The repair of pathological gene variants is an ultimate aim for treating genetic diseases; however, the development of different therapeutic reagents for each of the many variants that can occur in a gene may not be scalable. Here, we investigated whether base editing to introduce a gain-of-function variant in blood coagulation factor IX (FIX) can increase FIX activity as a targeted therapeutic approach for hemophilia B. We engineered a G:C to A:T substitution at c.1151 of F9 by cytosine base editing to generate R338Q, known as the Shanghai F9 variant, which markedly potentiates coagulation factor activity. An adeno-associated virus vector harboring the base editor converted more than 60% of the target G:C to A:T and increased FIX activity in HEK293 cells harboring patient-derived F9 variants, as well as in knock-in mice harboring a human F9 cDNA. Furthermore, administration of lipid nanoparticles embedded with the base editor mRNA and gRNA increased FIX activity in mice. These data indicate that cytosine base editing to generate R338Q in FIX is a broadly applicable genome editing approach for hemophilia B with residual FIX activity.

GPRC5D has emerged as a promising therapeutic target in relapsed/refractory multiple myeloma (RRMM), particularly following progression after BCMA-directed CAR T-cell therapies. RD118 is a novel CAR T-cell therapy incorporating a fully human single-domain antibody fragment (VHH) targeting GPRC5D. In this phase 1 study, 18 relapsed/refractory patients (17 multiple myeloma, 1 with a history of primary plasma cell leukemia [pPCL]) received a single infusion of RD118 at 1.0, 2.0, or 3.0 × 106 CAR+ T cells/kg. At a median follow-up of 17.0 months, the overall response rate (ORR) was 94.4%, including 72.2% complete or stringent complete responses. Among seven patients previously exposed to BCMA-directed CAR T-cell therapy, ORR reached 85.7%. Median progression-free survival (PFS) was 18.2 months (95% CI, 14.4-not estimable), with 12-month PFS and overall survival (OS) rates of 82.1% and 93.3%, respectively. Cytokine release syndrome occurred in 88.9% of patients, primarily grade 1-2. One patient developed grade 3 immune effector cell-associated neurotoxicity which resolved within 72 hours. No cerebellar toxicities or treatment-related deaths were reported. These findings support that RD118 is a highly effective and safe therapeutic option for heavily pretreated RRMM. This trial is registered at ClinicalTrials.gov as NCT05759793 and NCT05219721.

Myeloid leukemia in children with Down Syndrome (ML-DS) is associated with an excellent prognosis but high treatment-related toxicity and mortality. The ML-DS 2018 trial aimed to maintain the excellent event-free survival achieved in the previous ML-DS 2006 trial while further reducing treatment intensity. Children aged 6 months to 6 years with ML-DS were eligible. Intensity-reduced induction and reinduction therapy with cytarabine, idarubicin ± etoposide of the ML-DS 2006 trial was replaced with CPX-351 (66 U/m² on three days in course 1 and two days in course 2). Risk stratification was based on flow cytometric measurable residual disease after first induction. High-risk patients received high-dose cytarabine (3 g/m²/12h) in consolidation; standard-risk patients received 1 g/m²/12h. Thirty-five patients were enrolled until the trial was halted due to an unexpectedly high relapse rate. A per protocol interim analysis revealed a significantly lower 24-month event-free survival compared to the ML-DS 2006 trial (69% vs. 90%, P<.001). In contrast to previous studies, most relapsed patients responded to salvage therapy, resulting in a comparable 24-month overall survival of 88% (vs. 92% in ML-DS 2006, P=.612). CPX-351 demonstrated a favorable toxicity profile with no treatment-related mortality. Positive measurable residual disease by error-corrected GATA1 next-generation sequencing, as well as the presence of trisomy 8 or a complex karyotype, were associated with an increased risk of relapse. In conclusion, replacing intensity-reduced induction therapy with CPX-351 in ML-DS resulted in significantly lower event-free survival, highlighting the need for dose optimization to balance efficacy and toxicity in this sensitive patient population. EudraCT: 2018-002988-25.

