The H3K36me2 methyltransferase NSD2 is deleted in Wolf-Hirschhorn syndrome and aberrantly expressed in 10-15% of multiple myeloma (MM) due to a t(4;14) translocation. Although NSD2 is thought to be a primary driver in MM, the exact molecular mechanisms by which it regulates transcription remain unclear. We applied the dTAG system to acutely degrade NSD2 and used this, in combination with time-resolved SLAM-seq, to identify 307 transcriptional targets of NSD2. Reconstitution with either wild-type NSD2 or a catalytically inactive mutant (NSD2Y1179A) showed that NSD2's transcriptional effects are almost exclusively dependent on its SET domain activity. Mechanistically, H3K36me2 deposition by NSD2 antagonizes H3K27me3 levels, and treatment with two distinct PRC2 inhibitors demonstrated that approximately half of NSD2 target genes are regulated in an H3K27me3-dependent manner. CUT&Tag analysis showed that upon NSD2 depletion there was an increase in H3K27me3 that occurred at genome-wide intergenic regions, rather than at the promoters or gene bodies of NSD2 target genes. These data suggest that NSD2, via H3K36me2, antagonizes H3K27me3 deposition likely at distal regulatory elements including enhancers, creating a chromatin landscape favorable for target gene transcription. Importantly, NSD2 target genes were enriched for key oncogenic pathways, and 24 transcription factors implicated in neurodevelopment and acute leukemia, consistent with its role in Wolf-Hirschhorn syndrome and MM. Eight of these transcription factors are known oncogenic drivers in acute leukemia or MM, highlighting a novel molecular mechanism for NSD2's role in t(4;14) MM.

Relapsed or refractory (R/R) disease occurs in up to 40% of diffuse large B-cell lymphoma (DLBCL) patients following first-line immunochemotherapy. However the molecular mechanisms underlying drug persistence remain incompletely defined. Here, we performed single-cell RNA and B-cell receptor sequencing on paired diagnostic and R/R samples from eight patients who were either treatment-refractory or relapsed after remission, and validated our findings in three independent patient cohorts. We found that drug-persistent cells exhibited a transcriptional profile indicative of a less-differentiated state and adopted a memory B cell-like program with enhanced stem-like properties, which correlated with unfavorable clinical outcomes across multiple DLBCL cohorts. Functionally, drug-persistent cells showed significantly increased in vitro clonogenicity and in vivo tumor-initiating capacity. Mechanistically, the WNT signaling regulator CSNK1E was upregulated in these stem-like drug-persistent cells, in part through the activation of the APRIL-TNFRSF13B axis. Notably, CSNK1E inhibition impaired the growth and tumor-initiating capacity of drug-persistent cells and potentiated the efficacy of R-CHOP-based treatment both in vitro and in vivo. Together, our study reveals the stem-like transcriptional and functional properties of drug-persistent cells, and identifies CSNK1E as a critical mediator and therapeutic vulnerability to improve the efficacy of standard immunochemotherapy in DLBCL.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for hematologic malignancies. The primary non-relapse complication after allo-HSCT is graft-versus-host disease (GVHD). The use of T regulatory (Treg) cells to prevent GVHD has emerged as a promising allogeneic T cell immunotherapy in the form of Orca-T. However, the precise differences in immune activation which may influence infection, GVHD, and relapse after Orca-T compared with unmanipulated peripheral blood stem cell (PBSC) grafts remain unexplored. Using peripheral blood specimens longitudinally collected between 3 weeks and one year after leukemia treatment, we report single-cell mRNA sequencing (scRNA-seq) and flow cytometric analysis of 51 HLA-matched patients receiving either Orca-T or unmanipulated PBSC grafts. Orca-T recipients exhibited increased frequencies of effector memory CD4+ T cells 3 weeks after transplant and this difference persisted through six months after treatment. scRNA-seq analysis 3 weeks post-transplant identified increased expression of FOXP3 and Helios amongst CD4+CD25- T conventional (Tcon) cells in Orca-T treated patients. Using flow cytometry, we then confirmed the increased frequency of this novel population of CD4+CD25-FOXP3+Helios+ Tcons 3 weeks post-treatment in patients receiving Orca-T. Further, we discovered that this T cell subset possessed a regulatory-like phenotype and correlated significantly with the frequencies of activated CD4+ and CD8+ T cell populations 3 months post-treatment, regardless of which therapy patients received. Overall, this study identifies a novel T cell subset which is enriched very early after cellular therapy for leukemia and may be predictive of long-term immune activation after Orca-T and PBSC-derived T cell infusion.

