Hematology
Abstract
Ecotropic viral integration site 1 (EVI1/MECOM) is frequently upregulated in myeloid malignancies. Here, we present an Evi1-transgenic mouse model with inducible expression in hematopoietic stem/progenitor cells (HSPCs). Upon induction of Evi1 expression, mice displayed anemia, thrombocytopenia, lymphopenia, and erythroid and megakaryocyte dysplasia with a significant expansion of committed myeloid progenitor cells, resembling human myelodysplastic syndrome/myeloproliferative neoplasm–like (MDS/MPN–like) disease. Evi1 overexpression prompted HSPCs to exit quiescence and accelerated their proliferation, leading to expansion of committed myeloid progenitors while inhibiting lymphopoiesis. Analysis of global gene expression and Evi1 binding site profiling in HSPCs revealed that Evi1 directly upregulated lysine demethylase 6b (Kdm6b). Subsequently, Kdm6b-mediated H3K27me3 demethylation resulted in activation of various genes, including Laptm4b. Interestingly, KDM6B and LAPTM4B are positively correlated with EVI1 expression in patients with MDS. The EVI1/KDM6B/H3K27me3/LAPTM4B signaling pathway was also identified in EVI1hi human leukemia cell lines. We found that hyperactivation of the LAPTM4B-driven mTOR pathway was crucial for the growth of EVI1hi leukemia cells. Knockdown of Laptm4b partially rescued Evi1-induced abnormal hematopoiesis in vivo. Thus, our study establishes a mouse model to investigate EVI1hi myeloid malignancies, demonstrating the significance of the EVI1-mediated KDM6B/H3K27me3/LAPTM4B signaling axis in their maintenance.
Authors
Qiong Wu, Chunjie Yu, Fang Yu, Yiran Guo, Yue Sheng, Liping Li, Yafang Li, Yutao Zhang, Chao Hu, Jue Wang, Tong-chuan He, Yong Huang, Hongyu Ni, Zhiguang Huo, Wenshu Wu, Gang Greg Wang, Jianxin Lyu, Zhijian Qian
Abstract
Despite the revolutionary achievements of chimeric antigen receptor (CAR) T cell therapy in treating cancers, especially leukemia, several key challenges still limit its therapeutic efficacy. Of particular relevance is the relapse of cancer in large part, as a result of exhaustion and short persistence of CAR-T cells in vivo. IL-2-inducible T cell kinase (ITK) is a critical modulator of the strength of T-cell receptor (TCR) signaling, while its role in CAR signaling is unknown. By electroporation of clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) ribonucleoprotein (RNP) complex into CAR-T cells, we successfully deleted ITK in CD19-CAR-T cells with high efficiency. Bulk and single-cell RNA sequencing (scRNA-seq) analyses revealed down-regulation of exhaustion and up-regulation of memory gene signatures in ITK-deficient CD19-CAR-T cells. Our results further demonstrated a significant reduction of T cell exhaustion and enhancement of T cell memory, with significant improvement of CAR-T cell expansion and persistence both in vitro and in vivo. Moreover, ITK-deficient CD19-CAR-T cells showed better control of tumor relapse. Our work provides a promising strategy of targeting ITK to develop sustainable CAR-T products for clinical use.
Authors
Zheng Fu, Zineng Huang, Hao Xu, Qingbai Liu, Jing Li, Keqing Song, Yating Deng, Yujia Tao, Huifang Zhang, Peilong Wang, Heng Li, Yue Sheng, Aijun Zhou, Lianbin Han, Yan Fu, Chen-Zhi Wang, Saurav Kumar Choudhary, Kaixiong Ye, Gianluca Veggiani, Zhihong Li, Avery August, Weishan Huang, Qiang Shan, Hongling Peng
Abstract
Proper control of inflammatory responses is essential for embryonic development, but the underlying mechanism is poorly understood. Here, we show that under physiological conditions, inactivation of ISG15, an inflammation amplifier, is associated with the interaction of Beclin 1 (Becn1), via its ECD domain, with STAT3 in the major fetal hematopoietic organ of mice. Conditional loss of Becn1 caused sequential dysfunction and exhaustion of fetal liver hematopoietic stem cells, leading to lethal inflammatory cell-biased hematopoiesis in the fetus. Molecularly, the absence of Becn1 resulted in the release of STAT3 from Becn1 tethering and subsequent phosphorylation and translocation to the nucleus, which in turn directly activated the transcription of ISG15 in fetal liver hematopoietic cells, coupled with increased ISGylation and production of inflammatory cytokines, whereas inactivating STAT3 reduced ISG15 transcription and inflammation but improved hematopoiesis potential, and further silencing ISG15 mitigated the above collapse in the Becn1 null hematopoietic lineage. The Becn1-STAT3-ISG15 axis remains functional in autophagy-disrupted fetal hematopoietic organs. These results suggest that Becn1, in an autophagy-independent manner, secures hematopoiesis and survival of the fetus by directly inhibiting STAT3-ISG15 activation to prevent cytokine storms. Our findings highlight a previously undocumented role of Becn1 in governing ISG15 to safeguard the fetus.
