Acute megakaryoblastic leukemia driven by the RBM15-MKL1 fusion protein (RM-AMKL) is encoded by the recurrent t(1;22) translocation. Dysregulation of m6A modification affects RNA fate and is linked to oncogenesis. Because RBM15 is critical for bringing the m6A writer complex to specific RNAs, we hypothesized that RM disrupts m6A modification, altering RNA fate to drive leukemogenesis in RM-AMKL. Using a multi-omic approach, we show for the first time that RM retains the RNA-binding and m6A-modifying functions of RBM15 while also selectively regulating distinct mRNA targets including Frizzled genes in the WNT signaling pathway. Treating murine RM-AMKL cells with the METTL3 inhibitor STM3675, which decreases m6A deposition, induced apoptosis in vitro and prolonged survival in transplanted mice. Frizzled genes were upregulated by RM and downregulated upon METTL3 inhibition, implicating an m6A-dependent mechanism for their dysregulation. Direct Frizzled knockdown reduced RM-AMKL growth in vitro and in vivo, highlighting Wnt signaling as a key oncogenic driver. Elevated Wnt pathway and Frizzled expression in multiple forms of human AMKL underscores the relevance of our findings. Together, our results establish RM-specific m6A modifications and Wnt pathway activation as critical drivers of RM-AMKL, identifying these pathways as potential therapeutic targets.

The continuous improvement in progression-free survival (PFS) of multiple myeloma (MM) patients raises interest in evaluating peripheral residual disease (PRD) towards more frequent readouts of tumor kinetics while preserving quality of life. Here we present BloodFlow, a new method that combines immunomagnetic enrichment of CD138+ circulating plasma cells in peripheral blood (PB) with next-generation flow (NGF), for the detection of PRD below the 2x10-6 NGF threshold. BloodFlow detected PRD in 55/644 (8.5%) PB samples collected from 295 MM patients. Of note, 29/55 (52.7%) PB samples were positive using BloodFlow and negative by NGF. The lowest level of PRD detected by BloodFlow was 6x10-8. Considering patients' minimal residual disease (MRD) status in bone marrow as the reference, BloodFlow showed positive and negative predictive values of 95.1% and 76.6%. Presence of PRD during maintenance or observation predicted dismal progression-free and overall survival (2-year rates of 0% and 62%). BloodFlow surpassed NGF in PB and retained independent prognostic value for PFS in multivariate analysis including transplant-eligibility, the Revised International Staging System, complete remission and MRD status in bone marrow. BloodFlow is the first flow cytometry method that detects tumor cells below the 10-6 threshold, which resulted in improved minimally-invasive monitoring of MM patients.

The majority of calreticulin (CALR) mutations in myeloproliferative neoplasms (MPNs) are classified as either type 1, a 52 base-pair deletion (CALRdel52), or type 2, a 5 base-pair insertion (CALRins5). Both are gain-of-function (GOF) mutations that generate an identical mutant C-terminal tail, which mediates the binding to and activation of the thrombopoietin receptor MPL. We recently reported that despite this shared GOF, CALRdel52 but not CALRins5 mutations cause loss of calcium binding function, leading to activation of and dependency on the IRE1a/XBP1 pathway of the unfolded protein response (UPR). This led us to ask whether CALRins5 mutations activate and depend on a different UPR pathway, and whether this is likewise mediated by a mutation type-specific loss-of-function (LOF). Here, we show that CALRins5 mutations lead to activation of the ATF6 pathway of the UPR due to loss of CALR chaperone function. This LOF is caused by interference of the CALRins5 mutant C-terminus with key chaperone residue H170. Further, we show that CALRins5 cells are partially dependent on ATF6 for cytokine-independent growth, and identify BCL-xL as a transcriptional target of ATF6 that promotes type 2 CALR mutant cell survival.

