The integrity of the hematopoietic stem cell (HSC) pool depends on effective long-term self-renewal and the timely elimination of damaged or differentiation-prone HSCs. Although the protein kinase R-like endoplasmic reticulum kinase (PERK) branch of the unfolded protein response has been shown to initiate proapoptotic signaling in response to endoplasmic reticulum (ER) stress in vitro, its role in regulating the HSC fate in vivo remains incompletely understood. Here, we demonstrated that PERK is dispensable for steady-state hematopoiesis and HSC self-renewal under homeostatic conditions. However, under ER stress induced by the disruption of ER-associated degradation (ERAD) through knockout of key components, such as Sel1L or Hrd1, PERK becomes activated and drives HSC proliferation and depletion. Notably, deletion of PERK or expression of a kinase-dead PERK mutant significantly rescued the HSC defects caused by Sel1L or Hrd1 loss. Mechanistically, an ERAD deficiency did not lead to increased HSC apoptosis or elevated reactive oxygen species, and PERK knockout had minimal impact on HSC apoptosis. Instead, PERK activation promoted aberrant mTOR (mammalian target of rapamycin) signaling and HSC hyperproliferation, ultimately compromising self-renewal capacity. This PERK-driven elimination of stressed HSCs may function as a protective mechanism to maintain the overall HSC pool integrity. Collectively, our findings reveal a previously unrecognized, proliferative, and apoptosis-independent role for PERK in regulating HSC fate under ER stress, highlighting a novel mechanism for preserving HSC homeostasis.

Inotuzumab ozogamicin (InO) is an antibody-calicheamicin conjugate with high efficacy in lymphoid malignancies. It targets the B-cell surface protein CD22, which is expressed in most B-cell acute lymphoblastic leukemia (B-ALL) cases, albeit with variable intensity. However, factors governing CD22 expression and thus leukemia sensitivity to InO remain incompletely understood. Using multiomic characterization of 196 human B-ALL samples, coupled with ex vivo InO sensitivity profiling, we showed that early leukemia differentiation arrest at the pre-pro-B stage is associated with resistance to InO. Screening of 1639 transcription factor genes identified early B-cell factor 1 (EBF1) as a key regulator of CD22 expression (false discovery rate of 7.1 × 10-4). When comparing the assay for transposase-accessible chromatin with sequencing profiling results of the most InO-sensitive and -resistant cases (50% lethal concentration <10th vs >90th percentile, n = 18), the binding motif for EBF1 was strikingly enriched in regions with differential open chromatin status (P = 8 × 10-174). CRISPR interference targeting EBF1 binding sites at the CD22 locus led to an ∼50-fold reduction in cell surface CD22 expression and, consequently, an ∼22-fold increase in InO resistance in ALL cell lines. Interestingly, within BCR::ABL1 ALL, we observed intrasubtype heterogeneity linked to EBF1 transcriptional downregulation (P = 1.1 × 10-15) and/or somatic alteration (P = .004), which led to reduced CD22 expression (P = 8.3 × 10-11) and ex vivo and in vivo resistance to InO. Collectively, these findings point to the direct impact of EBF1 on CD22 expression during B-cell development, which, in turn, contributes to interpatient variability in InO response, even within the same subtype of B-ALL.

RNA homeostasis is dysregulated in cancer and affects disease progression and therapy resistance. N6-methyladenosine (m6A), the most abundant epitranscriptomic modification in eukaryotic messenger RNA, plays a pivotal role in RNA biology, affecting transcript stability, translation, and splicing. Our study uncovers the extensive m6A changes in patients with T-cell acute lymphoblastic leukemia (T-ALL), to our knowledge, for the first time. It reveals m6A's regulatory role in the oncogenic MYC and cholesterol biosynthesis pathways. In addition, we discovered that T-ALL is highly dependent on the m6A reader heterogeneous nuclear ribonucleoprotein C (HNRNPC). HNRNPC is transcriptionally controlled by MYC and is an essential regulator of m6A-modified transcripts. Consequently, transcriptional silencing of HNRNPC profoundly impairs oncogenic pathways and critically diminishes leukemia cell growth. In addition, the levels of the m6A demethylase fat mass and obesity-associated protein (FTO) are significantly elevated in T-ALL cells compared with normal cells, and to other types of leukemia. Targeting FTO shows therapeutic potential in preclinical disease models and synergizes with clinically relevant therapeutics. Our findings underscore the integral role of RNA methylation in orchestrating cancer cell oncogene expression and metabolism and highlight promising novel therapeutic avenues for the treatment of T-cell leukemia.

