Hypodiploid (Hypo) and BCR::ABL1-positive (BCR-ABL+) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) confer a high risk of disease relapse. We investigated post-hematopoietic stem cell transplantation (HSCT) outcomes within the prospective FORUM trial, comparing these genetic subgroups to patients without these lesions. The use of pre- and post-HSCT add-on treatments, including tyrosine-kinase inhibitors (TKIs) and immunotherapies, was also assessed. Multivariate analysis evaluated associations with overall survival (OS), event-free survival (EFS), cumulative incidence of relapse (CIR). The FORUM trial enrolled 741 patients ≥4 years of age with BCP-ALL who underwent HSCT from HLA-matched donors (2013-2023). The 3-year OS (0.86 [95% CI, 0.76-0.92], 0.79 [0.65-0.87], and 0.79 [0.76-0.82]) and EFS (0.71 [0.59-0.80], 0.73 [0.59-0.83], and 0.67 [0.63-0.71]) did not differ significantly between BCR-ABL+, Hypo, and Neither patients, respectively. However, Hypo patients in second complete remission (CR2) showed inferior OS and EFS, driven by higher non-relapse mortality (NRM), which occurred exclusively in near-diploid cases. No NRM occurred in severe hypodiploid cases conditioned with TBI. MRD positivity at transplant predicted worse OS, EFS, and CIR in all genetic groups. Hypo patients were difficult to salvage post-relapse, even with CAR-T therapy. By contrast, BCR-ABL+ patients had favorable outcomes, even when MRD-positive prior HSCT. Prophylactic TKI use post-HSCT improved EFS and reduced CIR. BCR::ABL+ patients transplanted in CR2 had a 3-year OS of 96%. In conclusion, the standardized FORUM protocol yielded comparable outcomes across genetic subgroups. Post-transplant TKI maintenance improved outcomes in BCR-ABL+ BCP-ALL. EudraCT: 2012-0032-22; ClinicalTrials.gov: NCT01949129.

Acute myeloid leukemia (AML) patients have a poor five-year survival rate highlighting the need for the identification of new approaches to target this disease. AML is highly dependent on glutathione (GSH) metabolism for survival. While the metabolic role of GSH is well-characterized in AML, the contribution of protein glutathionylation-a reversible modification that protects protein thiols from oxidative damage-remains largely unexplored. Therefore, we sought to elucidate the role of protein glutathionylation in AML pathogenesis. Here, we demonstrate that protein glutathionylation is essential for AML cell survival. Specifically, the loss of glutaredoxin 2 (GLRX2), an enzyme that removes glutathione modifications, resulted in selective primary AML cell death while sparing normal human hematopoietic stem and progenitor cells. Unbiased proteomic analysis revealed increased mitochondrial protein glutathionylation upon GLRX2 depletion, accompanied by mitochondrial dysfunction, including impaired oxidative phosphorylation, reduced mitochondrial membrane potential, and increased opening of the mitochondrial permeability transition pore (mPTP). Further investigation identified ATP5PO, a key regulator of mPTP opening and a component of the ATP synthase complex, as a critical GLRX2 target. Disruption of ATP5PO glutathionylation partially restored mPTP function and rescued AML cell viability following GLRX2 depletion. Moreover, both genetic and pharmacologic inhibition of mPTP opening restored the leukemic potential of primary AML specimens in the absence of GLRX2. By disrupting glutathionylation-dependent mitochondrial homeostasis, this study reveals a novel vulnerability in AML that could inform future therapeutic strategies.

