Journal of Hematology & Oncology

IDH inhibitors promote differentiation and hematological improvement in myeloid malignancies by reversing epigenetic dysregulation. However, molecular predictors of response remain poorly defined. We retrospectively analyzed 79 patients with IDH mutated myeloid malignancies treated with either ivosidenib (IDH1; n=36) or enasidenib (IDH2; n=43). Hematologic benefit was assessed using a composite complete hematologic response (CHR), defined by neutrophil and platelet recovery, and transfusion independence. Targeted sequencing was performed at baseline and at best hematologic response. Associations between co mutations, hematologic response, and treatment duration were evaluated using multivariable models. Overall, 43% of patients achieved CHR, with similar rates between inhibitors. Ivosidenib treated patients achieving CHR demonstrated a significant reduction in IDH1 VAF (p=0.010), which was not observed in enasidenib-treated CHR patients. Within the CHR cohort, ivosidenib patients had a greater reduction in IDH VAF relative to enasidenib patients (mean VAF change -16.74% vs -0.05%, p=0.034). Ivosidenib responders demonstrated a trend toward more durable treatment duration compared with enasidenib responders (p = 0.0908). RUNX1 and BCOR mutations were significantly enriched among patients with incomplete hematologic response and were independently associated with shorter treatment duration after adjustment for clinical factors (RUNX1: HR 2.53, p=0.0024; BCOR: HR 2.31, p=0.0058). IDH1 inhibition was associated with clonal contraction and durable hematologic benefit, a pattern not observed with IDH2 inhibition. Co mutations in RUNX1 and BCOR identified patients unlikely to achieve sustained hematologic improvement, suggesting a secondary block to differentiation. These findings support the integration of mutational and molecular profiling to refine patient selection and guide combinatorial strategies in IDH mutated myeloid malignancies.

Cancer immunotherapy targeting B-cell specific CD19 antigen meant a major breakthrough in the treatment of B-cell malignancies. Yet, vast proportion of treated patients experience relapse and failure of the therapy. Although multiple mechanisms of CD19-immunotherapy failure have been described, CD19-negative relapses represent the major hurdle in achieving higher and durable response rates. Our established in vitro co-culture models revealed that suboptimal CAR-T cell performance, inefficient to mediate target cell killing, results in robust downregulation of CD19 target antigen. Using genome-wide CRISPR screening, we addressed the mechanisms responsible for such CD19 downregulation and identified Cullin-1 and CD81 playing instrumental role in negative and positive regulation of CD19 expression, respectively. Inhibiting Cullin-1 activity with pevonedistat prevents the loss of CD19 under immunotherapeutic pressure, results in higher CD19 surface levels and consequently enhances the efficacy of target cell killing by CD19-CAR-T cells, CD19-CAR-NK cells and CD19-targeting antibody treatment. Mechanistically, we show that pevonedistat blocks the degradation of CD19 upon its internalization and allows its recycling back to the plasma membrane. CD81 chaperone protein is critically involved in this process as the absence of CD81 abrogates the effect of pevonedistat. In summary, we identify Cullin-1 as a novel and druggable regulator of CD19 protein stability. Cullin-1 inhibition augments CD19 surface expression, thereby improving the efficiency of CD19-targeting immunotherapies, thus arguing for potential incorporation of pevonedistat into novel combination therapies. Key PointsO_LICullin-1 inhibition stabilizes CD19 surface expression, preventing its loss under immunotherapeutic pressure C_LIO_LIPevonedistat treatment enhances the efficacy of CD19-CAR-T cells, CAR-NK cells and CD19-targeting antibodies C_LI

The extent to which rare and common genetic variants jointly contribute to the risk of acute myeloid leukemia (AML) still remains relatively unexplored in large-scale biobank whole-genome sequencing cohorts. Here, we leverage the latest sequencing and phenotypic data from the All of Us Research Program to identify variants, genes, and gene-sets associated with AML. We performed set-based association tests for rare protein-coding variants (Ncases=265 and Ncontrols=169,706) and single-variant association tests for common variants (Ncases=265 and Ncontrols=169,705) utilizing the large European-like ancestry sample. For the rare-variant set-based tests conducted using SAIGE-GENE+, four genes were statistically significant: DNMT3A, TET2, SRSF2, and IDH2 (Bonferroni-corrected Cauchy p-value < 0.05). We also constructed multiple rare-variant burden risk scores using different gene-sets to identify those with a substantial rare-variant burden for AML. Gene-sets derived from Genomic Data Commons whole-genome sequencing data, comprising two distinct groups-genes observed to harbor somatic mutations in AML and genes observed to harbor somatic mutations across all cancer types-showed a statistically significant rare-variant burden (Bonferroni-corrected p-value < 0.05). Ultimately, these findings demonstrate that leveraging whole-genome sequencing in large-scale biobanks enables the identification of rare protein-coding variants, genes, and gene sets associated with AML.

