Self-renewing multipotent hematopoietic stem cells (HSCs) are a rare but important cell population that can reconstitute the entire blood and immune system after transplantation. Due to their rarity, it has been difficult to comprehensively study the mechanisms regulating HSC activity. However, recent improvements in hematopoietic stem and progenitor cell (HSPC) culture methods using polyvinyl alcohol-based media now facilitate large-scale ex vivo HSC expansion. Here, we performed a genome-wide CRISPR knockout (KO) screen in primary mouse HSPCs to discover novel regulators of ex vivo expansion. The screen identified Runx2 as a strong negative regulator of HSC expansion, which we validated using ex vivo and in vivo assays. Loss of Runx2 increased the frequency of immunophenotypic HSCs in HSPC cultures by approximately threefold. After expansion, these Runx2-KO HSCs engrafted at approximately fivefold higher levels in transplantation assays. Noncultured Runx2-KO HSCs also displayed enhanced reconstitution potential, but loss of Runx2 did not alter blood parameters. Notably, however, T-cell reconstitution was diminished from Runx2-KO HSCs, and we further validated an additional role for Runx2 in T-cell commitment using ex vivo and in vivo assays. In summary, we have identified a multifaceted role for Runx2 in HSCs, as a negative regulator of HSC self-renewal and as a facilitator of T-cell commitment. These results contribute to our understanding of the transcriptional regulation of hematopoiesis and HSC therapies.

RNA splicing and processing are critical for erythropoiesis, because dysregulation of RNA splicing ultimately disrupts protein synthesis. The RNA-binding protein Rbm38 is highly expressed during terminal erythropoiesis. Although in vitro studies have implicated Rbm38 as a key regulator of erythroid differentiation, the landscape of RNA splicing regulated by Rbm38 and its role in terminal erythropoiesis in vivo have not been fully elucidated. Here, we generated whole-body and conditional knockout mouse models for Rbm38 and found that mature red blood cell (RBC) production was impaired in the bone marrow of Rbm38-deficient mice. Rbm38-/- RBCs exhibited reduced hemoglobin content and increased susceptibility to oxidative stress-induced hemolysis. These mutant mice also developed microcytic hypochromic anemia, along with dysregulated iron homeostasis. Additionally, they exhibited decreased mitochondrial heme biosynthesis and accumulation of free protoporphyrin IX (PPIX) in erythrocytes and feces, resembling human erythropoietic protoporphyria (EPP). Mechanistically, Rbm38 regulates the incorporation of ferrous iron (Fe2+) into PPIX to form heme by modulating alternative splicing, messenger RNA decay, and translation of the porphyrin metabolic enzyme gene Ferrochelatase (Fech). Importantly, enforced expression of Fech largely restored erythroid differentiation defects and ameliorated anemia in Rbm38-/- transplants. We further demonstrated that genetic variants in the human RBM38 gene locus influence PPIX levels in erythrocytes from healthy cohorts. Our findings demonstrate that Rbm38 governs terminal erythropoiesis by orchestrating RNA splicing, stability, and translation during heme biosynthesis.

The progression of multiple myeloma (MM), an incurable malignancy of plasma cells, is often associated with the suppression of ferroptosis, a type of cell death driven by iron-dependent lipid peroxidation. The mechanisms underlying this suppression remain largely unknown. Here, we identified serine/threonine kinase 17b (STK17B) kinase as a critical suppressor of ferroptosis in MM. Elevated levels of STK17B are associated with poor overall survival in patients with MM, and STK17B expression is significantly higher in relapsed vs newly diagnosed MM cases. We found that inhibiting STK17B in MM cells increased the labile iron pool, enhanced lipid peroxidation, and sensitized cells to conventional anti-MM therapies. Notably, an orally available, in-house-generated STK17B inhibitor induced ferroptosis and significantly reduced tumor growth in MM xenograft mouse models. Mechanistically, proximity labeling assay combined with the phospho-proteomic analysis identified 2 major regulators of iron uptake and transport as direct targets of STK17B: iron-responsive element binding protein 2 (IREB2), and heat shock protein family B member 1 (HSPB1). We demonstrated that STK17B phosphorylates critical regulatory sites on IREB2 (S157) and HSPB1 (S15), thereby modulating the balance between IREB2 and HSPB1 downstream effectors, proferroptotic transferrin receptor, and antiferroptotic ferritin heavy chain proteins. Furthermore, we demonstrated that STK17B indirectly maintains activating phosphorylation of STAT3, a ferroptosis suppressor and a major driver of MM pathobiology. Our findings uncovered a clinically relevant and targetable STK17B-pIREB2S157/pHSPB1S15 signaling axis that suppresses ferroptosis and contributes to drug resistance in MM.