The regulation of the switch from fetal (HBG) to adult (HBB and HBD) b-globin gene expression has served as a paradigm for clinically relevant developmental transcriptional control. Mechanistic studies of this switch have predominantly focused on HBG repressors with comparatively little attention paid to potential HBG activators. We found that in adult type HUDEP2 erythroid cells, the ATP-dependent chromatin remodeler BRG1 preferentially activates the HBG genes as well as the minor adult HBD gene. BRG1 is a core catalytic subunit of three BAF complexes, canonical BAF, polybromo BAF, and non-canonical BAF (ncBAF) that regulate chromatin accessibility in distinct gene- and cell-type contexts. To dissect the specific BAF complex configuration mediating selective activation of HBG and HBD in erythroid cells, we performed CRISPR mediated targeting of individual subunits and pinpointed the regulatory activity to the ncBAF complex. Loss of the ncBAF complex subunits BRD9 and BAF60A preferentially decreased HBG and HBD transcription while accelerating terminal erythroid differentiation and hemoglobinization. Acute pharmacologic depletion of BRD9 in HUDEP2 and primary erythroid cells selectively reduced transcription of HBD and HBG, suggesting direct effects at these genes. Collectively, our unexpected findings demonstrate that the BAF complex, through distinct subcomplex configurations, can regulate selective gene expression within a multi-gene cluster. This expands the traditional view of BAF as a general co-activator, highlights its role in gene-specific regulation, and identifies a potential target for therapeutic manipulation of b-like globin genes in erythroid cell disorders.

We compared daunorubicin/AraC plus fractionated gemtuzumab (DAGO2) with CPX-351 (CPX) (1:2 randomisation) in 439 patients ≥60yrs (median age 68yrs) without known adverse-risk cytogenetics entering the NCRI AML18 version2 trial (NCT02272478). Median follow-up was 35 months. Patients not in MRD-negative remission after course-1 could enter a second randomization between standard versus intensified chemotherapy. Post course-1 response rates were greater after DAGO2 (CR+CRi, 60% vs 47.5%, OR 0.61 95%CI 0.41-0.91, p=0.016). Following course-2 the overall response was not significantly different, 85% for DAGO2 vs 78% for CPX (OR 0.64, 95%CI 0.39-1.09, P=0.095). More patients attained CR with MRD negativity post course-1 in the DAGO2 arm (47% vs 29% for CPX, OR 0.46 95%CI 0.29-0.72, p=0.004). We observed better 3yr EFS (34% vs 27%, HR 0.73 95%CI 0.57-0.93, P=0.012) and OS (52% vs 35%, HR 0.62, 95%CI 0.46-0.83, P=0.001) with DAGO2. In a stratified analysis, CPX did not provide a survival benefit in patients with MDS-related mutations (HR 1.40, 95%CI 0.97-2.03) and was associated with poorer survival in patients with NPM1 (HR 2.83 95%CI 1.17-6.82) and FLT3 mutations (HR 2.14, 95%CI 0.98-4.68). 37% of patients were transplanted in CR1 and this did not differ by randomization. Survival post-transplant did not differ between arms. For patients entering the course-2 randomisation (n=107) survival was equivalent between standard versus intensified CPX doses (P=0.565).In this population of older patients without known adverse-risk cytogenetics, DAGO2 resulted in superior survival compared to CPX. CPX did not benefit those with MDS-related mutations over DAGO2.

Only 30-50% of patients with immune thrombocytopenia (ITP) exhibit a sustained response upon thrombopoietin receptor agonists (TPO-RAs) withdrawal, underscoring the necessity for mechanistic elucidation. We enrolled 49 patients treated with TPO-RAs for four months and performed a follow-up study for three months, classifying them into sustained responders (ITP-sus, n=21), and non-sustained responders (ITP-non-sus, n=28). Compared with total TGF-β1 levels, activated TGF-β1 levels (3854±4380 vs. 943±1500 pg/ml, p＜0.001) were significantly elevated in sustained responders, with integrin αvβ8 regulating TGF-β1 activation and restoring immune tolerance. We established a passive ITP model using PF4-TGF-β1 conditional knockout (CKO) mice, which exhibited a shorter duration of sustained response compared to WT mice. CKO mice demonstrated a reduced Treg population, an increased M1/M2 macrophage ratio, and more severe megakaryocyte destruction following anti-CD41 injection. Exogenous administration of αvβ8 (250 ng/kg) effectively activated TGF-β1 and prolonged remission after TPO discontinuation in WT mice. Additionally, CD4+ T cells were transfected with lentiviral siRNA or shRNA to modulate integrin β8 expression and these were injected into SCID mice undergoing an active model of ITP. Results showed that β8 overexpression increased Treg cells and reduced megakaryocyte damage. Mechanistically, TPO-RAs modulated αvβ8-mediated TGF-β1 activation through the activator protein-1(AP-1) family and Smad-2 signaling pathways. Furthermore, D-mannose combined with TPO prolonged the response in ITP mice by upregulating αvβ8 and activating TGF-β1. Overall, the integrin αvβ8-mediated activation of TGF-β1 pathway represents a promising therapeutic target for ITP, with substantial potential for clinical application.

