Recent advances in acute myeloid leukemia (AML) come from studies investigating older adults aged <60 years, those aged ≥75 years, or less fit adults. Uncertainty exists for the management of otherwise healthy adults with AML in their 60s and 70s, which also represents a significant proportion of AML cases. We discuss current considerations in older, fit adults with AML, including determination of fitness, factors beyond fitness that should be assessed, and challenges and innovations to improve patient outcomes.

Leukopoiesis is lethally arrested in mice lacking the master transcriptional regulator PU.1. Depending on the animal model, subtotal PU.1 loss either induces acute myeloid leukemia or arrests early B-cell and dendritic-cell development. Although humans with absolute PU.1 deficiency have not been reported, a small cadre of congenital agammaglobulinemia patients with sporadic, inborn PU.1 haploinsufficiency was recently described. To better estimate the penetrance, clinical complications, immunophenotypic features, and malignancy risks of PU.1-mutated agammaglobulinemia (PU.MA), a collection of 134 novel or rare PU.1 variants from publicly available databases, institutional cohorts, previously published reports, and unsolved agammaglobulinemia cases were functionally analyzed. In total, 25 loss-of-function (LOF) variants were identified in 33 heterozygous carriers from 21 kindreds across 13 nations. Of individuals harboring LOF PU.1 variants, 22 were agammaglobulinemic, 5 displayed antibody deficiencies, and 6 were unaffected, indicating an estimated disease penetrance of 81.8% with variable expressivity. In a cluster of patients, disease onset was delayed, sometimes into adulthood. All LOF variants conveyed effects via haploinsufficiency, either by destabilizing PU.1, impeding nuclear localization, or directly interfering with transcription. PU.MA patient immunophenotypes consistently demonstrated B-cell, conventional dendritic-cell, and plasmacytoid dendritic-cell deficiencies. Associated infectious and noninfectious symptoms hewed closely to X-linked agammaglobulinemia and not monogenic dendritic-cell deficiencies. No carriers of LOF PU.1 variants experienced hematologic malignancies. Collectively, in vitro and clinical data indicate heterozygous LOF PU.1 variants undermine humoral immunity but do not convey strong leukemic risks.

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.

Previous studies have reported that chronic lymphocytic leukemia (CLL) shows a de novo chromatin activation pattern compared with normal B cells. Here, we explored whether the level of chromatin activation is related to the clinical behavior of CLL. We identified that, in some regulatory regions, increased de novo chromatin activation is linked to clinical progression, whereas, in other regions, it is associated with an indolent course. We next developed 2 prognostic scores for progressive and indolent disease, respectively, calculated a single score representing the balance between them, and further generated surrogate scores based on gene and protein expression of the target genes. The balance score outperformed the clinical impact of the 2 individual scores, because it seemed to capture the prognostic information provided by each of them. Biologically, CLLs with higher balance score showed increased activation of tumor necrosis factor alpha (TNF-α)/NF-κB and mTOR signaling pathways. Regulatory programs related to progression were predominantly activated in the lymph node microenvironment, whereas those linked to indolent disease appeared to be microenvironment independent. Finally, we thoroughly validated the balance score as a powerful and independent quantitative prognostic factor for time to first treatment across independent CLL cohorts and data modalities, such as chromatin, transcriptome, or proteome data. Our findings support the concept that de novo acquisition of chromatin changes in CLL cells plays a dual biological role, and the balance between proprogression and proindolence is a strong independent determinant of CLL prognosis.

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.

For patients with transplant-eligible newly diagnosed multiple myeloma, induction therapy with a quadruplet regimen before autologous transplant is the standard of care. The phase 3 IFM2020-02-MRD-adapted strategy (MIDAS) study assessed a minimal residual disease (MRD)-driven consolidation and maintenance strategy after induction with isatuximab, carfilzomib, lenalidomide, and dexamethasone (IsaKRD). We report safety and efficacy outcomes of six 28-day cycles of IsaKRD in 791 patients. The median age was 59 years; 13% had International Staging System (ISS) stage III, 5% Revised-ISS stage III, and 8% high-risk cytogenetics (Intergroupe Francophone du Myélome linear predictor cytogenetic score of >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. 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. This trial was registered at www.clinicaltrials.gov as #NCT04934475.

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.