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.

The Group for Research in Adult Acute Lymphoblastic Leukemia (GRAALL)-2014 trial (ClinicalTrials.gov: NCT02617004, NCT02619630) evaluated an intensive, age-adapted protocol for adults aged 18-59 years with Philadelphia chromosome-negative acute lymphoblastic leukemia (ALL). The trial was motivated by findings from the previous GRAALL-2005 study, which reported excessive toxicity from pediatric-inspired therapy in older patients and no added benefit from allogeneic hematopoietic stem cell transplantation (alloHSCT) in those with an early favorable response to treatment. Thus, the GRAALL-2014 protocol aimed to reduce treatment-related toxicity in patients aged ≥45 years and to limit alloHSCT to patients with poor measurable residual disease (MRD) responses. A total of 743 patients was included, and outcomes were compared to those of the GRAALL-2005 trial. The GRAALL-2014 demonstrated reduced early mortality and higher complete remission rates in patients aged ≥45 years. MRD-guided transplant decisions reduced alloHSCT indications by approximately 50%. While older patients experienced a higher cumulative incidence of relapse, no significant difference in disease-free survival (DFS) was observed compared to historical cohorts across age subgroups. The overall 4-year DFS was 57.1% (95% CI, 53.4-61.1). Notably, 4-year overall survival improved significantly, from 65.5% (95%CI, 61.7-69.8) to 71.7% (95%CI, 67.7-76.0) in younger patients (p=0.031) and from 49.6% (95%CI, 43.5-56.5) to 59.5% (95%CI, 53.5-66.3) in older patients (p=0.011). These findings highlight the value of individualized treatment strategies, balancing efficacy and safety. Future studies should investigate the integration of immunotherapy to further reduce treatment intensity and improve outcomes.