Invariant natural killer T (iNKT) cells can limit graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT), but their scarcity in peripheral blood limitstherapeutic development. Current clinical-grade human iNKT expansion protocols mainly rely on IL-2, require prior iNKT-cell sorting, last 6-8 weeks, and predominantly expand CD4+ iNKT cells, whereas human CD4- iNKT cells are more strongly associated with GVHD control in patients and uniquely regulate antigen-presenting cells and T-cell activation. We developed a scalable culture system to preferentially expand human CD4- iNKT cells directly from total peripheral blood mononuclear cells (PBMCs) using alpha galactosylceramide (-GalCer) and optimized cytokine conditions. IL-15 was the most effective cytokine. The optimized 14-day protocol generated a mean of 3.8x107 iNKT cells from 2x107 PBMCs, including 74% CD4- iNKT cells. Single-cell transcriptomic profiling identified eight major iNKT subsets, differentiation trajectories during expansion, and distinct IL-2- versus IL-15-associated transcriptional programs. IL-15-expanded iNKT cells induced apoptosis of monocyte-derived dendritic and leukemic cells in vitro, controlled xeno-GVHD, and preserved graft-versus-leukemia (GVL) activity in preclinical mouse models. This platform enables reproducible production of human CD4- iNKT cells at clinically relevant scale and position IL-15-expanded iNKT cells as a compelling immunotherapy candidate for allo-HSCT.

Chronic myelomonocytic leukemia (CMML) is an aggressive hematologic malignancy characterized by excess inflammatory signaling and clonal myeloproliferation. The relative contribution of neutrophils (PMNs) to the inflammatory milieu in CMML is poorly understood. In this study we sought to understand whether neutrophil extracellular trap (NET) formation, a key mediator of neutrophilic inflammation, is dysregulated in CMML and can be therapeutically targeted with a novel peptide inhibitor of NETosis called neonatal NET-inhibitory factor (nNIF). Here, we demonstrate that baseline NET formation is aberrantly increased in primary CMML PMNs transcriptionally primed for NETosis, and that soluble factors produced during CMML NET formation promote clonogenicity in CMML CD34+ hematopoietic cells matched to the same patient. Further, we show that nNIF and clinical agents under investigation in CMML effectively inhibit NETosis, warranting further study of NET inhibitory agents in this rare disease with limited treatment options.

Background: Translocation t(11;14) defines a biologically distinct subset of multiple myeloma (MM) enriched for BCL2 dependency. Treatment with venetoclax, selective BCL2 inhibitor, has shown varied responses in clinical trials and retrospective cohorts. Efficacy of venetoclax-based combination therapies and optimal timing of treatment in t(11;14) MM patients remain incompletely characterized. Methods: We have compared the overall survival of t(11;14) MM patients who received Venetoclax (N = 97) at any moment in time, to those who never did (N =284), in the largest retrospective cohort (N = 381) reported to date. We used longitudinal fluorescence in situ hybridization (FISH) to assess cytogenetic evolution and genomic complexity. We performed transcriptomic profiling of CD138 enriched tumors using RNA sequencing in a subset of samples and gene expression signatures (UAMS/HALLMARKS) were used to define molecular subtypes. Ex vivo drug sensitivity assays integrated with paired RNA sequencing were used to identify subtype specific therapeutic vulnerabilities and rational venetoclax based combination strategies. Results: Venetoclax exposure was associated with an improvement of median overall survival by nearly four years compared to non VEN exposed patients (p = 0.0003). Longitudinal cytogenetic analysis demonstrated stability of the primary t(11;14) translocation over time, while secondary abnormalities tend to accumulate, including those harboring high-risk secondary cytogenetic abnormalities such as del13q, amp/gain1q21, del17p, and del1p, resulting in increasing genomic complexity with disease progression. Transcriptomic analyses identified selective enrichment of CD1/CD2 signature (associated with t(11;14) NDMM) by single sample gene set enrichment analysis as a marker of prolonged progression free survival. However, patients with more than five prior lines of therapy were enriched for transcriptionally complex biology characterized by CD1/CD2 with either proliferative/hypermetabolic or inflammatory transcriptional programming which were associated with inferior outcomes with VEN based treatment. Ex vivo drug sensitivity profiling revealed subtype specific vulnerabilities, identifying daratumumab, lenalidomide, ixazomib, and panobinostat as rational partners for venetoclax depending on transcriptional context. Conclusion: Venetoclax-based therapy significantly improved overall survival in t(11;14) MM. Clinical benefit, defined by improved progression free survival, was greatest when venetoclax was administered earlier, preceding the emergence of transcriptomic reprogramming that reduces BCL2 dependency. These findings support transcriptomic biomarker guided, subtype specific venetoclax based treatment strategies to optimize outcomes in patients with t(11;14) MM.

