,789 PCs, 64,432 were genotypically identified as pPCs with frequencies decreasing over different disease stages, from 23.66% in monoclonal gammopathy of undetermined significance (MGUS) to 3.23% in MMs (p=0.00012). Both cPCs and pPCs had a comparable expression of typical lineage markers (i.e. CD38, CD138), while others were more variable (CD27, ITGB7). Only cPCs overexpressed oncogenes (e.g. CCND1/2, NSD2), but CCND3 was often expressed in pPCs. BCMA was expressed on both p- and cPCs, while GPRC5D was mostly upregulated in cPCs with implications for on-target, off-tumor activity of targeted immunotherapies. In comparison with HDs, pPCs from patients showed upregulated autophagy and disrupted interaction with TME. Importantly, interferon related pathways where significantly enriched in pPCs from patients vs HDs (p-adjusted < 0.05) showing an inflamed phenotype affecting genotypically normal PCs. Function of pPCs was consequently impacted and correlated with immunoparesis, driven by disrupted cellular interactions with TME. Leveraging our scRNAseq data, we derived a "healthy PC signature" that could be applied to bulk transcriptomics from the CoMMpass dataset and predicted significantly better PFS and OS (log rank p < 0.05 for both). Our findings show that genotypic, single-cell identification of pPCs in PC dyscrasias has relevant pathogenic and clinical implications.

Patients with TP53-mutated acute myeloid leukemia (AML) have an extremely poor prognosis, necessitating new treatments. The global, randomized, phase 3 ENHANCE-2 trial evaluated the anti-CD47 monoclonal antibody magrolimab plus azacitidine (Magro/Aza) for previously untreated TP53-mutated AML. Patients determined inappropriate for intensive therapy were randomized to receive Magro/Aza or venetoclax plus azacitidine (Ven/Aza); those appropriate for intensive therapy were randomized to receive Magro/Aza or 7+3 induction chemotherapy. Primary endpoint was overall survival (OS) in the non-intensive arm. At interim analysis, non-intensive-arm OS hazard ratio (HR) between treatment groups was 1.191 (95% CI, 0.744-1.906), meeting the study's definition for futility and resulting in study termination. At final analysis, median OS was 4.4 vs 6.6 months (HR, 1.132; 95% CI, 0.783-1.637; P = .5070) in the non-intensive arm (n = 205) and 7.3 vs 11.1 months (HR, 1.434; 95% CI, 0.635-3.239; P = .3798) in the intensive arm (n = 52) between Magro/Aza and control groups, respectively. Incidences of grade ≥3 adverse events (AEs) were similar across Magro/Aza and control groups (non-intensive, n = 194: 96.9% and 95.9%; intensive, n = 50: 92.6% and 95.7%), including grade ≥3 anemia (non-intensive: 27.1% and 23.5%; intensive: 25.9% and 21.7%). Grade ≥3 infections were observed in 50.0% and 53.1% of patients in the non-intensive arm and 44.4% and 65.2% of intensive arm patients. ENHANCE-2 did not meet its primary endpoint of OS in TP53-mutated AML but provides important data informing future studies in this challenging population. This trial was registered at www.clinicaltrials.gov as #NCT04778397.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that is characterized by an expansion of T-cell progenitors and DNA mutations that lead to overactive NOTCH1 signaling in >50% of T-ALL cases. Using synthetic models of human T-ALL, we report that NOTCH1 dimeric signaling was crucial for the leukemogenesis of human hematopoietic stem/progenitor cells (HSPCs) from cord blood. We also identified a Notch dimerization-dependent gene signature, including the HES4 transcription factor, which induced a proliferative advantage in human HSPCs and in Notch dimerization-dependent, patient-derived xenografts of T-ALL. Interestingly, in human T-ALL cells, HES4 enforced the expression of the Δ133p53 isoform with the concomitant block of proapoptotic p53 target genes and the induction of BCL2L1 gene expression and antiapoptotic B-cell lymphoma extra-large protein. In addition, through an integrated experimental approach that included genetically modified cell lines, RNA/chromatin immunoprecipitation sequencing, and single-cell RNA sequencing profiles of primary T-ALL samples, we revealed cell subsets with Notch dimerization-dependent gene signatures, which indirectly correlated with proapoptotic genes and directly associated with cell markers of poor clinical outcome in primary T-ALL samples. Taken together, these findings highlight the crucial role of NOTCH1 dimeric signaling in human T-cell leukemogenesis and T-ALL maintenance, suggesting that a possible benefit can be obtained with a therapeutic strategy that target NOTCH1 dimer signaling or its downstream effectors.

