Mosaic loss of chromosome Y (mLOY) in blood cells is one of the most frequent chromosome alterations in adult males. It is strongly associated with clonal hematopoiesis, hematopoietic malignancies, and other hematopoietic and nonhematopoietic diseases. However, whether there is a causal relationship between mLOY and human diseases is unknown. Here, we generated mLOY in murine hematopoietic stem and progenitor cells (HSPCs) with CRISPR/Cas9 genome editing. We found that mLOY led to dramatically increased DNA damage in HSPCs. Interestingly, HSPCs with mLOY displayed significantly enhanced reconstitution capacity and gave rise to clonal hematopoiesis in vivo. mLOY, which is associated with AML1-ETO translocation and p53 defects in patients with acute myeloid leukemia (AML), promoted AML in mice. Mechanistically, loss of KDM5D, a chromosome Y–specific histone 3 lysine 4 demethylase in both humans and mice, partially recapitulated mLOY in DNA damage and leukemogenesis. Thus, our study validates mLOY as a functional driver for clonal hematopoiesis and leukemogenesis.
Qi Zhang, Lei Zhao, Yi Yang, Shujun Li, Yu Liu, Chong Chen
BACKGROUND. Red blood cell (RBC) transfusion effectiveness varies due to donor, component, and recipient factors. Prior studies identified characteristics associated with variation in hemoglobin increments following transfusion. We extended these observations, examining donor genetic and non-genetic factors affecting transfusion effectiveness. METHODS. This is a multicenter retrospective study of 46,705 patients, and 102,043 evaluable RBC transfusions from 2013-2016 across 12 hospitals. Transfusion effectiveness was defined as hemoglobin, bilirubin, or creatinine increments following single RBC unit transfusion. Models incorporated a subset of donors with data on single nucleotide polymorphisms associated with osmotic and oxidative hemolysis in vitro. Mixed modelling accounting for repeated transfusion episodes identified predictors of transfusion effectiveness. RESULTS. Blood donor (sex, Rh status, fingerstick hemoglobin, smoking), component (storage duration, gamma irradiation, leukoreduction, apheresis collection, storage solution), and recipient (sex, body mass index, race, age) characteristics were associated with hemoglobin and bilirubin but not creatinine increments following RBC transfusions. Increased storage duration was associated with increased bilirubin and decreased hemoglobin increments, suggestive of in vivo hemolysis following transfusion. Donor G6PD-deficiency and polymorphisms in SEC14L4, HBA2, and MYO9B genes were associated with decreased hemoglobin increments. Donor G6PD-deficiency and polymorphisms in SEC14L4 were associated with increased transfusion requirements in the subsequent 48 hours. CONCLUSIONS. Donor genetic and other factors, such as RBC storage duration, affect transfusion effectiveness as defined by decreased hemoglobin or increased bilirubin increments. Addressing these factors will provide a precision medicine approach to improve patient outcomes, particularly for chronically-transfused RBC recipients, who would most benefit from more effective transfusion products.
Nareg H. Roubinian, Sarah E. Reese, Hannah Qiao, Colleen Plimier, Fang Fang, Grier P. Page, Ritchard G. Cable, Brian Custer, Mark T. Gladwin, Ruchika Goel, Bob Harris, Jeanne E. Hendrickson, Tamir Kanias, Steve Kleinman, Alan E. Mast, Steven R. Sloan, Bryan R. Spencer, Steven L. Spitalnik, Michael P. Busch, Eldad A. Hod
Leukemia stem cells (LSCs) promote the disease and seem resistant to therapy and immune control. Why LSCs are selectively resistant against elimination by cytotoxic CD8+ T cells (CTLs) is still unknown. In this study, we demonstrate that LSCs in chronic myeloid leukemia (CML) can be recognized and killed by CD8+ CTLs in vitro. However, Tregs, which preferentially localized close to CD8+ CTLs in CML bone marrow (BM), protected LSCs from MHC-class I dependent CD8+ CTL-mediated elimination in vivo. BM Tregs in CML were characterized by the selective expression of tumor necrosis factor receptor 4 (Tnfrsf4). Stimulation of Tnfrsf4-signaling did not deplete Tregs but reduced the capacity of Tregs to protect LSCs from CD8+ CTL-mediated killing. In the BM of newly diagnosed CML patients, TNFRSF4 mRNA levels were significantly increased and correlated with the expression of the Treg-restricted transcription factor FOXP3. Overall, these results identify Tregs as key regulator of immune escape of LSCs and TNFRSF4 as a potential target to reduce the function of Tregs and boost anti-leukemic immunity in CML.