The IELSG37 trial enrolled 545 patients with primary mediastinal B-cell lymphoma (PMBCL) and demonstrated that consolidation radiotherapy (RT) can be omitted in patients with complete metabolic response, defined by the Lugano classification as Deauville score (DS) 1 to 3. This report evaluates outcomes after different frontline rituximab- and doxorubicin-based immunochemotherapy regimens chosen according to local practice. Patients treated with R-CHOP21 (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone, administered every 21 days) showed a significantly higher percentage of DS 5 than those on other regimens (23.8% vs 8.2% average; P < .001) and a trend toward additional unplanned treatments (53.2% vs 46.9%; P = .30). The increased risk of poor response was confirmed in a multinomial logistic regression analysis adjusted for age, sex, international prognostic index score, and performance status. R-CHOP21 was also associated with smaller reductions in metabolic tumor volume and less pronounced decreases in maximum standardized uptake value. Patients with DS 5 more often received additional treatment (RT and/or salvage chemotherapy with or without autologous consolidation) after induction immunochemotherapy (96% vs 41%; P < .001) and experienced significantly poorer outcomes. Although differences in progression-free and overall survival between R-CHOP21 and more aggressive regimens were not statistically significant, R-CHOP21 may increase the risk of additional treatments and may be inadvisable as frontline therapy for PMBCL. This trial was registered at www.clinicaltrials.gov as #NCT01599559.

Human herpesvirus 6 reactivation in chimeric antigen receptor T-cell recipients is reported to be rare. Our patient cohort (n = 119) experienced a sevenfold higher incidence of viremia than a previous study and 2 cases of encephalitis. Further studies are needed to determine risk factors for infection.

Isatuximab is an immunoglobulin G1κ monoclonal antibody that binds with high affinity to CD38 expressed on plasma cells. Anti-CD38 antibodies have shown efficacy as monotherapy and in combination in a variety of settings for patients with multiple myeloma and light chain (AL) amyloidosis. This multicenter, cooperative group phase 2 trial was designed to evaluate hematologic response, organ response, and safety of isatuximab monotherapy for the treatment of relapsed AL amyloidosis. Isatuximab at 20 mg/kg was administered IV weekly during the first 28-day cycle, and then every other week during cycles 2 to 24. Forty-three patients were registered, with 35 patients being evaluable for response. The overall hematologic response rate was 77.1%, with 57% of patients achieving a very good partial response (VGPR) or better. The median time to partial response (PR) or better was 1.1 months. Renal response occurred in 50% (7/14) of patients with renal involvement, and cardiac response occurred in 57% (8/14) of patients who were evaluable utilizing N-terminal pro b-type natriuretic peptide (NT-proBNP) with cardiac involvement. The most common treatment-related grade ≥3 adverse events included lymphopenia (n = 3, 8.5%) and infection (n = 2, 6%). Isatuximab demonstrated substantial efficacy in previously treated patients with AL amyloidosis, and was associated with a good safety profile. This trial was registered at www.clinicaltrials.gov as #NCT03499808.

Hepcidin is the key hyposideremic hormone produced primarily by the liver. However, recent reports reveal extrahepatic functional sources of hepcidin, including the intestine, the site of dietary iron absorption. To determine whether intestinal hepcidin may play a role in plasma iron lowering, we generated transgenic mice overexpressing the peptide specifically in this tissue. At 1 month of age, transgenic mice exhibited severe iron deficiency along with decreased hematologic indices and a drastic suppression of liver hepcidin in response to hyposideremia. Mechanistically, we showed that intestinal hepcidin was produced in the intestine lumen, inducing a striking downregulation of divalent metal transporter 1 (DMT1) protein at the enterocyte. To confirm the capacity of hepcidin to decrease DMT1, we developed food-grade recombinant lactic acid bacteria (recLAB) genetically modified to deliver hepcidin directly into the intestinal lumen. These recLAB induced a rapid decrease of duodenal DMT1 and, most importantly, when daily orally administrated, protected against iron overload in a mouse model of hemochromatosis. Taken together, our data reveal a previously unrecognized role of intestinal hepcidin as a regulator of systemic iron homeostasis, acting on DMT1 on the apical side of enterocytes, with potential therapeutic relevance for hematologic or iron disorders.

Long-term maintenance of somatic stem cells relies on precise regulation of self-renewal and differentiation. Understanding the molecular framework for these homeostatic processes is essential for improved cellular therapies and treatment of myeloid neoplasms. CUX1 is a widely expressed, dosage-sensitive transcription factor crucial for development and frequently deleted in myeloid neoplasia in the context of -7/(del7q). Here, using novel mouse models and single-cell approaches, we report that dynamic and distinct CUX1 levels are integral to hematopoietic stem cell (HSC) activity. Knockdown of CUX1 reverses HSC differentiation and strikingly reendows progenitors with stem cell function, accompanied by restoration of the HSC transcriptome and DNA accessibility landscape. CUX1 mediates these activities, in part, via suppressing endogenous retroelements (EREs) and the ensuing interferon-stimulated gene expression program. Both EREs and the interferon response are upregulated in CUX1-deficient acute myeloid leukemia, suggesting a conserved role of CUX1 in regulating these elements. These data establish an unexpected entwinement between stem cell-intrinsic innate immune activation and the transcriptional programs of stem cell identity. Furthermore, we reveal the profound effects of transcription factor levels in cell fate.

