NPM1-mutated (NPM1-mut) acute myeloid leukemia (AML) is generally associated with a more favorable outcome, although the presence of additional gene mutations can influence patient prognosis. We analyzed intensively-treated adult NPM1-mut AML patients included in the HARMONY Alliance database. A newly developed risk classification, which included combinations of co-mutations in FLT3-ITD, DNMT3A, IDH1/IDH2, and TET2 genes, was applied to a training cohort of NPM1-mut AML patients included in clinical trials (n = 1001), an internal validation cohort more representative of real-world settings (n = 762), and an external validation cohort enrolled in UK-NCRI trials (n = 585). The HARMONY classification considered 51.8% of the NPM1-mut AML training cohort patients as favorable, 24.8% as intermediate, and 23.4% as adverse risk, with median overall survival (OS) of 14.4, 2.2, and 0.9 years, respectively; p < 0.001), thereby reclassifying 42.7% of NPM1-mut patients into a different European LeukemiaNet (ELN) 2022 risk category. These results were confirmed both in an internal and external validation cohort. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR1) showed the highest benefit in the NPM1-mut adverse-risk subgroup. The HARMONY classification provides the basis for a refined genetic risk stratification for adult NPM1-mut AML with potential clinical impact on allo-HSCT decision-making.

Neuronal specification, expansion and differentiation are tightly regulated by the concerted actions of transcription and chromatin modifying factors that are recruited to regulatory elements in the genome. Tissue-specific distal regulatory elements are typically located tens to hundreds of kilobases from the gene they regulate. To identify the distal enhancers that directly regulate a gene, information on the localisation of enhancers relative to the gene promoter in the nucleus is crucial. Cerebellar granule cell progenitors (GCps) are important transit amplifying neuronal progenitors, giving rise to the most abundant neuronal cell type in the brain. Many of the key factors that regulate fundamental developmental processes in GCps have been identified. For instance, the proneural transcription factor Atoh1 is essential for GCp specification, proliferation and differentiation and the ATP-dependent chromatin remodeller CHD7 is necessary for normal GCp proliferation and differentiation. However, both these factors are recruited to distal regulatory elements and the direct regulatory relationships between these factors, the enhancers they are recruited to, and the genes they regulate in GCps remain uncharacterised. To identify active, long-range gene regulatory interactions in GCps, we used promoter capture Hi-C (pcHi-C), together with ATAC-seq and ChIP-seq data. We present a rich dataset consisting of 46,428 interactions between 22,797 putative distal regulatory regions and 12,905 protein coding gene promoters in primary mouse GCps. Using VISTA-designated hindbrain enhancers as an example, we show that 80% of these enhancers are incorrectly annotated at present and identify the genes most likely regulated directly by these enhancers. Motif enrichment analyses showed a significant enrichment of proneural transcription factor motifs in CHD7-regulated enhancers. Further analyses revealed co-localisation of Atoh1 and CHD7 at gene enhancers, suggesting a novel regulatory relationship between Atoh1 and CHD7 in controlling the expression of key genes in the GCp lineage. We used our data to identify >1,500 Atoh-regulated enhancers, controlling the expression of 577 genes in GCps, and 197 enhancers of 22 genes that appear to be co-regulated by Atoh1 and CHD7. Co-immunoprecipitation experiments showed that Atoh1 and CHD7 interacted with each other. These findings support the emerging picture of CHD7 as an important gene regulatory co-factor for lineage-specific transcription factors. The pcHi-C data is presented as a useful resource to the community for investigating the function of long-range enhancers in the cerebellar GCp lineage.

