Acute myeloid leukemias (AMLs) have an overall poor prognosis with many high-risk cases co-opting stem cell gene regulatory programs, but the mechanisms through which these programs are propogated remain poorly understood. The increased expression of the stem cell transcription factor, MECOM, underlies a key driver mechanism in largely incurable AMLs. However, how MECOM results in such aggressive AML phenotypes remains unknown. To address existing experimental limitations, we engineered and applied targeted protein degradation with functional genomic readouts to demonstrate that MECOM promotes malignant stem cell-like states by directly repressing prodifferentiation gene regulatory programs. Remarkably and unexpectedly, a single node in this network, a MECOM-bound cis-regulatory element located 42 kilobase (kb) downstream of the myeloid differentiation regulator CEBPA is both necessary and sufficient for maintaining MECOM-driven leukemias. Importantly, the targeted activation of this regulatory element promotes differentiation of these aggressive AMLs and reduces leukemia burden in vivo. These findings suggest a broadly applicable approach for functionally dissecting oncogenic gene regulatory networks to inform improved therapeutic strategies.

The recent approval of adeno-associated virus (AAV)-based gene therapies for hemophilia A (HA) represents a major advancement in the management of this X-linked bleeding disorder, offering multiyear bleed protection and improved quality of life over factor VIII (FVIII) replacement. However, challenges remain, including concerns over long-term durability of expression and the difficulty of packaging the oversized FVIII transgene into AAV vectors. To address these limitations, we developed AAV8-Bi8, a liver-directed gene therapy encoding Bi8, a novel 54.5-kilodalton FVIII-mimetic antibody. Bi8 is expressed as a compact, single-chain tandem, single-chain fragment variable, and is delivered via a 4.4-kilobase expression cassette packaged within AAV8 capsids, well within the vector packaging capacity. In vitro, Bi8 demonstrated FVIII-mimetic activity, and effectively corrected FVIII-deficient human plasma to levels comparable with emicizumab, the current market standard. In vivo, a single administration of AAV8-Bi8 in FVIII-deficient mice resulted in dose-dependent, durable expression of Bi8, complete phenotypic correction of bleeding, and therapeutic equivalence to both emicizumab-treated and wild-type animals. Importantly, no toxicity or antidrug antibody responses were observed. This approach, based on delivering FVIII-mimetic antibodies through AAV rather than truncated FVIII transgenes, could provide a more flexible and efficient platform for gene therapy in HA. AAV8-Bi8 has the potential to offer sustained, lifelong hemostatic control, including in patients who have developed inhibitors to FVIII.

Talquetamab, a G protein-coupled receptor class C group 5 member D-targeting bispecific antibody for relapsed/refractory multiple myeloma (R/R MM), plus daratumumab, may lead to deeper and more durable responses than either therapy alone. In the phase 1b TRIMM-2 study, patients with R/R MM (at least 3 previous lines of therapy or double refractory to a proteasome inhibitor and an immunomodulatory drug) received subcutaneous talquetamab 0.4 mg/kg weekly (QW; "QW cohort") or 0.8 mg/kg every other week (Q2W cohort) plus daratumumab 1800 mg per the approved schedule. The primary end point was safety. Secondary end points included overall response and duration of response. Progression-free survival was an exploratory end point. Sixty-five patients (median 5 previous lines of therapy; 61.5% triple-class refractory; 24.6% bispecific antibody exposed) received talquetamab plus daratumumab (QW, n = 14; Q2W, n = 51; median follow-up of 18.6 months). Most common adverse events were oral events, skin events, cytokine release syndrome, and infections. Grade 3 or 4 events occurred in 81.5%. Two patients had dose-limiting toxicities, both in the Q2W cohort (grade 3 stomatitis/oral mucositis, and grade 3 maculopapular rash). Responses occurred in 71.4% (QW cohort) and 82.4% (Q2W cohort) of patients. Median progression-free survival was 23.3 and 21.2 months, respectively, in each cohort. Pharmacodynamic results suggest the immunomodulatory action of daratumumab contributes to a conducive environment for talquetamab by reducing immunosuppressive cells. Talquetamab plus daratumumab demonstrated promising efficacy outcomes in patients with heavily pretreated disease, with a safety profile consistent with each agent as monotherapy. This trial was registered at www.clinicaltrials.gov as #NCT04108195.