Hematopoietic stem cells (HSCs) responsible for blood cell production and their bone marrow regulatory niches undergo age-related changes, affecting immune responses and predisposing individuals to hematologic malignancies. Here, we show that the age-related alterations of the megakaryocytic niche and associated downregulation of platelet factor 4 (PF4) are pivotal mechanisms driving HSC aging. PF4-deficient mice display several phenotypes reminiscent of accelerated HSC aging, including lymphopenia, increased myeloid output, and DNA damage, mimicking physiologically aged HSCs. Remarkably, recombinant PF4 administration restored old HSCs to youthful functional phenotypes characterized by improved cell polarity, reduced DNA damage, enhanced in vivo reconstitution capacity, and balanced lineage output. Mechanistically, we identified low-density lipoprotein receptor and C-X-C motif chemokine receptor 3 as HSC receptors transmitting the PF4 signal, with double knockout mice exhibiting exacerbated HSC aging phenotypes similar to PF4-deficient mice. Furthermore, human HSCs across various age groups also respond to the youthful PF4 signaling, highlighting its potential for rejuvenating aged hematopoietic systems. These findings pave the way for targeted therapies aimed at reversing age-related HSC decline, with potential implications in the prevention or improvement of the course of age-related hematopoietic diseases.

Multiple myeloma (MM) continues to be an incurable malignancy, even with recent therapeutic advancements. Although epigenetic dysregulation at cis-regulatory elements is known to drive disease progression, the complete molecular mechanisms underlying these alterations are poorly understood. Using Assay for Transposase-Accessible Chromatin with high-throughput sequencing analysis combined with the computational footprinting of CD138+ cells from 55 patients with MM, we depicted the dynamic changes in chromatin accessibility during disease progression and identified nuclear respiratory factor 1 (NRF1) as a master regulator of vital MM survival pathways. We demonstrated that NRF1 maintains proteasome homeostasis by orchestrating the ubiquitination pathway, which is essential for MM cell survival. We discovered a novel enhancer element that physically interacts with the NRF1 promoter, sustaining its expression. Targeting this enhancer RNA reduced NRF1 levels and increased tumor cell sensitivity to bortezomib (BTZ), suggesting therapeutic potential. In xenograft models, we showed that antisense oligonucleotides targeting the NRF1 enhancer, either alone or combined with BTZ, significantly decreased tumor burden and improved survival. Our findings reveal a previously unknown NRF1-dependent mechanism regulating MM cell survival and present a promising therapeutic approach through the manipulation of its regulatory network.

Epcoritamab is a subcutaneous CD3×CD20 bispecific antibody approved as monotherapy for relapsed/refractory (R/R) follicular lymphoma (FL). We evaluated fixed-duration epcoritamab with rituximab plus lenalidomide (R2) in R/R FL in arm 2 of EPCORE NHL-2 (phase 1b/2). Patients received epcoritamab (2 step-up doses, then 48-mg full doses) for up to 2 years, and R2 for up to 12 cycles (28 days per cycle). The primary end point was overall response rate (ORR) per investigator assessment (Lugano criteria). As of 21 September 2024, 108 patients received ≥1 epcoritamab dose in expansion (median follow-up, 28.2 months). Median age was 65 years; 57% had 1 previous line of therapy. ORR and complete response (CR) rate were 96% and 88%, respectively; CR rates in patients with high-risk features were 90% (primary refractory), 82% (refractory to anti-CD20 and an alkylating agent), and 83% (disease progression within 24 months of first-line therapy). Two-year estimates for remaining in CR, progression-free survival, overall survival, and not starting next antilymphoma therapy were 82%, 76%, 90%, and 84%, respectively. Minimal residual disease negativity was observed in 86% of evaluable patients (clonoSEQ assay). Common treatment-emergent adverse events (TEAEs) included neutropenia (65%), COVID-19 (59%), and cytokine release syndrome (CRS; 51%). Grade ≥3 TEAEs occurred in 87% of patients; 5 had grade 5 TEAEs (all COVID-19). CRS events were mostly low grade (grade 1, 38%; grade 2, 11%; grade 3, 2%), all resolved, and none led to epcoritamab discontinuation. Fixed-duration epcoritamab plus R2 demonstrated deep, durable responses with manageable safety and favorable outcomes in R/R FL, irrespective of risk features. This trial was registered at www.ClinicalTrials.gov as #NCT04663347.