The major challenges of gene therapy for hemophilia A using adeno-associated virus (AAV) vectors are reducing vector doses and the long-term maintenance of stable factor VIII (FVIII). Here, we developed engineered human B-domain-deleted FVIIIs (FVIIISQs) with enhanced secretion and coagulation potential. Intracellular accumulation was markedly reduced in some engineered FVIIISQs, resulting in reduced unfolded protein responses. The administration of AAV vectors carrying engineered FVIIISQ to hemophilia A mice resulted in approximately eight-fold higher FVIII activity and four-fold higher FVIII antigen levels compared with wild-type FVIIISQ administration. The specific FVIII activity of the engineered FVIIISQ was 3.6 times higher than that of the wild-type FVIIISQ, and its binding to activated coagulation factor IX was significantly enhanced, which is supported by the structural analysis. In macaques, the administration of AAV5 vector carrying the engineered FVIIISQ without CpG sequences resulted in a supra-physiological increase in plasma FVIII activity at a dose one-thirtieth that of valoctocogene roxaparvovec (2 × 1012 vg/kg). The engineered FVIIISQ may thus provide stable, long-term therapeutic efficacy in AAV-mediated hemophilia A gene therapy even at low doses.

Self-renewal and differentiation are at the basis of hematopoiesis. While it is known that tight regulation of translation is vital for hematopoietic stem cells' (HSCs) biology, the mechanisms underlying translation regulation across the hematopoietic system remain obscure. Here we reveal a novel mechanism of translation regulation in the hematopoietic hierarchy, which is mediated by ribosomal RNA (rRNA) methylation dynamics. Using ultra-low input ribosome-profiling, we characterized cell-type-specific translation capacity during erythroid differentiation. We found that translation efficiency changes progressively with differentiation and can distinguish between discrete cell populations as well as to define differentiation trajectories. To reveal the underlying mechanism, we performed comprehensive mapping of the most abundant rRNA modification - 2'-O-methyl (2'OMe). We found that, like translation efficiency, 2'OMe dynamics followed a distinct trajectory during erythroid differentiation.Genetic perturbation of individual 2'OMe sites demonstrated their distinct roles in modulating proliferation and differentiation. By combining CRISPR screening, molecular and functional analyses, we identified a specific methylation site, 28S-Gm4588, which is progressively lost during differentiation, as a key regulator of HSC self-renewal. We showed that low methylation at this site led to translational skewing, mediated mainly by codon frequency, which promoted differentiation. Functionally, HSCs with diminished 28S-Gm4588 methylation exhibited impaired self-renewal capacity ex-vivo, and loss of fitness in-vivo in bone marrow transplantations.Extending our findings beyond the hematopoietic system, we also found distinct dynamics of 2'OMe profiles during differentiation of non-hematopoietic stem cells. Our findings reveal rRNA methylation dynamics as a general mechanism for cell-type-specific translation, required for cell function and differentiation.

Therapy resistance in acute myeloid leukemia (AML) remains a major clinical obstacle, particularly due to the persistence of leukemia stem cells (LSCs) capable of metabolic adaptation. While venetoclax (Ven) inhibits oxidative phosphorylation (OXPHOS), we found that Ven-resistant LSCs undergo glycolytic reprogramming to bypass OXPHOS inhibition. This metabolic shift is supported by enhanced ribosome biogenesis, sustained by upregulated de novo guanine nucleotide biosynthesis. Abundant guanine nucleotides suppress the impaired ribosome biogenesis checkpoint (IRBC), leading to TP53 destabilization and persistent MYC expression. Inhibition of inosine monophosphate dehydrogenases (IMPDH1/2) depletes guanine nucleotides, activates IRBC, stabilizes TP53, represses MYC, and impairs the metabolic shift to glycolysis. This metabolic rewiring disrupts LSC stemness and suppresses the reconstitution of human AML cells in xenotransplantation experiments. Notably, the suppression of LSC stemness was observed regardless of Ven resistance or the TP53 mutational status of AML cells. These findings reveal that mutation-independent TP53 inactivation is involved in resistant AML and suggest that targeting guanine nucleotide biosynthesis may offer a clinically actionable strategy to eradicate therapy-resistant LSCs.