Suppression of plasminogen activation and/or plasmin activity (PA) reduces blood loss and decreases hemorrhage-related death. However, whether the endogenous PA system is a biological mechanism to prevent intravascular thrombus formation is debated, and the potential that reduced PA may increase venous thrombosis/thromboembolism (VTE) risk cautions against the use of antifibrinolytic agents. We aimed to determine the contribution of PA to VTE. Type 1 plasminogen-deficient humans enrolled in the HISTORY registry (https://clinical trials.gov; NCT03797495) reported pathologic pseudomembrane formation, but not unprovoked VTE. When subjected to an experimental model of venous thrombosis, compared to Plg+/+ mice, neither partial (Plg+/-) nor complete (Plg-/-) deletion of plasminogen altered thrombus mass or thrombus nucleated cell, platelet, or fibrin(ogen) content at 24 or 6 hours after thrombus induction. Administration of tranexamic acid (TXA) to mouse plasma in vitro or healthy mice in vivo dose-dependently delayed and suppressed plasma plasmin generation for up to 3 hours. However, mice administered TXA did not have significantly altered thrombus mass or thrombus composition at 24 or 6 hours after thrombus induction, despite unexpectedly persistent TXA in plasma. In a genome-wide association study, variants in gene regions encoding PA pathway proteins were not significantly associated with VTE risk. In the UK Biobank repository, plasminogen protein levels were not significantly associated with VTE risk. These data from genetic, pharmacologic, and proteomic analyses of mice and humans indicate that perturbations in PA do not increase VTE risk. Collectively, these results suggest PA is not a molecular regulatory mechanism to protect against VTE.

Early recognition and treatment of heparin-induced thrombocytopenia (HIT) are crucial to prevent severe complications. While immunoassays offer rapid diagnosis, their sensitivity and specificity are suboptimal. Sequential combinations of quantitative immunoassay results improve their diagnostic accuracy. We aimed to validate two Bayesian approaches combining two rapid immunoassays and to compare their diagnostic performance with two other diagnostic approaches ("Hamilton", "TORADI-HIT" algorithms). We included 1194 patients with suspected HIT, of whom 6.0% had confirmed HIT. HemosIL Acustar HIT-IgG (CLIA) and HemosIL HIT-Ab (LIA) (Instrumentation Laboratory, Munich, Germany) were performed. Definite HIT confirmation or exclusion was made using heparin-induced platelet activation (HIPA) test and PF4-enhanced HIPA (PIPA). Our sequential approaches (CLIA first and LIA for unsolved cases or vice versa) correctly excluded HIT in 95.6% and 96.4%, predicted HIT in 95.8% and 97.2%, with 3.3% and 2.3% of cases remaining undetermined. There were 13 respectively 15 false positive and no false negative predictions. The modified version of the "Hamilton algorithm" correctly excluded HIT in 92.1% and predicted HIT in 97.2% with 88 false positive and two false negative results. The "TORADI-HIT algorithm" correctly excluded HIT in 97.9% and predicted HIT in 93.8% (10 false positives, three false negatives). In conclusion, a Bayesian approach sequentially employing two immunoassays is accurate for HIT diagnosis. Performing immunoassays simultaneously without considering clinical pre-test probability misses HIT cases. The "TORADI-HIT algorithm" offers better HIT exclusion with a 6% false-negative rate. Using our Bayesian approach, HIT exclusion or recognition can be achieved in ≥97% of cases within <1 hour.

Long noncoding RNAs (lncRNAs) are a significant yet largely uncharted component of the cancer transcriptome, with their isoform-specific functions remaining poorly understood. In this study, we employed RNA-targeting CRISPR-Cas13d to uncover and characterize hundreds of tumor-essential (te)-lncRNA isoforms with clinical relevance. Focusing on multiple myeloma (MM), we targeted the lncRNA transcriptome expressed in tumor cells from MM patients and revealed both MM-specific and pan-cancer dependencies across diverse cancer cell lines, which we further validated in animal models. Additionally, we mapped the subcellular localization of these te-lncRNAs, identifying over 30 cytosolic isoforms that proved essential when targeted by cytosol-localized Cas13d. Notably, a specific isoform of SNHG6, enriched in the endoplasmic reticulum, interacts with heat shock proteins to maintain cellular proteostasis. We also integrated functional and clinical data into the publicly accessible LongDEP Portal, providing a valuable resource for the research community. Our study offers a comprehensive characterization of te-lncRNAs, underscoring their oncogenic roles and therapeutic potential.

