Sickle cell disease (SCD) is a hereditary hemoglobinopathy characterized by painful vaso-occlusive crises (VOC) and chronic hemolysis. The mononuclear phagocyte system is pivotal to SCD pathophysiology, but the mechanisms governing monocyte/macrophage differentiation remain unknown. This study examined the influence of hemolysis on circulating monocyte trajectories in SCD. We discovered that hemolysis stimulated CSF-1 production, partly by endothelial cells via Nrf2, promoting classical monocyte (CMo) differentiation into blood patrolling monocytes (PMo) in SCD mice. However, hemolysis also upregulated CCL-2 through IFN-I, inducing CMo transmigration and differentiation into tissue monocyte-derived macrophages. Blocking CMo transmigration by anti-P-selectin antibody in SCD mice increased circulating PMo, corroborating that CMo-to-tissue macrophage differentiation occurs at the expense of CMo-to-blood PMo differentiation. We observed a positive correlation between plasma CSF-1/CCL-2 ratios and blood PMo levels in SCD patients, underscoring the clinical significance of these two opposing factors in monocyte differentiation. Combined treatment with CSF-1 and anti-P-selectin antibody more effectively increased PMo numbers and reduced stasis compared to single-agent therapies in SCD mice. Altogether, these data indicate that monocyte fates are regulated by the balance between two heme pathways, Nrf2-CSF-1 and IFN-I-CCL-2, and suggest that the CSF-1/CCL-2 ratio may present a diagnostic and therapeutic target in SCD.
Yunfeng Liu, Shan Su, Sarah Shayo, Weili Bao, Mouli Pal, Kai Dou, Patricia A. Shi, Banu Aygun, Sally Campbell-Lee, Cheryl A. Lobo, Avital Mendelson, Xiuli An, Deepa Manwani, Hui Zhong, Karina Yazdanbakhsh
Protease activated receptor (PAR) 4 (gene: F2RL3) harbors a functional dimorphism, rs773902 A/G (encoding Thr120/Ala120, respectively) and is associated with greater platelet aggregation. The A allele frequency is more common in Black individuals, and Black individuals have a higher incidence of ischemic stroke than White individuals. However, it is not recognized whether the A allele is responsible for worse stroke outcomes. To directly test the in vivo effect of this variant on stroke, we generated mice where F2rl3 was replaced by F2RL3, thereby expressing human PAR4 (hPAR4) with either Thr120 or Ala120. Compared to hPAR4 Ala120 mice, hPAR4 Thr120 mice had worse stroke outcomes, mediated in part by enhanced platelet activation and platelet-neutrophil interactions. Analyses of 7620 Black subjects with 487 incident ischemic strokes demonstrated the AA genotype was a risk for incident ischemic stroke and worse functional outcomes. In humanized mice, ticagrelor with or without aspirin improved stroke outcomes in hPAR4 Ala120 mice, but not in hPAR4 Thr120 mice. P-selectin blockade improved stroke outcomes and reduced platelet-neutrophil interactions in hPAR4 Thr120 mice. Our results may explain some of the racial disparity in stroke and support the need for studies of non-standard anti-platelet therapies for patients expressing PAR4 Thr120.
Frederik Denorme, Nicole D. Armstrong, Michelle L. Stoller, Irina Portier, Emilia A. Tugolukova, Rikki M. Tanner, Emilie Montenont, Seema Bhatlekar, Mark Cody, John L. Rustad, Abigail Ajanel, Neal D. Tolley, Darian C. Murray, Julie L. Boyle, Marvin T. Nieman, Steven E. McKenzie, Christian Con Yost, Leslie A. Lange, Mary Cushman, Marguerite R. Irvin, Paul F. Bray, Robert A. Campbell
RAS mutations are among the most prevalent oncogenic drivers in cancers. RAS proteins propagate signals only when associated with cellular membranes as a consequence of lipid modifications that impact their trafficking. Here, we discovered that RAB27B, a RAB family small GTPase, controlled NRAS palmitoylation and trafficking to the plasma membrane, a localization required for activation. Our proteomic studies revealed RAB27B upregulation in CBL- or JAK2-mutated myeloid malignancies, and its expression correlated with poor prognosis in acute myeloid leukemias (AMLs). RAB27B depletion inhibited the growth of CBL-deficient or NRAS-mutant cell lines. Strikingly, Rab27b deficiency in mice abrogated mutant but not WT NRAS–mediated progenitor cell growth, ERK signaling, and NRAS palmitoylation. Further, Rab27b deficiency significantly reduced myelomonocytic leukemia development in vivo. Mechanistically, RAB27B interacted with ZDHHC9, a palmitoyl acyltransferase that modifies NRAS. By regulating palmitoylation, RAB27B controlled c-RAF/MEK/ERK signaling and affected leukemia development. Importantly, RAB27B depletion in primary human AMLs inhibited oncogenic NRAS signaling and leukemic growth. We further revealed a significant correlation between RAB27B expression and sensitivity to MEK inhibitors in AMLs. Thus, our studies presented a link between RAB proteins and fundamental aspects of RAS posttranslational modification and trafficking, highlighting future therapeutic strategies for RAS-driven cancers.
