Hematology
Abstract
Excessive Erythrocytosis (EE) is a major hallmark of patients suffering from chronic mountain sickness (CMS, Monge’s disease) and is responsible for major morbidity and even mortality in early adulthood. We took advantage of unique populations, one living at high altitude (Peru) showing EE, while another population, at the same altitude and region, shows no evidence of EE (non-CMS). Through RNA-seq, we identified and validated the function of a group of long non-coding RNA (lncRNAs) that regulate erythropoiesis in Monge’s disease but not in the non-CMS population. Among these lncRNAs is HIKER (Hypoxia Induced Kinase-mediated Erythropoietic Regulator)/LINC02228 which we showed plays a critical role in erythropoiesis in CMS cells. Under hypoxia, HIKER modulated CSNK2B (the regulatory subunit of Casein kinase 2). A down-regulation of HIKER down-regulated CSNK2B, remarkably reducing erythropoiesis (<70% reduction of BFUs); furthermore, an up-regulation of CSNK2B on the background of HIKER down-regulation rescued erythropoiesis defects. Pharmacologic inhibition of CSNK2B drastically reduced erythroid colonies (50-75% reduction in BFU colonies) and knock-down of CSNK2B in zebrafish lead to a defect in hemoglobinization (<97% morphants show reduction in hemoglobin levels). We conclude that HIKER regulates erythropoiesis in Monge’s disease and acts through at least one specific target, CSNK2B, a casein kinase.
Authors
Priti Azad, Dan Zhou, Hung-Chi Tu, Francisco C. Villafuerte, David Traver, Tariq M. Rana, Gabriel G. Haddad
Abstract
Endothelial cells (ECs) are constitutively an anticoagulant surface but switch to support coagulation following pathogenic stimuli. This switch promotes thrombotic cardiovascular disease. To generate thrombin at physiologic rates, coagulation proteins assemble on a membrane containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid “scramblases”, such as TMEM16F. TMEM16F-dependent PS externalization is well-characterized in platelets. In contrast, how ECs externalize phospholipids to support coagulation is not understood. We employed a focused genetic screen to evaluate the contribution of transmembrane phospholipid transport on EC procoagulant activity. We identified two TMEM16 family members, TMEM16F, and its closest paralog, TMEM16E, which were both required to support coagulation on ECs via PS externalization. Applying an intravital laser-injury model of thrombosis, we observed, unexpectedly, that PS externalization was concentrated at the vessel wall, not on platelets. TMEM16E-null mice demonstrated reduced vessel-wall dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevented PS externalization and EC procoagulant activity and protected mice from thrombosis without increasing bleeding following tail transection. These findings indicate the activated endothelial surface is a source of procoagulant phospholipid contributing to thrombus formation. TMEM16 phospholipid scramblases may be a therapeutic target for thrombotic cardiovascular disease.
Authors
Alec A. Schmaier, Papa F. Anderson, Siyu M. Chen, Emale El-Darzi, Ivan Aivasovsky, Milan P. Kaushik, Kelsey D. Sack, H. Criss Hartzell, Samir M. Parikh, Robert Flaumenhaft, Sol Schulman
Abstract
Although certain human genetic variants are conspicuously loss-of-function, decoding the impact of many variants is challenging. Previously, we described a leukemia predisposition syndrome (GATA2-deficiency) patient with a germline GATA2 variant that inserts nine amino acids between the two zinc fingers (9aa-Ins). Here, we conducted mechanistic analyses using genomic technologies and a genetic rescue system with Gata2 enhancer-mutant hematopoietic progenitor cells to compare how GATA2 and 9aa-Ins function genome-wide. Despite nuclear localization, 9aa-Ins was severely defective in occupying and remodeling chromatin and regulating transcription. Variation of the inter-zinc finger spacer length revealed that insertions were more deleterious to activation than repression. GATA2-deficiency generated a lineage-diverting gene expression program and a hematopoiesis-disrupting signaling network in progenitors with reduced Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and elevated Interleukin-6 (IL-6) signaling. As insufficient GM-CSF signaling causes pulmonary alveolar proteinosis and excessive IL-6 signaling promotes bone marrow failure, GATA2-deficiency patient phenotypes, these results inform mechanisms underlying GATA2-linked pathologies.