The autoimmune response driving hematopoietic stem and progenitor cell (HSPC) destruction in immune-mediated aplastic anemia (AA) remains incompletely understood. We previously identified a disease-specific immune cell network involving T-, B-, and myeloid cells. However, the interactions within this network, the interaction with the microenvironment and the chronological events in AA development, remain unclear. In this study, we aimed to characterize the changes occurring during disease development and to define the interactions between potential autoreactive cells and their target. Using imaging mass cytometry, we analyzed bone marrow (BM) biopsies from AA patients at diagnosis and after treatment with horse-derived anti-thymocyte globulin (hATG), and six controls. Within the hypocellular BM architecture, we identified lymphoid-dominant 'immune hotspots' with high densities of pro-inflammatory lymphocytes, and macrophage-enriched hotspots that additionally contained activated macrophages in proximity to progenitors. These immune hotspots potentially represent sites where the active immune response resulting in HSPC destruction takes place. In BM regions depleted of progenitors, effector cells with a differentiated phenotype remain. Our data indicate that HSPC destruction in AA is mediated by coordinated interactions among specific immune cell subpopulations. As the immune response progresses and HSPCs are depleted, the immune composition shifts, with activated T- and B-cells differentiating into terminally differentiated T-cells and plasma cells. In patients with normalizing BM post-hATG treatment, most immune hotspots were depleted, underscoring their potential pathogenic role. Collectively, our study visualizes the complex interactions among immune cell subpopulations and, for the first time, reveals the order of events in the immune-mediated pathogenesis of AA.

Transfusion-related acute lung injury (TRALI) is a leading cause of blood transfusion triggered mortality. Recently, we demonstrated the critical role of Fc-dependent complement activation in anti-CD36-mediated murine TRALI. In this study, we found that C5-/- mice were protected and administration of anti-C5 rescued wild-type mice from anti-CD36-mediated TRALI. However, C5aR1-/- mice were not protected against anti-CD36-mediated TRALI, implying a possible role of C5b-9 (membrane attack complex [MAC]). Accordingly, elevated levels of MAC were detected in bronchoalveolar lavage fluid and lung tissue of mice with anti-CD36 induced TRALI. Inhibition of MAC formation by administration of anti-C7 blocking monoclonal antibody (mAb) alleviated TRALI in mice, suggesting the critical role of the MAC in the pathology of anti-CD36-mediated TRALI. Furthermore, anti-C7 treatment also led to favorable outcome in anti-MHC I-induced murine TRALI, indicating the potential broader applicability of MAC inhibitors in the treatment of antibody-mediated TRALI. Therefore, this approach may be promising to further explore for the treatment of TRALI patients.

Macrophage activation syndrome (MAS) is believed to be the result of inappropriate proliferation and activation of the mononuclear phagocytic system. Adult-onset Still's disease (AOSD) is characterized by neutrophil activation and a cytokine storm, which can lead to its severe and potentially life-threatening complication MAS. RNA sequencing revealed that neutrophils may play a distinct and enhanced role in innate immunity in AOSD patients with MAS (AOSD-MAS). In the CpG-induced secondary hemophagocytic lymphohistiocytosis (HLH) model, depletion of neutrophils significantly reduced cytokine levels, with effects comparable to monocyte depletion. Significant enrichment was observed in the type I/II interferon and JAK-STAT pathways in neutrophils from AOSD-MAS patients. Treatment of 10 refractory AOSD-MAS patients with ruxolitinib led to the resolution of inflammatory parameters and clinical symptoms. RNA sequencing and ex vivo assays confirmed that ruxolitinib suppressed aberrant NETosis and STAT3/STAT5 signaling. In vivo, PAD4 knockout further confirmed the pathogenic role of NETosis in secondary HLH model. Moreover, selective inhibition of STAT3 or STAT5 alleviated systemic inflammation. Ten functional variants were identified in genes related to the JAK-STAT pathway, although their clinical relevance requires further validation. These findings suggest the potential of ruxolitinib in achieving disease remission in refractory AOSD-MAS patients by broadly inhibiting JAK-STAT signaling and modulation of neutrophil activation and NETosis.

