We previously demonstrated that reduced intrinsic electron transport chain (ETC) activity predicts and promotes sensitivity to the B-cell lymphoma 2 (BCL-2) antagonist, venetoclax (Ven), in multiple myeloma (MM). Heme, an iron-containing prosthetic group and metabolite, is fundamental to maintaining ETC activity. Interrogation of the cyclin D1 group 2 subgroup of MM from the Relating Clinical Outcomes in MM to Personal Assessment of Genetic Profile (CoMMpass) trial (NCT01454297), which can be used as a proxy for Ven-sensitive MM (VS MM), shows reduced expression of the conserved heme biosynthesis pathway gene signature. Consistent with this, we identified that VS MM exhibits reduced heme biosynthesis and curiously elevated hemin (oxidized heme) uptake. Supplementation with hemin or protoporphyrin IX (heme lacking iron) promotes Ven resistance, whereas targeting ferrochetalase, the penultimate enzyme involved in heme biosynthesis, increases Ven sensitivity in cell lines and primary MM cells. Mechanistically, heme-mediated activation of prosurvival rapidly accelerated fibrosarcoma-rat sarcoma virus-mitogen-activated protein kinase (MEK) signaling and metabolic rewiring, increasing de novo purine synthesis, were found to contribute to heme-induced Ven resistance. Cotargeting BCL-2 and myeloid cell leukemia-1 suppresses heme-induced Ven resistance. Interrogation of the Multiple Myeloma Research Foundation CoMMpass study of patients shows increased purine and pyrimidine biosynthesis to corelate with poor progression-free survival and overall survival. Elevated heme and purine biosynthesis gene signatures were also observed in matched relapse refractory MM, underscoring the relevance of heme metabolism in therapy-refractory MM. Overall, our findings reveal, for the first time, a role for extrinsic heme, a physiologically relevant metabolite, in modulating proximity to the apoptotic threshold with translational implications for BCL-2 antagonism in MM therapy.

This article has been retracted; please see Elsevier's Article Correction, Retraction and Removal Policy (Article withdrawal | Elsevier policy).This article has been retracted at the request of the Editors.Within the paper, image duplications were identified in Figures 2 and 6 and supplemental Figure 4. Image duplications were also identified between Figure 1 and supplemental Figure 4 from this paper and a 2019 publication in another journal. In each case, the duplicated image was modified between versions, such as via rotation and/or shifting the field of view.The authors state that the duplications were image handling errors and that the adjustments were made to improve visual comparison and do not affect their conclusions.No authors approve the retraction.

Diffuse large B-cell lymphoma (DLBCL) is a clinically and molecularly heterogeneous disease. The increasing recognition and targeting of genetically defined DLBCLs highlight the need for robust classification algorithms. We previously characterized recurrent genetic alterations in DLBCL and identified 5 discrete subtypes, clusters 1 to 5 (C1-C5), with unique mechanisms of transformation, immune evasion, candidate treatment targets, and different outcomes after standard first-line therapy. Herein, we validate the C1 to C5 DLBCL taxonomy in an independent data set and use the expanded series of 699 primary DLBCLs to develop a probabilistic molecular classifier and confirm its performance in an independent test set. Using our previously assigned cluster labels as a reference, we systematically compared multiple machine learning models and strategies for input feature dimensionality reduction with a newly developed performance metric that captured the relationship between accuracy and confidence of class assignments. The winning neural network model, DLBclass, assigned all cases in the training/validation and independent test sets with 91% and 89% accuracies, respectively. In the 75% of cases with confidence >0.7, DLBclass assignments were accurate in 97% of the training/validation set and 98% of the test set. DLBclass enables robust prospective classification of single cases for inclusion in genetically guided clinical trials or practice and represents a framework for the development of genomics-based classification methods in other cancers.

