Hematology
Abstract
TCR repertoire diversification constitutes a foundation for successful immune reconstitution after allogeneic hematopoietic cell transplantation (allo-HCT). Deep TCR Vβ sequencing of 135 serial specimens from a cohort of 35 allo-HCT recipients/donors was performed to dissect posttransplant TCR architecture and dynamics. Paired analysis of clonotypic repertoires showed a minimal overlap with donor expansions. Rarefied and hyperexpanded clonotypic patterns were hallmarks of T cell reconstitution and influenced clinical outcomes. Donor and pretransplant TCR diversity as well as divergence of class I human leukocyte antigen genotypes were major predictors of recipient TCR repertoire recovery. Complementary determining region 3–based specificity spectrum analysis indicated a predominant expansion of pathogen- and tumor-associated clonotypes in the late post–allo-HCT phase, while autoreactive clones were more expanded in the case of graft-versus-host disease occurrence. These findings shed light on post–allo-HCT adaptive immune reconstitution processes and possibly help in tracking alloreactive responses.
Authors
Simona Pagliuca, Carmelo Gurnari, Sanghee Hong, Ran Zhao, Sunisa Kongkiatkamon, Laila Terkawi, Misam Zawit, Yihong Guan, Hassan Awada, Ashwin Kishtagari, Cassandra M. Kerr, Thomas LaFramboise, Bhumika J. Patel, Babal K. Jha, Hetty E. Carraway, Valeria Visconte, Navneet S. Majhail, Betty K. Hamilton, Jaroslaw P. Maciejewski
Hematologic and systemic metabolic alterations due to Mediterranean class II G6PD deficiency in mice
Abstract
Deficiency of Glucose 6 phosphate dehydrogenase (G6PD) is the single most common enzymopathy, present in approximately 400 million humans (e.g., 5% of humans). Its prevalence is hypothesized to be due to conferring resistance to malaria. However, G6PD deficiency also results in hemolytic sequelae from oxidant stress. Moreover, G6PD deficiency is associated with kidney disease, diabetes, pulmonary hypertension, immunological defects, and neurodegenerative diseases. To date, the only available mouse models have decreased levels of G6PD due to promoter mutations, but with stable G6PD. However, human G6PD mutations are missense mutations that result in decreased enzymatic stability. As such, this results in very low activity in red blood cells (RB that cannot synthesize new protein. To generate a more accurate model, the human sequence for a severe form of G6PD deficiency (Med -) was knocked into the murine G6PD locus. As predicted, G6PD levels were extremely low in RBCs and deficient mice have increased hemolytic sequalae to oxidant stress. Non-erythroid organs had metabolic changes consistent with mild G6PD deficiency, consistent with what has been observed in humans. Juxtaposition of G6PD deficient and wild-type mice revealed altered lipid metabolism in multiple organ systems. Together, these findings both establish a new mouse model of G6PD deficiency that more accurately reflects human G6PD deficiency and also advance our basic understanding of altered metabolism in this setting.
Authors
Angelo D’Alessandro, Heather L. Howie, Ariel M. Hay, Karolina H. Dziewulska, Benjamin C. Brown, Matthew J. Wither, Matthew Karafin, Elizabeth F. Stone, Steven L. Spitalnik, Eldad A. Hod, Richard O. Francis, Xiaoyun Fu, Tiffany Thomas, James C. Zimring
Abstract
Haploidentical hematopoietic stem cell transplantation (h-HSCT) represents an efficient curative approach for patients affected by hematologic malignancies in which the reduced intensity conditioning induces a state of immunologic tolerance between donor and recipient. However, opportunistic viral infections greatly affect h-HSCT clinical outcomes. Natural Killer (NK) cells are the first lymphocytes recovering after transplant and provide a prompt defense against human Cytomegalovirus (HCMV) infection/reactivation. By undertaking a longitudinal single cell computational profiling of multiparametric flow cytometry, we show that HCMV accelerates NK cell immune-reconstitution together with the expansion of CD158b1b2jpos/NKG2Aneg/NKG2Cpos/NKp30low NK cells. The frequency of this subset correlates with HCMV viremia, further increases in recipients experiencing multiple episodes of viral reactivations and persists for months after the infection. The transcriptional profile of FACS-sorted CD158b1b2jpos NK cells confirmed the ability of HCMV to de-regulate NKG2C, NKG2A and NKp30 gene expression, thus inducing the expansion of NK cells with adaptive traits. These NK cells are characterized by the down-modulation of several gene pathways associated with cell migration, cell-cycle, effector-functions and by a state of metabolic/cellular exhaustion. This profile reflects the functional impairments of adaptive NK cells to produce IFN-γ, a phenomenon also due to the viral-induced expression of LAG-3 and PD-1 checkpoint-inhibitors.