1Adoptive cell therapy using tumor antigen-targeting T cell receptors (TCRs) offers a compelling approach to treat both hematological cancers and solid tumors due to broad antigen accessibility and the ability to target cancer-specific neoantigens. However, unlike clinically validated second generation CAR-T cells bearing built-in co-stimulatory signaling modules (i.e. 41BB or CD28), TCR-T cells receive little to no co-stimulation within most tumor microenvironments leading to attenuated cellular responses. Additionally, CD4+ TCR-T cells engineered to express HLA-Class I restricted TCRs possess minimal T-helper cell activity and thus do not effectively mobilize CD8+ TCR-T cells or host anti-tumor immune responses. To address these limitations, we used CRISPR-Cas9 to engineer TCR-T cells with targeted integration of chimeric CD8 constructs containing intracellular co-stimulatory domains. We found that expression of wild-type CD8{beta}, but not CD8, could promote CD4+ T cell activities in HLA-Class I restricted TCR-T cells. However, this was insufficient to drive durable anti-tumor responses in challenging tumor mouse models when using a high-affinity WT1-directed TCR. To address this, several CD8 co-stimulatory fusion constructs containing CD28 or 41BB intracellular domains were designed and screened, identifying two CD8-41BB based chimeras that substantially increased TCR-T cell activity relative to wild-type CD8{beta}. WT1-TCR-T cells co-expressing the CD8-41BB fusions demonstrated not only enhanced CD4+ activity including strong and polarized Th1-type cytokine secretion, but also enhanced the proliferation, cytokine release, and cytotoxicity of CD8+ CTLs. Remarkably, when combined with TGFBR2 gene disruption, WT1-TCR-T cells co-expressing CD8-41BB receptors were able to completely regress established cell line-derived ovarian tumors, showed robust in vivo expansion and persistence, and provided long-term protection from tumor rechallenge. Importantly, the specificity profile of the WT1-TCR including its HLA-A*02:01 restriction and WT1 peptide recognition motif was preserved upon expression of CD8-41BB. To simplify cell engineering processes for clinical applications, we configured a homology directed repair (HDR) cassette to allow for efficient CRISPR-Cas9-based insertion of both the TCR and CD8-41BB transgenes in the TRAC locus in a single step with >80% efficiency. Lastly, the enhanced activity conferred by CD8-41BB expression was validated with a second clinically relevant TCR targeting PRAME, suggesting this platform can be a universal approach for enhancing the therapeutic potential of TCR-based cell therapies.

