Sickle cell disease (SCD) is an inherited blood disorder caused by a mutation in the β-globin gene, leading to hemoglobin polymerization under low oxygen conditions. This results in sickle-shaped red blood cells (erythrocytes), hemolysis, severe acute and chronic pain, and shortened erythrocyte lifespan. The severity of disease in SCD is linked to the type of hemoglobin mutation, with hemoglobin-SS genotype (HbSS) causing more frequent and severe than hemoglobin-SC genotype (HbSC). We previously identified mitochondrial retention and excessive reactive oxygen species (ROS) production in SCD erythrocytes. Here, we report that patients with SCD with the HbSS exhibit significantly higher erythrocyte mitochondrial retention and ROS levels than those with the HbSC. Mitochondrial retention positively correlates with serum bilirubin and lactate dehydrogenase (LDH), particularly in hydroxyurea-naïve patients. Gene expression analysis using a human autophagy array revealed upregulation of SNCA, GABARAP, GABRAPL2, MAP1LC3B, and CTSB in erythrocyte precursor cells from patients with SCD experiencing severe pain. Immunoblot analyses further confirmed accumulation of gamma-aminobutyric acid type-A receptor-associated protein (GABARAP), GABARAPL1, GABARAPL2, cathepsin-B, and alpha-synuclein in circulating erythrocytes and plasma from patients with SCD compared with controls. Our findings suggest a potential link between dysregulated autophagy proteins and erythrocyte mitochondrial retention in patients with SCD, opening new avenues for therapeutic interventions targeting these proteins to mitigate SCD pathogenesis.

Over the recent years, pigs have re-emerged as an alternative source of organs for xenotransplantation into humans with the promise to overcome a worldwide shortage of human donors. Xenotransplantation still faces critical issues with immune rejection that could be potentially solved by the generation of lymphohematopoietic chimeras. Moreover, pig hematopoietic stem and progenitor cells (HSPCs) can constitute themselves an unlimited source of HSPCs for lifesaving HSPC transplantation in bone marrow failure and post-chemotherapy, among other cell therapies. The generation of these hematopoietic chimeras requires a profound study of pig hematopoiesis including pig hematopoietic stem and progenitor cells (HSPCs). Importantly, via single cell RNA sequencing of pig bone marrow cells we identified pig HSPC populations transcriptionally similar to those in humans and many common transcriptional regulators of hematopoiesis evolutionarily preserved in erythromyeloid and lymphoid differentiation. This supports that hematopoiesis in pigs is hierarchically organized and regulated in a very similar fashion as in humans. We also provided a sorting strategy for the identification and isolation of several putative pig HSPC populations which should open a new means to functionally study pig hematopoiesis.

Human-derived induced pluripotent stem cells (iPSCs) are an invaluable resource in both two-dimensional (2D) and three-dimensional (3D) tissue engineering due to their multilineage potential in culture systems. To date, modeling red blood cell (RBC) disorders such as sickle cell disease (SCD) from iPSCs has been challenging due to the tendency for differentiation protocols to produce immature erythrocytes that lack robust β-globin expression and enucleate poorly. Here, we demonstrated an optimized three-stage erythroid differentiation protocol that generates enucleated, β-globin-expressing RBCs from somatically sourced iPSCs, derived from both healthy donors and patients with homozygous SCD. Induced RBCs (iRBCs) present phenotypically as GlyABand3CD71 and express adult hemoglobin tetramers. SCD iRBCs displayed sickling phenotypes in vitro when exposed to hypoxia. RNA-sequencing analysis of iPSC-derived SCD reticulocytes revealed dysregulated disease-relevant molecular pathways, suggesting future therapeutic avenues of investigation can be identified in this model. We further refined the protocol into a xeno-free formulation by replacing albumin sources with polyvinyl alcohol (PVA), significantly enhancing iRBC production without loss of terminal maturation. The ability to generate patient-specific iRBCs from somatic cell sources provides a valuable in vitro tool for the study of SCD and the development of novel treatments.