6521 Background: Intensive chemotherapy (IC; cytarabine plus an anthracycline) remains a standard first-line therapy for fit patients with acute myeloid leukemia (AML), a heterogeneous hematologic malignancy with poor long-term survival. IC induces composite complete remission (cCR) in ~60–70% of cases, and IC response variability persists within genetic/ELN risk strata. As emerging regimens increasingly challenge IC as a default therapy, biology-informed predictors are needed to identify patients unlikely to benefit from IC to avoid unnecessary toxicity. Mass spectrometry (MS)-based phosphoproteomics enables quantitative profiling of phosphopeptides (PPs) from patient samples, providing a functional readout of tumor biology. Thus, we sought to develop a PP-based clinically-deployable assay, orthogonal to genetic risk stratification, to predict response to IC in AML. Methods: 261 retrospective samples were collected from patients with newly-diagnosed AML (Table) treated with standard “7+3” IC in 9 centers in Europe, Australia, and North America. Response was assessed at the end of induction by the treating physician. Samples underwent phosphoproteomics using: 1) global MS for biomarker discovery or 2) targeted, clinically-compatible MS for validation. Predictive model performance was evaluated using rebalanced leave-one-out cross-validation. Results: During discovery, 3205 PPs were detected. Using Bayesian approaches, we identified an 80-PP multi-analyte signature of IC response (refractory vs cCR), and achieved an AUROC of 0.68 (95% CI 0.54-0.83). The signature was enriched for DNA damage signalling and repair (DNA-PK-S2612, ATM/cohesin, nucleotide excision and double-strand break repair proteins), and cellular stress pathways (phospho-p38, IL-16, AP-1/c-Jun). In an independent validation cohort, the targeted assay reliably detected the biomarker set and preserved its association with outcome (AUROC 0.69; 95% CI 0.53-0.84). Biomarker-based classification was associated with improved event-free survival (HR 0.45; 95% CI 0.26-0.78). Conclusions: We identified and validated a signature of response to IC in AML, and translated it into a targeted, clinically-deployable assay. This approach captures signaling states relevant to IC mechanisms of action that are not directly inferred from standard clinical or genetic variables. Ongoing analyses are evaluating its relationship to established genetic and ELN risk stratification. These findings support diagnostic phosphoproteomics and suggest functional biomarkers may complement existing approaches for treatment selection in AML. Cohort characteristics. Cohort Discovery Validation Median age at diagnosis (years, quartiles) 54 (23, 65) 59 (47, 68) Median diagnosis year (range) 2014 (1999, 2023) 2013 (2001, 2024) No of patients/samples 135/165 102/106 No of cCR/Refractory 94/41 79/23 PPs 3205 80

Background:Clonal hematopoiesis (CH) is associated with increased risks of diverse cardiovascular diseases, hematologic malignancies and mortality, yet no preventive therapies are approved. As emerging data implicate lipid pathways in CH pathogenesis, we investigated the association of statin use and genetically proxied inhibition of HMG-CoA reductase (HMGCR) with CH risk, and validated findings using primary peripheral blood mononuclear cells (PBMCs). Methods:We performed an observational analysis of 416,118 UK Biobank participants of European ancestry using multivariable logistic regression to compare CH prevalence among statin users and nonusers. Mendelian randomization (MR) analyses evaluated the causal association of genetically proxied lowering of low-density lipoprotein cholesterol (LDL-C) with risk of CH using two instruments; (i) the lead HMGCR variant (rs12916) which proxied LDL-C lowering by statins, and, (ii) 303 genome-wide LDL-C-lowering variants representing polygenic mechanisms. Summary statistics were obtained from the Global Lipid Genetics Consortium genome-wide association study (N = 842,634). Experimentally, primary PBMCs from a DNMT3A R882 hotspot mutation carrier were cultured in methylcellulose with pravastatin or vehicle control to evaluate colony-forming dynamics. Results:Among 416,118 individuals, 20,488 had CH, including 11,550 with single DNMT3A-mutant and 4,375 with single TET2-mutant CH. Pre-recruitment statin users had reduced odds of DNMT3A-mutant CH (OR=0.93; 95% CI:0.88-0.98; P=0.009), driven primarily by associations with DNMT3A R882-mutant (OR=0.78; 95% CI:0.66-0.92; P=0.003), but not TET2-mutant CH (OR=1.05; 95% CI:0.97-1.14; P=0.20). Similarly, genetically predicted HMG-CoA-reductase inhibition equivalent to a 1 SD reduction in circulating LDL-C levels was associated with lower odds of DNMT3A-mutant CH (OR=0.66; 95% CI:0.45-0.95; P=0.03) but not TET2-mutant CH (OR=1.34; 95% CI:0.76-2.36; P = 0.31). By contrast, polygenic estimation of LDL-C lowering was not associated with DNMT3A-mutant CH (OR=1.05; 95% CI:0.97-1.14; P=0.20), suggesting protective effects were independent of LDL-C lowering per se. Genetically predicted HMG-CoA reductase inhibition had wide effects on blood cell counts and indices, suggesting effects on bone marrow cell dynamics. In vitro, pravastatin selectively suppressed colony formation of primary human DNMT3A R882-mutant relative to wild-type cells (P=0.031). Conclusions:Statin therapy and genetically predicted lifelong inhibition of HMG-CoA reductase were significantly associated with reduced risk of DNMT3A-mutant CH, likely via LDL-C-independent mechanisms, which may be specific to DNMT3A-mutant CH. This provides a strong rationale for prospective trials evaluating the effect of statins on risk of developing DNMT3A-mutant CH, subsequent clonal expansion, and associated clinical sequelae.

