Hematopoietic stem cells (HSCs) responsible for blood cell production and their bone marrow regulatory niches undergo age-related changes, impacting 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 LDLR and CXCR3 as the HSC receptors transmitting the PF4 signal, with double knockout mice showing 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.

Epcoritamab is a subcutaneous CD3xCD20 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; NCT04663347). 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/cycle). Primary endpoint was overall response rate (ORR) per investigator assessment (Lugano criteria). As of September 21, 2024, 108 patients received ≥1 epcoritamab dose in expansion (median follow-up, 28.2 months). Median age was 65 years; 57% had 1 prior line of therapy. ORR and complete response (CR) rate were 96% and 88%; 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 (G) ≥3 TEAEs occurred in 87% of patients; 5 had G5 TEAEs (all COVID-19). CRS events were mostly low grade (38% G1, 11% G2, 2% G3), 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.

Because frail and patients ≥80 years with CLL are still underrepresented in clinical trials, the CLL-Frail trial aimed to evaluate the efficacy and safety of acalabrutinib in these patients. The primary endpoint was the overall response rate (ORR) after 6 cycles of treatment to test the null hypothesis of ORR ≤ 65%.53 patients were included into the trial and 34 patients are still on therapy. Adverse events (AEs) were the most frequent reason for early discontinuation (ten patients), whereas five patients stopped treatment because of death. Median age was 81 years and 47.2% of patients were frail. The ORR for the 46 patients receiving ≥3 cycles of treatment was 93.5% (95% confidence interval 82.1 - 98.6) meeting the primary endpoint of this trial (p < 0.001). The estimated 12-month progression-free and overall survival rates were 93.3% and 95.7%, respectively, after a median follow-up of 19 months. 53.5% of patients reported an improvement in their self-perceived frailty. Although all patients experienced AEs and severe (CTC° ≥ 3) events were reported in 63.5% of patients, there were no events of severe bleeding and atrial fibrillation was rare (two cases of CTC° 2 and 3). Five patients died, of which four deaths happened during or <28 days after treatment. Infections/COVID-19 were the cause of death in three cases. This is the first prospective trial in older and/or frail CLL patients demonstrating a high efficacy and safe treatment with acalabrutinib monotherapy. ClinicalTrials.gov ID: NCT04883749 EUDRA-CT: 2020-002142-17.

Venetoclax-obinutuzumab (Ven-Obi) is a standard first-line therapy for chronic lymphocytic leukemia (CLL). The impact of age, fitness, and dose reductions remains unclear. We analyzed patients treated with Ven-Obi in the CLL13 and CLL14 trials, excluding patients with TP53 aberrations. Fitness was assessed using the cumulative illness rating scale (CIRS, >6) and creatinine clearance (≤70 ml/min). Among 410 patients (median age 67), 55.7% were unfit (median age 72) and 44.3% fit (58). Overall response rate (ORR) was 89.5% in unfit and 96.1% in fit patients. Undetectable minimal residual disease (uMRD) <10-4 rates were 80.3% in unfit and 85.1% in fit patients. Progression-free survival (PFS) at 3 years was 86.4% vs 87.5% (HR 1.12, 95%-CI 0.70-1.81, p=0.63). Overall survival at 3 years was 91.8% vs 96.9% (HR 2.02, 95%-CI 0.90-4.55, p=0.088). Adverse events included neutropenia (62.7% unfit, 56.9% fit), infusion-related-reactions (44.3% unfit, 56.9% fit), fatigue (15.8% unfit, 35.9% fit) and infections (57.5% unfit, 69.6% fit). Venetoclax dose reductions <80% occurred in 39.6% of unfit and 17.6% of fit patients, with lower ORR (83.3% vs 98.2%) and uMRD rates (74.2% vs 87.9%) in those with reduced dose intensities, but similar PFS. Dose reductions <70% were associated with shorter PFS. Overall, this study shows comparable efficacy and toxicity of Ven-Obi in fit and unfit patients with CLL.