Authors
Wen Wei, Xueqin Gao, Jiawei Qian, Lei Li, Chen Zhao, Li Xu, Yanfei Zhu, Zhenzhen Liu, Nengrong Liu, Xueqing Wang, Zhicong Jin, Bowen Liu, Lan Xu, Jin Dong, Suping Zhang, Jiarong Wang, Yumu Zhang, Yao Yu, Zhanjun Yan, Yanjun Yang, Jie Lu, Yixuan Fang, Na Yuan, Jianrong Wang
Abstract
Severe congenital neutropenia (SCN) is frequently associated with dominant point mutations in ELANE, the gene encoding neutrophil elastase (NE). Chronic administration of granulocyte colony-stimulating factor (G-CSF) is a first-line treatment of ELANE-mutant (ELANEmut) SCN. However, some ELANEmut patients including patients with ELANE start codon mutations do not respond to G-CSF. Here, through directed granulopoiesis of gene-edited isogenic normal and patient-derived iPSCs, we demonstrate that ELANE start codon mutations suffice to induce G-CSF resistant granulocytic precursor cell death and refractory SCN. ELANE start codon mutated neutrophil precursors express predominantly nuclear N-terminal truncated alternate NE. Unlike G-CSF sensitive ELANE mutations that induce endoplasmic reticulum and unfolded protein response stress, we found that the mutation of the ELANE translation initiation codon resulted in NE aggregates and activated pro-apoptotic aggrephagy as determined by downregulated BAG1 expression, decreased BAG1/BAG3 ratio, NE co-localization with BAG3, and localized expression of autophagic LC3B. We found that SERF1, an RNA-chaperone protein, known to localize in misfolded protein aggregates in neurodegenerative diseases, was highly upregulated and interacted with cytoplasmic NE of mutant neutrophil precursors. Silencing of SERF1 enhanced survival and differentiation of iPSC-derived neutrophil precursors, restoring their responsiveness to G-CSF. These observations provide a mechanistic insight of G-CSF-resistant ELANEmut SCN, revealing targets for therapeutic intervention.
Authors
Ramesh C. Nayak, Sana Emberesh, Lisa Trump, Ashley Wellendorf, Abhishek Singh, Brice Korkmaz, Marshall S. Horwitz, Kasiani C. Myers, Theodosia A. Kalfa, Carolyn Lutzko, Jose A. Cancelas
CXCL8 secreted by immature granulocytes inhibits WT hematopoiesis in chronic myelomonocytic leukemia
Abstract
Chronic myelomonocytic leukemia (CMML) is a severe myeloid malignancy with limited therapeutic options. Single-cell analysis of clonal architecture demonstrates early clonal dominance with few residual WT hematopoietic stem cells. Circulating myeloid cells of the leukemic clone and the cytokines they produce generate a deleterious inflammatory climate. Our hypothesis is that therapeutic control of the inflammatory component in CMML could contribute to stepping down disease progression. The present study explored the contribution of immature granulocytes (iGRANs) to CMML progression. iGRANs were detected and quantified in the peripheral blood of patients by spectral and conventional flow cytometry. Their accumulation was a potent and independent poor prognostic factor. These cells belong to the leukemic clone and behaved as myeloid-derived suppressor cells. Bulk and single-cell RNA-Seq revealed a proinflammatory status of iGRAN that secreted multiple cytokines of which CXCL8 was at the highest level. This cytokine inhibited the proliferation of WT but not CMML hematopoietic stem and progenitor cells (HSPCs) in which CXCL8 receptors were downregulated. CXCL8 receptor inhibitors and CXCL8 blockade restored WT HSPC proliferation, suggesting that relieving CXCL8 selective pressure on WT HSPCs is a potential strategy to slow CMML progression and restore some healthy hematopoiesis.