Pediatric acute myeloid leukemia frequently harbors fusion oncogenes associated with poor prognosis, including KMT2A, NUP98, and GLIS2 rearrangements. Although murine models have demonstrated their leukemogenic activities, the steps from a normal human cell to leukemic blasts remain unclear. Here, we precisely reproduced the inversion of chromosome 16 resulting in the ETO2::GLIS2 fusion in human induced pluripotent stem cells (iPSCs). iPSC-derived ETO2::GLIS2-expressing hematopoietic cells showed differentiation alterations in vitro and efficiently induced in vivo development of leukemia that closely phenocopied human acute megakaryoblastic leukemia (AMKL), reflected by flow cytometry and single-cell transcriptomes. Comparison of iPS-derived cells with patient-derived cells revealed altered chromatin accessibility at early and later bona fide leukemia stages, with aberrantly higher accessibility and expression of the osteogenic homeobox factor DLX3 that preceded increased accessibility to ETS factors. DLX3 overexpression in normal CD34+ cells increased accessibility to ETS motifs and reduced accessibility to GATA motifs. A DLX3 transcriptional module was globally enriched in both ETO2::GLIS2 AMKL and some aggressive pediatric osteosarcoma. Importantly, DLX3 knockout abrogated leukemia initiation in this ETO2::GLIS2 iPSC model. Collectively, the characterization of a novel human iPSC-derived AMKL model revealed that hijacking of the osteogenic homeobox transcription factor DLX3 is an essential early step in chromatin changes and leukemogenesis driven by the ETO2::GLIS2 fusion oncogene.

Pediatric acute myeloid leukemia (pAML) is a clonal disease with recurrent genetic alterations that affect epigenetic states. However, the implications of epigenetic dysregulation in disease progression remain unclear. Here, we interrogated single-cell and clonal level chromatin accessibility of bone marrow samples from 28 patients with pAML representing multiple subtypes using mitochondrial single-cell assay for transposase-accessible chromatin with sequencing, which revealed distinct differentiation hierarchies and abnormal chromatin accessibility in a subtype-specific manner. Innate immune signaling was commonly enhanced across subtypes and related to improved advantage of clonal competition and unfavorable prognosis, with further reinforcement in a relapse-associated leukemia stem cell-like population. We identified a panel of 31 innate immunity-related genes to improve the risk classification of patients with pAML. By comparing paired diagnosis and postchemotherapy relapse samples, we showed that primitive cells significantly reduced major histocompatibility complex class II signaling, suggesting an immune evasion mechanism to facilitate their expansion at relapse. Key regulators orchestrating cell cycle dysregulation were identified to contribute to pAML relapse in drug-resistant clones. Our work establishes the single-cell chromatin accessibility landscape at clonal resolution and reveals the critical involvement of epigenetic disruption, offering insights into classification and targeted therapies of patients with pAML.

The pathophysiology of sickle cell disease (SCD) is characterized by hemolytic anemia and vaso-occlusion, although its impact on the adaptive immune responses remains incompletely understood. To comprehensibly profile the humoral immune responses, we immunized SCD mice with T-cell-independent (TI) and T-cell-dependent (TD) antigens (Ags). Our study showed that SCD mice have significantly enhanced type 2 TI (TI-2) immune responses in a manner dependent on the level of type I interferons (IFN-I), while maintaining similar or decreased TD immune responses depending on the route of Ag administration. Consistent with the enhanced TI-2 immune responses in SCD mice, the frequencies of B-1b cells (B-1 cells in humans), a major cell type responding to TI-2 Ags, were significantly increased in both the peritoneal cavity and spleens of SCD mice and in the blood of patients with SCD. In support of expanded B-1 cells, elevated levels of anti-red blood cell (anti-RBC) autoantibodies were detected in both SCD mice and patients. Both the levels of TI-2 immune responses and anti-RBC autoantibodies were significantly reduced after IFN-I receptor (IFNAR) antibody blockades and in IFNAR1-deficient SCD mice. Moreover, the alterations of B-1 cell subsets were reversed in IFNAR1-deficient SCD mice, uncovering a critical role for IFN-I in the enhanced TI-2 immune responses and the increased production of anti-RBC autoantibodies by modulating the innate B-1 cell subsets in SCD. Overall, our study provides experimental evidence that the modulation of B-1 cells and IFN-I can regulate TI immune responses and the levels of anti-RBC autoantibodies in SCD.