Authors
Elisa Zaghi, Michela Calvi, Simone Puccio, Gianmarco Spata, Sara Terzoli, Clelia Peano, Alessandra Roberto, Federica De Paoli, Jasper J.P. van Beek, Jacopo Mariotti, Chiara De Philippis, Barbara Sarina, Rossana Mineri, Stefania Bramanti, Armando Santoro, Vu Thuy Khanh Le-Trilling, Mirko Trilling, Emanuela Marcenaro, Luca Castagna, Clara Di Vito, Enrico Lugli, Domenico Mavilio
Abstract
Telomerase catalyzes chromosome end replication in stem cells and other long-lived cells. Mutations in telomerase or telomere-related genes result in diseases known as telomeropathies. Telomerase is recruited to chromosome ends by the ACD/TPP1 protein (TPP1 hereafter), a component of the shelterin complex that protects chromosome ends from unwanted end-joining. TPP1 facilitates end-protection by binding shelterin proteins POT1 and TIN2. TPP1 variants have been associated with telomeropathies, but remain poorly characterized in vivo. Disease variants and mutagenesis scans provide efficient avenues to interrogate the distinct physiological roles of TPP1. Here, we conduct mutagenesis in the TIN2- and POT1-binding domains of TPP1 to discover mutations that dissect TPP1’s functions. Our results extend upon current structural data to reveal that the TPP1-TIN2 interface is more extensive than previously thought, and highlight the robustness of the POT1-TPP1 interface. Introduction of separation-of-function mutants alongside known TPP1 telomeropathy mutations in mouse hematopoietic stem cells (mHSCs) lacking endogenous TPP1 demonstrated a clear phenotypic demarcation. TIN2- and POT1-binding mutants were unable to rescue mHSC failure resulting from end-deprotection. In contrast, TPP1 telomeropathy mutations sustained mHSC viability, consistent with them selectively impacting end-replication. These results highlight the power of scanning mutagenesis in revealing structural interfaces and dissecting multifunctional genes.
Authors
Sherilyn Grill, Shilpa Padmanaban, Ann Friedman, Eric Perkey, Frederick Allen, Valerie M. Tesmer, Jennifer Chase, Rami Khoriaty, Catherine E. Keegan, Ivan Maillard, Jayakrishnan Nandakumar
Abstract
The splenic microenvironment regulates hematopoietic stem and progenitor cell (HSPC) function, particularly during demand-adapted hematopoiesis, however practical strategies to enhance splenic support of transplanted HSPCs have proven elusive. We have previously demonstrated that inhibiting 15-hydroxyprostaglandin dehydrogenase (15-PGDH), using the small molecule (+)SW033291 (PGDHi), increases bone marrow (BM) prostaglandin E2 (PGE2) levels, expands HSPC numbers, and accelerates hematologic reconstitution following BM transplantation (BMT) in mice. Here we demonstrate that the splenic microenvironment, specifically 15-PGDH high-expressing macrophages (MΦs), megakaryocytes (MKs), and mast cells (MCs), regulates steady-state hematopoiesis and potentiates recovery after BMT. Notably, PGDHi-induced neutrophil, platelet, and HSPC recovery were highly attenuated in splenectomized mice. PGDHi induced non-pathologic splenic extramedullary hematopoiesis at steady-state, and pre-transplant PGDHi enhanced the homing of transplanted cells to the spleen. 15-PGDH enzymatic activity localized specifically to MΦs, MK lineage cells, and MCs, identifying these cell types as likely coordinating the impact of PGDHi on splenic HSPCs. These findings suggest that 15-PGDH expression marks novel HSC niche cell types that regulate hematopoietic regeneration. Therefore, PGDHi provides a well-tolerated strategy to therapeutically target multiple HSC niches and to promote hematopoietic regeneration and improve clinical outcomes of BMT.