The transcription factor BCL11A is a genetically and clinically validated regulator of the fetal-to-adult hemoglobin switch in human erythroid cells. CRISPR editing of an intronic enhancer within the BCL11A gene reactivates fetal hemoglobin (HbF) in adult erythroid cells, serving as the first CRISPR-based therapy for β-hemoglobinopathies. However, the molecular basis for the remarkable efficacy of CRISPR-mediated enhancer ablation remains elusive. Here, we describe a new genome architecture, an enhancer-dependent chromatin rosette, that is essential for epigenetic insulation and the developmentally regulated, hematopoietic lineage-specific expression of BCL11A. CRISPR-mediated disruption of the BCL11A erythroid enhancer impairs transcription of enhancer-driven RNAs and NIPBL-dependent cohesin loading, leading to destabilization of the rosette structure, loss of chromatin insulation, and epigenetic silencing of BCL11A. Moreover, targeted depletion enhancer RNAs using antisense oligonucleotide silences BCL11A by disrupting epigenetic insulation, causing HbF reactivation in adult erythroid cells. These findings uncover an essential role for enhancer-driven epigenetic insulation in transcriptional control, presenting a new strategy for therapeutic targeting of BCL11A.

Laboratory reference intervals must reflect population diversity for accurate medical decisions. The Duffy null variant lowers absolute neutrophil counts (ANC), but existing dedicated reference intervals are based on a single African American cohort. The impact across other ethnic groups and regions remains unclear, and no white blood cell count (WBC) intervals exist for Duffy null individuals. This study aimed to establish and compare Duffy null ANC and WBC reference intervals across four continents. A cross-sectional study was conducted assessing healthy Duffy null individuals from dedicated cohorts (blood donors in Namibia, Saudi Arabia, and the UK; primary care patients in the USA) and biobanks (participants from the UK and USA). Reference intervals were determined using Clinical & Laboratory Standards Institute guidelines. Among 8,018 participants (880 from dedicated cohorts, 7,138 from biobanks), novel ANC and WBC reference intervals were established: Namibia (820-6,370/µL; 2.51-9.85× 109/L), Saudi Arabia (1,140-5,290/µL; 3.72-10.71× 109/L), UK (1,185-5,462/µL; 3.1-8.8× 109/L) and the USA (1,210-5,390/µL; 3.00-9.66× 109/L), with no significant differences between cohorts. Institutional reference intervals misclassified 27.9% (Namibia), 50.9% (Saudi Arabia), 26.0% (UK) and 21.7% (USA) as neutropenic. Biobank analyses confirmed no significant difference in ANC between Black and non-Black Duffy null participants. Duffy null individuals consistently exhibit lower ANC and WBC across ethnic groups and regions. Current reference intervals overlook this variation, risking misdiagnosis and health inequities. Implementing Duffy-specific reference intervals is essential for equitable and accurate clinical decisions worldwide.

Acquired resistance to targeted, non-intensive therapies is common in myeloid malignancies. However, the kinetics of selection, the hematopoietic cell compartments where selection occurs, and the molecular mechanisms underlying selection remain open questions. To address this, we studied the kinetics of clonal and transcriptional responses to ivosidenib + venetoclax ± azacitidine combination therapy across hematopoiesis in 8 patients with IDH1-mutant myeloid malignancy. All 8 patients initially responded to treatment but 6 relapsed while 2 remained in sustained remission for >4 years. We performed combined high-sensitivity single-cell (sc) genotyping and scRNA-seq in index-sorted sequential patient samples. In all patients, clonal selection occurred rapidly, within 1-3 treatment cycles. Clonal selection preceded treatment failure by months to years. Relapse was associated with expansion of either clones harboring newly-detected myeloid driver mutations or pre-existing minor clones that underwent differentiation delay upon treatment exposure. In both cases, clonal selection occurred within immature cell populations previously shown to contain leukemic stem cell (LSC) potential. Different genetic alterations within relapse-associated clones converged onto common upregulated transcriptional programs of stemness, branched-chain amino acid catabolism, and genes sensitive to menin inhibition. Importantly, this relapse-associated transcriptional signature was selected within 3 cycles of therapy. In contrast, in both patients remaining in remission, leukemic clones were rapidly eradicated and replaced by clonal and wild-type hematopoiesis. Overall, in patients treated with ivosidenib combination therapy, rapid clonal selection occurs within the first treatment cycles. In those patients destined to relapse, genetically heterogeneous resistant clones are characterized by common transcriptional programs.