Fixed-duration venetoclax-rituximab (VenR) in patients with relapsed/refractory chronic lymphocytic leukemia (CLL) in the phase 3 MURANO trial resulted in superior progression-free survival (PFS) and overall survival (OS) vs bendamustine-rituximab (BR). We report the final analyses of MURANO (median follow-up, 7 years). Patients were randomized to VenR (venetoclax 400 mg daily for 2 years plus monthly rituximab for 6 months; n = 194) or BR (6 months; n = 195). In a substudy, patients with progressive disease (PD) received VenR as retreatment or crossover from BR. At the final data cut (3 August 2022), the median PFS with VenR was 54.7 months vs 17.0 months with BR. The 7-year PFS with VenR was 23.0%. The 7-year OS was 69.6% and 51.0%, respectively. Among VenR-treated patients with undetectable minimal residual disease (MRD; uMRD) and no PD at end of treatment (EOT; n = 83), the median PFS from EOT was 52.5 vs 18.0 months in patients with MRD at EOT (n = 35; P < .0001). Fourteen patients had enduring uMRD. Three distinct mutations in BCL2 in 4 patients were identified. In the substudy, 25 patients were retreated with VenR, and 9 patients crossed over to VenR; the median PFS was 23 and 27 months, and the best overall response rate was 72% and 89%, respectively. At the end of combination treatment (EOCT), after retreatment or crossover, 8 and 6 patients achieved uMRD, respectively. No new safety findings were observed. Overall, these final MURANO analyses support consideration of fixed-duration VenR therapy for patients with relapsed/refractory CLL. This trial was registered at www.clinicaltrials.gov as #NCT02005471.

T-cell-recruiting bispecific antibodies (BsAbs) are in clinical development for relapsed/refractory acute myeloid leukemia (AML). Despite promising results, early clinical trials have failed to demonstrate durable responses. We investigated whether activation of the innate immune system through stimulator of interferon (IFN) genes (STING) can enhance target cell killing by a BsAb targeting CD33 (CD33 bispecific T-cell engager molecule; AMG 330). Indeed, we show that cytotoxicity against AML mediated by AMG 330 can be greatly enhanced when combined with the STING agonist 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) or diamidobenzimidazole (diABZI). We used in vitro cytotoxicity assays, immunoblotting, transcriptomic analyses, and extensive CRISPR-Cas9 knockout experiments to investigate the enhancing effect of a STING agonist on the cytotoxicity of AMG 330 against AML. Importantly, we validated our findings with primary AML cells and in a xenograft AML model. Mechanistically, in addition to direct cytotoxic effects of STING activation on AML cells, activated T cells render AML cells more susceptible to STING activation through their effector cytokines, IFN-γ and tumor necrosis factor, resulting in enhanced type I IFN production and induction of IFN-stimulated genes. This feeds back to the T cells, leading to a further increase in effector cytokines and an overall cytotoxic T-cell phenotype, contributing to the beneficial effect of cGAMP/diABZI in enhancing AMG 330-mediated lysis. We established a key role for IFN-γ in AMG 330-mediated cytotoxicity against AML cells and in rendering AML cells responsive to STING agonism. Here, we propose to improve the efficacy of CD33-targeting BsAbs by combining them with a STING agonist.

