Hematology
Abstract
Diamond-Blackfan anemia syndrome (DBA) is a ribosomopathy associated with loss-of-function variants in more than 20 ribosomal protein (RP) genes. Here, we report the genetic, functional and biochemical dissection of two multigenerational pedigrees with variants in RPL17, a large ribosomal subunit protein-encoding gene. Affected individuals had clinical features and erythroid proliferation defects consistent with DBA. Furthermore, RPL17/uL22 depletion resulted in anemia and micrognathia in zebrafish larvae, and in vivo complementation studies indicated that RPL17 variants were pathogenic. Lymphoblastoid cell lines (LCLs) derived from patients displayed a ribosomal RNA maturation defect reflecting haploinsufficiency of RPL17. The proteins encoded by RPL17 variants were not incorporated into ribosomes, but 10-20% of 60S ribosomal subunits contained a short form of 5.8S rRNA (5.8SC), a species that is marginal in normal cells. These atypical 60S subunits were actively engaged in translation. Ribosome profiling showed changes of the translational profile, but those are similar to LCLs bearing RPS19 variants. These results link an additional RP gene to DBA. They show that ribosomes can be modified substantially by RPL17 haploinsufficiency, but support the paradigm that translation alterations in DBA are primarily related to insufficient ribosome production rather than to changes in ribosome structure or composition.
Authors
Florence Fellmann, Carol Saunders, Marie-Françoise O'Donohue, David W. Reid, Kelsey A. McFadden, Nathalie Montel-Lehry, Cong Yu, Mingyan Fang, Jianguo Zhang, Beryl Royer-Bertrand, Pietro Farinelli, Narjesse Karboul, Jason R. Willer, Lorraine Fievet, Zahurul Alam Bhuiyan, Alissa L.W. Kleinhenz, Julie Jadeau, Joy Fulbright, Carlo Rivolta, Raffaele Renella, Nicholas Katsanis, Jacques S. Beckmann, Christopher V. Nicchitta, Lydie Da Costa, Erica E. Davis, Pierre-Emmanuel Gleizes
Abstract
The most common subtype of lymphoma globally, diffuse large B-cell lymphoma (DLBCL) is a leading cause of cancer death in people with HIV (HIV+). The restructuring of the T-cell compartment due to HIV infection and antiretroviral therapy (ART) may have implications for modern treatment selection, but current understanding of these dynamic interactions is limited. Here, we investigated the T-cell response to DLBCL by sequencing the T-cell receptor (TCR) repertoire in a cohort of HIV-negative (HIV-), HIV+/ART-experienced and HIV+/ART-naïve DLBCL patients. HIV+/ART-naïve tumor TCR repertoires were more clonal and more distinct from each other than HIV- and HIV+/ART-experienced. Further, increased overlap between tumor and blood TCR repertoires was associated with improved survival and HIV/ART status. Our study describes TCR repertoire characteristics for the first time in an African DLBCL cohort and demonstrates contributions of HIV infection and ART exposure to the DLBCL TCR repertoire.
Authors
Sophia M. Roush, Jenny Coelho, Alexander M. Xu, Kaushik Puranam, Marriam Mponda, Edwards Kasonkanji, Maurice Mulenga, Tamiwe Tomoka, Jonathan Galeotti, Amy Brownlee, Hormas Ghadially, Maganizo Chagomerana, Blossom Damania, Matthew Painschab, Akil Merchant, Satish Gopal, Yuri Fedoriw
Abstract
This study lays the groundwork for future lentivirus-mediated gene therapy in patients with Diamond Blackfan anemia (DBA) caused by mutations in ribosomal protein S19 (RPS19), showing evidence of a new safe and effective therapy. The data show that, unlike patients with Fanconi anemia (FA), the hematopoietic stem cell (HSC) reservoir of patients with DBA was not significantly reduced, suggesting that collection of these cells should not constitute a remarkable restriction for DBA gene therapy. Subsequently, 2 clinically applicable lentiviral vectors were developed. In the former lentiviral vector, PGK.CoRPS19 LV, a codon-optimized version of RPS19 was driven by the phosphoglycerate kinase promoter (PGK) already used in different gene therapy trials, including FA gene therapy. In the latter one, EF1α.CoRPS19 LV, RPS19 expression was driven by the elongation factor alpha short promoter, EF1α(s). Preclinical experiments showed that transduction of DBA patient CD34+ cells with the PGK.CoRPS19 LV restored erythroid differentiation, and demonstrated the long-term repopulating properties of corrected DBA CD34+ cells, providing evidence of improved erythroid maturation. Concomitantly, long-term restoration of ribosomal biogenesis was verified using a potentially novel method applicable to patients’ blood cells, based on ribosomal RNA methylation analyses. Finally, in vivo safety studies and proviral insertion site analyses showed that lentivirus-mediated gene therapy was nontoxic.