Authors
Paul Deschamps, Margaux Wacheux, Axel Gosseye, Margot Morabito, Arnaud Pagès, Anne-Marie Lyne, Alexia Alfaro, Philippe Rameau, Aygun Imanci, Rabie Chelbi, Valentine Marchand, Aline Renneville, Mrinal M. Patnaik, Valerie Lapierre, Bouchra Badaoui, Orianne Wagner-Ballon, Céline Berthon, Thorsten Braun, Christophe Willekens, Raphael Itzykson, Pierre Fenaux, Sylvain Thépot, Gabriel Etienne, Emilie Elvira-Matelot, Francoise Porteu, Nathalie Droin, Leïla Perié, Lucie Laplane, Eric Solary, Dorothée Selimoglu-Buet
Abstract
BTK inhibitor therapy induces peripheral blood lymphocytosis in chronic lymphocytic leukemia (CLL) lasting for several months. It remains unclear whether non-genetic adaptation mechanisms exist, allowing CLL cells’ survival during BTK inhibitor-induced lymphocytosis and/or playing a role in therapy resistance. We show that in approximately 70 % of CLL cases, ibrutinib treatment in vivo increases Akt activity above pre-therapy levels within several weeks, leading to compensatory CLL cell survival and a more prominent lymphocytosis on therapy. Ibrutinib-induced Akt phosphorylation (pAktS473) is caused by the upregulation of FoxO1 transcription factor, which induces expression of Rictor, an assembly protein for mTORC2 protein complex that directly phosphorylates Akt at serine 473 (S473). Knock-out or inhibition of FoxO1 or Rictor led to a dramatic decrease in Akt phosphorylation and growth disadvantage for malignant B cells in the presence of ibrutinib (or PI3K inhibitor idelalisib) in vitro and in vivo. FoxO1/Rictor/pAktS473 axis represents an early non-genetic adaptation to BCR inhibitor therapy not requiring PI3Kδ or BTK kinase activity. We further demonstrate that FoxO1 can be targeted therapeutically, and its inhibition induces CLL cells’ apoptosis alone or in combination with BTK inhibitors (ibrutinib, acalabrutinib, pirtobrutinib) and blocks their proliferation triggered by T-cell factors (CD40L, IL-4, and IL-21).
Authors
Laura Ondrisova, Vaclav Seda, Krystof Hlavac, Petra Pavelkova, Eva Hoferkova, Giorgia Chiodin, Lenka Kostalova, Gabriela Mladonicka Pavlasova, Daniel Filip, Josef Vecera, Pedro Faria Zeni, Jan Oppelt, Zuzana Kahounova, Rachel Vichova, Karel Soucek, Anna Panovska, Karla Plevova, Sarka Pospisilova, Martin Simkovic, Filip Vrbacky, Daniel Lysak, Stacey M. Fernandes, Matthew S. Davids, Alba Maiques-Diaz, Stella Charalampopoulou, Jose I. Martin-Subero, Jennifer R. Brown, Michael Doubek, Francesco Forconi, Jiri Mayer, Marek Mraz
Abstract
Authors
Hajime Senjo, Daigo Hashimoto, Takanori Teshima
Abstract
Lung megakaryocytes (Mks) are largely extravascular with an immune phenotype (1). Because bone marrow (BM) Mks are short-lived it has been assumed that extravascular lung Mks are constantly ‘seeded’ from the BM. To investigate lung Mk origins and how that impacts their functions, we developed methods to specifically label lung Mks using CFSE dye and biotin delivered oropharyngeal. Labeled lung Mks were present for up to four months, while BM Mks had a <1 week lifespan. In a parabiosis model, lung Mks were partially replaced over 1-month from a circulating source. Unlike tissue-resident macrophages, using MDS1-Cre-ERT2 TdTomato mice, we found that lung Mks arise from hematopoietic stem cells. However, studies with FlkSwitch mTmG mice showed that lung Mks are derived from a Flt3-independent lineage that does not go through a multipotent progenitor. CFSE labeling to track lung Mk-derived platelets showed that about 10% of circulating platelets are lung resident Mk-derived at steady state, but in sterile thrombocytopenia this was doubled (about 20%). Lung-derived platelets were similarly increased in a malaria infection model (Plasmodium yoelii) typified by thrombocytopenia. These studies indicate that lung Mks arise from a Flt3-negative BM source, are long-lived, and contribute more platelets during thrombocytopenia.