Cardiovascular disease has been recognized as the main cause of death in adults with sickle cell disease (SCD). Although the exact mechanism linking SCD to cardiomyopathy remains elusive, a possible role of subclinical acute transient myocardial ischemia during acute sickle cell-related vaso-occlusive crises (VOCs) has been suggested. We approached SCD cardiomyopathy by integrated omics using humanized SS mice exposed to hypoxia/reoxygenation (H/R; 10 hours hypoxia followed by 3 hours reoxygenation) stress, mimicking acute VOCs. In sickle cell (SS) mice exposed to H/R, a neutrophil-driven cardiac hypertrophic response is initiated by cardiac proinflammatory pathways, intersecting proteins and micro RNA involved in profibrotic signaling. This response may be facilitated by local unresolved inflammation. We then examined the effect of 17(R)-resolvin D1 (17R-RvD1), a member of the specialized proresolving lipid mediator superfamily, administration on H/R-activated profibrotic and proangiogenic pathways. In SS mice, we found that 17R-RvD1 (1) modulates miRNAome; (2) prevents the activation of NF-κB p65; (3) protects against the H/R-induced activation of both platelet derived growth factor receptor and transforming growth factor (TGF)-β1/Smad2-3 canonical pathways; (4) reduces the expression of hypoxia-inducible factor-dependent proangiogenic signaling; and (5) decreases the H/R-induced proapoptotic cell signature. The protective role of 17R-RvD1 against H/R-induced maladaptive heart remodeling was supported by the reduction of galectin-3, procollagen C-proteinase enhancer-1, and endothelin-1 expression and perivascular fibrosis in SS mice at 3 days after H/R stress compared with vehicle-treated SS animals. Collectively, our data support the novel role of unresolved inflammation in pathologic heart remodeling in SCD mice in response to H/R stress. Our study provides new evidence for protective effects of 17R-RvD1 against SCD-related cardiovascular disease.

Venetoclax (Ven), when combined with intensive chemotherapy, shows promise for untreated acute myeloid leukemia (AML), but its integration with the 7+3 regimen remains underexplored. In a phase 1b study, we assessed the safety and efficacy of Ven with daunorubicin and cytarabine in patients with newly diagnosed AML. A total of 34 patients (median age, 59 years; 62% non-White) received Ven at escalating durations (8, 11, or 14 days). Adverse events included febrile neutropenia (100%), sepsis (29%), and enterocolitis (23.5%), but there were no induction deaths. The median recovery times for neutrophils (>1.0 × 103/μL) and platelets (>100 × 103/μL) were less than 30 days. Composite complete remission was achieved in 85.3% of patients, and 86.2% were negative for measurable residual disease (MRD). Responses spanned all European Leukemia Net 2022 risk categories. With a median follow-up of 9.6 (2-20) months, the median duration of response, event-free survival, and overall survival were not reached. Ven (400 mg), when combined with 7+3 chemotherapy, was safe and effective in achieving MRD-negative remissions across all durations. Ven dose optimization is being explored in the expansion phase of this trial. Future multicenter studies should confirm our findings. This trial was registered at clinicaltrials.gov as #NCT05342584.

Although described more than a decade ago, the mechanism by which the JAK2 46/1 haplotype increases the risk of developing JAK2-mutated myeloproliferative neoplasms (MPNs) remains unexplained. Inflammation and immunity are linked to MPN development and thus could be relevant to the mechanism by which 46/1 mediates its effect. Here, we show that programmed death-1 receptor ligand (PD-L1) expression is elevated in 46/1 haplotype, both in healthy carriers and in CD34+ cells from patients with MPN. Using circular chromosome conformation capture, we observed that PD-L1 and the neighboring PD-L2 loci physically interact with JAK2 in a manner that differs between 46/1 and nonrisk haplotypes. CRISPR/Cas9 genome editing identified a region within JAK2 intron 2 that influences both JAK2 and PD-L1 expression. We suggest that increased PD-L1 expression may be relevant to the mechanism by which 46/1 leads to an increased inherited risk of developing MPN.