Hematopoietic stem cells (HSCs) possess unique characteristics that distinguish them from other hematopoietic progenitor cells, including self-renewal capacity, multipotency, stress response, metabolism, and deep quiescence. Recent advances have significantly enhanced our understanding of the epigenomic states that define these properties. HSCs undergo profound changes in their three-dimensional (3D) genome reorganization throughout development, differentiation, and responses to stimuli. Recent advancements in chromatin conformation capture techniques that require only a small number of cells have provided detailed insights into these dynamic processes. This review explored the latest discoveries in the 3D genome reorganization in HSCs, with a focus on chromatin remodeling during key transitions, including fetal-to-adult development, quiescence-to-activation, differentiation, and aging. We discussed the roles of key transcription factors, epigenetic modifiers, and structural proteins in shaping the 3D genome landscape. Additionally, we examined how alterations in the 3D genome organization impact HSC function and dysfunction in hematologic disorders. Finally, we highlighted future directions in this rapidly evolving field, emphasizing the potential implications of 3D genome research for targeted therapies in hematology.

Myelodysplastic syndromes (MDS) are a group of malignant clonal disorders that are characterized by functional impairment of hematopoiesis, morphologic dysplasia, and genetic heterogeneity. While less likely to transform to acute leukemia, lower-risk MDS (LR-MDS) include patients with IPSS-M moderate low risk, low risk, and very low risk patients and have a limited median survival of 3 to 10 years. Further, there is growing interest in discovering translational targets of LR-MDS pathophysiology. Clonal populations within the hematopoietic stem and progenitor (HSPC) to myeloid differentiation spectrum are widely considered to be a major contributor to MDS pathophysiology. A granular assessment of cell-type and lineage-specific states that contribute to LR-MDS pathophysiology remains to be elucidated. Here, we leverage single-cell transcriptomics to characterize cell states across the HSPC-myeloid differentiation landscape in LR-MDS. We develop a 30-gene score to classify LR-MDS HSPCs and identify novel molecular features of LR-MDS. The genes in our score suggest dysfunction in vesicular trafficking, which we further resolve across the myeloid differentiation axis. The gene products of vesicular trafficking-related pathways may be suitable translational targets for LR-MDS.

Complement-dependent cytotoxicity (CDC) is one of the main mechanisms of action for approved therapeutic anti-CD20 IgG antibodies, including rituximab, ofatumumab, and ocrelizumab, in the treatment of B-cell lymphoma patients. However, resistance to these therapies inevitably develops in patients, and thus novel antibody approaches are needed. Here, we described the CDC activity of an anti-CD20 IgM in comparison to an anti-CD20 IgG. We applied live-cell imaging and kinetic analysis to measure CDC activity in real time. Through this imaging platform, we demonstrated that an IgM antibody exhibited more potent and faster target cell killing through CDC compared with an IgG antibody. Additionally, an IgM antibody was more effective at killing target cells with low antigen density, in low levels of complement, and in the presence of high complement inhibitor expression. An anti-CD20 IgM also showed superior CDC against ex vivo tumor samples from a patient with B-cell lymphoma. These preclinical studies demonstrated the potential of an anti-CD20 IgM-based therapeutic antibody having superior CDC in B-cell lymphoma compared with a traditional IgG antibody.

Hematopoietic stem cells (HSCs) within the bone marrow (BM) display significant molecular and functional heterogeneity. Deciphering intrinsic factors that govern HSC diversity is key to enriching specific HSC subtypes for predictable and clinically relevant differentiation outcomes. Here, we show that the mitochondrial protein Asrij/OCIAD1, a conserved regulator of hematopoietic homeostasis, contributes to HSC heterogeneity. Asrij depletion is known to cause loss of quiescence, myeloid bias, and aging-like changes in mouse BM HSCs. Interestingly, Asrij expression is inherently heterogeneous and enriched in only 47% of the HSC population. To investigate whether Asrij expression levels influence HSC fate, we generated a novel Asrij-mNeonGreen (mNG) knock-in reporter mouse using CRISPR-Cas9 technology. We show that the Asrij reporter faithfully recapitulates its heterogeneous expression in the BM HSCs, allowing isolation of live cells based on Asrij expression levels. Ex vivo culture of HSCs demonstrated that Asrij HSCs exhibit enhanced self-renewal capacity, whereas Asrij HSCs are primed for differentiation. Transplantation assays further revealed that Asrij HSCs have enhanced reconstitution in the BM hematopoietic stem and progenitor cell (HSPC) and myeloid cell compartments. Transcriptomic analysis uncovered signatures of quiescence in Asrij HSCs, whereas Asrij HSCs exhibit hallmarks of HSC activation. In summary, we show that Asrij levels impact the quiescence, self-renewal, and differentiation potential of HSCs, thereby contributing to the functional diversity of the HSC pool. Furthermore, the Asrij-mNG reporter mouse provides a powerful and versatile model for investigating the molecular underpinnings of functional diversity within the HSC compartment.

