Therapy resistance in acute myeloid leukemia (AML) remains a major clinical obstacle, particularly because of the persistence of leukemia stem cells (LSC) capable of metabolic adaptation. Although venetoclax (Ven) inhibits oxidative phosphorylation (OXPHOS), we found that Ven-resistant LSC undergo glycolytic reprogramming to bypass OXPHOS inhibition. This metabolic shift is supported by enhanced ribosome biogenesis, which is 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. The 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 LSC.

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 the destabilization of the rosette structure, loss of chromatin insulation, and epigenetic silencing of BCL11A. Moreover, targeted depletion of enhancer RNAs using antisense oligonucleotides 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 the 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 4 continents. A cross-sectional study was conducted assessing healthy Duffy-null individuals from dedicated cohorts (blood donors in Namibia, Saudi Arabia, and the United Kingdom; primary care patients in the United States) and biobanks (participants from the United Kingdom and the United States). Among 8018 participants (880 from dedicated cohorts and 7138 from biobanks), novel ANC and WBC reference intervals were established (Namibia [ANC, 820/μL to 6370/μL; WBC, 2.51 × 109/L to 9.85 × 109/L]; Saudi Arabia [ANC, 1140/μL to 5290/μL; WBC, 3.72 × 109/L to 10.71 × 109/L]; United Kingdom (ANC, 1185/μL to 5462/μL; WBC, 3.1 × 109/L to 8.8 × 109/L]; the United States [ANC, 1210/μL to 5390/μL; WBC, 3.00 × 109/L to 9.66 × 109/L]), with no significant differences between cohorts. Institutional reference intervals misclassified 27.9% (Namibia), 50.9% (Saudi Arabia), 26.0% (United Kingdom), and 21.7% (the United States) 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, nonintensive therapies is common in myeloid malignancies. However, the kinetics of selection, the hematopoietic cell compartments in which selection occurs, and the molecular mechanisms underlying selection remain open questions. To address this, we studied the kinetics of clonal and transcriptional responses to combinational therapy with ivosidenib plus venetoclax, with or without azacitidine, across hematopoiesis in 8 patients with IDH1-mutant myeloid malignancy. All 8 patients initially responded to treatment, but 6 relapsed, whereas 2 remained in sustained remission for >4 years. We performed combined high-sensitivity single-cell genotyping and scRNA sequencing in index-sorted sequential patient samples. In all patients, clonal selection occurred rapidly, within 1 to 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 preexisting 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 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 chemogenomics 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 requirement for 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 analogs, 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. Multiomic characterization of isogenic cell lines sensitive and resistant to PIK001 identified a catalytic domain mutation (PIKFYVE N1939K) and heterogenous alterations in autophagy capabilities. Importantly, we noted that PIK001 exposure also resulted in significantly increased cholesterol metabolism and upregulation of major histocompatibility complex (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 lay the foundation for further preclinical (particularly 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. Although promising efficacy was observed in early-phase clinical trials with several agents, subsequent randomized phase 3 trials failed to confirm improvements in complete response 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 patients with TP53-inactivated MDS and chronic myelomonocytic leukemia 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 discontinuation of investigational agent 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, antiprogrammed cell death protein 1 antibodies (aPD1) are increasingly incorporated into first-line treatment. However, the short- and long-term immune-related adverse event burden in this setting is insufficiently understood. Here, we review the currently available evidence on the 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 Trp53-knockout mouse AML models, combined with transcriptomic and proteomic analyses of AML samples from mice and humans, we identify the XPO7-NPAT (exportin 7-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 the 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, the analysis of publicly available AML data sets, primary AML samples, and single-cell intrapatient 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 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.

Brexucabtagene autoleucel (brexu-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for adults with relapsed/refractory (R/R) mantle cell lymphoma (MCL) based on the ZUMA-2 cohort 1 (ClinicalTrials.gov identifier: 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 previous 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 per kilogram. The primary end point was ORR assessed by independent radiology review committee (IRRC). As of 26 November 2023, 95 patients were enrolled, and 86 received brexu-cel; median follow-up was 15.5 months. The primary end point was met, with a 91% ORR (95% confidence interval [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, 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 previous BTKi therapy who have high-risk disease. This trial was registered at clinicaltrials.gov as #NCT04880434.

Thrombin is generated from prothrombin through cleavage at 2 sites by the enzyme prothrombinase, composed of factor Xa (fXa) 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 γ-carboxyglutamic acid domain of fXa. The main contacts involve the serine protease and epidermal growth factor-like domain 2 (EGF2) domains of fXa and the A2 and A3 domains of fVa, resulting in the burying of a total surface area of 4900 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 β-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 FMS-like tyrosine kinase-3 (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, such as the B-cell lymphoma 2 inhibitor venetoclax. Despite the critical need to inhibit RAS/MAPK signaling in AML, no targeted therapies have demonstrated a clinical benefit in RAS-driven AML. To address this unmet need, we investigated the preclinical activity of RMC-7977, a multiselective inhibitor of GTP-bound active (RAS[ON]) isoforms of mutant and wild-type RAS in AML models. RMC-7977 exhibited potent antiproliferative and proapoptotic activity across AML cell lines with MAPK-activating signaling mutations. In cell line models with acquired FLT3i resistance because of 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 because of activated RAS signaling.