We previously reported a chemo-genomics screen that unexpectedly identified Phosphatidylinositol-3-phosphate 5 kinase (PIKfyve) as a vulnerable target in multiple myeloma (MM). PIKfyve is an essential regulator of lysosomal function and autophagy. Given the high basal necessity of autophagy in MM for sustainable immunoglobulin synthesis, targeting autophagy holds clinical potential as a novel therapeutic avenue. Here, we report the development and characterization of PIK001 and analogues, potent and selective novel small-molecule inhibitors of PIKfyve. PIK001 demonstrated potent anti-MM activity in vitro, as well as synergistic activity with established anti-MM agents (including venetoclax and selinexor), while retaining efficacy in lenalidomide-resistant models. Multi-omic characterization of isogenic cell lines sensitive / resistant to PIK001 identified a catalytic domain mutation (PIKFYVEN1939K) and heterogenous alterations in autophagy capabilities. Importantly, we noted that PIK001 exposure also resulted in significantly increased cholesterol metabolism and upregulation of MHC Class I expression, with potential implications in tumor immunity. Beyond MM, PIKfyve inhibition also shows selective cytotoxicity in acute myeloid leukemia, melanoma, and renal cancer, highlighting broader therapeutic potential. These findings establish PIKfyve inhibition as a valid target for MM and other hematologic malignancies, provide insights into mechanisms of sensitivity and resistance, and a compelling foundation for further pre-clinical (particularly with respect to the role of cholesterol metabolism and tumor immunity) and clinical development.

Aside from allogeneic transplantation, the current standard of care approach for higher-risk myelodysplastic syndromes/neoplasms (HR-MDS) remains monotherapy with a hypomethylating agent (HMA) including azacitidine, decitabine, or oral decitabine/cedazuridine. Many attempts using HMA-based combinations have failed to improve upon HMA monotherapy. While promising efficacy was observed in early phase clinical trials with several agents, subsequent randomized phase 3 trials failed to confirm improvements in complete response (CR) rates or overall survival. In this review, we discuss lessons learned from the recently reported negative trials of azacitidine in combination with eprenetapopt (APR-246), magrolimab, pevonedistat, sabatolimab, tamibarotene, and venetoclax. First, we make a case for emphasizing biological classification rather than disease risk status alone to select patients for HR-MDS trials. Second, we argue that TP53 inactivated MDS and CMML patients should be treated in dedicated clinical trials. Alternatively, if TP53 inactivated MDS is included in HR-MDS trials, then randomization stratification by TP53 inactivation status should be considered. Third, we caution against ignoring signals of excessive toxicity and premature investigational agent discontinuation observed in early phase trials. Fourth, we show that the International Working Group (IWG) 2006 response criteria, long used in HR-MDS trials, can both overestimate and underestimate the true therapeutic benefit. Instead, we advocate for using the IWG 2023 response criteria to better capture clinically meaningful benefits in HR-MDS. Lastly, we emphasize the need for the scientific community to access patient-level data and samples from failed phase 3 trials in an efficient and expedited fashion to inform the development of subsequent trials.

Classic Hodgkin Lymphoma (cHL) is highly curable with risk-adapted first-line treatment. Due to exceptional efficacy, anti-programmed cell death protein 1 antibodies (aPD1) are increasingly incorporated into first-line treatment. The short- and long-term immune-related adverse event (irAE) burden in this setting, however, is insufficiently understood. Herein, we review the currently available evidence on feasibility and safety of aPD1 first-line cHL treatment. A more harmonized and complete reporting is critical to enable a detailed understanding and comprehensive assessment of aPD1-related morbidity.

