Uchida T, Shide K, Kameda T
… +11 more, Ozono Y, Kubuki Y, Tahira Y, Kamiunten A, Marutsuka K, Akizuki K, Karasawa M, Uehira Y, Ueno H, Yamaguchi H, Shimoda K
Bone marrow (BM) fibrosis in primary and post-polycythemia vera/essential thrombocythemia myelofibrosis (MF) has traditionally been considered a reactive process driven by cytokines, such as transforming growth factor (T...Bone marrow (BM) fibrosis in primary and post-polycythemia vera/essential thrombocythemia myelofibrosis (MF) has traditionally been considered a reactive process driven by cytokines, such as transforming growth factor (TGF)-β1, primarily produced by neoplastic megakaryocytes and platelets. These cytokines promote the differentiation of wild-type mesenchymal stromal cells into collagen- and fibronectin-producing myofibroblasts, thereby inducing BM fibrosis. However, hematopoietic-derived collagen-producing cells of monocyte lineage, termed fibrocytes, have also been implicated in this process. Here, we demonstrate that fibrocytes constitute a major collagen-producing cell population in the BM of patients with JAK2V617F-mutated MF, with additional contributions from myofibroblasts. Analysis of BM samples from patients with JAK2V617F-mutated myeloproliferative neoplasms (MPNs) revealed that fibrocytes accounted for nearly two-thirds of collagen-producing cells, whereas myofibroblasts represented a smaller subset. Using BM-derived fibrocytes from Jak2V617F transgenic mice (Jak2V617F mice), we performed a high-throughput drug screen and identified statins as inhibitors of fibrocyte proliferation in vitro. In vivo, pitavastatin treatment reduced fibrocyte numbers, ameliorated BM fibrosis, and improved anemia in Jak2V617F mice. Pitavastatin also decreased TGF-β1 production by neoplastic fibrocytes, resulting in reduced myofibroblast expansion. Peripheral blood-derived fibrocytes from patients with JAK2V617F-mutated MPNs were similarly sensitive to pitavastatin in vitro. Together, these findings suggest that fibrocytes substantially contribute to BM fibrosis in JAK2V617F-mutated MF and support further investigation of pitavastatin as a potential antifibrotic strategy in this molecular subset.
Leukemia stem cells (LSCs) drive acute myeloid leukemia (AML) initiation, relapse, and chemoresistance, yet the core post-translational events sustaining LSC maintenance remain poorly defined. Here, through phosphoproteo...Leukemia stem cells (LSCs) drive acute myeloid leukemia (AML) initiation, relapse, and chemoresistance, yet the core post-translational events sustaining LSC maintenance remain poorly defined. Here, through phosphoproteomic profiling of normal hematopoietic stem and progenitor cells (HSPCs) versus LSC-enriched populations, we identify DEK phosphorylation as a critical modification during leukemogenesis. Functional studies in MLL-AF9- and HOXA9/MEIS1-driven AML mouse models, as well as patient-derived xenografts (PDXs), demonstrate that DEK deficiency impairs LSC maintenance and AML progression. Moreover, DEK deletion enhances LSC chemosensitivity to the standard-of-care combination of azacitidine and venetoclax (Aza/Ven), whereas DEK overexpression confers robust chemoresistance. Mechanistically, DEK recruits the transcription factor GABPA to upregulate the transcriptional cofactor PBX3, a key oncogenic driver in AML, thereby sustaining the leukemogenic transcriptional program. This DEK-GABPA interaction strictly depends on DEK phosphorylation at Ser301/303/306/307 (the 4S sites), which stabilizes the conformation of the DEK-GABPA complex. We identify casein kinase 2 (CK2) as the upstream kinase that directly phosphorylates DEK-4S sites. Importantly, blockade of DEK phosphorylation via 4S site mutagenesis or treatment with the clinical-stage CK2 inhibitor CX-4945 selectively depletes LSCs while sparing normal HSPCs. Furthermore, combining CX-4945 with venetoclax promotes LSC apoptosis and represses the PBX3-driven leukemogenic transcriptional program, exhibiting synergistic anti-AML effects both in vitro and in vivo. Collectively, our findings uncover a previously unrecognized phosphorylation event (DEK-4S phosphorylation) that sustains LSCs and establish the CK2-DEK axis as a promising LSC-specific therapeutic strategy for AML.