Authors
Alison C. Livada, Kathleen E. McGrath, Michael W. Malloy, Chen Li, Sara K. Ture, Paul D. Kingsley, Anne D. Koniski, Leah A. Vit, Katherine E. Nolan, Deanne Mickelsen, Grace E. Monette, Preeti Maurya, James Palis, Craig N. Morrell
Abstract
The ubiquitously expressed small GTPase Ras-related protein 1B (RAP1B) acts as a molecular switch that regulates cell signaling, cytoskeletal remodeling, and cell trafficking and activates integrins in platelets and lymphocytes. The residue G12 in the P-loop is required for the RAP1B-GTPase conformational switch. Heterozygous germline RAP1B variants have been described in patients with syndromic thrombocytopenia. However, the causality and pathophysiological impact remained unexplored. We report a boy with neonatal thrombocytopenia, combined immunodeficiency, neutropenia, and monocytopenia caused by a heterozygous de novo single nucleotide substitution, c.35G>A (p.G12E) in RAP1B. We demonstrate that G12E and the previously described G12V and G60R were gain-of-function variants that increased RAP1B activation, talin recruitment, and integrin activation, thereby modifying late responses such as platelet activation, T cell proliferation, and migration. We show that in our patient, G12E was a somatic variant whose allele frequency decreased over time in the peripheral immune compartment, but remained stable in bone marrow cells, suggesting a differential effect in distinct cell populations. Allogeneic hematopoietic stem cell transplantation fully restored the patient’s hemato-immunological phenotype. Our findings define monoallelic RAP1B gain-of-function variants as a cause for constitutive immunodeficiency and thrombocytopenia. The phenotypic spectrum ranged from isolated hematological manifestations in our patient with somatic mosaicism to complex syndromic features in patients with reported germline RAP1B variants.
Authors
Marta Benavides-Nieto, Frédéric Adam, Emmanuel Martin, Charlotte Boussard, Chantal Lagresle-Peyrou, Isabelle Callebaut, Alexandre Kauskot, Christelle Repérant, Miao Feng, Jean-Claude Bordet, Martin Castelle, Guillaume Morelle, Chantal Brouzes, Mohammed Zarhrate, Patricia Panikulam, Nathalie Lambert, Capucine Picard, Damien Bodet, Jérémie Rouger-Gaudichon, Patrick Revy, Jean-Pierre de Villartay, Despina Moshous
Abstract
Background. Donor cell engraftment is a pre-requisite of successful allogeneic hematopoietic stem cell transplantation. Based on peripheral blood analyses it is characterized by early myeloid recovery and T- and B-cells lymphopenia. However, cellular networks associated with bone marrow engraftment of allogeneic human cells have been poorly described. Methods. Mass cytometry and CITEseq analyses were performed on bone marrow cells, three months post-transplant in patients with acute myelogenous leukemia. Results. Mass cytometry in 26 patients and 20 healthy controls disclosed profound alterations in myeloid and B-cell progenitors, with a shift towards terminal myeloid differentiation and decreased B-cell progenitors. Unsupervised analysis separated recipients into 2 groups, one of them being driven by previous GVHD (R2 patients). We then used single-cell CITEseq to decipher engraftment, which resolved 36 clusters, encompassing all bone marrow cellular components. Hematopoiesis in transplant recipients was sustained by committed myeloid and erythroid progenitors in a setting of monocytes-, NK cells- and T-cells mediated inflammation. Gene expression disclosed major pathways in transplant recipients, namely, TNFα signaling via NFκ-B, and interferon-γ response. The hallmark of allograft rejection was consistently found in clusters from transplant recipients, especially in R2 recipients. Conclusion. Bone marrow cell engraftment of allogeneic donor cells is characterized by a state of emergency hematopoiesis in the setting of allogeneic response driving inflammation. Trial registration. Not applicable. Funding. This study has been supported by the French National Cancer Institute (Institut National du Cancer): PLBIO19-239 and by an unrestricted research grant by Alexion Pharmaceutical.
Authors
Jennifer Bordenave, Dorota Gajda, David Michonneau, Nicolas Vallet, Mathieu F. Chevalier, Emmanuelle Clappier, Pierre Lemaire, Stéphanie Mathis, Marie Robin, Aliénor Xhaard, Flore Sicre de Fontbrune, Aurélien Corneau, Sophie Caillat-Zucman, Regis PEFFAULT de LATOUR, Emmanuel Curis, Gerard Socie
Copyright © 2024 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)