Tumor infiltrating CD8+ T cells (TILs) progress to a state of terminal exhaustion (Ttex) which have impaired functionality and are nonrenewable. However their precursors (Tpex) are renewable and can generate efficient effector cells. We started from the observation that melanoma patients undergoing therapy with checkpoint inhibitors show increased survival when their T cells have low BCL11B mRNA. In line with this, ablation of Bcl11b in CD8+ TILs conferred a superior anti-tumor response in murine melanoma and ovarian cancer models. Bcl11b KO TILs failed to progress to the Ttex state and retained elevated stemness. Bcl11b exerted its role by repressing expression of essential transcription factors (TF) controlling stemness, and conversely by promoting expression of exhaustion-associated TFs and inhibitory receptor genes, through complex epigenetic control. In addition, Bcl11b KO CD8+ T cells showed increased Ag-specific cytolytic activity and elevated Gzmb and Prf1 proteins, but no increase in their mRNAs, however presented higher expression of genes with role in translation. Furthermore, CRISPR-CAS9-mediated deletion of BCL11B in human TILs from a patient with poor response to adoptive cell therapy with autologous TILs, improved their cytolytic activity and promoted expression of the stemness-associated TF TCF1, underlying its potential therapeutic use. HIGHLIGHTS- Adoptive transfer of Bcl11b KO CD8+ TILs surpasses WT in tumor burden reduction - Bcl11b ablation reprograms TILs and impairs the progression to Ttex state - Bcl11b KO CD8+ T cells have elevated cytotoxicity and kill only Ag-MHCI targets - BCL11B deletion in nonresponder ACT-TIL improves cytolytic activity and elevates TCF1 GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/742578v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@10040d4org.highwire.dtl.DTLVardef@1a045caorg.highwire.dtl.DTLVardef@145f790org.highwire.dtl.DTLVardef@8012ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous hematologic malignancy in which LMO2 {gamma}{delta}-like T-ALL represents a rare but clinically aggressive subtype associated with poor treatment response and inferior survival. Integrated transcriptomic analyses identified high SOX11 expression as a defining feature of high-risk LMO2 {gamma}{delta}-like T-ALL, where elevated SOX11 levels correlated with refractory disease and poor clinical outcome. To investigate the functional role of SOX11 in {gamma}{delta} T-cell biology and leukemogenesis, we generated a conditional R26-SOX11 mouse model enabling lineage-specific SOX11 overexpression in T-cell progenitors. SOX11 expression promoted expansion of the innate {gamma}{delta} T-cell compartment in thymus, spleen, and bone marrow, accompanied by transcriptional activation of {gamma}{delta} T-cell differentiation, activation, and cytotoxicity programs. However, SOX11 overexpression alone was insufficient to induce leukemia or confer thymocyte self-renewal capacity. In contrast, combined SOX11 and LMO2 overexpression markedly accelerated T-ALL development and strongly increased the incidence of {gamma}{delta}-like leukemias, thereby recapitulating the human high-risk LMO2 {gamma}{delta}-like T-ALL subtype. Mechanistically, SOX11 expanded the pre-leukemic DN3 thymocyte compartment in LMO2-driven mouse model while promoting differentiation toward the {gamma}{delta} lineage. Transcriptomic profiling identified activation of MYCN-associated transcriptional programs in SOX11/LMO2 pre-leukemic thymocytes. Consistently, MYCN was highly expressed in human LMO2 {gamma}{delta}-like T-ALL, and recurrent stabilizing MYCN P44L mutations were enriched in this subtype. Functional validation using genetic and transplantation-based mouse models demonstrated that SOX11 cooperates with MYCN to accelerate T-ALL onset. Together, these findings establish a cooperative SOX11-MYCN oncogenic axis driving {gamma}{delta}-like T-ALL and provide a novel preclinical model for investigating therapeutic vulnerabilities in this high-risk leukemia subtype.

Major histocompatibility complexes (MHC) govern antigen presentation and T cell receptor (TCR) selection. Accurate in vivo modeling of human immunity therefore requires physiological human MHC TCR interactions. Humanized NOD scid IL2null (NSG) mice engrafted with human CD34+ hematopoietic stem cells are widely used to provide preclinical platforms for the development of advanced therapies; however, interactions between murine MHC and human TCR can promote xenoreactivity and alter T cell development. Here, we investigated how elimination of murine MHC together with different conditioning regimens shapes human T cell maturation in vivo. CD34+ cells from ten cord blood donors were transplanted into conventional NSG mice or murine MHC deficient NSG derivatives (DKO) following either sublethal irradiation or myeloablative busulfan conditioning. Integrated analyses combining flow cytometry, plasma cytokine profiling, and bulk and single cell TCR sequencing revealed marked differences in T cell differentiation across models. Busulfan conditioned DKO mice developed highly proliferative, activated, and cytotoxic T cells together with clonally expanded TCR repertoires. In contrast, irradiated NSG mice preferentially accumulated naive, NKT, and regulatory T cell populations. Busulfan-conditioned DKO mice showed no evidence of xenogeneic graft versus host disease and represent a refined enabling platform for human T cell development and provide a foundation for future preclinical evaluation of advanced gene and cell therapies.