Oncogenic growth places great strain and dependence on protein homeostasis (proteostasis). This has made proteostasis pathways attractive therapeutic targets in cancer, but efforts to drug these pathways have yielded disappointing clinical outcomes. One exception is proteasome inhibitors, which are approved for frontline treatment of multiple myeloma. However, proteasome inhibitors are largely ineffective for treatment of other cancers at tolerable doses, including acute myeloid leukemia (AML), although reasons for these differences are unknown. Here, we determined that proteasome inhibitors are ineffective in AML due to inability to disrupt proteostasis. In response to proteasome inhibition, AML cells activated HSF1 and increased autophagic flux to preserve proteostasis. Genetic inactivation of HSF1 sensitized AML cells to proteasome inhibition, marked by accumulation of unfolded protein, activation of the PERK-mediated integrated stress response, severe reductions in protein synthesis, proliferation and cell survival and significant slowing of disease progression and extension of survival in vivo. Similarly, combined autophagy and proteasome inhibition suppressed proliferation, synergistically killed human AML cells, and significantly reduced AML burden and extended survival in vivo. Furthermore, autophagy and proteasome inhibition preferentially suppressed protein synthesis and colony formation, and induced apoptosis in primary patient AML cells, including AML stem/progenitor cells, compared to normal hematopoietic stem/progenitor cells. Combined autophagy/proteasome inhibition activated a terminal integrated stress response, which was surprisingly driven by Protein kinase R (PKR). These studies unravel how proteostasis pathways are co-opted to promote AML growth, progression and drug resistance, and reveal that disabling the proteostasis network is a promising strategy to therapeutically target AML.

Acute myeloid leukemia (AML) is a polyclonal malignancy marked by high relapse rates despite initial morphologic remission. Although measurable residual disease (MRD) is an established prognostic tool, increasing evidence supports a role for pre-emptive, MRD-directed therapy. AML monitoring is hampered by absence of a universal MRD marker, necessitating a more personalized approach. NPM1 is suited to an MRD-directed strategy as the mutation is AML-defining and monitoring methods highly sensitive. Critically, rising NPM1mut levels portend clinical relapse with high fidelity and recent studies demonstrate that venetoclax-based regimens induce rapid and deep MRD responses in a high proportion of patients with NPM1mut MRD relapse. The range of MRD-directed treatment options are expanding and include FLT3 and menin inhibitors for MRD relapse driven by FLT3-ITD and KMT2A rearrangements, respectively. To overcome the logistical issue of multiple MRD markers and associated therapies, we have developed a multi-target, multi-arm platform trial named INTERCEPT. Novel features include the potential to adaptively expand the range of MRD markers and directed therapies, seamless transition of patients from a local to centralized MRD monitoring framework, a clinical decision rules approach to allocate treatment in a hierarchical and pre-specified manner, creation of parallel protocol appendices to enable multiple industry partners to co-exist with commercial independence, and development of approaches to minimize the "MRD relapse to treatment" time interval. The future success of MRD-directed therapy will depend on rapid diagnostic turnaround, coordinated logistics and innovative clinical trial designs able to keep pace with advances in MRD-detection technologies and associated targeted therapies.

The BCR::ABL1 tyrosine kinase inhibitors (TKIs) in CML represent a paradigm for molecularly targeted therapy. However, clinical outcomes - rate/depth of response, treatment-free remission (TFR), progression to blast crisis (BC) - and adverse events vary among patients. While additional somatic mutations have been invoked to explain varying clinical outcomes, we here propose a complementary perspective based on single-cell omics approaches that have enabled unprecedented resolution of the cellular ecosystems, including their composition, interactions, and activity. In treatment-naïve chronic phase (CP) patients, this has revealed differences in the growth-rate of BCR::ABL1+ clones, ratio of TKI-insensitive leukemic stem cells (LSCs) to residual hematopoietic stem cells (HSCs), and immune cell composition - factors that collectively contribute to variability in therapy efficacy. Together these findings suggest that cellular heterogeneity serves as a foundation of clinical outcome in CML. Patients who remain in CP exhibit an erythroid signature in LSCs, while those progressing to BC manifest an inflammatory profile, additional mutations, and expansion of early progenitors. Deep responders with active natural killer, and regulatory T-cells are more likely to sustain TFR. Similarly, the outcomes of donor lymphocyte infusion after allogenic stem cell transplantation are heterogenous and reflect differences in pre-existing T-cell clonotypes, their expansion and interaction with leukemic cells in responders versus non-responders. Here, we summarize key insights from sc-omics in CML and propose an actionable roadmap to further leverage these technologies. This includes mechanistically explaining heterogeneity, predicting therapy response and BC, tracking leukemogenic clones longitudinally, targeting TKI-insensitive LSCs, and restoring hematopoiesis from residual HSCs.

The European LeukemiaNet has periodically issued guidelines for the diagnosis and management of acute myeloid leukemia (AML) in adults. These consensus recommendations, most recently updated in 2022, incorporate recent advances in genomic testing, disease detection methods, target identification, and response assessment. Whilst similarities exist between AML in children and adults, pediatric AML is frequently characterized by unique cytogenetic and molecular features, which require distinct genetic and immunophenotypic diagnostics, therapeutic approaches, response assessment criteria, and supportive care strategies. To address these specific needs, an international panel of pediatric hematologist-oncologists, biologists, geneticists, and laboratory medicine scientists convened to develop recommendations for the diagnosis and management of AML in children, adolescents, and young adults (hereafter termed pediatric AML) that are discussed in this special report.