Leukaemia arises through the stepwise transformation of healthy haematopoietic cells, yet the asymptomatic premalignant phase and its progression to overt disease remain poorly understood. To model this process, we engineered a patient-derived CEBPA mutation into Hoxb8-FL multipotent murine progenitors and transplanted them into syngeneic mice, capturing a clinically silent premalignant stage. All recipients developed overt disease after ~12 months with 100% penetrance and all acquired secondary RTK-RAS mutations, often with identical amino acid changes to those in patients. Single-cell transcriptomics and phenotypic profiling showed that premalignant mutant cells adopt a plasmacytoid dendritic progenitor-like state in vitro which generates both myeloid and B-lymphoid lineages during premalignancy in vivo, with individual tumours restricted to one lineage. The specificity for RTK-RAS mutations coupled with ongoing differentiation, reflects clinically relevant biological contexts thus providing a tractable model of myeloid neoplasm for mechanistic studies and drug discovery.

Protein tyrosine kinases activate signaling pathways by catalyzing the phosphorylation of tyrosine residues in their substrates. Mounting evidence suggests that, in addition to recognizing phosphorylated tyrosine (pTyr) residues through specific phosphobinding modules, many protein kinases selectively recognize pTyr directly adjacent to the tyrosine residue they phosphorylate and catalyze the formation of twin pTyr-pTyr sites. Here, we demonstrate the importance of this phosphopriming-driven twin pTyr signaling in promoting cell cycle progression through the cell cycle-inhibitory protein p27Kip1. We identify, structurally resolve, and tune two distinct molecular determinants driving the selective recognition of pTyr directly N- and C-terminal to the target phospho-acceptor tyrosine site. We further show structural and biochemical conservation in this recognition, and identify cancer-associated alterations to these determinants that are unable to recognize phosphoprimed substrates. Finally, using an in vivo mouse model of leukemia we show that Bcr-Abl mutants unable to recognize phosphoprimed substrates paradoxically result in enhanced tumor development and progression. These data indicate that Bcr-Abl, like other proto-oncogenes such as Ras or Myc, engages both pro- and anti-oncogenic programs - but in the case of Bcr-Abl, this is accomplished through a mechanism involving traditional and phosphoprimed substrate recognition.

B-cell acute lymphoblastic leukemia (B-ALL) is a leading cause of death in childhood and outcomes in adults remain dismal. There is therefore an urgent clinical need for therapies that target the highest risk cases. Mutations in the histone acetyltransferase CREBBP confer high-risk and increased chemoresistance in ALL. Performing a targeted drug-screen in isogenic human cell lines, we identify a number of small molecules that specifically target CREBBP-mutated B-ALL, the most potent being the BCL2-inhibitor Venetoclax. Of note, this acts through a non-canonical mechanism resulting in ferroptotic rather than apoptotic cell death. CREBBP-mutated cell lines show differences in cell-cycle, metabolism, lipid composition and response to oxidative stress, predisposing them to ferroptosis, which are further dysregulated upon acquisition of Venetoclax resistance. Lastly, small-molecule inhibition of CREBBP pharmacocopies CREBBP-mutation, sensitizing B-ALL cells, regardless of genotype, to Venetoclax-induced ferroptosis in-vitro and in-vivo, providing a promising drug combination for broader clinical translation in B-ALL.