Quadruplet therapy with isatuximab, bortezomib, lenalidomide, and dexamethasone (Isa-VRd) followed by Isa-Rd in the randomized Phase 3 IMROZ study provided significant PFS benefit to transplant-ineligible, newly diagnosed multiple myeloma (NDMM) patients. In the primary analysis, more Isa-VRd/Isa-Rd-treated patients achieved MRD-negativity and MRD-negative complete response (CR) at any timepoint compared with VRd triplet therapy followed by Rd. Here, we report results from landmark analyses of MRD-negativity over time and its impact on clinical outcomes in IMROZ. Treatment with Isa-VRd/Isa-Rd led to deeper responses, with higher rates of MRD-negativity and MRD-negative CR at both the end of the initiation phase and during maintenance vs. VRd/Rd, up to 60 months of follow-up. Benefit with Isa-VRd/Rd was observed across key patient subgroups, including older (>70 years) and frail patients. The deeper responses achieved throughout treatment were associated with improved outcomes, with a significant prolongation in time-to-progression (TTP) in favor of Isa-VRd/Rd vs. VRd/Rd in patients who converted from MRD-positive to MRD-negative. TTP in patients who converted from MRD-negative at the end of initiation to MRD-positive also favored Isa-VRd. Evaluation of MRD status for individual patients at more than one timepoint may thus be useful to support decisions on treatment selection and treatment continuation/discontinuation. Our findings on the extent of MRD-negativity and MRD-negative CR benefit achieved in the initiation and maintenance phases by patients receiving Isa-VRd/Isa-Rd vs. VRd/Rd extend the IMROZ primary analyses and further support the Isa-VRd quadruplet regimen as a standard-of-care for front-line treatment of transplant-ineligible NDMM patients. Clinical Trial Information: ClinicalTrials.gov, NCT03319667.

Altered lipid metabolism enables growth of acute myeloid leukemia (AML) cells. While mitochondrial lipid oxidation is well characterized, the contribution of peroxisomal fatty acid oxidation (pFAO) is unclear. In this study, we demonstrate that AML cells upregulate the peroxisomal very-long-chain fatty acid (VLCFA) transporter ABCD1 and increase endogenous levels of pFAO relative to healthy hematopoietic cells. Genetic silencing or pharmacological inhibition of ABCD1, with eicosenol, impairs pFAO causing accumulation of VLCFAs and selective AML cell death in vitro and in vivo. Loss of ABCD1 disrupts peroxisomal fatty acid import and lipid homeostasis in AML, while normal progenitors remain viable by upregulating glycolysis. In murine models, ABCD1 inhibition with eicosenol reduces leukemia burden and prolongs survival without toxicity. These findings identify ABCD1 as a regulator of pFAO and a novel anti-AML therapeutic target.

Bispecific antibodies (bsAbs) such as glofitamab represent a promising therapeutic approach for relapsed/refractory B-cell non-Hodgkin's lymphoma (R/R B-NHL), but resistance mechanisms remain poorly understood. This study aimed to identify predictive markers of bsAbs resistance based on the response of 3D patient-derived lymphoma spheroids (PDLS) established from 39 R/R B-NHL samples. PDLS were treated with glofitamab for 3 days and B-cell depletion was quantified to assess the ex-vivo treatment response. Comprehensive immune profiling was performed on patient samples using multiparametric flow cytometry, single-cell RNA sequencing, CODEX spatial proteomics and functional assays. High responders to glofitamab possessed CD8+ T-cells with consistently higher cytotoxic and activation signatures across effector differentiation states, while low responders showed enrichment of exhausted CD8+ T-cell with enhanced expression of exhaustion markers (TIGIT, LAG3, PD1). Furthermore, low responders exhibited elevated functional CD4+ T-follicular helper (Tfh) cells in close proximity to malignant B-cell thus promoting their survival through IL21 and CXCL13 signaling pathways. Analysis of pretreatment RNA-seq data from 48 R/R B-NHL patients confirmed that high Tfh abundance is associated with poor glofitamab response. In PDLS, anti-TIGIT co-treatment enhanced glofitamab efficacy in low responders, and Tfh depletion experiments confirmed that reducing Tfh activity increased B-cell depletion. Together, these findings identify CD8+ T-cell exhaustion and functionally activated Tfh cells as key factors associated with glofitamab resistance in R/R B-NHL. This work supports their potential use as predictive biomarkers for selecting patients with higher probability of response and provides a foundation for future combination therapeutic strategies.