Wang C, Geng X, Abdelrahman S
… +12 more, Fu Y, Kady N, Rauf MA, Hristov A, Tejasvi T, Murga-Zamalloa C, Tsoi L, Welch J, Fox J, Carty SA, Gudjonsson JE, Wilcox RA
Primary cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of extranodal non-Hodgkin lymphomas. Outcomes for patients with advanced-stage disease are suboptimal, as few complete and durable responses are achieve...Primary cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of extranodal non-Hodgkin lymphomas. Outcomes for patients with advanced-stage disease are suboptimal, as few complete and durable responses are achieved with currently available therapeutic agents. However, improved understanding of oncogenic transcriptional programs in malignant T cells and their engagement with the tumor microenvironment (TME) may unveil therapeutic vulnerabilities. Therefore, we have compiled the largest available scRNA-seq CTCL atlas that includes >2 million skin and blood cells from 116 individual patients. We identified recurrent transcriptional programs in malignant T cells, a subset of which are associated with GATA-3 dependent transcriptional programs, especially in the setting of large cell transformation and advanced-stage disease. Many of the transcriptional programs identified are therapeutically targetable with clinically available agents, including HDAC, XPO1, CDK9, JAK/CSF1R, and IKZF1/IKZF3 antagonists. The CTCL TME is dominated by infiltrating and exhausted effector and cytotoxic T cells that are restrained by a robust infiltrate of regulatory T cells, transcriptionally polarized monocytes/macrophages, and cancer-associated fibroblasts. Collectively, these findings have significant implications for the rationale design of combinatorial strategies targeting immune checkpoints, including PD-1. We have identified transcriptional programs, driven by oncogenic transcription factors, and constituents of the TME as therapeutic vulnerabilities in CTCL, and hope that the CTCL atlas constructed will provide a valuable resource for future studies exploiting these therapeutic vulnerabilities.
The Phase 3 TRANSFORM-1 study (NCT04472598) evaluated ruxolitinib (RUX) in combination with navitoclax (NAV) or placebo (PBO) in Janus-kinase-inhibitor-naïve adults with intermediate-2 or high-risk myelofibrosis and East...The Phase 3 TRANSFORM-1 study (NCT04472598) evaluated ruxolitinib (RUX) in combination with navitoclax (NAV) or placebo (PBO) in Janus-kinase-inhibitor-naïve adults with intermediate-2 or high-risk myelofibrosis and Eastern Cooperative Oncology Group performance status ≤2. Patients were randomized 1:1 to NAV (200 mg/day starting dose or 100 mg escalated to 200 mg/day) or PBO, with RUX dosed per label. The primary endpoint was ≥35% spleen volume reduction (SVR) at Week 24 (SVR35W24). Secondary endpoints included change from baseline in Total Symptom Score (TSS) at Week 24 and SVR35 at any time. A total of 252 patients (NAV+RUX, n=125; PBO+RUX, n=127; median follow-up 20.3 months) were randomized; >80% had intermediate-2 risk and nearly 50% were high-molecular risk (HMR). SVR35W24 was achieved by 63.2% with NAV+RUX versus 31.5% with PBO+RUX (P<0.0001). Mean change in TSS at Week 24 was not significantly different between NAV+RUX and PBO+RUX (-10.2 vs -11.6; P=0.2852). SVR35 at any time was achieved in 76.8% with NAV+RUX versus 44.1% with PBO+RUX (nominal P<0.0001). A ≥20% variant allele frequency reduction (exploratory endpoint) occurred in 58.5% (95% CI: 49.0-67.5) with NAV+RUX and 45.5% (95% CI: 36.4-54.8) with PBO+RUX. NAV+RUX showed higher hematologic toxicity versus PBO+RUX (Grade 3/4 thrombocytopenia: 54.0% vs 19.2%; Grade 3/4 neutropenia: 40.3% vs 8.8%); diarrhea (any grade: 41.9% vs 16.8%) was also more common. Cytopenias were generally manageable and reversible with dose adjustments.