X-linked sideroblastic anemia (XLSA) is a congenital anemia caused by mutations in ALAS2, a gene responsible for heme synthesis. Treatments are limited to pyridoxine supplements and blood transfusions, offering no definitive cure except for allogeneic hematopoietic stem cell transplantation, only accessible to a subset of patients. The absence of a suitable animal model has hindered the development of gene therapy research for this disease. We engineered a conditional Alas2-knockout (KO) mouse model using tamoxifen administration or treatment with lipid nanoparticles carrying Cre-mRNA and conjugated to an anti-CD117 antibody. Alas2-KOBM animals displayed a severe anemic phenotype characterized by ineffective erythropoiesis (IE), leading to low numbers of red blood cells, hemoglobin, and hematocrit. In particular, erythropoiesis in these animals showed expansion of polychromatic erythroid cells, characterized by reduced oxidative phosphorylation, mitochondria's function, and activity of key tricarboxylic acid cycle enzymes. In contrast, glycolysis was increased in the unsuccessful attempt to extend cell survival despite mitochondrial dysfunction. The IE was associated with marked splenomegaly and low hepcidin levels, leading to iron accumulation in the liver, spleen, and bone marrow and the formation of ring sideroblasts. To investigate the potential of a gene therapy approach for XLSA, we developed a lentiviral vector (X-ALAS2-LV) to direct ALAS2 expression in erythroid cells. Infusion of bone marrow (BM) cells with 0.6 to 1.4 copies of the X-ALAS2-LV in Alas2-KOBM mice improved complete blood cell levels, tissue iron accumulation, and survival rates. These findings suggest our vector could be curative in patients with XLSA.

Anemia of inflammation is a prevalent comorbidity in patients with chronic inflammatory disorders. Inflammation causes hypoferremia and iron-restricted erythropoiesis by limiting ferroportin (FPN)-mediated iron export from macrophages that recycle senescent erythrocytes. Macrophage cell surface expression of FPN is reduced by hepcidin-induced degradation and/or by repression of FPN (Slc40a1) transcription via cytokine and Toll-like receptor (TLR) stimulation. Although the mechanisms underlying hepcidin-mediated control of FPN have been extensively studied, those inhibiting Slc40a1 messenger RNA (mRNA) expression remain unknown. We applied targeted RNA interference and pharmacological screens in macrophages stimulated with the TLR2/6 ligand FSL1 and identified critical signaling regulators of Slc40a1 mRNA repression downstream of TLRs and NF-κB signaling. Interestingly, the NF-κB regulatory hub is equally relevant for Slc40a1 mRNA repression driven by the TLR4 ligand lipopolysaccharide, the cytokine tumor necrosis factor β/lymphotoxin-alpha (LTA), and heat-killed bacteria. Mechanistically, macrophage stimulation with heat-killed Staphylococcus aureus recruits the histone deacetylases (HDACs) HDAC1 and HDAC3 to the antioxidant response element (ARE) located in the Slc40a1 promoter. Accordingly, pretreatment with a pan-HDAC inhibitor abrogates Slc40a1 mRNA repression in response to inflammatory cues, suggesting that HDACs act downstream of NF-κB to repress Slc40a1 transcription. Consistently, recruitment of HDAC1 and HDAC3 to the Slc40a1 ARE after stimulation with heat-killed S aureus is dependent on NF-κB signaling. These results support a model in which the ARE integrates the transcriptional responses of Slc40a1 triggered by signals from redox, metabolic, and inflammatory pathways. This work identifies the long-sought mechanism of Slc40a1 transcriptional downregulation upon inflammation, paving the way for therapeutic interventions at this critical juncture.

We identified 347 pregnancies in patients with β-thalassemia minor. Hemoglobin was <9 g/dL in 31% during third trimester and 7.6% at delivery. Postpartum hemorrhage occurred in 8.9%. Forty-six percent of IV iron administration was to iron-replete patients.

Early intervention in smoldering multiple myeloma (SMM) may delay progression to MM. Here, we present the final analysis of the phase 2 CENTAURUS study. In total, 123 patients with intermediate/high-risk SMM were randomized to IV daratumumab 16 mg/kg after a long-intense (n = 41), intermediate (n = 41), or short-intense (n = 41) dosing schedule. At a combined median follow-up of 85.2 months, in the long-intense, intermediate, and short-intense arms complete response or better rates were 4.9%, 9.8%, and 0%; overall response rates were 58.5%, 53.7%, and 37.5%; progressive disease/death rates were 0.096, 0.102, and 0.109 (P < .0001 for all arms); and median progression-free survival was not reached, 84.4, and 74.1 months, respectively. Median overall survival was not reached in any arm. Thirty-six patients in the long-intense or intermediate arms continued daratumumab in an optional extension phase after completing 20 cycles of per-protocol treatment. The median duration of study treatment was 44.0 (range, 1.0-91.6), 35.2 (range, 1.9-90.6), and 1.6 (range, 0.1-1.9) months in the long-intense, intermediate, and short-intense arms, respectively. No new safety signals were observed. With extended follow-up (median, ∼7 years), these data highlight the tolerability of daratumumab and support ongoing trials investigating daratumumab as an early intervention for SMM. This trial was registered at www.ClinicalTrials.gov as #NCT02316106.