The 3' untranslated region (3'UTR) of mRNA is crucial for post-transcriptional gene regulation, primarily through miRNAs. However, the overall role of the Gata1 3'UTR in mammals remains unclear. In this study, we knocked out the Gata1 3'UTR and observed a defect in erythropoiesis in mutant mice, evidenced by macrocytic anemia at baseline. The deletion of the Gata1 3'UTR also caused deficiencies in erythropoiesis within fetal livers. Mechanistically, removing the Gata1 3'UTR destabilizes Gata1 mRNA, leading to decreased levels of GATA1 protein. This reduced stability results from the dissociation of AU-rich elements in the 3'UTR from a trans-acting factor called ELAVL1. Specifically, we conducted an RNA pulldown assay followed by mass spectrometry to identify proteins that bind to the Gata1 3'UTR. Gene Ontology analysis revealed that ELAVL1 is a binding partner across nearly all categories related to mRNA stabilization. Western blotting, RNA immunoprecipitation, and mutagenesis assays confirmed the direct interaction between the Gata1 3'UTR and ELAVL1. Modulating ELAVL1 activity or protein levels with the small molecule inhibitor Dihydro-tanshinone-I, or through ectopic expression in erythroid cells, validated ELAVL1 as a stabilizing factor for Gata1 mRNA. Our results highlight the important role of the Gata1 mRNA 3'UTR in erythroid development. Teaser Abstract: The role of the Gata1 3'UTR in mammals remains unclear. In this study, we generated Gata1 3'UTR knockout mice and observed a defect in erythropoiesis, evidenced by macrocytic anemia at baseline. Mechanistically, removing the Gata1 3'UTR destabilizes Gata1 mRNA and causes reduced expression of GATA1 protein. This low mRNA stability results from the dissociation of AU-rich elements in the 3'UTR from trans-acting factor ELAVL1, rather than from loss of miRNA binding or loss of poly A sequences. Our results highlight the crucial role of the Gata1 3'UTR in erythroid development.

We investigated the clinical significance of a rare germline SH2B3 variant (c.232G>A; p.Glu78Lys) identified by targeted next-generation sequencing (NGS) in patients with myeloproliferative neoplasms (MPNs).

Cytoreductive treatment is a main strategy to reduce thrombotic complications and ameliorate symptoms in Phi-negative myeloproliferative neoplasms (MPNs) comprising essential thrombocythemia, polycythemia vera, and primary myelofibrosis. Based on the observation of differences in platelet size during our routine microscopic analysis of blood smears from patients with MPN, in this work we studied whether these differences could be dependent on the type of cytoreductive drug used for patients' treatment and whether changes in platelet size could be induced by the effect of these drugs on thrombopoiesis. Maximum platelet diameter (MPD) was measured in 120 patients with MPN. The effect of drugs on thrombopoiesis was evaluated in normal megakaryocytes (MKs) obtained from cord blood-derived CD34+ hematopoietic progenitors. Anagrelide (ANA), α-interferon (IFN), and ruxolitinib (Ruxo) increased, whereas hydroxyurea (HU) decreased platelet size. MK incubation with these drugs revealed that ANA and IFN-induced abnormal proplatelet (PP) architecture and affected microtubular structure, but only ANA altered actin organization, whereas neither Ruxo nor HU modified MK cytoskeleton. By bioinformatic analysis, RANTES downregulation was identified as a candidate responsible for ANA-induced abnormalities. RANTES downregulation was confirmed in MK incubated with ANA but not with IFN. Addition of recombinant RANTES reverted ANA-induced cytoskeletal abnormalities. Evaluation of RANTES plasmatic levels and platelet RNA expression in patients with MPN showed that RANTES decreased in both samples during ANA treatment, suggesting that in vitro findings could reflect ANA action in vivo. In conclusion, this study demonstrates the influence of cytoreductive drugs on platelet size and reveals their differential mechanisms of action during platelet production.