Authors
Julianne N.P. Smith, Dawn M. Dawson, Kelsey F. Christo, Alvin P. Jogasuria, Mark J. Cameron, Monika I. Antczak, Joseph M. Ready, Stanton L. Gerson, Sanford D. Markowitz, Amar B. Desai
Abstract
Multiple myeloma (MM) is characterized by an accumulation of malignant plasma cells (PCs) within the bone marrow (BM). The BM microenvironment supports survival of the malignant cells and is comprised of cellular fractions that foster myeloma development and progression by suppression of the immune response. Despite major progress in understanding the biology and pathophysiology of MM, this disease is still incurable and requires aggressive treatment with significant side effects. CD84 is a self-binding immuno-receptor belonging to the signaling lymphocyte activating molecule (SLAM) family. Previously, we showed that CD84 bridges between chronic lymphocytic leukemia cells and their microenvironment, and regulates T cell function. In the current study, we investigated the role of CD84 in MM. Our results show that MM cells express low levels of CD84. However, these cells secrete the cytokine macrophage migration inhibitory factor (MIF), which induces CD84 expression on cells in their microenvironment. Its activation leads to an elevation of expression of genes regulating differentiation to M/G- myeloid derived suppressor cells (MDSCs) and upregulation of PD-L1 expression on MDSCs, which together suppress T cell function. Downregulation of CD84 or its blocking reduces MDSC accumulation, resulting in elevated T cell activity and reduced tumor load. Our data suggest that CD84 might serve as a novel therapeutic target in MM.
Authors
Hadas Lewinsky, Emine Gulsen Gunes, Keren David, Lihi Radomir, Matthias P. Kramer, Bianca Pellegrino, Michal Perpinial, Jing Chen, Ting-fang He, Anthony Mansour, Kun-Yu Teng, Supriyo Bhattacharya, Enrico Caserta, Estelle Troadec, Peter P. Lee, Mingye Feng, Jonathan J. Keats, Amrita Krishnan, Michael Rosenzweig, Jianhua Yu, Michael A. Caligiuri, Yosef Cohen, Olga Shvetz, Shirly Becker-Herman, Flavia Pichiorri, Steven Rosen, Idit Shachar
Abstract
The cohesin complex plays an essential role in chromosome maintenance and transcriptional regulation. Recurrent somatic mutations in the cohesin complex are frequent genetic drivers in cancer including myelodysplatic syndromes (MDS) and acute myeloid leukemia (AML). Here, using genetic dependency screens of STAG2-mutant AML, we identified DNA damage repair and replication as genetic dependencies in cohesin-mutant cells. We demonstrated increased levels of DNA damage and sensitivity of cohesin-mutant cells to PARP inhibition. We developed a mouse model of MDS in which Stag2 mutations arise as clonal secondary lesions in the background of clonal hematopoiesis driven by Tet2 mutations, and demonstrated selective depletion of cohesin-mutant cells with PARP inhibition in vivo. Finally, we demonstrated a shift from STAG2- to STAG1-containing cohesin complexes in cohesin-mutant cells, which is associated with longer DNA loop extrusion, more intermixing of chromatin compartments, and increased interaction with PARP and RPA proteins. Our findings inform the biology and therapeutic opportunities for cohesin-mutant malignancies.