In this response-adapted clinic trial with daratumumab monotherapy for older patients with newly diagnosed multiple myeloma (MM), we identified target antigen expression, a plasma cell phenotype, and an activated immune tumor microenvironment (iTME) as critical features associated with response to CD38 monoclonal antibody therapy. Here, patients achieving a partial response after 2 cycles continued daratumumab, otherwise lenalidomide or bortezomib was added. This strategy resulted in an overall response rate of 97% and low rates of adverse events, with 37% of patients able to continue daratumumab monotherapy. Importantly, we found that higher CD38 expression, plasma cell gene expression programming, and an activated iTME were associated with patients who were able to continue daratumumab therapy alone. In contrast, patients requiring the addition of lenalidomide or bortezomib had increased expression of adhesion, tumor necrosis factor signaling, KRAS signaling, and B-cell programs, as well as an immunosuppressed iTME. Tracking of clonal dynamics illustrated the selection of subclones enriched for de novo resistance gene expression programs after only 2 cycles of daratumumab monotherapy. Upon relapse, daratumumab refractory MM cells were characterized by the expansion of preexisting minor subclones with mixed transcriptomic programs containing the plasma cell phenotype with decreased CD38 expression and maintenance of resistance programs, suggesting development of acquired resistance involves an uncoupling of transcriptional programs present in therapy-naïve tumors. To our knowledge, this is the first study to demonstrate the effectiveness of response-adapted daratumumab treatment and describe critical biomarkers of single-agent daratumumab sensitivity in vulnerable patients with therapy-naïve MM. This trial was registered at www.ClinicalTrials.gov as #NCT04151667.

Telomere length shortening has been associated with genomic instability and acquisition of molecular lesions, but these processes have not been systematically studied across large cohorts of myeloid neoplasia (MN). As proof of concept for a novel, cross-validated whole-genome sequencing-based method of telomere content (TC) determination combined with mutations, transcriptomics, and functional assays, we studied TC in correlation with specific molecular features of a large cohort (N = 1804) of patients with MN, including acute myeloid leukemia (AML) and myelodysplastic syndrome. When compared with healthy participants and patients with nonclonal diseases such as persistent polyclonal B-cell lymphocytosis, both MN and nonmalignant controls with clonal disease, such as paroxysmal nocturnal hemoglobinuria and aplastic anemia, exhibited decreased TC. Furthermore, we show that TC is lowered in adult MN abrogating correlation with age with considerable TC diversification among certain morphologic and molecular subtypes. For instance, AML harbored the lowest TC. Furthermore, MN originating from a more mature cell of origin (eg, acute promyelocytic leukemia) or characterized by hyperproliferative driver mutations (eg, RAS pathway genes) had lower TC, possibly indicating a loss of telomere maintenance capacity. In contrast, compared with other mutations, MN subtypes arising in a context of profound genetic alterations, such as TP53 mutations and complex karyotype, exhibited a relatively higher/preserved TC. This phenomenon did not involve alternative lengthening processes but was rather consistent with an increased TC due to preserved activity of the telomerase complex. Our results describe a common and genotype-specific telomeric makeup of a large cohort of patients with MN providing a molecular benchmark for future therapeutic targeting of the telomere machinery.