Stem cell antigen-1 (SCA1) is widely used to identify mouse hematopoietic stem cells (HSC) and multipotent progenitors (MPP) among lineage-negative KIT (LK) cells. However, SCA1 is expressed only in a few inbred mouse strains and becomes strongly upregulated on LK cells following in vivo challenge with interferons, lipopolysaccharide (LPS) or pathogens leading to incorrect analysis of HSC function subsets and delineation of HSC, MPP and lineage-restricted progenitor subsets. Endothelial protein C receptor CD201can be used as an alternative marker for mouse and even human HSC. However, whether CD201 expression changes following infectious challenge is unknown. Unlike SCA1, CD201 expression did not change on mouse LK cells in response to LPS in vivo. Long-term competitive transplantations with CD201, CD201 or SCA1 LK cells showed that most reconstituting HSCs are within the LK CD201 population after LPS challenge. However long-term competitive repopulation potential of LK SCA1 cells from LPS-treated mice was much more severely reduced than that of LK CD201 cells from the same LPS-treated donors suggesting that the LK SCA1 population in challenged donors becomes contaminated with CD201 progenitors devoid of long-term repopulation potential. Based on CD201 gating strategy, we re-assessed the effect of LPS on HSC and MPP cycling and mobilization, and their dependency on MY88 and TRIF adaptors. In conclusion, CD201 enables a more accurate analysis of mouse HSC and MPP subsets in all inbred strains in septic conditions or steady-state.

Gene rearrangements of the human mixed lineage leukemia (MLL) gene (also known as KMT2A) generate multiple fusion oncoproteins, which cause leukemia with poor prognosis. MLL is an epigenetic regulator that reads and writes epigenetic information and has an evolutionarily conserved role in maintaining expression of Homeotic (HOX) genes during embryonic development. Most MLL gene rearrangements found in leukemia generate a constitutively active version of the wild-type protein, which causes overexpression of HOX and other genes and leukemic transformation of normal hematopoietic progenitors. Elucidating the molecular mechanisms underlying how MLL activates gene expression and how gene rearrangements affect this gene-regulating activity provided therapeutic opportunities to block fusion oncoprotein-specific activities. One uniform molecular dependency of MLL fusion oncoproteins is its interaction with the chromatin-binding partner MENIN that is essential to maintain leukemic transformation. MENIN inhibitors that interfere with the MLL-MENIN interaction have been developed and are now entering clinical practice. Also, the MLL complex physically interacts with several histone acetyl transferases (HATs), including MOZ/MORF, HBO1, and EP300/CREBBP to effect MLL-MENIN-dependent gene activation. Aberrant recruitment of these HATs and other transcriptional effector complexes are key differences between MLL and MLL fusion oncoproteins. In this review, we first summarized our current understanding of wild-type MLL function and the aberrant function of its oncogenic variants. We then discussed in detail how chromosomal translocations generate constitutive-active forms of MLL and categorize them into five major classes. We touched on the collaborative gene activation by MLL and specific interacting HATs. Lastly, we discussed how these mechanistic insights have led to the development of the first-in-class MENIN inhibitors and discussed efforts to anticipate and treat both genetic and nongenetic mechanisms of resistance.

The utilization of alteplase for the management of intracerebral thromboembolism is associated with an elevated risk of intracerebral hemorrhage. To mitigate this risk, we investigated the fibrinolytic effect of alteplase-loaded platelets as a preliminary step in the development of a biocompatible drug delivery system (DDS). In this context, we demonstrated that platelet loaded with alteplase exhibited thrombolytic efficacy. Therefore, a drug DDS that employs alteplase-loaded platelets may offer a promising avenue for the development of a more biocompatible and less invasive thrombolytic therapy.