High-grade B-cell lymphoma with 11q-aberration (HGBCL-11q) is a rare pediatric non-Hodgkin lymphoma. This study assessed outcome in 90 children with HGBCL-11q. With survival rates ≥95%, patients with HGBCL-11q and no predisposition are candidates for deescalated therapy in future prospective trials.

Acute myeloid leukemia (AML) with TP53 mutations is almost universally refractory to chemotherapy, molecular-targeted therapies, and hematopoietic stem cell transplantation, leading to dismal clinical outcomes. The lack of effective treatments underscores the urgent need for novel therapeutic strategies. Using genome-wide CRISPR/Cas9 dropout screens in isogenic Trp53-wild-type (WT) and knockout (KO) mouse AML models, combined with transcriptomic and proteomic analyses of mouse and human AML samples, we identify the XPO7 (exportin 7)-NPAT (nuclear protein, coactivator of histone transcription) pathway as essential for TP53-mutated AML cell survival. In TP53-WT AML, XPO7 functions as a tumor suppressor by regulating nuclear abundance of p53 protein, particularly when basal levels of functional p53 are high. However, in TP53-mutated AML, XPO7 drives leukemia proliferation by retaining NPAT, an XPO7-associated protein predominantly expressed in TP53-mutated AML, within the nucleus. NPAT depletion induces genome-wide histone loss, compromises genomic integrity, and triggers replication catastrophe in TP53-mutated AML cells. Notably, analysis of publicly available AML datasets, primary AML samples, and single-cell intra-patient mRNA profiles further reveals elevated XPO7 and NPAT expression in TP53-mutated AML. Finally, we validate the XPO7-NPAT pathway as a critical driver of leukemia progression in vivo using patient-derived xenograft (PDX) models of TP53-WT and TP53-mutant AML. Our study delineates key molecular mechanisms underlying TP53-mutated AML pathogenesis and identifies the XPO7-NPAT axis as a critical vulnerability in this refractory leukemia subtype.

Brexu-cel is an autologous anti-CD19 CAR T-cell therapy approved for adults with R/R MCL in the US, and after ≥2 lines of prior therapy, including a BTKi in the EU. Approval was based on the ZUMA-2 Cohort 1 (NCT02601313) study in which brexu-cel demonstrated a 93% objective response rate (ORR) and 67% complete response (CR) rate in patients with R/R MCL and prior BTKi therapy (N=60). Here we report the primary results of ZUMA-2 Cohort 3, brexu-cel in patients with BTKi-naive R/R MCL. Adults received brexu-cel at 2×106 anti-CD19 CAR T cells/kg. The primary endpoint was ORR assessed by independent radiology review committee (IRRC). As of November 26, 2023, 95 patients were enrolled, and 86 received brexu-cel; median follow-up was 15.5 months. The primary endpoint was met, with a 91% ORR (95% CI, 82.5-95.9, P<.0001; N=86) and a CR rate of 73% (95% CI, 62.6-82.2). Estimated 12-month progression-free survival (PFS), duration of response (DOR), and overall survival (OS) rates were 75%, 80% and 90%, respectively. Among 95 enrolled patients, the ORR was 82%, the CR rate was 66%, and the 12-month PFS and OS rates (95% CI) were 73% (62.1-80.8) and 85% (75.6-90.7), respectively. Most patients (88%) experienced treatment-related Grade ≥3 adverse events, including 4 treatment-related Grade 5 events. Consistent with Cohort 1, brexu-cel demonstrated a high ORR and similar safety profile. These results support the continued use of brexu-cel in patients with R/R MCL, and consideration in some patients without prior BTKi therapy who have high-risk disease. This trial is registered at ClinicalTrials.gov as #NCT04880434.