No standard of care for elderly patients with aggressive adult T-cell leukemia/lymphoma (ATL) has been established. We evaluated the efficacy of CHOP every 2 weeks with mogamulizumab (Moga) (Moga-CHOP-14) for untreated elderly patients with ATL. In this multicenter phase 2 trial, patients aged ≥66 years and those aged 56-65 years ineligible for transplantation received 6 cycles of Moga-CHOP-14, followed by 2 cycles of Moga monotherapy. The primary endpoint was 1-year progression-free survival (PFS). Secondary endpoints were the complete response (CR) rate, overall response rate (ORR), overall survival (OS), 1-year event-free survival (EFS), and safety. We also investigated the impact of CCR4 mutation and Moga-associated cutaneous adverse events (cAEs) on PFS and OS. The study protocol was amended to allow the dosing interval to be extended to 21 days at the physician's discretion. Among 48 evaluable patients, the 1-year PFS was 36.2% (90% confidence interval [CI], 24.9-47.6), with a median follow-up of 1.6 years. One-year OS and EFS were 66.0% (95% CI, 50.6-77.6) and 29.9% (95% CI, 17.6-43.2), respectively. CR and ORR were 64.6% (95%CI, 49.5-77.8) and 91.7% (95% CI, 80.0-97.7). No unexpected toxicities were observed. Of the 47 patients who received ≥2 cycles of CHOP, 20 (42.6%) received CHOP-14, among whom 12 (25.5%) completed 6 cycles. CCR4 mutation and Moga-associated cAE were associated with better OS. This study showed that Moga-CHOP significantly improved PFS, though the optimal interval for CHOP remains undetermined. Moga-CHOP is now considered a preferable first-line treatment for these patients. Clinical Trial Identifier: jRCTs041180130.

Myeloproliferative neoplasms (MPNs) are hematopoietic stem cell-driven malignancies marked by excessive myelopoiesis and high risk of myelofibrosis, which remains therapeutically challenging. Senescent neutrophils home daily to the bone marrow (BM) to be cleared by macrophages. This avoids their accumulation, which can increase the risk of chronic inflammation or oncogenesis. Neutrophils carrying the most common oncogenic MPN driver (JAK2V617F) are protected from apoptosis, which may prolong their lifespan and enhance their pro-inflammatory activity. On the other hand, abnormal interactions of neutrophils with megakaryocytes ("emperipolesis") have been associated with BM fibrosis in disparate hematological disorders, including MPN and grey platelet syndrome; however, the underlying pathophysiology remains unclear. We investigated neutrophil homeostasis and cellular interactions in MPN. We found that senescent neutrophils evade homeostatic clearance and accumulate in JAK2V617F MPN, but not in MPN caused by the second most prevalent mutations affecting Calreticulin (CALR) gene. This is explained by GM-CSF-JAK2-STAT5-dependent upregulation of the "don't-eat-me" signal CD24 in neutrophils. Mechanistically, JAK2V617F CD24hi neutrophils evade efferocytosis, invade megakaryocytes and increase active TGF-b. Collectively, JAK2V617F neutrophil-megakaryocyte interactions promote platelet production in a humanized bioreactor and myelofibrosis in mouse models. Notably, chronic antibody blockade or genetic loss of CD24 restores clearance of senescent neutrophils, reduces emperipolesis and active TGF-b. Consequently, CD24 blockade improves thrombocytosis and prevents myelofibrosis in MPN mice. Taken together, these findings reveals defective neutrophil clearance as a cause of pathogenic microenvironmental interactions of inflammatory neutrophils with megakaryocytes, associated with myelofibrosis in MPN. Our study postulate CD24 as a candidate innate immune checkpoint in MPN.

As evidenced by the excellent survival outcomes, chronic myeloid leukemia (CML) treatment in the era of tyrosine kinase inhibitors (TKIs) is often successful. However, when response milestones are not met or lost, treatment decision-making may be challenging. The availability for first-, second- or subsequent-line use of six different TKIs, each with definite and often non-overlapping features in terms of mechanism of action, potency, activity against resistance mutations and tolerability profile provides a reassuring opportunity to rescue an optimal response, but it must be exploited carefully to avoid hasty or inappropriate choices. When and how to sequence TKIs, and if and when to consider transplant are very important issues. 'One for all' rules cannot be formulated, since for each individual patient the decision process requires investigation and integration of a series of clinical and biological factors. After discussing how resistance is defined, we here aim to provide practical guidance to therapeutic reassessment, discussing which laboratory investigations should be performed, how they should be interpreted, which additional clinical considerations are mandatory, and how these factors should be weighed and reasonably concur to the final decision.