Purine nucleoside phosphorylase (PNP) deficiency causes inadequate purine metabolite detoxification, which leads to combined immunodeficiency and variable neurologic symptoms. Hematopoietic stem cell transplantation (HSCT) cures the immunodeficiency, but large studies on the long-term outcomes are lacking. In a retrospective study of the European Society for Blood and Marrow Transplantation, we investigated 46 patients with PNP deficiency from 21 centers. We analyzed the presenting clinical signs and outcomes after HSCT. Cognition (0-3), hearing (0-3), interaction (0-4), movement (0-4), and occupation (0-3) (CHIMO) were scored at the 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. The patients presented with infections (41%), neurological dysfunction (39%), both (15%), or autoimmune disease (5%). At the time of HSCT (median age, 26 [2-192] months), neurological abnormalities were observed in 88% of patients. 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) probabilities of 86% (confidence interval [CI], 77%-97%)/75% (CI, 64%-89%), respectively. High-level (>50%-100%)/low-level donor chimerism (11%-50%) was observed in 85%/15% of patients, respectively, leading to resolution of T lymphopenia. The median overall CHIMO score was 14 (6-17), while the median scores for each component were 3 (0-3), 3 (1-3), 4 (1-4), 3 (1-4), and 2 (0-3), respectively. Patients who underwent HSCT before 24 months after the initial presentation demonstrated superior OS (P = .049). Neurological symptoms that occurred before 11 months of age were associated with reduced OS (P = .027). While the overall results were satisfactory, earlier diagnosis could further improve outcomes.

The transcription factor lymphoid enhancer-binding factor 1 (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 messenger RNA levels remain stable, patients with clinically aggressive disease 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 signaling. Importantly, we uncovered a dual, context-dependent role for LEF1 that is determined by its protein levels. Low LEF1 protein, characteristic of indolent disease, supports B-cell activation, whereas 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. Although 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 posttranscriptionally 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 cross-linking 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 cross-links is restricted to the fibrin γ-chain and α-chain and follows thrombin-driven fibrin polymerization. Fibrinogen is also cross-linked by tissue transglutaminase (TG2) in a reaction favoring intramolecular and intermolecular α-γ cross-linking. Emerging evidence indicates that fibrinogen is a relevant substrate of TG2 in conditions of acute tissue damage. Remarkably, beyond detection of prototypical FXIII-directed cross-links (ie, α-α, γ-γ), we identified entirely novel covalent cross-links involving the fibrinogen β-chain (ie, β-α, 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 cross-linking proteomic analysis of in vitro clots recapitulated the precise TG2-directed β-chain cross-links observed in clots made using plasma from patients following traumatic injury. The results indicate in vitro and ex vivo cross-linking 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 patients with JMML 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-to-2021 period. Most patients carried mutations in CBL (43%), NRAS (34%), or homozygous germ line SH2B3 (14%). Over a median follow-up of 6.5 years, 30 of 35 (86%) achieved long-term survival with partial or complete resolution of myeloproliferative symptoms, although clonal hematopoiesis persisted in nearly all survivors (18/20). Disease progression occurred in 5 patients (CBL, n = 3; NRAS, n = 1; PTPN11, n = 1), mostly within 2 years after diagnosis. Overall, in the W&W cohort, the 5-year overall and event-free survivals were 93.1% and 84.5%, respectively. In NRAS-mutated cases, age of <30 months, normal to slightly elevated fetal hemoglobin, platelet counts of >45 × 109/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 germ line SH2B3. These findings support W&W as a viable alternative in up to 30% of patients with JMML, potentially sparing them from HSCT-associated risks. Given the persistence of clonal hematopoiesis and the risk of extrahematological complications, long-term monitoring remains essential.

Glutamine dependence of cancer cells reduces local glutamine availability, which hinders antitumor T-cell functionality and facilitates immune evasion. We thus speculated that glutamine deprivation might be limiting efficacy of chimeric antigen receptor (CAR) T-cell therapies in patients with cancer. We have seen that antigen-specific T cells are unable to proliferate or produce interferon gamma (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 B-cell maturation antigen (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 the metabolic fitness of CAR T cells by upregulating the mechanistic target of rapamycin complex 1 gene signature, modifying the solute carrier transporter repertoire, and improving both basal oxygen consumption rate and glycolytic function, thereby enhancing CAR T-cell persistence in vivo. Accordingly, expression of Asct2 increased the efficacy of Bcma-CAR T cells in syngeneic and genetically engineered mouse models of MM, which prolonged mouse survival. In patients, higher-level 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 antitumor 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.