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.