Obesity is a major health issue and a risk factor for venous thromboembolic disease (VTE). Plasminogen activator inhibitor 1 (PAI-1), encoded by the gene SERPINE1, is a negative regulator of fibrinolysis and has been associated with obesity. The liver, which senses obesity-induced metabolic stress, is a key determinant of circulating PAI-1 levels. However, the mechanisms underlying the increased PAI-1 expression in obesity are unclear. This study investigated the upstream regulation of PAI-1 and its role in fibrinolysis and deep vein thrombosis (DVT). Compared with lean mice, diet-induced obesity (DIO) mice presented significantly shorter fibrinolysis times and larger venous thrombi, due largely to increased hepatocyte expression of PAI-1. A public single-cell RNA-seq dataset from the livers of obese individuals suggested that increased PAI-1 expression may be related to reduced hepatocyte FXR signaling. FXR activation also suppressed Serpine1 mRNA and PAI-1 protein expression levels in both mice and primary mouse hepatocytes (MPHs), but a decrease in PAI-1 in MPHs of Fxr-null mice after FXR activation was not observed. Both Fxr-null mice and Fxrfl/fl mice with AAV8-TBG-Cre exhibited significantly elevated plasma PAI-1, resulting in further impaired fibrinolysis and increased DVT burden. Dual-luciferase reporter assays and chromatin immunoprecipitation (Ch-IP) suggested that FXR activation directly represses Serpine1 transcription. Importantly, tropifexor treatment of obese mice lowered plasma PAI-1 levels and further alleviated fibrinolysis and the DVT load. These findings suggest that targeting FXR in hepatocytes may improve fibrinolysis and reduce DVT risk.

Acute myeloid leukemia (AML) is characterized by a low five-year survival rate. Despite having many clinical metrics to assess patient prognosis, there remain opportunities to improve risk stratification. We hypothesized that an underexplored resource to examine AML patient prognosis is the plasma metabolome. Circulating metabolites are influenced by patients' clinical status and can serve as accessible cancer biomarkers. To establish a resource of circulating metabolites in genetically diverse AML patients, we performed an unbiased metabolomic and lipidomic analysis of 231 diagnostic AML plasma samples prior to treatment with intensive chemotherapy. Intriguingly, circulating metabolites were highly associated with the mutation status within the AML cells. Further, lipids were associated with refractory status. We established a machine learning algorithm trained on chemo-refractory associated lipids to predict patient survival. Cox regression and Kaplan-Meier analysis demonstrated that the high-risk lipid signature predicted overall survival in this patient cohort. Impressively, the top lipid in the high-risk lipid signature, sphingomyelin (d44:1), was sufficient to predict overall survival in the original and an independent validation dataset. Overall, this research underscores the potential of circulating metabolites to capture AML heterogeneity and lipids to be used as potential AML biomarkers.

Until recently, the JAK1/2 inhibitor ruxolitinib (Jakafi) was the only therapy for steroid-refractory acute graft-versus-host disease approved by the US Food and Drug Administration (FDA) for use in patients aged >12 years. The FDA has now approved a potent mesenchymal stromal cell product, remestemcel-L-rknd (Ryoncil), for children aged ≤18 years, showing 70% response rates and ∼70% 6-month survival. In this spotlight, we highlight this important advance in the field.

Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy that increasingly affects the elderly population, with its post-transcriptional landscape remaining largely elusive. Establishing a stable proteomics-based classification system and systematically screening age-related proteins and regulatory networks are crucial for understanding the pathogenesis and outcomes of AML. In this study, we leveraged a multi-omics cohort of 374 newly diagnosed AML patients, integrating proteome, phosphoproteome, genome, transcriptome, and drug screening data. Through similarity network fusion clustering, we established eight proteomic subtypes with distinct clinical and molecular properties, including S1 (CEBPA mutations), S3 (myelodysplasia-related AML), S4 (PML::RARA), S5 (NPM1 mutations), S6 (PML::RARA and RUNX1::RUNX1T1), S8 (CBFB::MYH11), S2 and S7 (mixed), aligning well with and adding actionable value to the latest World Health Organization nomenclature of AML. Hematopoietic lineage profiling of proteins indicated that megakaryocyte/platelet- and immune-related networks characterized distinct aging patterns in AML, which were consistent with our recent findings at the RNA level. Phosphosites also demonstrated distinct age-related features. The high protein abundance of megakaryocytic signatures was observed in S2, S3, and S7 subtypes, which were associated with advanced age and dismal prognosis of patients. A hematopoietic aging score with an independent prognostic value was established based on proteomic data, where higher scores correlated with myelodysplasia-related AML, NPM1 mutations, and clonal hematopoiesis-related gene mutations. Collectively, this study provides an overview of the molecular circuits and regulatory networks of AML during the aging process, advancing current classification systems and offering a comprehensive perspective on the disease.