TP53-mutated acute myeloid leukemia (AML) has dismal outcomes with current treatments and represents a critical unmet need. TP53-mutated AML is proposed to be susceptible to immunotherapeutic approaches but, to date, there is no established immunotherapy for this sub-group. Expression of stimulator of interferon genes (STING), a key innate immune driver that activates interferon (IFN) signaling, is decreased by epigenetic silencing or mutation in many cancers, including those with TP53 mutations. Here, we report that response to the next-generation synthetic STING agonist C92 is potentiated in AML cell lines and primary cells with TP53-mutated versus wild-type (WT) cells, representing a previously undescribed vulnerability of these leukemia cells to STING small molecule therapies. Moreover, combining treatment with the DNA methyltransferase inhibitor (DNMTi) decitabine (DAC), significantly increases STING activation, with marked transcriptome-wide increase in repetitive elements (REs) and upregulation of a critical set of interferon-related genes. Cell death in TP53 KO versus WT AML is specifically dependent on innate immune zinc finger NFX1-type containing 1 (ZNFX1) and Z-DNA-binding protein 1 (ZBP1) driving increased cleavage and activation of Receptor-Interacting-Serine/Threonine-Protein Kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL), suggesting mechanisms of necroptosis. Finally, C92 and DAC combination significantly reduces leukemia burden in humanized AML mouse models, accompanied by increased immune responses, including cytokines and cytotoxic T lymphocytes in the leukemia microenvironment. These results support development of clinical trial strategies combining STING agonists with DNMTis for patients with TP53-mutated AML. SummaryO_LITP53-mutated AML potentiates effects of novel next-generation STING agonist C92, with unique allosteric and non-cyclic dinucleotide (non-CD) mechanism of action, inducing increased STING activation and cytokine release C_LIO_LISTING agonists and DNMTis, synergistically increase STING activation with marked transcriptome-wide increase in repetitive elements (REs) and upregulation of a critical set of interferon-related genes in TP53-mutated AML C_LIO_LISTING agonists induce necroptosis via a STING-ZNFX1-ZBP1-necroptosis axis in TP53-mutated AML. C_LIO_LIThis drug combination reduces leukemia burden, activates immune responses in AML models and supports translation for high-risk AML patients. C_LI Statement of Translational RelevanceThis pre-clinical study identifies a novel therapeutic vulnerability in (TP53)-mutated acute myeloid leukemia (AML), a poor prognosis subtype with a critical unmet need. Novel next-generation STING agonist C92, with unique allosteric and non-cyclic dinucleotide (non-CD) mechanism of action, induces increased STING activation and cytokine release, compared with WT TP53 in AML cell lines and primary cells, and has superior STING activity with respect to several STING agonists currently in clinical studies. Combining C92 treatment with the DNA methyltransferase inhibitor (DNMTi) decitabine (DAC) synergistically increases STING activation, with marked transcriptome-wide increase in repetitive elements (REs) and upregulation of a critical set of interferon-related genes, driving ZNFX1-driven inflammatory necroptotic cell death. Utilizing humanized mouse models, C92 in combination with DAC significantly reduces leukemia burden and enhances cytotoxic T-cell responses in the tumor microenvironment, supporting clinical translation for high-risk AML patients.

Multiple myeloma (MM) is a plasma cell malignancy that frequently harbors activating mutations in NRAS and KRAS oncogenes. Previous clinical trials targeting the Ras/MAPK oncogenic pathway with MEK inhibitors (MEKi) were met with limited efficacy, and newer generation of Ras inhibitors (RASi) have not been specifically evaluated in MM patients. To investigate the vulnerabilities of Ras-mutant MM to targeted therapies, we examined the sensitivity of a panel of human MM cell lines to the RASi RMC-6236 (daraxonrasib) and the MEKi trametinib. Although Ras-mutant MM cells are responsive to oncogenic Ras signaling and are sensitive to RAS inhibition, their sensitivity to MEK inhibition is heterogeneous. Mechanistic studies revealed that c-Myc protein is destabilized by MEK inhibition only in MEKi-sensitive MM cells but not in MEKi-resistant cells, and pharmacological and genetic stabilization of c-Myc is sufficient to confer MEKi resistance. In contrast, Ras inhibition reduced c-Myc protein across all MM cell lines tested, regardless of their dependency on the MAPK pathway, and c-Myc expression was insufficient to promote RASi resistance. Together, these findings demonstrate that c-Myc protein stability differentiates the response of Ras-mutant MM cells to Ras and MEK inhibition, and suggest that direct targeting of the Ras oncoprotein, rather than its downstream MAPK pathway, may present a more effective strategy.