The contribution of interleukin-10 (IL-10), secreted by tumoral B cells, to the progression and shaping of the microenvironment in diffuse large B-cell lymphoma (DLBCL) with activated B-cell-like (ABC) phenotype is not yet completely understood. To shed light on this issue, we generated an immunocompetent mouse model of ABC-DLBCL with conditional knockout of IL-10 specifically in malignant B cells. Paradoxically, these mice had significantly worse overall survival when left untreated but experienced increased sensitivity to conventional anti-CD20 immunotherapy or regulatory T-cell depletion. We identified various immunomodulatory mechanisms involved in this behavior. In particular, we show that IL-10-deficient lymphomas acquire a highly immunosuppressed and T-cell-exhausted microenvironment with increased angiogenesis that results in a more aggressive phenotype, which is refractory to PD-1 immune checkpoint blockade. However, the response of IL-10-deficient mice to anti-CD20 immunotherapy was greatly enhanced by upregulation of calcium channels in B cells. In general, IL-10 autocrine signaling promotes the survival of malignant B cells, whereas the paracrine action of B-cell-derived IL-10 maintains an immunoreactive microenvironment that influences the efficacy of emerging immunotherapy strategies targeting the lymphoma microenvironment. Furthermore, IL-10-associated transcriptional signatures derived from our studies may correctly predict clinical outcomes of patients with DLBCL treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone). Thus, our work provides important functional and mechanistic insights into the role of B-cell-derived IL-10 in the biology of ABC-DLBCL.

Most patients with diffuse large B-cell lymphoma (DLBCL) treated with immunotherapies such as bispecific antibodies (BsAbs) or chimeric antigen receptor (CAR) T cells fail to achieve durable treatment responses, underscoring the need for a deeper understanding of mechanisms that regulate the immune environment and response to treatment. Here, an integrative multiomics approach was applied to multiple large independent data sets to characterize DLBCL immune environments and to define their association with tumor cell-intrinsic genomic alterations and outcomes to CD19-directed CAR T-cell and CD20 × CD3 BsAb therapies. This approach effectively segregated DLBCLs into 4 immune quadrants (IQs) defined by cell-of-origin and immune-related gene set expression scores. These quadrants consisted of activated B cell-like (ABC) hot, ABC cold, germinal center B cell-like (GCB) hot, and GCB cold DLBCLs. Recurrent genomic alterations were enriched in each IQ, suggesting that lymphoma cell-intrinsic alterations contribute significantly to orchestrating unique DLBCL immune environments. For instance, SOCS1 loss-of-function mutations were significantly enriched among GCB hot DLBCLs, identifying a putative subset of inflamed DLBCLs that may be inherently susceptible to immunotherapy. In patients with relapsed/refractory DLBCL, DLBCL-IQ assignment correlated significantly with clinical benefit with a CD20 × CD3 BsAb (N = 74), but not with CD19-directed CAR T cells (Stanford, N = 51; Memorial Sloan Kettering Cancer Center, N = 69). Thus, DLBCL-IQ provides a new framework to conceptualize the DLBCL immune landscape and suggests the endogenous immune environment has a more significant impact on outcomes to BsAb than CAR T-cell treatment.

Desmopressin (1-desamino-8-d-arginine vasopressin [DDAVP]) can be used to prevent or stop bleeding. However, large interindividual variability is observed in DDAVP response and determinants are largely unknown. In this systematic review and meta-analysis, we aimed to identify the response to DDAVP and the factors that determine DDAVP response in patients. We included studies with patients with any bleeding disorder receiving DDAVP. First and second screening round and risk of bias assessment were performed by independent reviewers. The main outcome was proportion of patients with complete (factor level >50 U/dL) or partial (30-50 U/dL) response to DDAVP. Determinants of response including disease type, age, sex, von Willebrand factor (VWF) and factor VIII (FVIII) mutations, and baseline factor levels were investigated. In total, 591 articles were found and 103 were included. Of these, 71 articles (1772 patients) were suitable for the study's definition of response. Meta-analysis showed a pooled response proportion of 0.71 (0.64; 0.78) and a significant difference in response between disease subtypes. For hemophilia A, baseline FVIII activity (FVIII:C) was a borderline significant determinant of response. In patients with von Willebrand disease (VWD) type 1, VWF antigen (VWF:Ag), VWF activity, and FVIII:C were significant determinants. A large variation in response was observed for specific mutations in VWF and FVIII. Response to DDAVP varied between disease subtypes and was largely determined by the baseline levels of FVIII:C for hemophilia A and VWF:Ag for VWD. Our findings highlight the significant differences in response and emphasize the need for a standardized response definition and further research into response mechanisms.