Blood clotting is triggered in hemostasis and thrombosis when the membrane-bound tissue factor (TF)/factor VIIa (FVIIa) complex activates factor X (FX). There are no structures of TF/FVIIa on membranes, with or without FX. Using cryo-EM to address this gap, we assembled TF/FVIIa complexes on nanoscale membrane bilayers (nanodiscs), bound to XK1 and an antibody fragment. XK1 is a FX mimetic whose protease domain is replaced by the first Kunitz-type (K1) domain of tissue factor pathway inhibitor, while 10H10 is a non-inhibitory, anti-TF antibody. We determined a cryo-EM structure of a TF/FVIIa/XK1/10H10/nanodisc complex with a resolution of 3.7 Å, allowing us to model all the protein backbones. TF/FVIIa extends perpendicularly from the membrane, interacting with a "handle shaped" XK1 at two locations: the K1 domain docks into FVIIa's active site, while the γ-carboxyglutamate-rich (GLA) domain binds to TF's substrate-binding exosite. The FX and FVIIa GLA domains also contact each other and the membrane surface. Except for a minor contact between the first epidermal growth factor (EGF)-like domain of XK1 and TF, the rest of the FX light chain does not interact with TF/FVIIa. The structure reveals a previously unrecognized, membrane-dependent allosteric activation mechanism between FVIIa and TF where a serine-rich loop in TF that partially obscures the TF exosite must undergo a shift to allow access of the FX GLA domain to its final binding location on the membrane-bound TF/FVIIa complex. This mechanism also provides a novel explanation for the otherwise puzzling phenomenon of TF encryption/decryption on cell surfaces.

Aberrant activation of BCL11B (BCL11B-a) defines a subtype of lineage-ambiguous leukemias with T-lymphoid and myeloid features, co-occurring activating FLT3 mutations, and a stem/progenitor immunophenotype and gene expression profile. Similar to other lineage-ambiguous leukemias, optimal treatment is unclear, and there are limited targeted therapeutic options. Here, we investigated the efficacy of B-cell lymphoma 2 (BCL-2) and FMS-like tyrosine kinase 3 (FLT3) inhibition with venetoclax and gilteritinib, respectively, in preclinical models of BCL11B-a leukemia. Despite variation in response to single-agent therapies, the combination of venetoclax plus gilteritinib (VenGilt) was highly effective in all models evaluated. BH3 profiling suggested that resistance to venetoclax monotherapy was due to the tumor-intrinsic dependence on additional BCL-2 family proteins before drug treatment. Longitudinal single-cell RNA sequencing analysis identified mitochondrial pathways and a pro-lymphoid gene expression signature as potential drivers of rare cell survival on VenGilt therapy. These data support clinical evaluation of venetoclax in combination with gilteritinib in BCL11B-a lineage-ambiguous leukemias.

Understanding the roles of myeloid cells in the tumor microenvironment (TME) has emerged as a promising strategy to identify novel targets to counteract the immunosuppressive barriers protecting multiple myeloma (MM). Neutrophils are a new cancer research focus due to their potential to reduce the efficacy of immune-based therapies. This study aimed to deepen understanding of neutrophil function in MM by analyzing freshly isolated myeloid cells from paired focal lesions (FL) and bone marrow (BM) using single-cell RNA sequencing (scRNA-seq), immunofluorescence imaging, and functional assays. We describe three distinct CXCR2+ mature neutrophil subsets: TREM1+CD10+, RETN+LCN2+, and TNFAIP3+CXCL8+, each exhibiting unique phenotypes within the TME. Notably, the TREM1+CD10+ subset was highly prevalent, particularly in FL, demonstrating potent immunosuppressive effects on T cells. This subset's gene signature was correlated with shorter overall survival (OS) in a large MM patient dataset, underscoring its clinical significance. Targeted inhibition of neutrophil activity through CXCR2 blockade, alone or combined with standard anti-MM therapies, significantly reduced tumor burden, improving OS in preclinical MM models. These insights into neutrophil-mediated immunosuppression in MM provide valuable knowledge regarding mechanisms driving immune evasion and reveal new therapeutic approaches to enhance the efficacy of MM treatment.