Drug resistance remains a critical barrier in effective cancer therapy. Previously, we demonstrated that expression of anti-apoptotic protein XIAP, contributes to the development of TRAIL resistance in chronic myeloid leukemia (CML) cells. However, upon acquiring drug resistance (K562R and KCL22R), XIAP degradation shifted from the lysosomal to the proteasomal pathway. Consistently, XIAP expression was markedly elevated in tumor samples compared to normal controls and was significantly higher in Imatinib-failure (IMA-FL) patients than in Imatinib-responsive (IMA-RP) counterparts within the patient cohort. Moreover, we have found that proteasomal activity increased in imatinib resistance cells and lysosomal pathway is inhibited. Mechanistically, we found that H₂O₂-induced activation of the ERK-mTOR axis suppressed autophagy in resistant cells, facilitating this shift in degradation pathway. Very interestingly, dual intervention by restoring autophagic flux via mTOR inhibition and inducing XIAP degradation using HO reverted Imatinib resistance in K562R cells. Thus, our findings uncover a novel ERK-mTOR-axis for upregulation of proteasomal degradation of XIAP which could be targeted to overcome Imatinib-resistance by combinatorial inhibition of mTOR and XIAP in CML. This study holds the promise of a new therapeutic strategy for overcoming drug resistance in cancer.

Secondary hemophagocytic lymphohistiocytosis (sHLH) is a life-threatening hyperinflammatory syndrome with high early mortality. This retrospective study of 96 adult patients with sHLH aimed to identify prognostic factors for early death, with a focus on lymphocyte subsets and clinical biomarkers. Using univariate and multivariate Cox regression analyses for 30, 60, and 90-day mortality, we demonstrated that CD3⁻CD16/CD56⁺ cells <2% were a strong independent predictor of early mortality across all time points. Furthermore, CD3⁺ cells (<50%) independently predicted 60-day mortality, whereas hemoglobin concentration (<70 g/L) emerged as an independent risk factor specifically for 30-day mortality. Elevated ferritin concentration (>10,000 ng/mL) was also significantly associated with poor outcomes, consistent with established literature. Importantly, lymphoma- or Epstein-Barr virus (EBV)-associated sHLH remained an independent predictor of early mortality after multivariate adjustment. These findings indicate that reduced CD3⁻CD16/CD56⁺ cells, combined with CD3⁺ cell depletion, anemia, and hyperferritinemia, provide valuable prognostic information for risk stratification and targeted therapeutic interventions to reduce early mortality in patients with sHLH.

During the development of the immune system, there is a progressive shift from fast-acting innate-like lymphocytes to slower-acting adaptive lymphocytes. This developmental shift is evident in B cells, γδ T cells, and αβ T cells, with the more innate-like lineages (B1a, B1b, Vδ1, virtual memory CD8+, iNKT, and CD8αα) being produced before the more adaptive lineages (B2, Vγ9Vδ2, and conventional CD8+ and CD4+ αβ T cells). However, immunologists have historically viewed the development of B and γδ T cells differently than αβ T cells. Whereas it is well accepted that the functions of B and γδ T cells are linked to their derivation from distinct hematopoietic progenitors that arise throughout ontogeny, the same phenomenon has largely been ignored for αβ T cells. Instead, the prevailing view is that all αβ T cells are made from the same hematopoietic stem cells (HSCs), and any diversity in the αβ T-cell compartment comes from stochastic expression of different TCRs and random environmental cues encountered in the thymus. In this review, we discussed the evidence that αβ T-cell lineage decisions are not solely determined by thymic selection and that hematopoietic origin also intrinsically biases development toward innate-like T cells in early life.

