In patients with transplant-eligible newly diagnosed multiple myeloma, induction therapy with a quadruplet regimen prior to autologous transplant is the standard of care. The phase III IFM2020-02-MIDAS study (NCT04934475) assessed a minimal residual disease (MRD)-driven consolidation and maintenance strategy following induction with isatuximab, carfilzomib, lenalidomide, and dexamethasone (IsaKRD). Here, we report safety and efficacy outcomes of six 28-day cycles of IsaKRD. Between December 2021 and July 2023, 791 patients were enrolled across 72 centers. The median age was 59 years; 13% had ISS-stage III, 5% had R-ISS-stage III, and 8% had high-risk cytogenetics (IFM Linear Predictor cytogenetic score >1). Overall, 96% (N=757) of patients completed induction. The median CD34+ cell yield was 7 × 106/Kg, with 94% of patients able to proceed with a potential tandem transplant. The best overall response rate was 95%. In the intent-to-treat population, 91% achieved a very good partial response or better after induction, with MRD-negativity rates of 63% at 10-5 and 47% at 10-6. MRD-negativity rates differed across ISS stages and cytogenetic subgroups. During induction, 7 patients experienced disease progression, and 5 died due to disease progression (N=1), cardiac events (N=2), or other causes (N=2). The most common grade 3/4 adverse events were neutropenia (25%), thrombocytopenia (5%), and infections (7%); only 13% of patients reported any grade peripheral neuropathy. IsaKRD induction yielded deep responses and high MRD-negativity rates while ensuring successful stem cell collection, with no new safety signals. Continued follow-up of this ongoing study is required to confirm these findings.

Stemness-associated cell states are linked to chemotherapy resistance in acute myeloid leukemia (AML). We uncovered a direct mechanistic link between expression of the stem cell transcription factor GATA2 and drug resistance. The GATA-binding protein 2 (GATA2) plays a central role in blood stem cell generation and maintenance. We find substantial intrapatient and interpatient variability in GATA2 expression across samples from patients with AML. GATA2 expression varies by molecular subtype and has been linked to outcome. In a murine model, KMT2A-MLL3-driven AML originating from a stem cell or immature progenitor cell population has higher Gata2 expression and is more resistant to the standard AML chemotherapy agent doxorubicin. Deletion of Gata2 resulted in a more robust induction of p53 after exposure to doxorubicin. Chromatin immunoprecipitation sequencing, RNA sequencing, and functional studies revealed that GATA2 regulates the expression of RASSF4, a modulator of the p53 inhibitor MDM2 (mouse double minute 2). GATA2 and RASSF4 are anticorrelated in human cell lines and in bulk and single-cell expression data sets from patients with AML. Knockdown of Rassf4 in Gata2-low cells resulted in doxorubicin or nutlin-3 resistance. Conversely, overexpression of Rassf4 results in sensitization of cells expressing high levels of Gata2. Finally, doxorubicin and nutlin-3 are synergistic in Gata2-high murine AML and in samples from patients with AML. We discovered a previously unappreciated role for GATA2 in dampening p53-mediated apoptosis via transcriptional regulation of RASSF4, a modulator of MDM2. This role for GATA2 directly links the expression of a stemness-associated transcription factor to chemotherapy resistance.