The current approach to minimize transplant-associated complications, including graft-versus-host disease (GVHD) includes long-term pharmacological immune suppression frequently accompanied by unwanted side effects. Advances in targeted immunotherapies regulating alloantigen responses in the recipient continue to reduce the need for pan-immunosuppression. Here, in vivo targeting of the TNF superfamily receptor TNFRSF25 and the high affinity IL-2 receptor with a TL1A-Ig fusion protein and low dose IL-2, respectively was used to pre-treat recipient mice prior to allogeneic-HSCT (aHSCT). Pre-treatment induced Treg expansion persisting 1-2 weeks post-HSCT leading to diminished GVHD and improved transplant outcomes. Expansion was accompanied by an increase in the frequency of stable and active Tregs creating a suppressive tissue environment in the colon, liver and eye. Importantly, pre-treatment supported epithelial cell function/integrity, a diverse microbiome including reduction of pathologic bacteria outgrowth and promotion of butyrate producing bacteria, while maintaining physiologic levels of obligate/facultative anaerobes. Notably, using a sphingosine 1-phosphate receptor agonist to sequester T cells in lymphoid tissues, it was found that the increased tissue Treg frequency included resident CD69+CD103+FoxP3+ hepatic Tregs. In contrast to infusion of donor Treg cells, the strategy developed here resulted in the presence of immunosuppressive target tissue environments in the recipient prior to the receipt of donor allo-reactive T cells and successful perseveration of GVL responses. We posit strategies that circumvent the need of producing large numbers of Tregs ex-vivo through manipulating this recipient compartment in vivo, can provide translational approaches to improve aHSCT outcomes.

T-cell engagers (TCEs) are transformative therapeutics in hematologic malignancies, including non-Hodgkin lymphoma. Initially approved for relapsed/refractory disease settings, TCEs are now explored in first-line and second-line settings, often combined with standard-of-care (SOC) treatments, including chemotherapy and antibody-drug conjugates. This study investigates glofitamab (CD20×CD3 TCE) combinations in preclinical humanized lymphoma models, addressing heterogeneity of tumor antigen expression, immune evasion, and T-cell exhaustion. Combining glofitamab with R-CHP-Pola (rituximab, cyclophosphamide, doxorubicin, prednisone, and polatuzumab vedotin) chemotherapy or Pola demonstrated strong synergistic antitumor efficacy with rapid tumor regression and reduced tumor cell proliferation. Glofitamab combination with gemcitabine/oxaliplatin also demonstrated strong efficacy, enhancing intratumor T-cell number, activation, and reduced exhaustion. These combinations were particularly advantageous in models with low and heterogeneous CD20 expression, facilitating rapid tumor debulking and elimination of CD20-low/CD20- cells. Translational studies with patient-derived peripheral blood mononuclear cells receiving glofitamab combination with chemotherapies demonstrated sustained T-cell functionality throughout extended treatment cycles. Novel chemotherapy-free combinations, including CD19-targeted 4-1BBL and CD19-CD28, amplified glofitamab activity, especially in CD20 high- and homogenous-expressing tumor models, with dual costimulatory approaches revealing synergy. In addition, the combination with checkpoint inhibitors (programmed cell death protein 1/Lag3-bispecific antibody) and regulatory T-cell depletion (α-CD25) emerged as promising approaches for enhanced efficacy and to sustain T-cell functionality. These findings highlight the versatility of glofitamab when integrated with SOC and innovative combinations, addressing resistance and improving patient outcomes. The preclinical investigations provide a strong foundation for ongoing and future clinical trials, emphasizing the need to tailor TCE-based combination therapies to maximize efficacy while minimizing toxicity in lymphoma treatment. These trials were registered at www.clinicaltrials.gov as #NCT04408638 and NCT03467373.