Magdalena Hinterbrandner, Viviana Rubino, Carina Stoll, Stefan Forster, Noah Schnüriger, Ramin Radpour, Gabriela M. Baerlocher, Adrian F. Ochsenbein, Carsten Riether
Myelodysplastic syndromes (MDS) are hematopoietic stem and progenitor cell (HSPC) malignancies characterized by ineffective hematopoiesis and an increased risk of leukemia transformation. Epigenetic regulators are recurrently mutated in MDS, directly implicating epigenetic dysregulation in MDS pathogenesis. Here, we identified a tumor suppressor role of the acetyltransferase p300 in clinically relevant MDS models driven by mutations in the epigenetic regulators TET2, ASXL1, and SRSF2. The loss of p300 enhanced the proliferation and self-renewal capacity of Tet2-deficient HSPCs, resulting in an increased HSPC pool and leukemogenicity in primary and transplantation mouse models. Mechanistically, the loss of p300 in Tet2-deficient HSPCs altered enhancer accessibility and the expression of genes associated with differentiation, proliferation, and leukemia development. Particularly, p300 loss led to an increased expression of Myb, and the depletion of Myb attenuated the proliferation of HSPCs and improved the survival of leukemia-bearing mice. Additionally, we show that chemical inhibition of p300 acetyltransferase activity phenocopied Ep300 deletion in Tet2-deficient HSPCs, whereas activation of p300 activity with a small molecule impaired the self-renewal and leukemogenicity of Tet2-deficient cells. This suggests a potential therapeutic application of p300 activators in the treatment of MDS with TET2 inactivating mutations.
Na Man, Gloria Mas, Daniel L. Karl, Jun Sun, Fan Liu, Qin Yang, Miguel Torres-Martin, Hidehiro Itonaga, Concepcion Martinez, Shi Chen, Ye Xu, Stephanie Duffort, Pierre-Jacques Hamard, Chuan Chen, Beth E. Zucconi, Luisa Cimmino, Feng-Chun Yang, Mingjiang Xu, Philip A. Cole, Maria E. Figueroa, Stephen D. Nimer
Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm characterized by hyperactivation of JAK/STAT signaling and dysregulation of the transcription factor GATA1 in megakaryocytes (MKs). TGFβ plays a pivotal role in the pathobiology of MF by promoting bone marrow fibrosis and collagen deposition and by enhancing the dormancy of normal hematopoietic stem cells (HSCs). In this study, we show that MF MKs elaborated significantly greater levels of TGFβ1 than TGFβ2 and TGFβ3 to a varying degree, and evaluated the ability of AVID200 a potent TGFβ1/3 protein trap, to block the excessive TGFβ signaling. Treatment of human mesenchymal stromal cells (MSCs) with AVID200 significantly reduced their proliferation, decreased phosphorylation of SMAD2, and interfered with the ability of TGFβ1 to induce collagen expression. Moreover, treatment of MF mononuclear cells (MNCs) with AVID200 led to increased numbers of progenitor cells (PC) with wild type JAK2 rather than mutated JAK2V617F. This effect of AVID200 on MF PC was attributed to its ability to block TGFβ1-induced p57Kip2 expression and SMAD2 activation thereby allowing normal rather than MF PCs to preferentially proliferate, and form hematopoietic colonies. To assess the in vivo effects of AVID200, Gata1low mice, a murine model of MF, were treated with AVID200 resulting in the reduction in bone marrow (BM) fibrosis and an increase in BM cellularity. AVID200 treatment also increased the frequency and numbers of murine progenitor cells as well as short and long term HSCs. Collectively, these data provide the rationale for TGFβ1 blockade with AVID200 as a therapeutic strategy for MF patients.
Lilian Varricchio, Camelia Iancu-Rubin, Bhaskar Upadhyaya, Maria Zingariello, Fabrizio Martelli, Paola Verachi, Cara Clementelli, Jean-Francois Denis, Adeeb H. Rahman, Gilles Tremblay, John Mascarenhas, Ruben A. Mesa, Maureen O'Connor-McCourt, Anna Rita Migliaccio, Ronald Hoffman
In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture–conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/μg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements.
Sara Elgamal, Emanuele Cocucci, Ellen J. Sass, Xiaokui M. Mo, Angela R. Blissett, Edward P. Calomeni, Kerry A. Rogers, Jennifer A. Woyach, Seema A. Bhat, Natarajan Muthusamy, Amy J. Johnson, Karilyn T. Larkin, John C. Byrd
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.
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
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.
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
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.
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
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.
Sherilyn Grill, Shilpa Padmanaban, Ann Friedman, