Hypodiploid (Hypo) and BCR::ABL1-positive (BCR-ABL+) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) confer a high risk of disease relapse. We investigated post-hematopoietic stem cell transplantation (HSCT) outcomes within the prospective FORUM trial, comparing these genetic subgroups to patients without these lesions. The use of pre- and post-HSCT add-on treatments, including tyrosine-kinase inhibitors (TKIs) and immunotherapies, was also assessed. Multivariate analysis evaluated associations with overall survival (OS), event-free survival (EFS), cumulative incidence of relapse (CIR). The FORUM trial enrolled 741 patients ≥4 years of age with BCP-ALL who underwent HSCT from HLA-matched donors (2013-2023). The 3-year OS (0.86 [95% CI, 0.76-0.92], 0.79 [0.65-0.87], and 0.79 [0.76-0.82]) and EFS (0.71 [0.59-0.80], 0.73 [0.59-0.83], and 0.67 [0.63-0.71]) did not differ significantly between BCR-ABL+, Hypo, and Neither patients, respectively. However, Hypo patients in second complete remission (CR2) showed inferior OS and EFS, driven by higher non-relapse mortality (NRM), which occurred exclusively in near-diploid cases. No NRM occurred in severe hypodiploid cases conditioned with TBI. MRD positivity at transplant predicted worse OS, EFS, and CIR in all genetic groups. Hypo patients were difficult to salvage post-relapse, even with CAR-T therapy. By contrast, BCR-ABL+ patients had favorable outcomes, even when MRD-positive prior HSCT. Prophylactic TKI use post-HSCT improved EFS and reduced CIR. BCR::ABL+ patients transplanted in CR2 had a 3-year OS of 96%. In conclusion, the standardized FORUM protocol yielded comparable outcomes across genetic subgroups. Post-transplant TKI maintenance improved outcomes in BCR-ABL+ BCP-ALL. EudraCT: 2012-0032-22; ClinicalTrials.gov: NCT01949129.

Acute myeloid leukemia (AML) patients have a poor five-year survival rate highlighting the need for the identification of new approaches to target this disease. AML is highly dependent on glutathione (GSH) metabolism for survival. While the metabolic role of GSH is well-characterized in AML, the contribution of protein glutathionylation-a reversible modification that protects protein thiols from oxidative damage-remains largely unexplored. Therefore, we sought to elucidate the role of protein glutathionylation in AML pathogenesis. Here, we demonstrate that protein glutathionylation is essential for AML cell survival. Specifically, the loss of glutaredoxin 2 (GLRX2), an enzyme that removes glutathione modifications, resulted in selective primary AML cell death while sparing normal human hematopoietic stem and progenitor cells. Unbiased proteomic analysis revealed increased mitochondrial protein glutathionylation upon GLRX2 depletion, accompanied by mitochondrial dysfunction, including impaired oxidative phosphorylation, reduced mitochondrial membrane potential, and increased opening of the mitochondrial permeability transition pore (mPTP). Further investigation identified ATP5PO, a key regulator of mPTP opening and a component of the ATP synthase complex, as a critical GLRX2 target. Disruption of ATP5PO glutathionylation partially restored mPTP function and rescued AML cell viability following GLRX2 depletion. Moreover, both genetic and pharmacologic inhibition of mPTP opening restored the leukemic potential of primary AML specimens in the absence of GLRX2. By disrupting glutathionylation-dependent mitochondrial homeostasis, this study reveals a novel vulnerability in AML that could inform future therapeutic strategies.

The regulation of the switch from fetal (HBG) to adult (HBB and HBD) b-globin gene expression has served as a paradigm for clinically relevant developmental transcriptional control. Mechanistic studies of this switch have predominantly focused on HBG repressors with comparatively little attention paid to potential HBG activators. We found that in adult type HUDEP2 erythroid cells, the ATP-dependent chromatin remodeler BRG1 preferentially activates the HBG genes as well as the minor adult HBD gene. BRG1 is a core catalytic subunit of three BAF complexes, canonical BAF, polybromo BAF, and non-canonical BAF (ncBAF) that regulate chromatin accessibility in distinct gene- and cell-type contexts. To dissect the specific BAF complex configuration mediating selective activation of HBG and HBD in erythroid cells, we performed CRISPR mediated targeting of individual subunits and pinpointed the regulatory activity to the ncBAF complex. Loss of the ncBAF complex subunits BRD9 and BAF60A preferentially decreased HBG and HBD transcription while accelerating terminal erythroid differentiation and hemoglobinization. Acute pharmacologic depletion of BRD9 in HUDEP2 and primary erythroid cells selectively reduced transcription of HBD and HBG, suggesting direct effects at these genes. Collectively, our unexpected findings demonstrate that the BAF complex, through distinct subcomplex configurations, can regulate selective gene expression within a multi-gene cluster. This expands the traditional view of BAF as a general co-activator, highlights its role in gene-specific regulation, and identifies a potential target for therapeutic manipulation of b-like globin genes in erythroid cell disorders.