Authors
Mabel Minji Jung, Siqi Shen, Giovanni A. Botten, Thomas Olender, Koichi R. Katsumura, Kirby D. Johnson, Alexandra A. Soukup, Peng Liu, Qingzhou Zhang, Zena D. Jensvold, Peter W. Lewis, Robert A. Beagrie, Jason K.K. Low, Lihua Yang, Joel P. Mackay, Lucy A. Godley, Marjorie Brand, Jian Xu, Sunduz Keles, Emery H. Bresnick
Abstract
Bruton’s tyrosine kinase (BTK) is a proven target in mantle cell lymphoma (MCL), an aggressive subtype of non-Hodgkin lymphoma. However, resistance to BTK inhibitors is a major clinical challenge. We here report that MALT1 is one of the top overexpressed genes in ibrutinib-resistant MCL cells, while expression of CARD11, which is upstream of MALT1, is decreased. MALT1 genetic knockout or inhibition produced dramatic defects in MCL cell growth regardless of ibrutinib sensitivity. Conversely, CARD11-knockout cells showed antitumor effects only in ibrutinib-sensitive cells, suggesting that MALT1 overexpression could drive ibrutinib resistance via bypassing BTK/CARD11 signaling. Additionally, BTK knockdown and MALT1 knockout markedly impaired MCL tumor migration and dissemination, and MALT1 pharmacological inhibition decreased MCL cell viability, adhesion, and migration by suppressing NF-κB, PI3K/AKT/mTOR, and integrin signaling. Importantly, cotargeting MALT1 with safimaltib and BTK with pirtobrutinib induced potent anti-MCL activity in ibrutinib-resistant MCL cell lines and patient-derived xenografts. Therefore, we conclude that MALT1 overexpression associates with resistance to BTK inhibitors in MCL, targeting abnormal MALT1 activity could be a promising therapeutic strategy to overcome BTK inhibitor resistance, and cotargeting of MALT1 and BTK should improve MCL treatment efficacy and durability as well as patient outcomes.
Authors
Vivian Changying Jiang, Yang Liu, Junwei Lian, Shengjian Huang, Alexa Jordan, Qingsong Cai, Ruitao Lin, Fangfang Yan, Joseph McIntosh, Yijing Li, Yuxuan Che, Zhihong Chen, Jovanny Vargas, Maria Badillo, John Nelson Bigcal, Heng-Huan Lee, Wei Wang, Yixin Yao, Lei Nie, Christopher R. Flowers, Michael Wang
Abstract
Myeloproliferative neoplasms (MPNs) are characterized by the activated JAK2-STAT pathway. Pleckstrin-2 (Plek2) is a downstream target of the JAK2-STAT pathway and overexpressed in patients with MPNs. We previously revealed that Plek2 plays critical roles in the pathogenesis of JAK2 mutated MPNs. The non-essential roles of Plek2 under physiologic conditions makes it an ideal target for MPN therapy. Here we identified first-in-class Plek2 inhibitors through an in silico high-throughput screening and cell-based assays followed by the synthesis of analogs. The Plek2 specific small molecule inhibitors showed potent inhibitory effects on cell proliferation. Mechanistically, Plek2 interacts with and enhances the activity of Akt through the recruitment of downstream effector proteins. The Plek2 signaling complex also includes Hsp72 that protects Akt from degradation. These functions were blocked by Plek2 inhibitors via their direct binding to Plek2 DEP domain. The role of Plek2 in activating the Akt signaling was further confirmed in vivo using a hematopoietic specific Pten knockout mouse model. We next tested Plek2 inhibitors alone or in combination with an Akt inhibitor in various MPN mouse models, which showed significant therapeutic efficacies similar to the genetic depletion of Plek2. The Plek2 inhibitor was also effective in reducing proliferation of CD34 positive cells from MPN patients. Our studies reveal a Plek2-Akt complex that drives cell proliferation and can be targeted by a new class of anti-proliferative compounds for MPN therapy.