CD30-directed chimeric antigen receptor T-cell therapy (CART30) has limited efficacy in relapsed or refractory patients with CD30+ lymphoma, with a low proportion of durable responses. We have developed an academic CART30 cell product (HSP-CAR30) by combining strategies to improve performance. HSP-CAR30 targets a proximal epitope within the nonsoluble part of CD30, and the manufacturing process includes a modulation of ex vivo T-cell activation, as well as the addition of interleukin-21 (IL-21) to IL-7 and IL-15 to promote stemness of T cells. We translated HSP-CAR30 to a phase 1 clinical trial of 10 patients with relapsed/refractory classic Hodgkin lymphoma (HL) or CD30+ T-cell non-Hodgkin lymphoma. HSP-CAR30 was mainly composed of memory stem-like (TSCM-like) and central memory (TCM) CAR30+ T cells (87.5% ± 5%). No dose-limiting toxicities were detected. Six patients had grade 1 cytokine release syndrome, and no patient developed neurotoxicity. The overall response rate was 100%, and 5 of 8 patients with HL achieved complete remission (CR). An additional patient with HL achieved CR after a second HSP-CAR30 infusion. Remarkably, 60% of patients have ongoing CR after a mean follow-up of 34 months. CAR30+ T cells at expansion peak had a predominance of TSCM and TCM cells, and CAR30+ T cells remained detectable in 3 of 5 evaluable patients at least 12 months after infusion. Our study shows that selection of the epitope targeting CD30 and ex vivo preservation of less-differentiated memory T cells may enhance the efficacy of CART30 in patients with refractory HL. This trial is registered at www.clinicaltrials.gov (NCT04653649).

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.

Thrombomodulin (TM) expressed on endothelial cells regulates coagulation. Specific nonsense variants in the TM gene, THBD, result in high soluble TM levels causing rare bleeding disorders. In contrast, although THBD variants have been associated with venous thromboembolism, this association remains controversial. A multigene panel was used to diagnose 601 patients with inherited bleeding or thrombotic disorders. This resulted in the identification of 8 THBD variants for 6 patients with a thrombotic (C175S, A282P, L433P, P501L, G502R, and P508L) and 2 patients with a bleeding (P260A and T478I) phenotype. These were all classified as variants of uncertain significance, and we here aimed to assess their functional role in coagulation. For this purpose, soluble and cell membrane-bound recombinant TM were produced in Expi293F cells. L433P TM showed a marked decrease in the inhibition of thrombin generation and complete inhibition of protein C and thrombin activatable fibrinolysis inhibitor (TAFI) activation. Soluble C175S TM showed decreased inhibition of thrombin generation and protein C activation, whereas no effect was observed for cell membrane-bound recombinant TM. For the other TM variants, no effect on thrombin generation, protein C, or TAFI activation could be observed. Surface plasmon resonance analysis showed no thrombin-TM binding in the presence of L433P because this residue is located at their interaction site. In conclusion, our study shows the functional effects of L433P TM and potentially C175S TM, which are compatible with an increased thrombosis risk. THBD variants are rare but can be relevant to both bleeding and thrombosis. Functional assays for these variants are critical to understand their roles.

Recurrent isocitrate dehydrogenase (IDH) mutations catalyze nicotinamide adenine dinucleotide phosphate (NADPH)-dependent production of oncometabolite (R)-2-hydroxyglutarate (R-2HG) for tumorigenesis. IDH inhibition provides clinical response in a subset of acute myeloid leukemia (AML) cases; however, most patients develop resistance, highlighting the need for more effective IDH-targeting therapies. By comparing transcriptomic alterations in isogenic leukemia cells harboring CRISPR base-edited IDH mutations, we identify the activation of adhesion molecules including CD44, a transmembrane glycoprotein, as a shared feature of IDH-mutant leukemia, consistent with elevated CD44 expression in IDH-mutant AML patients. CD44 is indispensable for IDH-mutant leukemia cells through activating pentose phosphate pathway and inhibiting glycolysis by phosphorylating glucose-6-phosphate dehydrogenase and pyruvate kinase muscle isozyme M2, respectively. This metabolic rewiring ensures efficient NADPH generation for mutant IDH-catalyzed R-2HG production. Combining IDH inhibition with CD44 blockade enhances the elimination of IDH-mutant leukemia cells. Hence, we describe an oncogenic feedforward pathway involving CD44-mediated metabolic rewiring for oncometabolite production, representing a potentially targetable dependency of IDH-mutant malignancies.