Immunomodulatory drug (IMiD) resistance is a key clinical challenge in myeloma treatment. Previous data suggest almost one-third of myeloma patients acquire genetic alteration of the key IMiD effector cereblon (CRBN) by the time they are pomalidomide refractory. Some events, including stop codons/frameshift mutations and copy loss, have clearly explicable effects on CRBN protein function. Missense mutations have also been reported throughout the length of CRBN but their functional impact has not been systematically studied. This study modeled selected missense mutations and examined their effect on CRBN function also analyzing whether any mutations deleterious to IMiD action could be overcome using the novel cereblon E3 ligase modulators (CELMoDs). Three patterns of response to missense mutations were apparent: mutations that led to complete loss of CRBN function for all agents, those that had no effect on CRBN function, and those with agent-dependent effect on CRBN function. The latter group of 4 mutations were profiled in more detail with confirmatory experiments demonstrating an ability of the more potent CELMoDs to lead to neosubstrate degradation and cell death even though IMiDs were not active. Dynamic modeling based on a newly generated crystal structure of the DDB1/CRBN/lenalidomide complex, with greater resolution than those published to date, helped to understand the impact of these mutations. These results have important implications for the interpretation of CRBN sequencing results from patients for future therapy decisions, particularly differentiating those who may, despite relapsing on IMiDs with CRBN mutations, have the potential to still benefit from the use of CELMoD agents.

Leukemic stem cells (LSCs) fuel acute myeloid leukemia (AML) growth and relapse, but therapies tailored toward eradicating LSCs without harming normal hematopoietic stem cells (HSCs) are lacking. FMS-like tyrosine kinase 3 (FLT3) is considered an important therapeutic target due to frequent mutation in AML and association with relapse. However, there has been limited clinical success with FLT3 drug targeting, suggesting either that FLT3 is not a vulnerability in LSC or that more potent inhibition is required, a scenario where HSC toxicity could become limiting. We tested these possibilities by ablating FLT3 using CRISPR/Cas9-mediated FLT3 knockout (FLT3-KO) in human LSCs and HSCs followed by functional xenograft assays. FLT3-KO in LSCs from FLT3-internal tandem duplication (ITD)-mutated but not FLT3-wild-type AMLs resulted in short-term leukemic grafts of FLT3-KO edited cells that disappeared by 12 weeks. By contrast, FLT3-KO in HSCs from the fetal liver, cord blood, and adult bone marrow did not impair multilineage hematopoiesis in primary and secondary xenografts. Our study establishes FLT3 as an ideal therapeutic target where ITD-positive LSCs are eradicated upon FLT3 deletion whereas HSCs are spared. These findings support the development of more potent FLT3-targeting drugs or gene-editing approaches for LSC eradication to improve clinical outcomes.

Platelets are crucial players in hemostasis and thrombosis but also contribute to immune regulation and host defense, using different receptors, signaling pathways, and effector functions, respectively. Whether distinct subsets of platelets specialize in these diverse tasks is insufficiently understood. Here, we used a pulse-labeling method in Mus musculus models for tracking in vivo platelet aging and its functional implications. Using in vitro and in vivo assays, we reveal that young, reticulated platelets show heightened responses in the setting of clot formation, with corresponding, increased responses to agonists, adhesion, and retractile function. Unexpectedly, aged platelets lose their hemostatic proficiency but are more prone to react to inflammatory challenge: compared with reticulated platelets, this cohort was more likely to form platelet-leukocyte aggregates and showed increased adhesion to neutrophils in vitro, as well as enhanced bactericidal function. In vivo, this was reflected in increased pulmonary recruitment of aged platelets in an acute lung injury model. Proteomic analyses confirmed the upregulation of immune pathways in this cohort, including enhanced procoagulant function. In mouse models of prolonged platelet half-life, this resulted in increased pulmonary leukocyte infiltration and inflammation upon acute lung injury. Similarly, human platelet concentrates decreased their hemostatic function and elevated their putative immunomodulatory potential in vitro over time, and in a mouse model of platelet transfusion, aged platelet concentrates resulted in augmented inflammation. In summary, we show that platelets exhibit age-dependent phenotypic shifts, allowing them to fulfill their diverse tasks in the vasculature. Because functional alterations of aging platelets extend to platelet concentrates, this may hold important implications for transfusion medicine.