Dysregulated RNA modifications contribute to cancer progression and therapy resistance, yet the underlying mechanism often remains unknown. Here, we perform CRISPR-based synthetic lethality screens to systematically explore the role of RNA modifications in mediating resistance to anti-leukaemic drugs. We identify the TRMT5-mediated formation of N1-methylguanosine (m1G) in the tRNA anticodon loop as essential for mediating drug tolerance to cytarabine and venetoclax in acute myeloid leukemia (AML). TRMT5 methylates nearly all mitochondrial and nuclear tRNAs with a guanosine at position 37, but its role in promoting drug tolerance specifically depends on its mitochondrial function. TRMT5 is essential for the dynamic upregulation of mitochondrial mRNA translation and oxidative phosphorylation (OXPHOS), which are critical for sustaining drug tolerance in leukemia cells. This mitochondrial dependency correlates with therapy outcomes in leukemia patients: lower expression of electron transport chain genes is linked to poorer outcomes in a cohort of nearly 100 AML patients undergoing first induction therapy. Finally, we demonstrate that targeted depletion of TRMT5 protein using a conditional degron, in conjunction with cytarabine and venetoclax treatment, synergistically induces cell death in drug-tolerant AML cells. Thus, our study reveals TRMT5 as promising drug target for therapy-resistant leukemia.

Chronic active Epstein-Barr virus (EBV) infection (CAEBV) is an orphan disease characterized by the proliferation and infiltration of EBV-infected T/natural killer (NK) cells into multiple organs. Although CAEBV is a heterogeneous disease with diverse clinical courses, its pathogenesis remains poorly understood. In this study, we explored the molecular mechanisms underlying CAEBV by performing a comprehensive multiomics analysis, including genome, transcriptome, epigenome, and single-cell transcriptome and surface proteome analyses, of 65 patients with CAEBV. Methylation analysis identified 2 distinct subtypes of NK cell-type CAEBV based on the CpG island methylator phenotype (CIMP). In CIMP-positive CAEBV, regions associated with enhancer of zeste homolog 2 binding sites and histone H3 lysine 27 trimethylation exhibited increased DNA hypermethylation, resulting in downregulation of tumor suppressor and antiherpesvirus genes. CIMP-positive CAEBV had a particularly poor prognosis and displayed a "neoplastic" phenotype with a DNA methylation pattern similar to that of extranodal NK/T-cell lymphoma, a higher tumor mutation burden, and frequent copy number alterations. In addition, both in vitro and in vivo functional assays demonstrated that 5-azacytidine, a hypomethylating agent, was a potentially effective agent for high-risk CIMP-positive CAEBV. Finally, we established a method to effectively detect EBV-infected cells in single-cell analysis, suggesting that EBV-infected NK cells have tissue-resident properties and that innate and adaptive immunity to EBV is compromised in patients with CAEBV. The present findings provide insight into the complex molecular features of CAEBV and suggest potential molecular therapies.

With up to 10 years of follow-up, we report results from the final analysis of RESONATE- 2, a phase 3 study of first-line ibrutinib vs chlorambucil for the treatment of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Patients aged ≥65 years with previously untreated CLL/SLL without del(17p) were randomly assigned to receive either single-agent ibrutinib (420 mg/d; n = 136) or chlorambucil (0.5-0.8 mg/kg; ≤12 cycles; n = 133). With a median follow-up of 9.6 years in the ibrutinib arm, the median progression-free survival (PFS) was 8.9 years (95% confidence interval [CI], 7.0 to not estimable [NE]) vs 1.3 years (95% CI, 0.9-1.6) for the chlorambucil arm. Among patients with unmutated immunoglobulin heavy chain variable (uIGHV), del (11q), mutated TP53, or complex karyotype, the median PFS was 8.4 years (95% CI, 6.8 to NE) with ibrutinib and 0.7 years (95% CI, 0.4-1.2) with chlorambucil. Median overall survival (OS) with ibrutinib was not reached. The most common adverse events (AEs) of any grade included diarrhea (52%), fatigue (41%), cough (39%), nausea (32%), arthralgia (31%), peripheral edema (31%), and hypertension (30%). During the entire study period, 34 of 136 patients (25%) had an ibrutinib dose reduction due to AEs; these AEs improved in 30 of 34 patients (88%). At study completion, 27% of patients remained on first-line ibrutinib treatment. This landmark RESONATE-2 study defines median PFS and demonstrates continued OS benefit of first-line ibrutinib treatment for patients with CLL/SLL, including those with high-risk genomic features. Sustained efficacy and tolerability of ibrutinib reemphasize the favorable benefit-risk profile. This trial was registered at www.ClinicalTrials.gov as NCT01722487/NCT01724346.