Antiphospholipid syndrome (APS) is an immunothrombotic disorder, frequently attributed to autoantibodies that bind β2-glycoprotein I (β2GPI). A study showed that the platelet-specific chemokine, platelet factor 4 (PF4),...Antiphospholipid syndrome (APS) is an immunothrombotic disorder, frequently attributed to autoantibodies that bind β2-glycoprotein I (β2GPI). A study showed that the platelet-specific chemokine, platelet factor 4 (PF4), binds to β2GPI, enhancing recognition of β2GPI by APS antibodies. APS antibodies induce the release of neutrophil extracellular traps (NETs), webs of decondensed chromatin that bind both PF4 and b2GPI. We propose that PF4 bridges β2GPI to NETs (and other PF4-targeted polyanions), leading to the formation of prothrombotic PF4:b2GPI:NET immunotargets in APS. Dynamic light-scattering studies of isolated IgGs from four patients with triple-positive APS show formation of PF4:β2GPI:NET complexes that bind APS antibodies. NETs released in a microfluidic system bound b2GPI, but only in the presence of PF4, forming a multimolecular APS antigenic target. Whole blood infused through a photochemically-injured, endothelium-lined microfluidic channel formed platelet-, fibrin-, and complement- rich thrombi that bound APS antibody only in the presence of PF4. Thrombi were reduced in size if either ADAMTS13 or DNase1 was infused. In a murine APS model, wildtype and transgenic mice expressing platelet human PF4 ± FcgRIIA developed more intense neutrophil rolling along veins, and more extensive thrombus formation following laser injury to cremaster arterioles and venules, whereas mice lacking PF4 did not. Three antigenically distinct anti-hPF4 monoclonal antibodies blocked thrombosis in vitro, and neutrophil rolling and thrombosis in vivo. Our studies provide new insights into the basis of APS that has mechanistic parallels to other known PF4 immunothrombotic disorders and offer potential diagnostic and non-anticoagulant therapeutic strategies for clinical management.
Allogeneic hematopoietic cell transplantation (HCT) has benefited many patients with hematological malignancies and bone marrow failure states. However, graft vs host disease (GVHD) and immune incompetence remain signifi...Allogeneic hematopoietic cell transplantation (HCT) has benefited many patients with hematological malignancies and bone marrow failure states. However, graft vs host disease (GVHD) and immune incompetence remain significant challenges that limit the field to the treatment of patients with life threatening, high-risk disorders due to the significant non-relapse mortality (NRM) associated with the treatment. With a greater understanding of the pathophysiology of GVHD it has become apparent that this disorder represents a dysregulated immune reaction resulting in immune dysfunction further exacerbated by the treatment with immunosuppressive medications and injury to lymphoid tissues where immune recovery and tolerance develop. The emergence of fundamental mechanisms of immune regulation, whose pioneers received the 2025 Nobel Prize in Physiology and Medicine, have resulted in new concepts for improving outcomes for patients and the hope that with reduction in transplant related risk HCT can be offered to more patients with a broader range of immune mediated disorders. In this review the history, preclinical studies and successful translation of CD4+CD25+FoxP3+ regulatory T cells (Treg) in the context of allogeneic HCT will be discussed.
Iron deficiency is a highly prevalent nutrient deficiency and the most common cause of anemia. Although iron deficiency exacerbates cardiovascular disease, the direct impact of iron deficiency on the vasculature remains...Iron deficiency is a highly prevalent nutrient deficiency and the most common cause of anemia. Although iron deficiency exacerbates cardiovascular disease, the direct impact of iron deficiency on the vasculature remains unstudied. We assessed iron levels across the vascular endothelium in mouse and human endothelial cells and found resistance artery endothelial cells have the lowest iron stores, suggesting they may be especially impacted by iron deficiency. Anemia has previously been shown to increase arterial NO signaling in patients, and we have previously shown endothelial a-globin scavenges nitric oxide (NO) in the resistance artery endothelium. We hypothesized iron regulates vascular function through regulation of endothelial a-globin. To test this, we used a mouse model of iron deficiency anemia (IDA). In female mice, IDA increased NO signaling, which was rescued to control levels by repletion of vascular iron with iron dextran. Despite being similarly anemic and having a similar reduction in a-globin protein, there were no changes in NO signaling across groups in male mice. We further measured whether a-globin was in its heme-bound (holo a-globin) or heme-free (apo a-globin) state and found males did not fully lose its functional, heme-bound a-globin. Using endothelial specific a-globin knockout mice, we show loss of endothelial a-globin is necessary for increased NO signaling in IDA and for the rescue of NO signaling by iron dextran in female mice. Altogether, the data presented here demonstrate iron is a determinant of endothelial identity and modulates endothelial NO signaling through the regulation of a-globin.