Selection of a hematopoietic progenitor cell donor for allogeneic hematopoietic cell transplantation (allo HCT) is essential for treatment planning; however, the parameters that define an "optimal" donor in the modern era are not well defined. Historically, donor-recipient human leukocyte antigen (HLA) mismatching correlated strongly with risk for graft versus host disease (GVHD) and reduced survival. For this reason, donor selection was typically hierarchical: HLA matched related and unrelated donors were evaluated first, followed by HLA mismatched donors (or deferral of HCT altogether) in patients lacking an HLA matched donor. The advent of post-transplant cyclophosphamide (PTCy)-based GVHD prevention has changed this paradigm. Survival outcomes following HLA-mismatched donor HCT with PTCy, including from related haploidentical or HLA-mismatched unrelated donors, are not different than HLA matched donor recipients in recent clinical trials and retrospective studies. These encouraging results present a new challenge: In the PTCy era, how should donors be prioritized among the many potential sources available? Herein we review HLA and non-HLA parameters that inform donor selection and discuss approaches to increase donor availability. Case vignettes focusing on concepts that may be adapted to heterogenous clinical scenarios are presented.

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by age-related somatic mutations in hematopoietic stem and progenitor cells (HSC/Ps) and is correlated with an increased risk of myeloid malignancies, elevated inflammatory pathways in circulating myeloid cells, higher all-cause mortality, chronic kidney disease, and cardiovascular disease. The pathophysiology of inflammatory bowel disease (IBD) is intrinsically linked to heightened inflammation. Nevertheless, the presence of CHIP in IBD and its role in the pathophysiology of IBD remains poorly elucidated. In the UK Biobank, CHIP was associated with an increased incidence of IBD. Females with CHIP had a 1.33-fold higher risk, which was further validated in All of Us data base (ßOR = 1.29). For Crohn's disease, DNMT3A mutations conferred a 1.81-fold increased incidence in females compared to non-DNMT3A-carriers, which rose to 2.09 for large clones (variant allele fraction ≥10%). In contrast, for ulcerative colitis, TET2 large clones were significantly associated, and only among individuals under 45. These associations were further identified using two-sample Mendelian randomization. In a mouse model of CHIP-IBD, HSC/Ps with Dnmt3a mutation demonstrated significantly worse pathophysiology compared to controls, due in part to heightened expression of Apurinic/apyrimidinic endonuclease 1 (APE1) in the bone marrow and colon. Treatment with the APE1/Ref-1 inhibitor APX3330 ameliorated CHIP-IBD driven by the Dnmt3a mutation.

Truncating and missense mutations of the CREBBP gene are highly prevalent in follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL), the most common lymphoid malignancies. These mutations are acquired early during tumor evolution by a common precursor cell (CPC) and lead to either complete protein loss or single amino-acid substitutions in the acetyltransferase (KAT) domain. As a result, CREBBP is impaired in its ability to acetylate enhancer histones and non-histone proteins implicated in the germinal center (GC) reaction, the structure from which these tumors originate. However, whether truncating and KAT domain missense mutations are functionally equivalent in instructing the CPC remains unexplored. Using a conditional GC-specific knock-in mouse model for the highly frequent CREBBP-R1446H amino-acid change (CrebbpRH), here we show that, compared to complete Crebbp loss, missense mutants impose distinct quantitative and qualitative effects on the GC response. CrebbpRH controls unique transcriptional programs leading to the pre-neoplastic expansion of GCs with abnormal architecture, increased percentage of Tfh cells, and a skewed immune response toward memory B-cell differentiation. Expression of CrebbpRH, but not Crebbp loss, was by itself sufficient to initiate malignant transformation, indicating a stronger tumor-promoting activity. Of note, lymphoma cells with CREBBPRH and CREBBP loss showed distinct sensitivity to CREBBP/p300 small molecule inhibitors. Together with the differential distribution of missense and truncating mutations in FL and DLBCL, these findings have implications for the pathogenesis and therapeutic targeting of these cancers.