Gfi1 regulates endothelial-to-hematopoietic transition (EHT) and hematopoietic stem cell (HSC) maintenance; however, its expression dynamics remain unclear. We generated a non-disruptive Gfi1-T2A-mScarlet reporter to track Gfi1 expression. At E10.5, reporter selectively marked EHT in the dorsal aorta, umbilical artery, and vitelline artery. At E9.5, mScarlet-positive endothelial cells were present in the umbilical and vitelline arteries but were scarce in the dorsal aorta, indicating vascular-bed-specific differences in hemogenic activation. Gfi1 was broadly expressed in fetal liver HSCs, while higher expression levels identified HSCs with enhanced multilineage reconstitution and preferential T-cell output. Although Gfi1 expression declined during fetal-to-adult maturation, a subset of adult bone marrow HSCs retained expression and exhibited superior repopulating and self-renewal capacity. Bulk and single-cell transcriptomic analyses linked this subset to dormant and fetal-associated programs, including imprinted genes. Reduced chromatin accessibility at a conserved Gfi1 +26.5kb putative enhancer correlated with developmental Gfi1 downregulation. Thus, Gfi1 dynamics define EHT onset and functionally distinct HSC stemness.

The non-receptor protein-tyrosine phosphatases Shp1 (PTPN6) and Shp2 (PTPN11) play critical roles in hematopoietic signaling networks, yet their specific functions in human megakaryopoiesis and thrombopoiesis remain incompletely understood. While Shp2 is recognized in murine models as a positive regulator of thrombopoietin (Tpo)-mediated signaling through the Ras/MAPK and PI3K/AKT pathways, Shp1 has been implicated in RhoA-dependent cytoskeletal remodeling. However, the extent to which these roles translate to human megakaryocyte (MK) development and platelet production is not known. In this study, we systematically investigated the contributions of Shp1 and Shp2 to human MK development and function using CRISPR/Cas9-mediated gene deletion of PTPN6 and PTPN11 in CD34+ hematopoietic stem and progenitor cells (HSPCs), combined with pharmacological inhibition of Shp2 using the structurally-distinct allosteric inhibitors SHP099 and RMC-4550. Efficient gene editing of PTPN6 and PTPN11 resulted in efficient ablation of Shp1 and Shp2 in CD34+ HSPC-derived MKs. Genetic deletion or pharmacological inhibition of Shp2 markedly impaired MK proliferation, polyploidization, maturation, and proplatelet formation, whereas loss of Shp1 expression did not. Further, Shp2 inhibition significantly reduced platelet production in a 3-dimensional human bone marrow tissue model. Deletion and inhibition of Shp2 abrogated Tpo-induced ERK1/2 and AKT phosphorylation, confirming its essential role in Mpl receptor signaling. These findings demonstrate the distinct functional roles of Shp1 and Shp2 in MKs and establish Shp2 as a critical positive regulator of Mpl- mediated megakaryopoiesis and thrombopoiesis. Key PointsO_LIEfficient deletion of Shp1 and Shp2 in human CD34 progenitor cell-derived MKs using CRISPR/Cas9. C_LIO_LILoss of Shp2 expression impairs thrombopoietin-induced human MK maturation, proplatelet formation and Mpl signaling. C_LI