ABSTRACT:NPM1 is a multifunctional phosphoprotein with key roles in ribosome biogenesis among its many functions. NPM1 gene mutations drive 30% of acute myeloid leukemia (AML) cases. The mutations disrupt a nucleolar localization signal and create a novel nuclear export signal, leading to cytoplasmic displacement of the protein (NPM1c). NPM1c mutations prime hematopoietic progenitors to leukemic transformation, but their precise molecular consequences remain elusive. Here, we first evaluate the effects of isolated NPM1c mutations on the global proteome of preleukemic hematopoietic stem and progenitor cells (HSPCs) using conditional knockin Npm1cA/+ mice. We discover that many proteins involved in ribosome biogenesis are significantly depleted in these murine HSPCs, but also importantly in human NPM1-mutant AMLs. In line with this, we found that preleukemic Npm1cA/+ HSPCs display higher sensitivity to RNA polymerase I inhibitors, including actinomycin D (ActD), compared with Npm1+/+ cells. Combination treatment with ActD and venetoclax inhibited the growth and colony-forming ability of preleukemic and leukemic NPM1c+ cells, whereas low-dose ActD treatment was able to resensitize resistant NPM1c+ cells to venetoclax. Furthermore, using data from CRISPR dropout screens, we identified and validated TSR3, a 40S ribosomal maturation factor whose knockout preferentially inhibited the proliferation of NPM1c+ AML cells by activating a p53-dependent apoptotic response. Similarly, to low-dose ActD treatment, TSR3 depletion could partially restore sensitivity to venetoclax in therapy-resistant NPM1c+ AML models. Our findings propose that targeted disruption of ribosome biogenesis should be explored as a therapeutic strategy against NPM1-mutant AML.

Somatic DNMT3A-R882 codon mutations drive the most common form of clonal haematopoiesis (CH) and are associated with increased acute myeloid leukaemia (AML) risk1,2. Preventing expansion of DNMT3A-R882-mutant haematopoietic stem/progenitor cells (HSPCs) may therefore avert progression to AML. To identify DNMT3A-R882-mutant-specific vulnerabilities, we conducted a genome-wide CRISPR screen on primary mouse Dnmt3aR882H/+ HSPCs. Among the 640 vulnerability genes identified, many were involved in mitochondrial metabolism, and metabolic flux analysis confirmed enhanced oxidative phosphorylation use in Dnmt3aR882H/+ versus Dnmt3a+/+ (WT) HSPCs. We selected citrate/malate transporter Slc25a1 and complex I component Ndufb11, for which pharmacological inhibitors are available, for downstream studies. In vivo administration of SLC25A1 inhibitor CTPI2 and complex I inhibitors IACS-010759 and metformin suppressed post-transplantation clonal expansion of Dnmt3aR882H/+, but not WT, long-term haematopoietic stem cells. The effect of metformin was recapitulated using a primary human DNMT3A-R882 CH sample. Notably, analysis of 412,234 UK Biobank participants showed that individuals taking metformin had a markedly lower prevalence of DNMT3A-R882-mutant CH, after controlling for potential confounders including glycated haemoglobin, diabetes and body mass index. Collectively, our data propose modulation of mitochondrial metabolism as a therapeutic strategy for prevention of DNMT3A-R882-mutant AML.

Mutation of some genes drives uncontrolled cell proliferation and cancer. The Philadelphia chromosome in chronic myeloid leukaemia (CML) provided the very first such genetic link to cancer 1,2 . However, little is known about the trajectory to CML, the rate of BCR::ABL1 clonal expansion and how this affects disease. Using whole-genome sequencing of 1,013 haematopoietic colonies from nine patients with CML aged 22 to 81 years, we reconstruct phylogenetic trees of haematopoiesis. Intronic breaks in BCR and ABL1 were not always observed, and out-of-frame exonic breakpoints in BCR , requiring exon skipping to derive BCR::ABL1 , were also noted. Apart from ASXL1 and RUNX1 mutations, extra myeloid gene mutations were mostly present in wild-type cells. We inferred explosive growth attributed to BCR::ABL1 commencing 3–14 years (confidence interval 2–16 years) before diagnosis, with annual growth rates exceeding 70,000% per year. Mutation accumulation was higher in BCR::ABL1 cells with shorter telomere lengths, reflecting their excessive cell divisions. Clonal expansion rates inversely correlated with the time to diagnosis. BCR::ABL1 in the general population mirrored CML incidence, and advanced and/or blast phase CML was characterized by subsequent genomic evolution. These data highlight the oncogenic potency of BCR::ABL1 fusion and contrast with the slow and sequential clonal trajectories of most cancers.