Authors
Yari Giménez, Manuel Palacios, Rebeca Sánchez-Domínguez, Christiane Zorbas, Jorge Peral, Alexander Puzik, Laura Ugalde, Omaira Alberquilla, Mariela Villanueva, Paula Río, Eva Gálvez, Lydie Da Costa, Marion Strullu, Albert Catala, Anna Ruiz-Llobet, Jose Carlos Segovia, Julián Sevilla, Brigitte Strahm, Charlotte M. Niemeyer, Cristina Beléndez, Thierry Leblanc, Denis L.J. Lafontaine, Juan Bueren, Susana Navarro
Abstract
Multiple myeloma is a largely incurable and life-threatening malignancy of antibody-secreting plasma cells. An effective and widely available animal model that recapitulates human myeloma and related plasma cell disorders is lacking. We show that busulfan-conditioned hIL-6 transgenic NSG mice (NSG+hIL6) reliably support the engraftment of malignant and pre-malignant human plasma cells including from patients diagnosed with monoclonal gammopathy of undetermined significance, pre- and post-relapse myeloma, plasma cell leukemia, and AL amyloidosis. Consistent with human disease, NSG+hIL6 mice engrafted with patient-derived myeloma cells, developed serum M spikes, and a majority developed anemia, hypercalcemia, and/or bone lesions. Single cell RNA sequencing showed non-malignant and malignant cell engraftment, the latter expressing a wide array of mRNAs associated with myeloma cell survival and proliferation. Myeloma engrafted mice given CAR T-cells targeting plasma cells or bortezomib experienced reduced tumor burden. Our results established NSG+hIL6 mice as an effective patient derived xenograft model for study and preclinical drug development of multiple myeloma and related plasma cell disorders.
Authors
Zainul S. Hasanali, Alfred L. Garfall, Lisa Burzenski, Leonard D. Shultz, Yan Tang, Siddhant Kadu, Neil C. Sheppard, Wei Liu, Derek Dopkin, Dan T. Vogl, Adam D. Cohen, Adam J. Waxman, Sandra P. Susanibar-Adaniya, Martin Carroll, Edward A. Stadtmauer, David Allman
Abstract
Thrombosis and inflammation are intimately linked and synergistically contribute to the pathogenesis of numerous thromboinflammatory diseases, including sickle cell disease (SCD). While platelets are central to thrombogenesis and inflammation, the molecular mechanisms of crosstalk between the 2 remain elusive. High-mobility group box 1 (HMGB1) regulates inflammation and stimulates platelet activation through Toll-like receptor 4. However, it remains unclear whether HMGB1 modulates other thrombotic agonists to regulate platelet activation. Herein, using human platelets, we demonstrate that HMGB1 significantly enhanced ADP-mediated platelet activation. Furthermore, inhibition of the purinergic receptor P2Y12 attenuated HMGB1-dependent platelet activation. Mechanistically, we show that HMGB1 stimulated ADP secretion, while concomitantly increasing P2Y12 levels at the platelet membrane. We show that in SCD patients, increased plasma HMGB1 levels were associated with heightened platelet activation and surface P2Y12 expression. Treatment of healthy platelets with plasma from SCD patients enhanced platelet activation and surface P2Y12, and increased sensitivity to ADP-mediated activation, and these effects were linked to plasma HMGB1. We conclude that HMGB1-mediated platelet activation involves ADP-dependent P2Y12 signaling, and HMGB1 primes platelets for ADP signaling. This complementary agonism between ADP and HMGB1 furthers the understanding of thromboinflammatory signaling in conditions such as SCD, and provides insight for therapeutic P2Y12 inhibition.