Myelodysplastic syndromes (MDS) are age-related diseases characterized by bone marrow (BM) dysfunction and an increased risk for developing acute leukemia. Although there is growing evidence that highlight the crucial role of the BM microenvironment (BMME) in MDS, the specific influence of inflammation on BMME changes and the potential benefits of targeting cytokines therapeutically remain to be elucidated. We previously found that interleukin-1 (IL-1) is a driver of aging phenotypes of the BMME and hematopoietic stem and progenitor cells (HSPCs). In this study, BM samples from patients with MDS demonstrated upregulated levels of IL-1 family cytokines, including IL-18. Using highly purified primary BM-derived mesenchymal stromal cells (MSCs), both IL-1b and IL-18 were found to exert direct effects on MSCs, thus influencing their ability to support HSPCs and erythroid progenitors. This confirms the significant involvement of both these IL-1 family cytokines in regulating the BM niche. Furthermore, targeting IL-1 receptor type 1 mitigated these aging phenotypes in older mice. We subsequently employed an age-appropriate murine model of MDS by transplanting NUP98-HOXD13 transgenic mice (NHD13Tg) cells into aged wild-type mice. Treatment with inhibitors that targeted IL-1 receptor-associated kinase 4 (IRAK4) and NOD-like receptor family pyrin domain containing 3 (NLRP3) reversed the proliferation of dysfunctional MSCs and enhanced their functionality. In addition, IRAK4 inhibition selectively suppressed MDS clonal cells while sparing non-MDS cells in the BM. These findings suggest that targeting IL-1 signaling holds promise for MDS treatment by addressing the underlying myeloid malignancy and restoring the altered BMME via BM-MSCs.

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.

Loss of anticancer natural killer (NK) cell function in patients with acute myeloid leukemia (AML) is associated with fatal disease progression and remains poorly understood. Here, we demonstrate that AML blasts isolated from patients rapidly inhibit NK cell function and escape NK cell-mediated killing. Transcriptome analysis of NK cells exposed to AML blasts revealed increased CREM expression and transcriptional activity, indicating enhanced cyclic adenosine monophosphate (cAMP) signaling, confirmed by uniform production of the cAMP-inducing prostanoid prostaglandin E2 (PGE2) by all AML-blast isolates from patients. Phosphoproteome analysis disclosed that PGE2 induced a blockade of lymphocyte-specific protein tyrosine kinase (LCK)-extracellular signal-regulated kinase signaling that is crucial for NK cell activation, indicating a 2-layered escape of AML blasts with low expression of NK cell-activating ligands and inhibition of NK cell signaling. To evaluate the therapeutic potential to target PGE2 inhibition, we combined Fcγ-receptor-mediated activation with the prevention of inhibitory PGE2 signaling. This rescued NK cell function and restored the killing of AML blasts. Thus, we identify the PGE2-LCK signaling axis as the key barrier for NK cell activation in 2-layered immune escape of AML blasts that can be targeted for immune therapy to reconstitute anticancer NK cell immunity in patients with AML.

A mixed phenotype (MP) is a characteristic of de novo MP acute leukemia (MPAL), but it can also be found in other leukemias. It poses substantial classification and management dilemmas. Herein, we report a large cohort of acute leukemia with MP and define acute myeloid leukemia with MP (AML-MP) and MPAL as 2 distinct groups by characterizing clinical, genetic, and transcriptomic features. Clinically, patients with AML-MP and MPAL were both treated with either AML- or acute lymphoblastic leukemia (ALL)-directed induction regimens. AML-MP has inferior responses (hazard ratio, 12.5; 95% confidence interval, 2.72-57.8; P = .001), whereas MPAL has better responses to ALL-directed treatment. Genetically, AML-MP harbors more frequent RUNX1 (23/52 [44%]) and TP53 (12/52 [23.1%]) mutations. In contrast, RUNX1 mutations are less frequent in MPAL (8/35 [23%]; P = .01 vs AML-MP) and TP53 mutations as a driver are virtually absent in MPAL. Transcriptionally, AML-MP shows enrichment for stemness signatures and a relative deficit of transcription factors critical for myeloid and lymphoid differentiation. Furthermore, AML-MP rarely switches to a lymphoid immunophenotype after treatment, in contrast to MPAL (1/40 [2.5%] vs 10/28 [35.7%]; P = .0003). Last, a genomic classification framework is proposed for future studies. Together, these data support the designation of AML-MP as a diagnosis distinct from MPAL and provide novel insights into the pathogenesis and therapies of acute leukemia with MP.