Attempting to induce a complete remission before allogeneic hematopoietic cell transplant (alloHCT) is current practice in patients with acute myeloid leukemia (AML). However, benefit of remission induction strategy (RIST) before alloHCT has never been proven in a prospective trial. Potent conditioning regimens exist that allow for successful alloHCT in patients with active AML. Therefore, the ASAP trial was conducted to test RIST by salvage chemotherapy before alloHCT against immediate transplant after intensified conditioning. In total, 281 patients with AML with poor response after first induction or untreated first relapse were randomized 1:1 to RIST with high-dose cytarabine plus mitoxantrone vs immediate alloHCT with sequential conditioning after nonintensive disease control (DisC) measures, preferentially watchful waiting only. Overall survival at 5 years from randomization analyzed according to intention-to-treat was 46.1% for DisC vs 47.5% for RIST (P = .82). In multivariable Cox regression analysis, genetic AML risk according to European LeukemiaNet criteria (P < .0001), age (P = .001), and comorbidities (P = .046) predicted survival, but not treatment arm (hazard ratio, 1.08 for DisC vs RIST; P = .67). In conclusion, long-term follow-up of the ASAP trial showed no survival advantage for standard salvage chemotherapy before alloHCT as opposed to immediate alloHCT. The trial results question the general concept of RIST with intensive standard salvage therapy before alloHCT for all patients, because immediate alloHCT may reduce time in hospital and health care expenses. Novel bridging therapies that are well tolerated, and posttransplant maintenance with targeted drugs are urgently warranted, especially for adverse-risk AML, to improve outcomes after alloHCT. This trial was registered at www.ClinicalTrials.gov as #NCT02461537.

Genetic alterations alone cannot account for the diverse phenotypes of cancer cells. Even cancers with the same driver mutation show significant transcriptional heterogeneity and varied responses to therapy. However, the mechanisms underpinning this heterogeneity remain underexplored. Here, we find that novel enhancer usage is a common feature in acute lymphoblastic leukemia (ALL). In particular, KMT2A::AFF1 ALL, an aggressive leukemia with a poor prognosis and a low mutational burden, exhibits substantial transcriptional heterogeneity between individuals. Using single-cell multiome analysis and extensive chromatin profiling, we reveal that much transcriptional heterogeneity in KMT2A::AFF1 ALL is driven by novel enhancer usage. By generating high-resolution Micro Capture-C data in primary patient samples, we identify patient-specific enhancer activity at key oncogenes such as MEIS1 and RUNX2, driving high levels of expression of both oncogenes in a patient-specific manner. Overall, our data show that enhancer heterogeneity is highly prevalent in KMT2A::AFF1 ALL and may be a mechanism that drives transcriptional heterogeneity in cancer more generally.

Leukemias with NUP98 rearrangements exhibit heterogeneous phenotypes such as acute myeloid leukemia, T-cell acute lymphoblastic leukemia (T-ALL), or myelodysplastic syndrome/neoplasms associated with fusion partners, whereas the mechanism responsible for this heterogeneity is poorly understood. Through genome-wide mutational and transcriptional analyses of 177 NUP98-rearranged leukemias, we show that cooperating alterations are associated with differentiation status even among leukemias sharing the same NUP98 fusions, such as NUP98::KDM5A acute megakaryocytic leukemia with RB1 loss or T-ALL with NOTCH1 mutations. CUT&RUN profiling of in vitro cord blood CD34+ cell (cbCD34) models of major NUP98 fusions revealed that NUP98-fusion oncoproteins (FOs) directly regulate differentiation-related genes contributing to the disease phenotypes, represented by NUP98::KDM5A binding to MEIS2 or GFI1B for megakaryocyte (MK) differentiation. In patient samples, NUP98-FO binding patterns are heterogeneous, potentially shaped by somatic mutations and differentiation status. Using cbCD34 models and CRISPR/Cas9 gene editing, we show that RB1 loss cooperates with NUP98::KDM5A by blocking terminal differentiation toward platelets and expanding MK-like cells, whereas WT1 frameshift mutations skew differentiation toward dormant lymphoid-myeloid primed progenitor cells and cycling granulocyte-monocyte progenitor cells, providing evidence for NUP98-rearranged leukemia phenotypes affected by cooperating alterations. NUP98::KDM5A cbCD34 models with RB1 or WT1 alterations have different sensitivities to menin inhibition, suggesting that cellular differentiation provides stage-specific menin dependencies and resistance mechanisms that can be leveraged for future treatment strategies for NUP98-rearranged leukemia.