Purine nucleoside phosphorylase (PNP) deficiency causes inadequate purine metabolite detoxification, leading to combined immunodeficiency and variable neurological symptoms. Hematopoietic stem cell transplantation (HCT) cures the immunodeficiency, but large studies on long-term outcomes are lacking. In a retrospective EBMT study, we investigated 46 patients with PNP deficiency from 21 centers. We analyzed the presenting clinical signs and outcomes after HCT. Cognition (0-3), hearing (0-3), interaction (0-4), movement (0-4) and occupation (0-3) (CHIMO-score) were scored at last follow-up (FU) visit (no impairment: 17; mild: 15-16, moderate: 12-14, and severe impairment: <12). The median age at initial presentation was 7.5 (1-48) months, with 41% of cases involving infectious, 39% neurological, 15% infectious/neurological, and 5% autoimmune symptoms. At timepoint of HCT (median age: 26 (2-192) mo.), 88% of patients exhibited neurologic abnormalities. After a median FU of 7.9 (1.0-22.3) years, 40 patients were alive with a 3-year overall survival (OS)/event-free survival (EFS) probability of 86% (CI 77-97%)/75% (CI 64-89%), respectively. At FU, high-level donor chimerism (>50-100%) was observed in 85% of patients, and low-level (11-50%) in 15% of patients resulting in resolution of T lymphopenia. The median scores for cognition, hearing, interaction, movement, and occupation were 3 (0-3), 3 (1-3), 4 (1-4), 3 (1-4), and 2 (0-3), respectively, with a median CHIMO-score of 14 (6-17). Patients who underwent HCT <24 months after initial presentation demonstrated superior OS (p=0.049). Neurological symptoms occurring <11 months of age were associated with reduced OS (p=0.027). While the overall results were satisfactory, earlier diagnosis could further improve outcomes.

The transcription factor LEF1 is aberrantly expressed across all subtypes and stages of chronic lymphocytic leukemia (CLL), yet the molecular mechanisms underlying its contribution to CLL pathogenesis remain poorly defined. Here, we conducted a comprehensive mechanistic dissection of LEF1 function in CLL using extensive functional analyses of patient-derived samples. We identified that, although LEF1 mRNA levels remain stable, clinically aggressive cases show elevated LEF1 protein levels due to enhanced protein stability. LEF1 protein abundance is selectively modulated by lymph node-derived stimuli, including T-cell interactions and B-cell receptor (BCR) signaling. Importantly, we uncovered a dual, context-dependent role for LEF1 that is determined by its protein levels. Low LEF1 protein, characteristic of indolent cases, supports B-cell activation, while increased protein abundance in aggressive disease promotes proliferation through the binding and induction of cell cycle and metabolic gene networks. We further showed that LEF1 exon 6 skipping is enriched in proliferative and aggressive CLL. Both in vitro and in vivo experiments revealed that LEF1-driven proliferation is mediated by these short, alternative spliced isoforms. While all LEF1 isoforms bind to a core set of proliferation- and activation-related genes, they induce distinct transcriptional programs: full-length LEF1 promotes a quiescence gene signature and limits leukemic growth, whereas exon 6-skipping isoforms drive proliferation. Our findings establish LEF1 as an oncogenic transcription factor in CLL whose biological and clinical effects are modulated post-transcriptionally by both protein abundance and isoform composition.

For over sixty years, blood researchers have been counting clones with every tool at their disposal. Inspired by phage and fly geneticists, Till and McCulloch irradiated mice to induce chromosomal aberrations. Using this labeling strategy, they demonstrated that different types of blood cells shared the same mutation in every spleen colony, thereby proving the existence of hematopoietic stem cells. Since their breakthrough, technological advances have enabled researchers to quantify hematopoiesis at single-cell resolution in increasingly complex samples across both mice and humans. With these modern sophisticated lineage tracing methods, our foundational understanding of the blood system is being reshaped. For instance, we now interpret hematopoietic architecture as arising from stem and progenitor cells of diverse developmental origins, each with distinct fate biases encoded by unique regulatory states. Interacting with this regulatory layer, genetic mutations and epimutations arise, expanding clonally and becoming pervasive with age. Together, clonal heterogeneity and age-driven clonal selection may underlie the perplexing diversity of therapy responses in cancer and beyond. As these paradigm-shifting insights gain traction, clonal tracing is being adopted across dozens of biological and clinical studies. Here, we review the modern toolbox of clonal tracking technologies, with a focus on next-generation sequencing-based approaches, and provide a practical guide for matching specific research questions with optimal experimental strategies.