Immunotherapies for acute myeloid leukemia (AML) and other cancers are limited by a lack of tumor-specific targets. Here we discover that RNA-binding proteins and glycosylated RNAs (glycoRNAs) form precisely organized nanodomains on cancer cell surfaces. We characterize nucleophosmin (NPM1) as an abundant cell surface protein (csNPM1) on a variety of tumor types. With a focus on AML, we observe csNPM1 on blasts and leukemic stem cells but not on normal hematopoietic stem cells. We develop a monoclonal antibody to target csNPM1, which exhibits robust anti-tumor activity in multiple syngeneic and xenograft models of AML, including patient-derived xenografts, without observable toxicity. We find that csNPM1 is expressed in a mutation-agnostic manner on primary AML cells and may therefore offer a general strategy for detecting and treating AML. Surface profiling and in vivo work also demonstrate csNPM1 as a target on solid tumors. Our data suggest that csNPM1 and its neighboring glycoRNA-cell surface RNA-binding protein (csRBP) clusters may serve as an alternative antigen class for therapeutic targeting or cell identification.

Transcription is regulated in a multitude of ways to ensure lineage- and context-specific gene expression in a coordinated fashion. Hematopoiesis is an exemplary process for studying the mechanisms of tightly regulated activation and repression of gene expression programs through transcription and gene regulatory complexes. These complexes act by posttranslational modification of histones and nonhistone proteins, epigenetic modifications of DNA, ATP-dependent chromatin remodeling, scaffolding and recruitment of combinatorial protein complexes, and alteration of three-dimensional genome conformation to bring about lineage-specific gene expression. This review will focus on the function of these gene regulatory complexes in hematopoiesis and how they are hijacked in acute myeloid leukemia, highlighting therapeutic progress and opportunities.

The chromatin regulator MLL2 (KMT2B) is the primary histone 3 lysine 4 (H3K4) trimethyltransferase acting at bivalent promoters in embryonic stem cells (ESCs) and is required for differentiation toward neuroectoderm. Here, we demonstrate that this requirement occurs during exit from naive pluripotency, days before neuroectoderm differentiation is impaired. During exit, the effect of MLL2 on transcription is subtle, increasing the expression of a few important neuroectodermal transcription factors. In contrast, MLL2's effect on chromatin architecture is substantial, stabilising loops associated with bivalent promoters in primed ESCs. MLL2 H3K4 catalytic activity is dispensable for stabilising these loops during ESC exit and for neuroectoderm differentiation. We therefore identify a non-catalytic function for MLL2 in stabilising 3D chromatin architecture, which has implications for lineage specification. Because MLL2 shares features with all four MLLs, we propose that chromatin tethering, rather than H3K4 methylation, represents a primary function for MLLs during lineage commitment decisions. ### Competing Interest Statement The authors have declared no competing interest.