Targeting mitochondrial oxidative phosphorylation (OXPHOS) enhances the effects of standard chemotherapy and overcomes treatment resistance in pre-clinical models of acute myeloid leukaemia (AML). So far, the few clinically available OXPHOS inhibitors have shown adverse effects or limited potency in clinical trials, therefore, identification of safe and effective drugs that can target mitochondrial metabolism in AML is critical. Here, we performed a high-throughput drug-repurposing screen, designed to identify clinically applicable OXPHOS-specific inhibitors through nutrient sensing. We uncover itraconazole, an FDA-approved antifungal compound, as a potent OXPHOS inhibitor in AML cells. Mechanistically, through stable isotope-assisted metabolomics and functional studies, we reveal that CYP51A1, which is part of the cytochrome P450 family and the prime target of azole antifungals, is involved in mitochondrial respiration and ETC complex I activity in AML cells. Critically, we demonstrate that itraconazole and related azole antifungals interfere with tricarboxylic acid cycle activity and inhibit OXPHOS through the inhibition of electron transport chain complex I activity. Over-expression of yeast NADH dehydrogenase-1 (NDI1) restored mitochondrial NADH oxidation and complex I activity upon itraconazole treatment. Using patient-derived cells and pre-clinical xenograft models, we demonstrate that itraconazole targets therapy-resistant leukaemic stem cells (LSCs) when used in combination with cytarabine, highlighting the repurposing potential for itraconazole as a clinically safe and effective therapeutic option for AML LSC eradication.

Menin inhibitors (MI) disrupt the binding of Menin to MLL1 leading to repression of MLL1 or MLL1-fusion protein (FP) target genes, including reduced levels of HOXA9 and MEIS1 in AML with mutant (mt) NPM1 or MLL1-rearrangement (r). While MIs are relatively well-tolerated and induce clinical remissions, these are often short-lived due to development of resistance followed by AML relapse. Through repeated shocks with the MI SNDX-50469, a precursor tool compound to revumenib, followed by recovery, we developed MI-resistant (MITR) AML MV4-11 and OCI-AML3 cells. Present studies show that, compared to MI-sensitive parental cells, MITR cells exhibit an altered epigenome, transcriptome and proteome, without Menin mutations. Through a CRISPR screen, novel druggable MI co-enrichments were identified and targeted, including BRD4, SMARCA4, and CREBBP. Co-treatment with the MI and the SMARCA4/SMARCA2 (BRG1/BRM) inhibitor FHD-286 or the BET proteins inhibitor OTX015 (birabresib), synergistically induced in vitro lethality in MITR and MI-resistant AML cells expressing the mutant Menin (M327I), as well as in patient-derived (PD) AML cells with MLL1-r or mtNPM1 that exhibited ex vivo resistance to MI. Compared to each drug alone, co-treatment with SNDX-5613 (revumenib) and FHD-286 or OTX015 and FHD-286 significantly reduced the in vivo AML burden and improved survival of the immune depleted mice, without inducing significant toxicity, in the xenograft models of MITR and MI-resistant PD MLL1-r AML cells. These findings highlight novel, targeted, drug combinations that overcome MI resistance in AML cells with MLL1-r or mtNPM1.

Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated curative potential in B cell malignancies, yet translating this success to chronic infections like human immunodeficiency virus (HIV) remains a major challenge. In people living with HIV (PLWH) on suppressive antiretroviral therapy (ART), low antigen levels limit CAR-T cell expansion and persistence. We previously reported data from a pilot study which suggested that HIV-targeted CD4CAR-T cells could overcome this barrier through exogenous antigen supplementation, leading to robust in vivo expansion. Here, we sought to comprehensively confirm and expand on those findings. We tested a broad array of strategies to enhance CD4CAR-T cell efficacy, including CRISPR-Cas9-mediated gene editing of immune checkpoint and HIV-associated genes, single and pooled competitive infusions of engineered CAR-T cells, distinct CAR constructs incorporating either CD28 or 4-1BB costimulatory domains, and exogenous antigen boosting. We also developed highly sensitive droplet digital PCR (ddPCR) assays both to quantify CAR-T cell frequency and corroborate flow cytometry-based quantification of CD4CAR T-cell expansion. We evaluated these new approaches across multiple NHP models of HIV, including both simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected, ART-suppressed NHPs. Although CD4CAR-T cell products exhibited antigen-specific proliferation and cytotoxicity ex vivo, they failed to expand, persist, or control viremia in vivo. We were also unable to confirm previously observed CD4CAR T cell expansions from our earlier studies, which will be retracted. Together these data highlight the need for alternative strategies to potentiate anti-HIV CD4CAR-T cells in the immunocompetent setting.