Myeloproliferative neoplasms (MPN) are chronic hematologic malignancies characterized by clonal myeloid expansion, inflammation, oxidative stress, and progressive bone marrow (BM) remodeling that may culminate in fibrosis and secondary acute leukemia. Here, we evaluated the therapeutic efficacy and the underlying mechanisms of melatonin (MT) and liposomal melatonin (nano-MT) in preclinical MPN models. MT selectively inhibited clonogenic growth of patient-derived peripheral blood mononuclear cells and induced pluripotent stem cell-derived CD34 hematopoietic stem and progenitor cells in comparison to healthy controls. This effect was associated with increased apoptosis, reduced reactive oxygen species (ROS), and decreased glucose uptake, independently of MT receptor signaling. Transcriptomic profiling of primary MPN CD34 cells revealed suppression of MYC targets, G2M checkpoint signaling, ROS, and glycolysis pathways. In co-culture models, MT reduced stromal -smooth muscle actin and phosphorylated SMAD2/3, indicating inhibition of TGF-{beta}-driven mesenchymal stromal cell-to-myofibroblast formation. In tamoxifen-inducible SclCreER;JAK2V617F mice, nano-MT achieved efficient spleen and BM targeting. Therapeutically, nano-MT reduced erythrocytosis, myeloid progenitor expansion, and BM IL-1{beta} levels. Longitudinal micro-computed tomography and histological analyses demonstrated normalization of BM architecture, reduced osteosclerotic remodeling and splenomegaly, decreased reticulin deposition and megakaryocyte numbers. In a dose-escalation study, nano-MT restored erythrocyte, hematocrit, and platelet counts and normalized megakaryocyte-erythroid progenitors. Combination treatment with ruxolitinib further reduced leukocytosis, neutrophilia, and monocytosis. Collectively, these findings demonstrate that (nano-)MT attenuates MPN and BM remodeling by targeting metabolic, inflammatory, and fibrotic pathways. This study provides the first evidence for a therapeutic benefit of nano-MT in MPN and establishes a rationale for further translational evaluation.

Hematopoietic stem cells(HSCs) maintain their pools by stem-stem division and produce mature blood cells through stem-progenitor or progenitor-progenitor division. A paired daughter cell(PDC) assay combined with single cell transplantation is a powerful method to compare the lineage outputs of two HSC daughter cells. However, single-cell transplantation precludes large-scale analysis of daughter-cell pairs, as only one cell can be transplanted per recipient. Here, we developed a DNA barcoding-based PDC assay to overcome this limitation, enabling simultaneous analysis of 476 daughter pairs from individual HSC divisions and revealing that daughter-cell fates are coordinated and that HSC division patterns are biased toward stem-stem and progenitor-progenitor divisions rather than stem-progenitor divisions. These findings indicate that HSC fate outcomes are directed toward symmetric division outcomes. Integration of single-cell RNA sequencing with DNA barcoding revealed a continuum of HSC states--from balanced HSCs to myeloid-biased HSCs and ultimately to a low-output HSC subset--in which progressively reduced production of mature blood cells relative to stem cell expansion, a proxy for stem-stem division bias, exhibits distinct activities of transcription factors and signaling pathways. Overall, our analysis uncovers characteristic patterns of HSC division and links stem maintenance with distinct molecular features.

Although B-cell acute lymphoblastic leukemia (B-ALL) is highly responsive to antigen-directed immunotherapies, treatment resistance remains a major barrier to achieving durable responses in patients. We recently developed a novel VpreB1 (CD179a)-directed antibody-drug conjugate with calicheamicin (VpreB1-ADC) that exploits the restricted expression of VpreB1 within the surrogate light chain in early B cells, including B-ALL. In the present work, we investigated mechanisms of resistance to the VpreB1-ADC. Mechanisms of resistance were evaluated using a TCF3::HLF B-ALL model, assessing target engagement parameters including VpreB1 surface expression and antibody internalization. The role of the multidrug resistance transporter ABCB1 (P-glycoprotein) was evaluated via pharmacologic inhibition, using tariquidar and zosuquidar, and enforced overexpression across multiple B-ALL cell lines. Sensitivity to alternative non-ABCB1 substrate payloads exatecan and PNU-159682 was also assessed. Resistant TCF3::HLF cells retained VpreB1 expression and efficient antibody internalization. Instead, resistance was driven by elevated ABCB1 expression and activity. ABCB1 inhibition with tariquidar or zosuquidar restored VpreB1-ADC sensitivity. Conversely, enforced ABCB1 overexpression conferred ADC resistance, which was reversed by ABCB1 inhibition. Cells with high ABCB1 activity remained fully sensitive to alternative payloads, including exatecan and PNU-159682, which are not ABCB1 substrates. ABCB1-mediated drug efflux drives intrinsic resistance to calicheamicin-conjugated ADCs in B-ALL. Combining ADCs with ABCB1 inhibitors or selecting payloads non-susceptible to ABCB1 efflux offer viable strategies to overcome resistance and optimize future ADC therapies.