Hemophilia A (HA) phenotype is predicted by factor VIII (FVIII) activity. Most HA adeno-associated virus (AAV) trials incorporate the B-domain-deleted FVIII-SQ variant and one-stage assay (OSA) FVIII activity exceeds chr...Hemophilia A (HA) phenotype is predicted by factor VIII (FVIII) activity. Most HA adeno-associated virus (AAV) trials incorporate the B-domain-deleted FVIII-SQ variant and one-stage assay (OSA) FVIII activity exceeds chromogenic substrate assay (CSA) values by 1.5-2-fold. This contrasts with recombinant FVIII-SQ (rFVIII-SQ), suggests altered biochemical properties, and highlights the need to determine which assay reflects hemostatic function. In gene therapy treated mice and SPK-8011 trial participants, AAV-derived FVIII-SQ (AAV-FVIII-SQ) activation and function within the intrinsic tenase enzyme complex was compared to rFVIII-SQ. In both species, AAV-FVIII-SQ demonstrated normal cofactor function and A2-domain stability. In mice, the assay discrepancy persisted without von Willebrand factor (vWF), indicating it is not driven by altered vWF interactions. Both species demonstrated enhanced thrombin-mediated activation of AAV-FVIII-SQ, detectable by OSA but not CSA. Negative binomial regression analysis of 23 SPK-8011 participants, representing 99 cumulative patient-years, trended toward better prediction of annualized bleeding rate with OSA than CSA. In vivo evaluation of AAV-FVIII-SQ in mice demonstrated that OSA activity better correlated with hemostatic function and corresponded to rFVIII-SQ function. These findings support that OSA FVIII activity reflects AAV-FVIII-SQ function within the intrinsic tenase complex, captures enhanced activation not detected by CSA, and best predicts clinical outcome.
Hu B, Edwards J, Modi H
… +26 more, Gamez J, Echeagaray OE, Hess K, Ren Y, Anderson D, Larrayoz M, Zhu J, Johnson SA, Deb G, Jankeel D, Janardhanan P, Leisten J, Peng S, Christoforou A, Stong N, Fontanillo C, Bjorklund CC, Hagner PR, Gandhi AK, Martínez-Climent JA, Narla RK, Lopez-Girona A, Rolfe M, Bence N, Mortensen DS, Groocock L
The t(4;14) chromosomal translocation drives overexpression of the histone methyltransferase NSD2 and defines a high-risk segment of multiple myeloma (MM) patients. Herein, we report the discovery of NSD2-LDD, a cereblon...The t(4;14) chromosomal translocation drives overexpression of the histone methyltransferase NSD2 and defines a high-risk segment of multiple myeloma (MM) patients. Herein, we report the discovery of NSD2-LDD, a cereblon-recruiting and PWWP1-mediated ligand directed degrader (LDD) that selectively and potently eliminates full length and PWWP1 domain containing NSD2 protein isoforms. NSD2-LDD treatment induces global loss of H3K36me2 leading to promoter-proximal spreading of H3K27me3 and re-wiring of cis-regulatory interactions that reverse t(4;14) transcriptional programs. These effects suppress MM disease-associated phenotypes including stromal adhesion, three-dimensional colony growth and paracrine signaling. By integrating patient single cell profiles with model 3D epigenomic and spatial transcriptomics, we delineate t(4;14) disease state together with the tumor-intrinsic reprogramming and resultant remodeling of the bone marrow microenvironment upon NSD2 degradation. In cell line derived xenografts and genetically engineered mouse models of t(4;14), NSD2-LDD extends median survival accompanied by tumoral H3K36me2 loss and niche re-modelling. Although the NSD2-LDD response is restricted to PWWP1-containining models, collectively this work validates NSD2 as a tractable dependency and supports clinical development of NSD2 degradation as a novel, targeted therapeutic strategy in high-risk MM.