Jian-Gang Ren, Bowen Xing, Kaosheng Lv, Rachel A. O’Keefe, Mengfang Wu, Ruoxing Wang, Kaylyn M. Bauer, Arevik Ghazaryan, George M. Burslem, Jing Zhang, Ryan M. O’Connell, Vinodh Pillai, Elizabeth O. Hexner, Mark R. Philips, Wei Tong
During emergency hematopoiesis, hematopoietic stem cells (HSCs) rapidly proliferate to produce myeloid and lymphoid effector cells, a response that is critical against infection or tissue injury. If unresolved, this process leads to sustained inflammation which can cause life-threatening diseases and cancer. We have identified a novel role of Dpf2 in inflammation. Dpf2 is a defining subunit of the hematopoietic-specific BAF (SWI/SNF) chromatin-remodeling complex, and it is mutated in multiple cancers and neurological disorders. We uncover that hematopoietic-specific Dpf2 knock-out mice develop leukopenia, severe anemia and lethal systemic inflammation characterized by histiocytic and fibrotic tissue infiltration, resembling a clinical hyper-inflammatory state. Dpf2 loss impairs the polarization of macrophages responsible for tissue repair, induces unrestrained activation of T helper cells, and generates an emergency-like state of HSC hyperproliferation and myeloid-biased differentiation. Mechanistically, Dpf2 deficiency results in the loss of the BAF catalytic subunit Brg1 from Nrf2-controlled enhancers, impairing the anti-oxidant and anti-inflammatory transcriptional response needed to modulate inflammation. Finally, pharmacological reactivation of Nrf2 can suppress the inflammation-mediated phenotypes and lethality of Dpf2Δ/Δ mice. Our work establishes the essential role of the Dpf2/BAF complex in licensing Nrf2-dependent gene expression in HSCs and immune effector cells to prevent chronic inflammation.
Gloria Mas, Na Man, Yuichiro Nakata, Concepcion Martinez-Caja, Daniel L. Karl, Felipe Beckedorff, Francesco Tamiro, Chuan Chen, Stephanie Duffort, Hidehiro Itonaga, Adnan K. Mookhtiar, Kranthi Kunkalla, Alfredo M. Valencia, Clayton K. Collings, Cigall Kadoch, Francisco Vega, Scott C. Kogan, Lluis Morey, Daniel Bilbao, Stephen D. Nimer
Characterized by the accumulation of somatic mutations in blood cell lineages, clonal hematopoiesis (CH) of indeterminate potential (CHIP) is frequent in ageing, involves expansion of mutated hematopoietic stem and progenitor cells (HSC/Ps) that leads to an increased risk of hematologic malignancy. However, risk factors that contribute to CHIP-associated CH are poorly understood. Obesity induces a pro-inflammatory state and fatty bone marrow (FBM), which may influence CHIP-associated pathologies. We analyzed exome sequencing and clinical data from 47,466 individuals with validated CHIP in UK Biobank. CHIP was present in 5.8% of the study population and was associated with a significant increase in waist-to-hip ratio (WHR). Mouse models of obesity and CHIP driven by heterozygosity of Tet2, Dnmt3a, Asxl1 and Jak2 resulted in exacerbated expansion of mutant HSC/Ps due in part to excessive inflammation. Our results show that obesity is highly associated with CHIP and a pro-inflammatory state can potentiate progression of CHIP to more significant hematologic neoplasia. Calcium channel blocker, nifedipine or SKF-96365, either alone or in combination with metformin, MCC950 or anakinra (IL-1 receptor antagonist), suppressed the growth of mutant CHIP cells and partially restored normal hematopoiesis. Targeting CHIP mutant cells with these drugs could be a potential therapeutic approach to treat CH and its associated abnormalities in obese individuals.
Santhosh Kumar Pasupuleti, Baskar Ramdas, Sarah S. Burns, Lakshmi Reddy Palam, Rahul Kanumuri, Ramesh Kumar, Taruni R. Pandhiri, Utpal Dave, Nanda Kumar Yellapu, Xinyu Zhou, Chi Zhang, George E. Sandusky, Zhi Yu, Michael C. Honigberg, Alexander G. Bick, Gabriel K. Griffin, Abhishek Niroula, Benjamin L. Ebert, Sophie Paczesny, Pradeep Natarajan, Reuben Kapur
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.
Priti Azad, Dan Zhou, Hung-Chi Tu, Francisco C. Villafuerte, David Traver, Tariq M. Rana, Gabriel G. Haddad
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.
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
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.
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
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.
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
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.
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