Although key for pathogen immobilization, neutrophil extracellular traps (NETs) often cause severe bystander cell/tissue damage. This was hypothesized to depend on their prolonged presence in the vasculature, leading to cytotoxicity. Imaging of NETs (histones, neutrophil elastase, and extracellular DNA) with intravital microscopy in blood vessels of mouse livers in a pathogen-replicative-free environment (endotoxemia) led to detection of NET proteins attached to the endothelium for months despite the early disappearance of extracellular DNA. Intravascular liver macrophages (Kupffer cells) and neutrophils, but not monocytes, were involved in NET removal. They used scavenger receptors (SRs; SR-A) and Toll-like receptors (TLRs; TLR2/4) to recognize NET components. Despite the absence of further stimuli, 14 days later a second wave of NET formation occurred, initiated by remnants of NETs from the first wave. The second burst of NET production was triggered by histones, which induced an inflammatory milieu interleukin-1β and activated platelets and coagulation-related events, including factor VII-activating protease activity. This, in turn, recruited and activated neutrophils to release the second wave of NETs. In peptidyl arginine deiminase-deficient mice, not forming NETs, inflammation and liver damage were reduced compared with their wild-type counterparts. When mice were challenged with methicillin-resistant Staphylococcus aureus 14 or 165 days after the second NETs, the course of infection/injury was diminished and exacerbated, respectively. Our study demonstrates that the complete removal of NETs in vivo takes much longer than hypothesized, and a vicious cycle of NET formation/disassembly affects subsequent infection, depending on the time elapsed since its occurrence.

Current intensive treatment of pediatric T-cell acute lymphoblastic leukemia (T-ALL) has substantial side effects, highlighting a need for novel biomarkers to improve risk stratification. Canonical biomarkers, such as genetics and immunophenotype, are largely not used in pediatric T-ALL stratification. This study aimed to validate the prognostic relevance of DNA methylation CpG island methylator phenotype (CIMP) risk stratification in 2 pediatric T-ALL patient cohorts: the Nordic Society of Paediatric Haematology (NOPHO) ALL2008 T-ALL study cohort (n = 192) and the Dutch Childhood Oncology Group (DCOG) ALL-10/ALL-11 validation cohorts (n = 156). Both cohorts revealed that combining CIMP classification at diagnosis with measurable residual disease (MRD) at treatment day 29 (D29) or 33 (D33) significantly improved outcome prediction. The poor prognosis subgroup, characterized by CIMP low/D29 or D33 MRD ≥ 0.1%, had a cumulative incidence of relapse (pCIR5yr) of 29% and 23% and overall survival (pOS5yr) of 59.7% and 65.4%, in NOPHO and DCOG, respectively. Conversely, a good prognosis subgroup was also identified representing CIMP high/D29 or D33 MRD < 0.1% with pCIR5yr of 0% and 3.4% and pOS5yr of 98.2% and 94.8%, in NOPHO and DCOG, respectively. For NOPHO, MRD was also evaluated on D15, and the relapse prediction accuracy of CIMP/D29 MRD (0.79) and CIMP/D15 MRD (0.75) classification was comparable, indicating potential for earlier stratification. The evaluation of the biology behind the CIMP subgroups revealed associations with transcriptome profiles, genomic aberrations, and mitotic history, suggesting distinct routes for leukemia development. In conclusion, integrating MRD assessment with the novel CIMP biomarker has the potential to improve risk stratification in pediatric T-ALL and guide future therapeutic decisions.