Covalent crosslinking of fibrin by the plasma transglutaminase coagulation factor XIII (FXIII) is a key determinant of blood clot stability and function. FXIII-catalyzed formation of ε-N-(γ-glutamyl)-lysyl crosslinks is restricted to the fibrin γ- and α-chains and follows thrombin driven fibrin polymerization. Fibrinogen is also crosslinked by tissue transglutaminase (TG2) in a reaction favoring intra- and intermolecular a-g crosslinking. Emerging evidence points to fibrinogen as a relevant substrate of TG2 in conditions of acute tissue damage. Remarkably, beyond detection of prototypical FXIII-directed crosslinks (i.e., a-a, g-g), we identified entirely novel covalent crosslinks involving the fibrinogen β chain (i.e., b-a, via FGB-Q82). Addition of TG2 to in vitro clotting reactions and analysis of fibrin(ogen) in reducing conditions revealed loss of β chain polypeptide paired with formation of high-molecular weight β chain species. Mass spectrometry-based crosslinking proteomic analysis of in vitro clots recapitulated the precise TG2-directed β chain crosslinks observed in clots made using the plasma of trauma patients. The results are the first to document in vitro and ex vivo crosslinking of the fibrin β chain and highlight a novel example of TG2 emerging as a relevant plasma transglutaminase.

Juvenile myelomonocytic leukemia (JMML) is a rare, aggressive pediatric myeloproliferative neoplasm for which hematopoietic stem cell transplantation (HSCT) is currently the only established curative therapy. However, a watch-and-wait (W&W) approach has shown promise for long-term survival in selected cases. In this real-world study, we analyzed outcomes of JMML patients initially managed with a W&W strategy within a nationwide cohort of 161 genetically characterized cases. W&W was chosen for 35 patients, with increasing adoption over time, reaching 39% in the 2016-2021 period. Most patients carried mutations in CBL (43%), NRAS (34%), or homozygous germline SH2B3 (14%). Over a median follow-up of 6.5 years, 86% (30/35) achieved long-term survival with partial or complete resolution of myeloproliferative symptoms, although clonal hematopoiesis persisted in nearly all survivors (28/30). Disease progression occurred in five patients (CBL: n=3, NRAS: n=1, PTPN11: n=1), mostly within two years post-diagnosis. Overall, in the W&W cohort, the 5-year OS and EFS were 93.1% and 84.5%. In NRAS-mutated cases, age <30 months, normal to slightly elevated fetal hemoglobin, platelet >45x109/L, the absence of additional somatic mutations and low DNA methylation profile were associated with favorable outcomes. In CBL-driven JMML, no predictive factor of adverse evolution was identified. Notably, W&W was effective in all patients with homozygous germline SH2B3, regardless of clinical or biological presentation. These findings support W&W as a viable alternative in up to 30% of JMML patients, potentially sparing them from HSCT-associated risks. Given the persistence of clonal hematopoiesis and the risk of extra-hematological complications, long-term monitoring remains essential.

Glutamine-dependence of cancer cells reduces local glutamine availability, which hinders anti-tumor T-cell functionality and facilitates immune evasion. We thus speculated that glutamine deprivation might be limiting efficacy of CAR T-cell therapies in cancer patients. We have seen that antigen-specific T cells are unable to proliferate or produce IFN-γ in response to antigen stimulation when glutamine concentration is limited. Using multiple myeloma (MM) as a glutamine-dependent disease model, we found that murine CAR-T cells selectively targeting BCMA in MM cells were sensitive to glutamine deprivation. However, CAR-T cells engineered to increase glutamine uptake by expression of the glutamine transporter Asct2 exhibited enhanced proliferation and responsiveness to antigen stimulation, increased production of IFN-γ, and heightened cytotoxic activity, even under conditions of low glutamine concentration. Mechanistically, Asct2 overexpression reprogrammed CART cell metabolic fitness of CART cells by upregulating the mTORC1 gene signature, modifying the Solute Carrier transporter (SLC) repertoire, and improving both basal oxygen consumption rate and glycolytic function thereby enhancing CART cell persistence in vivo. Accordingly, expression of Asct2 increased the efficacy of BCMA-CART cells in syngeneic and genetically-engineered mouse models of MM, which prolonged mouse survival. In patients, reduced expression of Asct2 by MM cells predicted poor outcome to combined immunotherapy and BCMA-CAR T-cell therapy. Our results indicate that reprogramming glutamine metabolism may enhance anti-tumor CAR T-cell functionality in MM. This approach may also be effective for other cancers that depend on glutamine as a key energy source and metabolic hallmark.

FLAG-IDA (fludarabine, cytarabine, granulocyte colony-stimulating factor, idarubicin) with venetoclax shows promise as frontline pediatric acute myeloid leukemia therapy. In 12 patients treated at MD Anderson, most achieved remission with good early survival outcomes, and many proceeded to transplant. Common toxicities included cytopenias, comparable to previous regimens.