Prizloncabtagene autoleucel (prizlon-cel), a novel bispecific chimeric antigen receptor T cell, targets and eliminates CD19/CD20-positive tumor cells. This phase 1, open-label study investigated the safety and efficacy of prizlon-cel in patients with relapsed/refractory B-cell non-Hodgkin lymphoma (R/R B-NHL). Patients with CD19 and/or CD20-positive R/R B-NHL received a 3-day lymphodepletion (cyclophosphamide: 300 mg/m2 per day; fludarabine: 30 mg/m2 per day) followed by an IV dose of prizlon-cel. The primary end points were dose-limiting toxicity (DLT) and incidence and severity of treatment-emergent adverse events (TEAEs). Secondary end points included overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Of the 48 patients infused prizlon-cel, 44 had large B-cell lymphoma (LBCL). No patient experienced DLT. Cytokine release syndrome occurred in 93.8% of the patients, with only 1 case of grade 3. Immune effector cell-associated neurotoxicity syndrome occurred in 6.3% of patients, with no grade 3 or higher events. The most common grade 3 or higher TEAEs were neutropenia (83.3%) and leukopenia (50%). The ORR and complete response (CR) rates in all patients were 91.5% and 85.1%, respectively, and in LBCL patients, ORR was 90.7% with 86.0% CR. With median follow-up of 30.0 months, median DOR, PFS, and OS were all not reached. Kaplan-Meier estimate of 2-year DOR, PFS, and OS rates were 66.0%, 62.6%, and 76.5%, respectively. Prizlon-cel had a favorable safety profile and a high and durable response in patients with R/R B-NHL, suggesting a promising treatment option for patients with R/R B-NHL. These trials were registered at www.clinicaltrials.gov as #NCT04317885, #NCT04655677, #NCT04696432, and #NCT04693676.

Apolipoprotein E (APOE) has multiple functions in metabolism and immunoregulation. Its common germ line variants APOE2, APOE3, and APOE4 give rise to 3 functionally distinct gene products. Previous studies reported yin-yang roles of APOE2 and APOE4 in immunological processes, but their effects in hematopoietic stem cell transplantation (HSCT) have never been studied. We performed APOE genotyping in 2 contemporary cohorts of 348 and 447 patients with acute myeloid leukemia who had received allogeneic HSCT and evaluated the associations of recipient and donor APOE genetic variations with posttransplant outcomes. Patients who carried at least 1 APOE2 allele had a higher risk of posttransplant death than APOE4 carriers in the discovery (hazard ratio [HR], 2.09; P = .024) and validation cohorts (HR, 1.96; P = .040). Detrimental APOE2 effects were driven by an increased risk of severe chronic graft-versus-host disease (GVHD; adjusted HR [HRadj], 1.85; P = .034) and nonrelapse death (HRadj, 1.72; P = .044). In non-APOE2 recipients, transplantation of an APOE2-positive allograft was associated with an increased incidence of grade 3 to 4 acute GVHD (HRadj, 2.82; P = .012) and severe chronic GVHD (HRadj, 2.54; P = .022) compared with APOE2-negative grafts. In summary, the APOE2 allele, typically considered a longevity gene in the general population, was associated with a higher risk of acute GVHD (HRadj, 2.75; P = .002), chronic GVHD (HRadj, 2.57; P = .001), and posttransplant mortality (HRadj, 1.79; P = .004), when present in either the host or transplanted from the donor.