Hematologies
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
Abstract
In chronic myeloid leukemia (CML), combination therapies with tyrosine kinase inhibitors (TKIs) aim to improve the achievement of deep molecular remission that would allow therapy discontinuation. IFN-α is one promising candidate, as it has long-lasting effects on both malignant and immune cells. In connection with a multicenter clinical trial combining dasatinib with IFN-α in 40 patients with chronic-phase CML (NordCML007, NCT01725204), we performed immune monitoring with single-cell RNA and T cell receptor (TCR) sequencing (n = 4, 12 samples), bulk TCRβ sequencing (n = 13, 26 samples), flow cytometry (n = 40, 106 samples), cytokine analyses (n = 17, 80 samples), and ex vivo functional studies (n = 39, 80 samples). Dasatinib drove the immune repertoire toward terminally differentiated NK and CD8+ T cells with dampened functional capabilities. Patients with dasatinib-associated pleural effusions had increased numbers of CD8+ recently activated effector memory T (Temra) cells. In vitro, dasatinib prevented CD3-induced cell death by blocking TCR signaling. The addition of IFN-α reversed the terminally differentiated phenotypes and increased the number of costimulatory intercellular interactions and the number of unique putative epitope-specific TCR clusters. In vitro IFN-α had costimulatory effects on TCR signaling. Our work supports the combination of IFN-α with TKI therapy, as IFN-α broadens the immune repertoire and restores immunological function.
Authors
Jani Huuhtanen, Mette Ilander, Bhagwan Yadav, Olli M.J. Dufva, Hanna Lähteenmäki, Tiina Kasanen, Jay Klievink, Ulla Olsson-Strömberg, Jesper Stentoft, Johan Richter, Perttu Koskenvesa, Martin Höglund, Stina Söderlund, Arta Dreimane, Kimmo Porkka, Tobias Gedde-Dahl, Björn T. Gjertsen, Leif Stenke, Kristina Myhr-Eriksson, Berit Markevärn, Anna Lübking, Andreja Dimitrijevic, Lene Udby, Ole Weis Bjerrum, Henrik Hjorth-Hansen, Satu Mustjoki
Abstract
Infantile (fetal and neonatal) megakaryocytes have a distinct phenotype consisting of hyperproliferation, limited morphogenesis, and low platelet production capacity. These properties contribute to clinical problems that include thrombocytopenia in neonates, delayed platelet engraftment in recipients of cord blood stem cell transplants, and inefficient ex vivo platelet production from pluripotent stem cell-derived megakaryocytes. The infantile phenotype results from deficiency of the actin-regulated coactivator, MKL1, which programs cytoskeletal changes driving morphogenesis. As a strategy to complement this molecular defect, we screened pathways with potential to affect MKL1 function and found that Dyrk1a kinase inhibition dramatically enhanced megakaryocyte morphogenesis in vitro and in vivo. Dyrk1 inhibitors rescued enlargement, polyploidization, and thrombopoiesis in human neonatal megakaryocytes. Megakaryocytes derived from induced pluripotent stem cells responded in a similar manner. Progenitors undergoing Dyrk1 inhibition demonstrated filamentous actin assembly, MKL1 nuclear translocation, and modulation of MKL1 target genes. Loss of function studies confirmed MKL1 involvement in this morphogenetic pathway. Ablim2, a stabilizer of filamentous actin, increased with Dyrk1 inhibition, and Ablim2 knockdown abrogated the actin, MKL1, and morphogenetic responses to Dyrk1 inhibition. These results thus delineate a pharmacologically tractable morphogenetic pathway whose manipulation may alleviate clinical problems associated with the limited thrombopoietic capacity of infantile megakaryocytes.
Authors
Kamaleldin E. Elagib, Ashton Brock, Cara M. Clementelli, Gohar Mosoyan, Lorrie L. Delehanty, Ranjit K. Sahu, Alexandra Pacheco-Benichou, Corinne Fruit, Thierry Besson, Stephan W. Morris, Koji Eto, Chintan Jobaliya, Deborah L. French, Paul Gadue, Sandeep Singh, Xinrui Shi, Fujun Qin, Robert Cornelison, Hui Li, Camelia Iancu-Rubin, Adam N. Goldfarb
Abstract
Myelodysplastic syndromes (MDS) are age-related myeloid neoplasms with increased risks of progression to acute myeloid leukemia (AML). The mechanisms of MDS to AML transformation are poorly understood, especially in relation to the aging microenvironment. We previously established a mDia1/miR-146a double knockout (DKO) mouse model phenocopying MDS. These mice develop age-related pancytopenia with over-secretion of pro-inflammatory cytokines. Here, we found that most of the DKO mice underwent leukemic transformation at 12-14 months of age. These mice showed myeloblast replacement of a fibrotic bone marrow and widespread leukemic infiltration. Strikingly, depletion of IL-6 in these mice largely rescued the leukemic transformation and markedly extended the survival. Single cell RNA sequencing analyses revealed that DKO leukemic mice had increased monocytic blasts that were reduced with IL-6 knockout. We further revealed that the levels of surface and soluble IL-6 receptor (IL-6R) in the bone marrow were significantly increased in high risk MDS patients. Similarly, IL-6R was also highly expressed in older DKO mice. Blocking of IL-6 signaling significantly ameliorated AML progression in the DKO model and clonogenicity of CD34 positive cells from MDS patients. Our study establishes a mouse model of age-related MDS to AML progression and indicates the clinical significance of targeting IL-6 signaling in treating high risk MDS.
Authors
Yang Mei, Kehan Ren, Yijie Liu, Annabel Ma, Zongjun Xia, Xu Han, Ermin Li, Hamza Tariq, Haiyan Bao, Xinshu Xie, Cheng Zou, Dingxiao Zhang, Zhaofeng Li, Lili Dong, Amit Verma, Xinyan Lu, Yasmin Abaza, Jessica K. Altman, Madina Sukhanova, Jing Yang, Peng Ji
Copyright © 2023 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)