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a recently discovered autoinflammatory disorder linked to somatic mutations in the UBA1 gene, resulting in a profound cytoplasm-restricted defect in ubiquitylation. The disease is characterized by a macrocytic anemia that remains poorly understood. To investigate the erythroid lineage in VEXAS, we conducted a comprehensive study combining in vivo assessments of patients' mature red cells and marrow erythroblasts, alongside in vitro base-editing models of erythropoiesis. Here we show that mature red cells do not exhibit ubiquitylation defects, and patient-derived bone marrow erythroblasts lack UBA1 mutations beyond the basophilic stage of erythroid differentiation. In vitro base editing of UBA1 variants in CD34+ primary cells resulted in high mortality during early erythroid differentiation, but not during monocytic differentiation. Edited erythroid precursors displayed TP53 overexpression linked to defective ubiquitylation and anomalies in ribosome biogenesis, reminiscent of Diamond-Blackfan anemia. We propose that VEXAS-associated anemia should be considered as a mosaic erythroblastopenia, where the severity of anemia is influenced by the quality and quantity of the UBA1-WT compartment. Our findings offer new insights into the physiopathology of VEXAS and may suggest new potential therapeutic options.

Immune driven fibrotic skin diseases including scleroderma/systemic sclerosis (SSc) and chronic graft-versus-host-disease (cGvHD) cause skin stiffening that has major impact on patient quality of life and associated patient mortality. Therapies to improve sclerotic skin resulting from these diseases are largely ineffective. We previously showed that EREG, a DC3 dendritic cell-derived EGFR ligand, is elevated in the skin and lung of patients with SSc and required for maintenance of skin fibrosis. Here, we developed a fully human anti-EREG neutralizing antibody that has both high affinity and specificity. We found this therapeutic antibody to be functional and safe in vivo using human EREG knock-in mice. To understand the antifibrotic mechanism of targeting EREG, we aligned skin single-cell transcriptomic profiles of SSc, morphea (localized scleroderma) and SclGvHD with disease biomarkers. EREG expression in the skin was elevated in all three fibrotic diseases and a driver of TNC production by myofibroblasts in all three fibrotic diseases. TNC is a pro-inflammatory extracellular glycoprotein that functions as an endogenous TLR4 ligand which induces expression of TLR4 target genes CCL2 and IL6. Examination of skin explants from patients with active SclGvHD treated with anti-EREG therapeutic antibody by spatial transcriptomics demonstrated upregulation of matrix degradation by increased MMP and decreased TIMP1 expression. Protein measurements showed reduced secretion of EREG targets TNC, CCL2, and TIMP1 in all patients, and type I collagen and FN1 in 3/4 patients. Thus, sclerotic skin treated with the anti-EREG therapeutic antibody reduced inflammatory and fibrosis biomarkers associated with EGFR and TLR4 signaling.

Marginal zone lymphoma (MZL) encompasses biologically heterogenous group of indolent B-cell lymphomas that remains substantially underrepresented in clinical research. Despite recent and significant therapeutic advancess in B-cell malignancies, trial design in MZL continues to face persistent challenges including diagnostic heterogeneity, inconsistent control arms, suboptimal endpoints, and economic barriers. In this narrative review, we examine these key obstacles and discuss emerging strategies to overcome them, such as the standardization of diagnostic criteria, implementation of subtype-specific treatment approaches, validation of surrogate endpoints, and integration of novel response assessment modalities such as metabolic imaging (positron emission tomography), minimal residual disease assessment in flow cytometry or single cell molecular evaluation, and circulating tumor DNA measurement, but need to be evaluated and harmonized for full appreciation We contend that MZL should be understood as a methodological paradigm, rather than as a clinical exception. This may facilitate the refinement of trial design and ultimately accelerate therapeutic innovation across the broad spectrum of indolent lymphomas.

Self-renewing multipotent hematopoietic stem cells (HSCs) are a rare but important cell population which can reconstitute the entire blood and immune system following 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 ~3-fold. Following expansion, these Runx2-KO HSCs engrafted at ~5-fold higher levels in transplantation assays. Non-cultured 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 will contribute understanding transcriptional regulation of hematopoiesis and improve HSC therapies.

RNA splicing and processing are critical for erythropoiesis, as dysregulation of RNA splicing ultimately disrupts protein synthesis. The RNA-binding protein Rbm38 is highly expressed during terminal erythropoiesis. While 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 production was impaired in the bone marrow of Rbm38-deficient mice. Rbm38-/- red blood cells 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 (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, mRNA 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.