Thrombin is generated from prothrombin through cleavage at two sites by the enzyme prothrombinase, composed of factor (f) Xa and fVa. The affinity of fXa for fVa is low, with assembly and function dependent on phospholipid (PL) membranes. Some snakes have evolved venom versions of fXa that bind to fVa with high affinity and efficiently activate prothrombin in the absence of PL. We created a similar high-affinity, PL-independent human prothrombinase with 17 mutations to human fXa (M17). The increase in affinity enabled cryogenic electron microscopy (cryo-EM) structure determination of M17-prothrombinase to a resolution of 3.3 Å. All protein domains were well resolved in the map, except for the Gla domain of fXa. The main contacts involve the serine protease and EGF2 domains of fXa and the A2 and A3 domains of fVa, resulting in the burying of a total surface area of 4,900 Å2. The map is of sufficient quality to resolve side chain interactions, including several key M17 mutations. To aid in the placement of the loop C-terminal to the A2 domain (a2-loop), we solved a high-resolution crystal structure of fXa in complex with a synthetic a2-peptide. The acidic a2-loop interacts with the basic heparin binding site of fXa, involving a conserved antiparallel b-strand interaction. The M17-prothrombinase structure is compatible with data from biochemical and mutagenesis research, and provides important new insights into the assembly and function of the prothrombinase complex.

Aberrant activation of RAS/MAPK signaling limits the clinical efficacy of several targeted therapies in acute myeloid leukemia (AML). In FLT3-mutant AML, the selection of clones harboring heterogeneous RAS mutations drives resistance to FLT3 inhibitors (FLT3i). RAS activation is also associated with resistance to other AML targeted therapies, including the BCL2 inhibitor venetoclax. Despite the critical need to inhibit RAS/MAPK signaling in AML, no targeted therapies have demonstrated clinical benefit in RAS-driven AML. To address this unmet need, we investigated the preclinical activity of RMC-7977, a multi-selective inhibitor of GTP-bound active [RAS(ON)] isoforms of mutant and wild-type RAS in AML models. RMC-7977 exhibited potent antiproliferative and pro-apoptotic activity across AML cell lines with MAPK-activating signaling mutations. In cell line models with acquired FLT3i resistance due to secondary RAS mutations, treatment with RMC-7977 restored sensitivity to FLT3i. Similarly, RMC-7977 effectively reversed resistance to venetoclax in RAS-addicted cell line models with both RAS wild-type and mutant genetic backgrounds. In murine patient-derived xenograft models of RAS-mutant AML, RMC-7977 was well tolerated and significantly suppressed leukemic burden in combination with gilteritinib or venetoclax. Our findings strongly support clinical investigation of broad-spectrum RAS(ON) inhibition in AML to treat and potentially prevent drug resistance due to activated RAS signaling.

Acute myeloid leukemia (AML) carrying chromosomal rearrangements involving the lysine methyltransferase 2A (KMT2A) gene frequently relapse after allogeneic hematopoietic cell transplantation (allo-HCT). Pharmacological blockade of the menin-KMT2A interaction disrupts the assembly of oncogenic KMT2A complexes on chromatin, thereby attenuating aberrant self-renewal and inducing myeloid differentiation. We found that beyond this anti-leukemic mechanism, menin-inhibition induced CIITA and MHC-II expression in KMT2A-rearranged and NPM1-mutated AML cells in vitro and in vivo. Increased MHC-II expression sensitized AML cells to T-cell mediated elimination after allo-HCT in mice. Menin-inhibition also increased MHC-II expression on primary human AML cells and enhanced the graft-versus-leukemia (GVL) effect in human xenograft models. Mechanistically, menin-inhibition increased expression of multiple human endogenous retroviruses (HERVs) leading to consecutive interferon-stimulated gene (ISG) upregulation and enhanced MHC-II expression. Additionally, menin-inhibition directly promoted anti-tumor effector functions of donor T-cells causing increased TNF-α, IFN-ү, perforin and granzyme A/B production and cytolytic activity. T-cell exhaustion and menin-KMT2A binding to genes encoding for negative regulators of T-cell activation were reduced by menin-inhibition. These findings indicate that menin-inhibition enhances the GVL-effect via the HERV/MHC-II axis in AML cells and promotes cytotoxicity of donor T-cells, which provides a rationale for a clinical trial using menin-inhibition as maintenance after allo-HCT.