The recurrent t(1;22) translocation in acute megakaryoblastic leukemia (AMKL) encodes the RBM15-MKL1 fusion protein. Dysregulation of the N6-methyladenosine (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 the m6A modification, thereby altering the RNA fate to drive leukemogenesis in RM-AMKL. Using a multiomics approach, we showed for the first time, to our knowledge, that RM retains the RNA-binding and m6A-modifying functions of RBM15 while also selectively regulating distinct messenger RNA targets, including Frizzled genes, in the Wnt signaling pathway. Treating murine RM-AMKL cells with the methyltransferase 3 (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 in 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 activity and Frizzled expression in multiple forms of human AMKL underscores the relevance of our findings. Together, our results establish that RM-specific m6A modifications and Wnt pathway activation are critical drivers of RM-AMKL, thereby identifying these pathways as potential therapeutic targets.

The continuous improvement in progression-free survival (PFS) of patients with multiple myeloma (MM) raises interest in evaluating peripheral residual disease (PRD) toward more frequent readouts of tumor kinetics while preserving quality of life. We present BloodFlow, a new method combining immunomagnetic enrichment of CD138+ circulating plasma cells in peripheral blood (PB) with next-generation flow (NGF), for the detection of PRD below the 2 × 10-6 NGF threshold. BloodFlow detected PRD in 55 of 644 PB samples (8.5%) from 295 patients. Of note, 52.7% PB samples were positive using BloodFlow and negative by NGF. The lowest PRD detected by BloodFlow was 6 × 10-8. Using bone marrow measurable residual disease (MRD) status as the reference, BloodFlow showed positive and negative predictive values of 95.1% and 76.6%, respectively. Detectable PRD during maintenance or observation predicted dismal PFS and overall survival (2-year rates of 0% and 62%, respectively). 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. BloodFlow is the first flow cytometry method that detects tumor cells below the 10-6 threshold, enabling improved minimally invasive monitoring of patients with MM.

Most calreticulin (CALR) mutations in myeloproliferative neoplasms 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 myeloproliferative leukemia protein (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 inositol-requiring enzyme 1/X-box binding protein 1 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 activating transcription factor 6 (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. Furthermore, we show that CALRins5 cells are partially dependent on ATF6 for cytokine-independent growth, and identify B-cell lymphoma extra large 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. Although immunoassays offer rapid diagnosis, their sensitivity and specificity are suboptimal. Sequential combinations of quantitative immunoassay results improve their diagnostic accuracy. We aimed to validate 2 Bayesian approaches combining 2 rapid immunoassays and to compare their diagnostic performance with 2 other diagnostic approaches (Hamilton and TORADI-HIT algorithms). We included 1194 patients with suspected HIT, of whom 6.0% had confirmed HIT. HemosIL Acustar HIT-IgG (chemiluminescent immune assay [CLIA]) and HemosIL HIT-Ab (latex immune-turbidimetric assay [LIA]) (Instrumentation Laboratory, Munich, Germany) were performed. Definite HIT confirmation or exclusion was made using heparin-induced platelet activation (HIPA) test and platelet factor 4-enhanced HIPA (PIPA). Our sequential approaches (CLIA first and LIA for 15-20% 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 no false-negative predictions, and 13 and 15 false-positive predictions, respectively The modified version of the Hamilton algorithm correctly excluded HIT in 92.1% and predicted HIT in 97.2% with 88 false-positive and 2 false-negative results. The TORADI-HIT algorithm correctly excluded HIT in 97.9% and predicted HIT in 93.8% (10 false positives, 3 false negatives). In conclusion, a Bayesian approach sequentially using 2 immunoassays is accurate for HIT diagnosis. Performing immunoassays simultaneously without considering clinical pretest 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 used RNA-targeting CRISPR-Cas13d to uncover and characterize hundreds of tumor-essential lncRNA (te-lncRNA) isoforms with clinical relevance. Focusing on multiple myeloma (MM), we targeted the lncRNA transcriptome expressed in tumor cells from patients with MM 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 >30 cytosolic isoforms that proved essential when targeted by cytosol-localized Cas13d. Notably, a specific isoform of small nucleolar RNA host gene 6, 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 patients with AA at diagnosis and after treatment with horse-derived anti-thymocyte globulin (hATG), and 6 controls. Within the hypocellular BM architecture, we identified lymphoid-dominant "immune hot spots" with high densities of proinflammatory lymphocytes, and macrophage-enriched hot spots that additionally contained activated macrophages in proximity to progenitors. These immune hot spots potentially represent sites in which 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 after hATG treatment, most immune hot spots were depleted, underscoring their potential pathogenic role. Collectively, our study visualizes the complex interactions among immune cell subpopulations and reveals, to our knowledge, for the first time, the order of events in the immune-mediated pathogenesis of AA.