Acute myeloid leukemia (AML) is sustained by leukemic stem cells (LSCs), which must be eradicated for durable remission yet remain therapy-resistant. Here, we present a scalable platform to identify compounds that eliminate LSCs by performing the first large high-throughput drug screen directly on human LSC-enriched cells. Using this system, we screened 11 142 compounds and identified 20 inhibitors selective for LSCs. For three candidates, BIO-acetoxime, SJB2-043 and UMxxxxx03, we confirmed anti-LSC activity across multiple primary patient samples and validated efficacy through xenotransplantation assays. Single-cell RNA sequencing uncovered convergent and distinct anti-LSC mechanisms of action, including USP1 inhibition, which suppressed stemness and cell-cycle programs while promoting metabolic and inflammatory stress responses. Collectively, the LSC-enriched fraction was eliminated through apoptosis or differentiation. Together, this work establishes the first platform to directly screen LSCs at scale and identifies novel vulnerabilities and therapeutics capable of eliminating the root of AML.

Despite the success of chimeric antigen receptor (CAR) T cell therapy for treatment of B cell acute lymphoblastic leukemia (B-ALL), its translation to acute myeloid leukemia (AML) has been hindered by limited efficacy and significant toxicity. Interferon-gamma (IFN{gamma}) blockade with emapalumab has recently emerged as a promising strategy to mitigate CAR T cell-related toxicities in B cell malignancies, based on evidence that IFN{gamma} is largely dispensable for optimal CAR T cell activity in B-ALL. Whether IFN{gamma} signaling is similarly non-essential in the AML context remains unclear. Here, we demonstrate that disruption of the IFN{gamma} axis impedes anti-AML CAR T cell function and prevents upregulation of target antigen CD123, the apoptotic mediator Fas, and the adhesion molecule ICAM-1 on AML cells. Conversely, exogenous IFN{gamma} enhances CAR T cell cytotoxicity and increases CAR T cell avidity for AML targets. These findings identify IFN{gamma} as a critical mediator of CAR T cell efficacy against AML by promoting increased target antigen expression, enhanced cytotoxicity, and stable CAR T/tumor interactions. Our results suggest that therapeutic IFN{gamma} blockade, including with emapalumab, may compromise CAR T cell responses in AML and should be approached with caution in this disease context.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy associated with a poor prognosis and an unmet need for more effective treatment options, particularly after relapse. One potential approach to address this need, which has been used successfully in solid tumors, is adoptive transfer of T cells engineered to express specific T cell receptors (TCRs). Particularly attractive are TCRs that can target epitopes derived from oncogenes on which cancer cells depend while sparing normal cells that do not harbor the mutation. One promising oncogenic target in AML is mutated nucleophosmin 1 (mtNPM1), present in up to 30% of AML cases. Most NPM1 mutations result in a common frameshifted sequence that generates peptides presented by several class I human leukocyte antigens (HLAs), such as HLA-A*11:01. In this study, we vaccinated HLA-A*11:01-transgenic mice with the mtNPM1 peptide AVEEVSLRK and isolated six functional TCRs from mtNPM1-reactive lymphocytes. When engineered into human T cells, two of these TCRs consistently mediated robust and specific lysis of an AML cell line and primary AML cells co-expressing HLA-A*11:01 and mtNPM1. Neither TCR recognized related human peptides, HLA-A*11:01-expressing normal peripheral blood mononuclear cells or stem/progenitor cells, or cells expressing diverse allogeneic HLAs, suggesting a low risk of cross-reactivity and alloreactivity. In an AML xenograft model, both TCRs controlled AML in hematopoietic organs, although extramedullary tumors emerged during treatment, potentially reflecting the aggressive behavior of the model that may not be representative of AML in patients. Together, these data suggest that the two TCRs represent strong candidates for further preclinical development in adoptive T cell therapy for NPM1-mutated, HLA-A*11:01-positive AML. These results also support the use of HLA-transgenic mice as a platform for generating TCRs targeting oncogene-derived epitopes in hematologic malignancies.