We compared daunorubicin/AraC plus fractionated gemtuzumab (DAGO2) with CPX-351 (CPX) (1:2 randomisation) in 439 patients ≥60yrs (median age 68yrs) without known adverse-risk cytogenetics entering the NCRI AML18 version2 trial (NCT02272478). Median follow-up was 35 months. Patients not in MRD-negative remission after course-1 could enter a second randomization between standard versus intensified chemotherapy. Post course-1 response rates were greater after DAGO2 (CR+CRi, 60% vs 47.5%, OR 0.61 95%CI 0.41-0.91, p=0.016). Following course-2 the overall response was not significantly different, 85% for DAGO2 vs 78% for CPX (OR 0.64, 95%CI 0.39-1.09, P=0.095). More patients attained CR with MRD negativity post course-1 in the DAGO2 arm (47% vs 29% for CPX, OR 0.46 95%CI 0.29-0.72, p=0.004). We observed better 3yr EFS (34% vs 27%, HR 0.73 95%CI 0.57-0.93, P=0.012) and OS (52% vs 35%, HR 0.62, 95%CI 0.46-0.83, P=0.001) with DAGO2. In a stratified analysis, CPX did not provide a survival benefit in patients with MDS-related mutations (HR 1.40, 95%CI 0.97-2.03) and was associated with poorer survival in patients with NPM1 (HR 2.83 95%CI 1.17-6.82) and FLT3 mutations (HR 2.14, 95%CI 0.98-4.68). 37% of patients were transplanted in CR1 and this did not differ by randomization. Survival post-transplant did not differ between arms. For patients entering the course-2 randomisation (n=107) survival was equivalent between standard versus intensified CPX doses (P=0.565).In this population of older patients without known adverse-risk cytogenetics, DAGO2 resulted in superior survival compared to CPX. CPX did not benefit those with MDS-related mutations over DAGO2.

Only 30-50% of patients with immune thrombocytopenia (ITP) exhibit a sustained response upon thrombopoietin receptor agonists (TPO-RAs) withdrawal, underscoring the necessity for mechanistic elucidation. We enrolled 49 patients treated with TPO-RAs for four months and performed a follow-up study for three months, classifying them into sustained responders (ITP-sus, n=21), and non-sustained responders (ITP-non-sus, n=28). Compared with total TGF-β1 levels, activated TGF-β1 levels (3854±4380 vs. 943±1500 pg/ml, p＜0.001) were significantly elevated in sustained responders, with integrin αvβ8 regulating TGF-β1 activation and restoring immune tolerance. We established a passive ITP model using PF4-TGF-β1 conditional knockout (CKO) mice, which exhibited a shorter duration of sustained response compared to WT mice. CKO mice demonstrated a reduced Treg population, an increased M1/M2 macrophage ratio, and more severe megakaryocyte destruction following anti-CD41 injection. Exogenous administration of αvβ8 (250 ng/kg) effectively activated TGF-β1 and prolonged remission after TPO discontinuation in WT mice. Additionally, CD4+ T cells were transfected with lentiviral siRNA or shRNA to modulate integrin β8 expression and these were injected into SCID mice undergoing an active model of ITP. Results showed that β8 overexpression increased Treg cells and reduced megakaryocyte damage. Mechanistically, TPO-RAs modulated αvβ8-mediated TGF-β1 activation through the activator protein-1(AP-1) family and Smad-2 signaling pathways. Furthermore, D-mannose combined with TPO prolonged the response in ITP mice by upregulating αvβ8 and activating TGF-β1. Overall, the integrin αvβ8-mediated activation of TGF-β1 pathway represents a promising therapeutic target for ITP, with substantial potential for clinical application.