Authors
Xu Han, Yang Mei, Rama K. Mishra, Honghao Bi, Atul D. Jain, Gary E. Schiltz, Baobing Zhao, Madina Sukhanova, Pan Wang, Arabela A. Grigorescu, Patricia C. Weber, John J. Piwinski, Miguel A. Prado, Joao A. Paulo, Len Stephens, Karen E. Anderson, Charles S. Abrams, Jing Yang, Peng Ji
Abstract
The mTORC1 pathway coordinates nutrient and growth factor signals to maintain organismal homeostasis. Whether nutrient signaling to mTORC1 regulates stem cell function remains unknown. Here, we show that SZT2 — a protein required for mTORC1 downregulation upon nutrient deprivation — is critical for hematopoietic stem cell (HSC) homeostasis. Ablation of SZT2 in HSCs decreased the reserve and impaired the repopulating capacity of HSCs. Furthermore, ablation of both SZT2 and TSC1 — 2 repressors of mTORC1 on the nutrient and growth factor arms, respectively — led to rapid HSC depletion, pancytopenia, and premature death of the mice. Mechanistically, loss of either SZT2 or TSC1 in HSCs led to only mild elevation of mTORC1 activity and reactive oxygen species (ROS) production. Loss of both SZT2 and TSC1, on the other hand, simultaneously produced a dramatic synergistic effect, with an approximately 10-fold increase of mTORC1 activity and approximately 100-fold increase of ROS production, which rapidly depleted HSCs. These data demonstrate a critical role of nutrient mTORC1 signaling in HSC homeostasis and uncover a strong synergistic effect between nutrient- and growth factor–mediated mTORC1 regulation in stem cells.
Authors
Na Yin, Gang Jin, Yuying Ma, Hanfei Zhao, Guangyue Zhang, Ming O. Li, Min Peng
Abstract
Platelets and megakaryocytes are critical players in immune responses. Recent reports suggest infection and inflammation alter the megakaryocyte and platelet transcriptome to induce altered platelet reactivity. We examined if non-viral sepsis induces differential platelet gene expression and reactivity. Non-viral sepsis upregulated IFITM3, an interferon responsive gene that restricts viral replication. As IFITM3 has been linked to clathrin-mediated endocytosis, we examined if IFITM3 promoted endocytosis of alpha granule proteins. Interferon stimulation enhanced fibrinogen endocytosis in megakaryocytes and platelets from Ifitm+/+ mice, but not Ifitm-/- mice. IFITM3 overexpression or deletion in megakaryocytes demonstrated IFITM3 was necessary and sufficient to regulate fibrinogen endocytosis. Mechanistically, IFITM3 interacts with clathrin and αIIb and altered their plasma membrane localization into lipid rafts. In vivo interferon administration increased fibrinogen endocytosis, platelet reactivity, and thrombosis in an IFITM-dependent manner. In contrast, Ifitm-/- mice were completely rescued from interferon-induced platelet hyperreactivity and thrombosis. During murine sepsis, platelets from Ifitm+/+ mice demonstrated increased fibrinogen content and platelet reactivity, which was dependent on interferon-alpha and IFITMs. Platelets from patients with non-viral sepsis had increases in platelet IFITM3 expression, fibrinogen content, and hyperreactivity. These data identify IFITM3 as a regulator of platelet endocytosis, hyperreactivity, and thrombosis during inflammatory stress.
Authors
Robert A. Campbell, Bhanu Kanth Manne, Meenakshi Banerjee, Elizabeth A. Middleton, Abigail Ajanel, Hansjorg Schwertz, Frederik Denorme, Chris Stubben, Emilie Montenont, Samantha Saperstein, Lauren Page, Neal D. Tolley, Diana L. Lim, Samuel M. Brown, Colin K. Grissom, Douglas W. Sborov, Anandi Krishnan, Matthew T. Rondina
Abstract
A major complication of hemophilia A therapy is the development of alloantibodies (inhibitors) that neutralize intravenously administered coagulation factor VIII (FVIII). Immune tolerance induction therapy (ITI) by repetitive FVIII injection can eradicate inhibitors, and thereby reduce morbidity and treatment costs. However, ITI success is difficult to predict and the underlying immunological mechanisms are unknown. Here, we demonstrated that immune tolerance against FVIII under non-hemophilic conditions was maintained by programmed death (PD) ligand 1 (PD-L1)-expressing regulatory T cells (Treg) that ligated PD-1 on FVIII-specific B cells, causing them to undergo apoptosis. FVIII-deficient mice injected with FVIII lacked such Treg and developed inhibitors. Using an ITI mouse model, we found that repetitive FVIII injection induced FVIII-specific PD-L1+ Tregs and re-engaged removal of inhibitor-forming B cells. We demonstrated the existence of FVIII-specific Tregs also in humans and showed that such Tregs upregulated PD-L1 after successful ITI. Simultaneously, FVIII-specific B cells upregulated PD-1 and became killable by Tregs. In summary, we showed that PD-1-mediated B cell tolerance against FVIII operated in healthy individuals and in hemophilia A patients without inhibitors, and that ITI re-engaged this mechanism. These findings may impact monitoring of ITI success and treatment of hemophilia A patients.