Cohesin gene mutations occur in many malignancies, including acute myeloid leukemia (AML). Loss-of-function mutations in the four major cohesin complex genes (RAD21, SMC3, SMC1a, and STAG2) occur across most major genetic subtypes of AML but are notably absent in AML harboring CBFB::MYH11, suggesting that cohesin mutations yield distinct biological outcomes dependent on the genetic AML driver. We hypothesized that CBFB::MYH11-expressing leukemias would be dependent on intact cohesin genes given their near-mutual exclusivity. To investigate this, we combined either germline or inducible heterozygous deletions in cohesin genes Smc3 or Rad21, respectively, with an inducible murine model of Cbfb::MYH11 AML. This approach allowed us to evaluate the effects of cohesin haploinsufficiency on leukemia development, chromatin accessibility, and transcriptional output. We demonstrated that intact cohesin function is dispensable for Cbfb::MYH11-driven leukemia. Instead, Cbfb::MYH11 expression is the primary driver of the transcriptional program in transformed leukemic cells. Furthermore, we observed differential effects of Rad21 and Smc3 deletion on leukemia development and secondary engraftment despite only minor differences in gene expression. These results demonstrate that cohesin mutations are not only tolerated in Cbfb::MYH11-expressing cells, but they also likely do not confer a strong selective advantage and are therefore not preferentially selected for during clonal evolution of this leukemia.

Neutrophils serve as the first line of defense against invasive bacterial and fungal pathogens. The loss of circulating neutrophils leaves patients at a critical risk of life-threatening infections. In this study, we optimized conditions for expanding human precursor neutrophils ex vivo while preserving the functional capacity of mature neutrophils. We evaluated several CD34+ hematopoietic stem cells (HSCs) from various sources, including umbilical cord blood (UCB), adult bone marrow (BM), and cadaveric sources. UCB-derived CD34+ cells consistently demonstrated the highest expansion capacity, achieving an additional two cell divisions compared with BM-derived cells. Surface receptor profiling demonstrated that all sources resulted in mature neutrophil differentiation, although UCB-derived cell sources exhibited higher expression of maturation markers CD11b, CD15, and CD66b, in conditions expanded with the small molecule UM729. Functionally, neutrophils derived from all cell sources retained the ability to phagocytose and produce reactive oxygen species (ROS), with enhanced activity following antibody-dependent opsonization. To better understand the impact of opsonization, Fc receptor expression levels were assessed in addition to profiling changes in complement and adhesion receptor expression. Single-cell expression analysis confirmed that ex vivo differentiation was consistent with known patterns of myeloid differentiation, leading to distinct neutrophil subpopulations. Notably, mature neutrophils generated ex vivo were transcriptionally distinct from freshly isolated primary cells. Overall, our findings demonstrate that UCB-derived precursors offer the highest expansion potential for generating neutrophil precursors, able to mature into fully functional neutrophils. These results provide valuable insights into optimizing human neutrophil production as a promising cellular therapy for neutropenic individuals.

Inhibitors of the menin-KMT2A interaction are promising agents for the treatment of KMT2A-rearranged leukemias. We evaluated menin inhibition in patient-derived xenografts of KMT2A-rearranged leukemias with high-risk features. Three acute myeloid leukemias with high-risk fusion partners (mixed-lineage leukemia-10 [MLLT10] and mixed-lineage leukemia-4 [MLLT4]) and two infant acute lymphocytic leukemia (ALL) samples were sensitive to menin inhibition. We also evaluated serial samples from two patients with multiply relapsed ALL. We found that highly pretreated KMT2A::AFF1 ALL samples were much less sensitive compared with cells obtained earlier in the same patients' disease course. Because none of the patients had been treated with a menin inhibitor, resistance in these highly pretreated samples was acquired in the absence of menin-inhibitor exposure. Transcriptomic analysis documented sustained on-target efficacy toward the canonical targets of the menin inhibitor in resistant cells. Targeted genomic analysis documented the emergence of multiple comutations, including RAS pathway and TP53 mutations, although neither was sufficient to induce menin-inhibitor resistance in vitro. Downregulation of KMT3D may account for resistance in one patient; inactivation of KMT2C/D has been reported to result in menin-inhibitor resistance, and KMT2C-edited cells from this patient were selected for in menin-inhibitor-containing growth conditions. Future studies will need to clarify more broadly which genomic/epigenomic alterations drive upfront resistance. Regardless of mechanism, our data support using menin inhibitors upfront or in early lines of therapy before substantial genomic or epigenomic evolution has occurred.