The World Health Organization fifth edition and International Consensus Classification for myeloid neoplasms both incorporate empirical numerical thresholds to morphologic and molecular features defining certain disease entities. However, the clinical implications of these thresholds remain unclear. We analyzed a large cohort (N = 6,976) of patients with myeloid neoplasms to evaluate the impact of proposed yet different numerical thresholds for variant allele frequency of genetic mutations or hematologic parameters set forth by the World Health Organization fifth edition and International Consensus Classification for classification of SF3B1-mutated myelodysplastic neoplasms, NPM1-mutated acute myeloid leukemia (AML), and oligomonocytic chronic myelomonocytic leukemia. Our analysis demonstrated that the clonal burden of SF3B1 mutation in myelodysplastic neoplasms informs classification and prognosis. Our findings support the notion that NPM1 mutation should be AML-defining regardless of blast percentage and highlight the adverse prognostic impact of the cumulative number of myelodysplasia-related mutations in NPM1-mutated AML. Finally, we provide evidence that integrating specific molecular signatures could improve the accuracy of oligomonocytic chronic myelomonocytic leukemia classification. SIGNIFICANCE:Using comprehensive clinical and molecular profiling, this study provides a data-driven approach for evaluating numerical thresholds of variant allele frequency or hematologic parameters (i.e., blast percentage and absolute monocyte count) included in current classification schemas across a spectrum of myeloid malignancies, enabling refinement of disease classification and prognostication.

Cardiac fibrosis is mediated by the persistent activity of myofibroblasts, which differentiates from resident cardiac fibroblasts in response to tissue damage and stress signals. The signaling pathways and transcription factors regulating fibrotic transformation have been thoroughly studied. In contrast, the roles of chromastin factors in myofibroblast differentiation and their contribution to pathogenic cardiac fibrosis remain poorly understood. Here, we combined bulk and single-cell CRISPR screens to characterize the roles of chromatin factors in the fibrotic transformation of primary cardiac fibroblasts. We uncover strong regulators of fibrotic states including Srcap and Kat5 chromatin remodelers. We confirm that these factors are required for functional processes underlying fibrosis including collagen synthesis and cell contractility. Using chromatin profiling in perturbed cardiac fibroblasts, we demonstrate that pro-fibrotic chromatin complexes facilitate the activity of well-characterized pro-fibrotic transcription factors. Finally, we show that KAT5 inhibition alleviates fibrotic responses in patient-derived human fibroblasts.

Background Myelofibrosis (MF) is a chronic myeloproliferative neoplasm causing progressive splenomegaly, cytopenias, systemic symptoms and mortality. Ruxolitinib (rux), a JAK 1/2 inhibitor, is an approved treatment for MF which effectively controls disease related symptoms and splenomegaly in some patients. However, disease control is often inadequate and there is eventual disease progression. A major unmet medical need remains. In mouse models of MF, the effects of rux are complemented by epigenetic inhibitors targeting BET (bromodomain and extra-terminal motif) proteins and combinations of BET and JAK inhibitors have shown promising initial clinical results. The PROMise clinical study was designed to test the hypothesis that the short-acting BET inhibitor OPN-2853 (previously called PLX2853) (T1/2 = 1.3 hrs) would combine with rux to restore disease control in patients with inadequate disease control with single agent rux. Preclinical studies of OPN-2853 revealed potent ability to inhibit splenomegaly in a Ba/F3 model, and combinability with rux to delay disease in a SET-2 MF model. Design and Methods PROMise is a phase I, multicentre, dose finding trial being conducted in the UK to identify a safe and tolerable recommended Phase II Dose (RP2D) of OPN-2853 in combination with rux and to assess the efficacy of this combination in reducing spleen size in patients with high or intermediate-2 risk MF who are not adequately responding to rux alone. PROMise investigates three potential daily dose levels of OPN-2853: 20 mg, 40 mg and 80 mg. Eligible patients must be ≥ 16 years, have been on rux for at least 24 weeks, with a stable dose for at least 4 weeks, and must have persistent splenomegaly extending at least 5cm below the costal margin. A maximum of 60 patients will be recruited across three rux dose groups: Low-Dose (5-20 mg daily), Mid-Dose (25-45 mg daily), and High-Dose (≥50 mg daily). Each rux group is evaluated separately, with dose recommendations made using the continual reassessment method, once 2 to 4 evaluable patients in the group have completed the first 21-day cycle. A co-primary outcome for the trial is a >50% reduction in palpable spleen size from screening at the end of 8 cycles. Spleen size is also evaluated by serial ultrasound. Serial blood and bone marrow samples are collected to measure molecular response, on-target transcriptional/epigenetic responses in myeloid and stem cells and other translational research exploratory endpoints. Additionally, pharmacokinetic samples are taken at specified time points to establish the PK properties of OPN-2853 in combination with rux. Current Status The PROMise trial is currently ongoing and open to recruitment. As of 12-Feb-2024, 16 patients have been evaluated; 6 were on low-dose rux, 8 on mid-dose and 2 on high-dose. Median age was 71 years with a median spleen size of 9 cm at screening. Median haemoglobin was 102 g/L. 56% of patients have primary myelofibrosis, 44% secondary myelofibrosis, 18% and 82% with MF-2 and MF-3, respectively. Whilst the RP2D is still being determined, early findings indicate that the combination therapy is well-tolerated with the majority of patients (10 of 16) completing 8 cycles of combination treatment. Two dose limiting toxicities have occurred to date (thrombocytopenia and raised liver transaminases) and all study arms are now recruiting at 80 mg dose level. Notably, grade 3 or above adverse events were infrequent with platelet count reduction (n=5, 31%) and anemia (n=2, 12.5%) the most common in patients receiving combination treatment. The median(range) spleen size, calculated as the change from baseline to minimum post-baseline spleen size, has reduced by 5 (0, 10) cm. Conclusion The ongoing PROMise study (EudraCT 2019-000916-27) combines a daily dose of OPN-2853 with standard of care ruxolitinib to test the hypothesis that a continuous daily dosing regimen of oral agents will improve disease burden. Encouraging levels of spleen reduction have been observed in the context of a well tolerated agent.