Authors
Deirdre Nolfi-Donegan, Gowtham K. Annarapu, Claudette St. Croix, Michael Calderon, Cheryl A. Hillery, Sruti Shiva
Abstract
Plasma cell-free DNA (cfDNA), a marker of disease severity in sepsis, is a recognized driver of thromboinflammation and a potential therapeutic target. In sepsis, plasma cfDNA is mostly derived from neutrophil extracellular trap (NET) degradation. Proposed NET-directed therapeutic strategies include preventing NET formation or accelerating NET degradation. However, NET digestion liberates pathogens and releases cfDNA that promote thrombosis and endothelial cell injury. We propose an alternative strategy of cfDNA and NET stabilization with chemokine platelet factor 4 (PF4, CXCL4). We previously showed that human PF4 (hPF4) enhances NET-mediated microbial entrapment. We now show that hPF4 interferes with thrombogenicity of cfDNA and NETs by preventing their cleavage to short-fragment and single-stranded cfDNA that more effectively activates the contact pathway of coagulation. In vitro, hPF4 also inhibits cfDNA-induced endothelial tissue factor surface expression and von Willebrand factor release. In vivo, hPF4 expression reduced plasma thrombin-antithrombin (TAT) levels in animals infused with exogenous cfDNA. Following lipopolysaccharide challenge, Cxcl4–/– mice had significant elevation in plasma TAT, cfDNA, and cystatin C levels, effects prevented by hPF4 infusion. These results show that hPF4 interacts with cfDNA and NETs to limit thrombosis and endothelial injury, an observation of potential clinical benefit in the treatment of sepsis.
Authors
Anh T.P. Ngo, Abigail Skidmore, Jenna Oberg, Irene Yarovoi, Amrita Sarkar, Nate Levine, Veronica Bochenek, Guohua Zhao, Lubica Rauova, M. Anna Kowalska, Kaitlyn Eckart, Nilam S. Mangalmurti, Ann Rux, Douglas B. Cines, Mortimer Poncz, Kandace Gollomp
Abstract
Patients with Down syndrome (DS, trisomy 21, T21) are at increased risk of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia (ML-DS). Both TAM and ML-DS require prenatal somatic mutations in GATA1, resulting in the truncated isoform GATA1s. The mechanism by which individual chromosome 21 (HSA21) genes synergize with GATA1s for leukemic transformation is challenging to study, in part due to limited human cell models with wild type GATA1 or GATA1s. HSA21-encoded DYRK1A is overexpressed in ML-DS and may be a therapeutic target. To determine how DYRK1A influences hematopoiesis in concert with GATA1s, we used gene editing to disrupt all 3 alleles of DYRK1A in isogenic T21 induced pluripotent stem cells (iPSCs) with and without the GATA1s mutation. Unexpectedly, hematopoietic differentiation revealed that DYRK1A loss combined with GATA1s leads to increased megakaryocyte proliferation and decreased maturation. This proliferative phenotype was associated with upregulation of D-type cyclins and hyperphosphorylation of Rb to allow E2F release and de-repression of its downstream targets. Notably, DYRK1A loss had no effect in T21/wtGATA1 megakaryocytes. These surprising results suggest that DYRK1A and GATA1 may synergistically restrain megakaryocyte proliferation in Trisomy 21 and that DYRK1A inhibition may not be a therapeutic option for GATA1s-associated leukemias.