Epstein-Barr virus (EBV) infects over 90% of the global population and drives multiple aggressive B cell malignancies, including Burkitt lymphoma (BL), diffuse large B cell lymphoma (DLBCL), and Hodgkin lymphoma. Standard chemoimmunotherapy regimens can be highly effective, yet EBV-positive lymphomas sometimes exhibit poorer responses, higher resistance, and worse survival compared with EBV-negative counterparts. This reflects the virus's ability to drive immune evasion, alter cell death pathways, and exploit host immune dysfunction, underscoring the potential value of EBV-directed strategies. Withaferin A (WA), a steroidal lactone with known anti-cancer and anti-inflammatory properties, was evaluated for its efficacy against EBV-associated B cell non-Hodgkin lymphomas (B-NHLs). Across a panel of lymphoma cell lines, WA demonstrated selective cytotoxicity toward EBV-positive B-NHLs in part through proteasome-dependent degradation of EBV nuclear antigen 1 (EBNA1) and subsequent loss of viral episomes, alongside additional effects on cellular stress and survival pathways. Mechanistic studies revealed that WA collapses antioxidant defenses, drives oxidative stress, and suppresses NF-κB signaling, creating a multi-pronged disruption of viral and host survival pathways. In primary B cell models and a cord-blood humanized mouse model of EBV-driven lymphomagenesis, WA inhibited B cell transformation, reduced splenomegaly and tumor burden, and significantly prolonged survival, without evidence of increased viral replication. These findings establish WA as a potent preclinical candidate that selectively targets vulnerabilities unique to EBV-transformed B cells, supporting further optimization and evaluation for EBV-positive B cell malignancies.

The peptide hepcidin is produced by the liver and serves as the central negative regulator of iron trafficking. Recently, drugs that affect the hepcidin pathway have been evaluated as potential treatment options for both controlling the degree of erythrocytosis in polycythemia vera (PV) patients as well as correcting anemia associated with myelofibrosis (MF). Under normal conditions, increased hepcidin levels limit iron absorption from the gastrointestinal tract and iron recycling from liver and splenic macrophages, thus decreasing plasma iron levels and restricting iron availability for erythropoiesis. In PV, however, unrestricted erythropoiesis occurs despite low systemic iron levels. Since hepcidin levels are relatively low in PV patients, hepcidin agonists (rusfertide, divesiran, sapablursen) are undergoing clinical development to control PV associated erythrocytosis, thereby reducing the need for therapeutic phlebotomies and myelosuppressive therapeutic options. By contrast, hepcidin levels are increased in MF patients leading to the trapping of iron in tissue macrophages which creates a picture which resembles the anemia of chronic inflammation. A number of strategies to lower hepcidin levels (the JAK2 inhibitors pacritinib and momelotinib, anti-hemojuvelin monoclonal antibody DISC-0974C) are currently undergoing clinical development to make systemic iron available for erythropoiesis and alleviate the degree of MF associated anemia. These new therapeutic options that modulate iron trafficking in PV and MF patients represent the application of greater knowledge of iron trafficking to create novel therapeutic options to treat patients with hematological malignancies.

Tissue factor (TF), encoded by F3, binds factor VII/VIIa to initiate blood coagulation. Because standard clinical assays do not measure endogenous TF directly, the extent to which human F3 variants impact blood coagulation is unknown. We sought to determine the effect of the human TF missense variants with the highest allele frequency as well as additional rare variants occurring at sites predicted to perturb the initiation of blood coagulation. The variants with the highest allele frequency did not impact coagulation activation. By contrast, some rare human TF missense substitutions did profoundly impact TF-initiated plasma clotting time and the activation of factors IX and X by two distinct mechanisms: by precluding TF interaction with factor VIIa or by altering the TF exosite to prevent macromolecular but not amidolytic substrate cleavage. Individuals heterozygous for the rare p.Gly196Arg variant have reduced basal factor VIIa-antithrombin complex and D-dimer levels but no major differences in TF or factor VII levels. Gly196Arg supported impaired factor VII autoactivation in vitro. These data demonstrate that rare missense variants in F3 can impair the activation of factors VII, IX and X and suggest these variants impair the basal activation of blood coagulation in humans.

We describe the first case of NMNAT3 deficiency, a novel red cell enzymopathy leading to reduced NAD levels, causing mild hemolytic anemia which improved upon supplementation with NAD-precursors. We therefore propose testing for NMNAT3 variants in unexplained hereditary hemolytic anemia.