Authors
Janine Becker-Gotot, Mirjam Meissner, Vadim Kotov, Blanca Jurado-Mestre, Andrea Maione, Andreas Pannek, Thilo Albert, Chrystel Flores, Frank A. Schildberg, Paul A. Gleeson, Birgit M. Reipert, Johannes Oldenburg, Christian Kurts
Abstract
BACKGROUND AND METHODS. The functional and transcriptional features of immune effector senescence and their influence on therapeutic response were investigated in independent AML clinical cohorts comprising 1,896 patients treated with chemotherapy and/or immune checkpoint blockade (ICB). RESULTS. We show that senescent-like bone marrow CD8+ T cells were impaired in killing autologous AML blasts, and that their proportion negatively correlated with overall survival (OS). We defined new immune effector dysfunction (IED) signatures using two gene expression profiling platforms and report that IED scores correlated with adverse-risk molecular lesions, stemness, and poor outcomes as a potentially more powerful predictor of OS than 2017-ELN risk or leukemia stem cell (LSC17) scores. IED expression signatures also identified an ICB-unresponsive tumor microenvironment and predicted significantly worse OS. CONCLUSION. The newly described IED scores provided improved AML risk stratification and could facilitate the delivery of personalized immunotherapies to patients who are most likely to benefit.
Authors
Sergio Rutella, Jayakumar Vadakekolathu, Francesco Mazziotta, Stephen Reeder, Tung-On Yau, Rupkatha Mukhopadhyay, Benjamin Dickins, Heidi Altmann, Michael Kramer, Hanna A. Knaus, Bruce R. Blazar, Vedran Radojcic, Joshua F. Zeidner, Andrea Arruda, Bofei Wang, Hussein A. Abbas, Mark D. Minden, Sarah K. Tasian, Martin Bornhäuser, Ivana Gojo, Leo Luznik
Abstract
SAMD9 and SAMD9L germline mutations have recently emerged as a new class of predispositions to pediatric myeloid neoplasms. Patients commonly have impaired hematopoiesis, hypocellular marrows, and a greater risk of developing clonal chromosome 7 deletions leading to MDS and AML. We recently demonstrated that expressing SAMD9 or SAMD9L mutations in hematopoietic cells suppresses their proliferation and induces cell death. Here we generated a mouse model that conditionally expresses mutant Samd9l to assess the in vivo impact on hematopoiesis. Using a range of in vivo and ex vivo assays, we showed that cells with heterozygous Samd9l mutations have impaired stemness relative to wild-type counterparts, which was exacerbated by inflammatory stimuli, and ultimately led to bone marrow hypocellularity. Genomic and phenotypic analyses recapitulated many of the hematopoietic cellular phenotypes observed in patients with SAMD9 or SAMD9L mutations, including lymphopenia, and pinpointed TGF-β as a potential targetable pathway. Further, we observed non-random genetic deletion of the mutant Samd9l locus on mouse chromosome 6, mimicking chromosome 7 deletions observed in patients. Collectively, our study has enhanced our understanding of mutant Samd9l hematopoietic phenotypes, emphasized the synergistic role of inflammation in exaggerating the associated hematopoietic defects, and provided insights into potential therapeutic options for patients.
Authors
Sherif Abdelhamed, Melvin E. Thomas III, Tamara Westover, Masayuki Umeda, Emily Xiong, Chandra Rolle, Michael P. Walsh, Huiyun Wu, Jason R. Schwartz, Virginia Valentine, Marcus Valentine, Stanley Pounds, Jing Ma, Laura J. Janke, Jeffery M. Klco
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)