Authors
Ying Ting Sit, Kaoru Takasaki, Hyun Hyung An, Yan Xiao, Christian Hurtz, Peter Gearhart, Zhe Zhang, Paul Gadue, Deborah French, Stella T. Chou
Abstract
Hereditary spherocytosis (HS) is the most common non-immune hereditary chronic hemolytic anemia after hemoglobinopathies. The genetic defects in membrane function causing HS leads to perturbation of red cell metabolome, with altered glycolysis. In mice genetically lacking protein 4.2 (4.2-/-, Epb42), a murine model of HS, we show increased expression of pyruvate kinase (Pk) isoforms in whole and fractioned red cells in conjunction with abnormalities in the glycolytic pathway and in GSH system. Mitapivat, a PKs activator, metabolically re-programs 4.2-/- mouse red cells with amelioration of glycolysis and GSH cycle. This results in improved osmotic fragility, reduced phosphatidyl-serine (PS) positivity and decrease in erythroid vesicles release in vitro. Mitapivat treatment significantly decreases erythrophagocytosis and beneficially impacts iron homeostasis. In mild/moderate HS, the beneficial effect of splenectomy is still controversial. Here, we show that splenectomy improves anemia in 4.2-/- mice and that mitapivat is non-inferior to splenectomy. An additional benefit of mitapivat treatment is lower expression of markers of inflammatory vasculopathy in 4.2-/- mice with or without splenectomy, indicating a multi-systemic action of mitapivat. These findings support the notion that mitapivat treatment should be considered for symptomatic HS.
Authors
Alessandro Matte, Anand Babu Wilson, Federica Gevi, Enrica Federti, Antonio Recchiuti, Giulia Ferri, Anna Maria Brunati, Mario Angelo Pagano, Roberta Russo, Christophe Leboeuf, Anne Janin, Anna Maria Timperio, Achille Iolascon, Elisa Gremese, Lenny Dang, Narla Mohandas, Carlo Brugnara, Lucia De Franceschi
Abstract
Overexpression of Phosphatases of Regenerating Liver 2 (PRL2), detected in numerous diverse cancers, is often associated with increased severity and poor patient prognosis. PRL2-catalyzed tyrosine dephosphorylation of the tumor suppressor PTEN results in increased PTEN degradation, and has been identified as a mechanism underlying PRL2 oncogenic activity. Overexpression of PRL2, coincident with reduced PTEN protein, is frequently observed in Acute Myeloid Leukemia (AML) patients. In the current study, a PTEN-knockdown AML animal model was generated to assess the impact of conditional PRL2 inhibition on the level of PTEN protein and the development and progression of AML. Inhibition of PRL2 resulted in a significant increase in median animal survival, from 40 weeks to greater than 60 weeks. The prolonged survival reflected delayed expansion of aberrantly differentiated hematopoietic stem cells into leukemia blasts, resulting in extended time required for clinically relevant leukemia blast accumulation in the bone marrow niche. Leukemia blast suppression following PRL2 inhibition was correlated with an increase in PTEN, and downregulation of AKT/mTOR regulated pathways. These observations directly established, in a disease model, the viability of PRL2 inhibition as a therapeutic strategy for improving clinical outcomes in AML and potentially other PTEN-deficient cancers by slowing cancer progression.
Authors
Colin Carlock, Yunpeng Bai, Allison Paige-Hood, Qinglin Li, Frederick Nguele Meke, Zhong-Yin Zhang
Abstract
Thrombosis is a common complication of advanced cancer. Yet the cellular mechanisms linking malignancy to thrombosis are poorly understood. The unfolded protein response (UPR) is an ER stress response associated with advanced cancers. A proteomic evaluation of plasmas from gastric and non-small cell lung cancer patients who were monitored prospectively for venous thromboembolism demonstrated increased levels of UPR-related markers in plasmas of patients who developed clots compared to those who did not. Release of procoagulant activity into supernatants of gastric, lung, and pancreatic cancer cells was enhanced by UPR induction and blocked by antagonists of the UPR receptors IRE1α or PERK. Release of extracellular vesicles bearing tissue factor (EVTF) from pancreatic cancer cells was inhibited by siRNA-mediated knockdown of IRE1α/XBP1 or PERK pathways. Induction of UPR did not increase TF synthesis, but rather stimulated localization of TF to the cell surface. UPR-induced TF delivery to EVTFs was inhibited by Arf1 knockdown or GBF1 antagonism, confirming the role of vesicular trafficking. Our findings show that UPR activation results in increased vesicular trafficking leading to release of prothrombotic EVTFs, thus providing a mechanistic link between ER stress and cancer-associated thrombosis.
Authors
Oluwatoyosi Muse, Rushad Patell, Christian G. Peters, Moua Yang, Emale El-Darzi, Sol Schulman, Anna Falanga, Marina Marchetti, Laura Russo, Jeffrey I. Zwicker, Robert Flaumenhaft
No posts were found with this tag.