Authors
Zuzana Tothova, Anne-Laure Valton, Rebecca Gorelov, Mounica Vallurupalli, John M. Krill-Burger, Amie Holmes, Catherine C. Landers, J. Erika Haydu, Edyta Malolepsza, Christina R. Hartigan, Melanie Donahue, Katerina D. Popova, Sebastian H. J. Koochaki, Sergey V. Venev, Jeanne F. Rivera, Edwin Chen, Kasper Lage, Monica Schenone, Alan D. D'Andrea, Steven A. Carr, Elizabeth A. Morgan, Job Dekker, Benjamin L. Ebert
Abstract
Computational models based on recent maps of the red blood cell proteome suggest that mature erythrocytes may harbor targets for common drugs. This prediction is relevant to red blood cell storage in the blood bank, in which the impact of small molecule drugs or other xenometabolites deriving from dietary, iatrogenic or environmental exposures (“exposome”) may alter erythrocyte energy and redox metabolism and, in so doing, affect red cell storage quality and post-transfusion efficacy. To test this prediction, here we provide a comprehensive characterization of the blood donor exposome, including the detection of common prescription and over-the-counter drugs in 250 units donated by healthy volunteers from the REDS-III RBC Omics study. Based on high-throughput drug screenings of 1,366 FDA-approved drugs, we report a significant impact of ~65% of the tested drugs on erythrocyte metabolism. Machine learning models built using metabolites as predictors were able to accurately predict drugs for several drug classes/targets (bisphosphonates, anticholinergics, calcium channel blockers, adrenergics, proton-pump inhibitors, antimetabolites, selective serotonin reuptake inhibitors, and mTOR) suggesting that these drugs have a direct, conserved, and significant impact on erythrocyte metabolism. As a proof of principle, here we show that the antiacid ranitidine – though rarely detected in the blood donor population – has a strong effect on RBC markers of storage quality in vitro. We thus show that ranitidine supplementation to blood units could improve erythrocyte metabolism and storage quality when supplemented to blood bags, through mechanisms involving sphingosine 1-phosphate-dependent modulation of erythrocyte glycolysis and/or direct binding to hemoglobin.
Authors
Travis Nemkov, Davide Stefanoni, Aarash Bordbar, Aaron Issaian, Bernhard O. Palsson, Larry J. Dumont, Ariel M. Hay, Anren Song, Yang Xia, Jasmina S. Redzic, Elan Z. Eisenmesser, James C. Zimring, Steve Kleinman, Kirk C. Hansen, Michael Busch, Angelo D’Alessandro
Abstract
Effective treatment for AML is challenging due to the presence of clonal heterogeneity and the evolution of polyclonal drug resistance. Here, we report that TP-0903 has potent activity against protein kinases related to STAT, AKT, and ERK signaling, as well as cell cycle regulators in biochemical and cellular assays. In vitro and in vivo, TP-0903 was active in multiple models of drug-resistant FLT3 mutant AML, including those involving the F691L gatekeeper mutation and bone marrow microenvironment–mediated factors. Furthermore, TP-0903 demonstrated preclinical activity in AML models with FLT3-ITD and common co-occurring mutations in IDH2 and NRAS genes. We also showed that TP-0903 had ex vivo activity in primary AML cells with recurrent mutations including MLL-PTD, ASXL1, SRSF2, and WT1, which are associated with poor prognosis or promote clinical resistance to AML-directed therapies. Our preclinical studies demonstrate that TP-0903 is a multikinase inhibitor with potent activity against multiple drug-resistant models of AML that will have an immediate clinical impact in a heterogeneous disease like AML.
Authors
Jae Yoon Jeon, Daelynn R. Buelow, Dominique A. Garrison, Mingshan Niu, Eric D. Eisenmann, Kevin M. Huang, Megan E. Zavorka Thomas, Robert H. Weber, Clifford J. Whatcott, Steve L. Warner, Shelley J. Orwick, Bridget Carmichael, Emily Stahl, Lindsey T. Brinton, Rosa Lapalombella, James S. Blachly, Erin Hertlein, John C. Byrd, Bhavana Bhatnagar, Sharyn D. Baker
Abstract
The nonimmune roles of Tregs have been described in various tissues, including the BM. In this study, we comprehensively phenotyped marrow Tregs, elucidating their key features and tissue-specific functions. We show that marrow Tregs are migratory and home back to the marrow. For trafficking, marrow Tregs use S1P gradients, and disruption of this axis allows for specific targeting of the marrow Treg pool. Following Treg depletion, the function and phenotype of both mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) was impaired. Transplantation also revealed that a Treg-depleted niche has a reduced capacity to support hematopoiesis. Finally, we found that marrow Tregs are high producers of IL-10 and that Treg-secreted IL-10 has direct effects on MSC function. This is the first report to our knowledge revealing that Treg-secreted IL-10 is necessary for stromal cell maintenance, and our work outlines an alternative mechanism by which this cytokine regulates hematopoiesis.
Authors
Virginia Camacho, Victoria R. Matkins, Sweta B. Patel, Jeremie M. Lever, Zhengqin Yang, Li Ying, Ashley E. Landuyt, Emma C. Dean, James F. George, Henry Yang, Paul Brent Ferrell, Craig L. Maynard, Casey T. Weaver, Heth R. Turnquist, Robert S. Welner
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