Outcomes are poor in patients with higher-risk myelodysplastic syndromes (HR MDS) and frontline treatment options are limited. This phase 1b study investigated safety and efficacy of venetoclax, a selective B-cell lymphoma 2 inhibitor, at the recommended phase 2 dose (RP2D; 400 mg for 14 days per 28-day cycle), in combination with azacitidine (75 mg/m2 for 7 days per 28-day cycle) for treatment-naive HR MDS. Safety was the primary outcome, and complete remission (CR) rate was the primary efficacy outcome. Secondary outcomes included rates of modified overall response (mOR), hematologic improvement (HI), overall survival (OS), and time to next treatment (TTNT). As of May 2023, 107 patients received venetoclax and azacitidine combination at the RP2D. Best response of CR or marrow CR was observed in 29.9% and 50.5% (mOR, 80.4%), respectively. Median OS was 26.0 months, with 1- and 2-year survival estimates of 71.2% and 51.3%, respectively. Among 59 patients with baseline red blood cell and/or platelet transfusion-dependence, 24 (40.7%) achieved transfusion independence on study, including 11 (18.6%) in CR. Fifty-one (49.0%) of 104 evaluable patients achieved HI. Median TTNT excluding transplantation was 13.4 months. Adverse events reflected known safety profiles for venetoclax and azacitidine, including constipation (53.3%), nausea (49.5%), neutropenia (48.6%), thrombocytopenia (44.9%), febrile neutropenia (42.1%), and diarrhea (41.1%). Overall, venetoclax plus azacitidine at the RP2D was well tolerated and had favorable outcomes. A phase 3 study (NCT04401748) is ongoing to confirm survival benefit of this combination. This trial was registered at www.clinicaltrials.gov as #NCT02942290.

Anti-CD19 chimeric antigen receptor (CAR)-engineered T and natural killer (NK) cell therapies have revolutionized the treatment of B-cell malignancies, but challenges including CD19 antigen loss greatly hinder their full therapeutic potential. Here, we revealed that cotreatment with anti-CD19 monoclonal antibody enhances antitumor activity of anti-CD19 CAR-T and -NK cells. Even though the treated antibody interferes with CD19 antigen binding of CAR, it significantly induces rapid detachment of anti-CD19 CAR effector cells from target cells, facilitating improved serial killing. This reduced interaction between CAR effector cells and target cells also leads to the alleviation of CAR-mediated trogocytosis. Interestingly, cotreatment with anti-CD19 antibody reveals time-dependent effects on the antitumor activity of anti-CD19 CAR-T cells, characterized by a reduction in early T cell activation followed by sustained high activity during prolonged exposure to target cells. This temporal modulation ultimately results in enhanced antitumor potency in vivo. These findings underscore the improved therapeutic efficacy achieved by combining anti-CD19 antibody with anti-CD19 CAR-T or -NK cells against B-cell malignancies.

The transcription factor GATA-binding protein 3 (GATA-3) and the transcriptional program it regulates have emerged as oncogenic drivers across diverse T-cell lymphomas (TCLs), many of which are resistant to conventional chemotherapeutic agents and characterized by recurrent losses of key tumor suppressor genes, including TP53 and PTEN, both of which are clients of the nuclear export protein XPO1. Here, we demonstrated that XPO1 is highly expressed by malignant T cells expressing GATA-3 and by lymphoma-associated macrophages (LAMs) within their tumor microenvironment (TME). Using complementary genetically engineered mouse models, we demonstrated that TP53- and/or phosphate and tensin homolog (PTEN)-deficient TCLs, and LAMs within their TME, are sensitive to the selective exportin-1 (XPO1) antagonist selinexor. In an effort to identify TP53- and PTEN-independent mechanisms, we used complementary and orthogonal approaches to investigate the role of eIF4E and XPO1-dependent messenger RNA nuclear export in these TCLs. We identified a novel role for eIF4E/XPO1 in exporting GATA-3 and GATA-3-dependent transcripts from the nucleus in TCLs, and in the export of therapeutically relevant transcripts, including colony-stimulating factor-1 receptor, from LAMs. Therefore, XPO1 antagonism, by impairing oncogenic transcriptional programs in TCLs and depleting LAMs from their TME, is a novel approach to target 2 independent dependencies in a group of therapeutically challenging TCLs.