Asciminib is a potent and selective inhibitor of BCR::ABL1, with potential to avoid toxicity resulting from off-target kinase inhibition. Forty-nine patients treated with asciminib under a managed access program in the UK were evaluated for toxicity and response. Intolerance, rather than resistance (65% vs. 35%), was the most common reason for cessation of the last-line of treatment but asciminib was well tolerated, with most patients (29, 59%) remaining on treatment at a median of 14 months follow-up, and only 6 (12%) stopping for intolerance. Of 44 patients assessable for response, 29 (66%) achieved a complete cytogenetic response (CCyR) or better, with poorer responses seen in those stopping their last-line of therapy for resistance. Fewer patients with a prior history of a non-T315I-BCR::ABL1 single nucleotide variant (BSNV), or a non-T315I-BSNV detectable at baseline achieved CCyR. Serial tracking of BSNV by next generation sequencing demonstrated clonal expansion of BSNV-harbouring populations, which in some settings was associated with resistance (E459K, F317L, F359I), while in others was seen in the context of ongoing response, often with intensified dosing (T315I, I502F). These data suggest that asciminib exerts selective pressure on some BSNV-harbouring populations in vivo, some of which may respond to intensified dosing.

Although NPM1-mutated acute myeloid leukemia (AML) carries a generally favorable prognosis, many patients still relapse and die. Previous studies identified several molecular and clinical features associated with poor outcome, however only FLT3-ITD mutation and adverse karyotype are currently used for risk stratification due to inconsistent results and uncertainty around how other factors should influence treatment, particularly given the strong prognostic impact of post-induction measurable residual disease (MRD). Here we analyzed a large group of patients with NPM1mut AML enrolled in prospective trials (NCRI AML17 and AML19, n=1357) to delineate the impact of baseline molecular and clinical features, post induction MRD status and treatment intensity on outcome. FLT3-ITD (HR 1.28, 95%CI 1.01-1.63), DNMT3A (HR 1.65, 95%CI 1.32-2.05), WT1 (HR 1.74, 95%CI 1272-2.38) and non-ABD NPM1 mutations (HR 1.64, 95%CI 1.22-2.21) were independently associated with poorer overall survival (OS). These factors were also strongly associated with MRD positivity. For patients achieving MRD negativity, these mutations (except FLT3-ITD) were associated with an increased cumulative incidence of relapse (CIR) and poorer OS. However, apart from the few patients with adverse cytogenetics, we could not identify any group of MRD negative patients with a CIR >40% or with benefit from allograft in first remission. Intensified chemotherapy with the FLAG-Ida regimen was associated with improved outcomes in all subgroups, with greater benefits observed in the highest risk molecular subgroups.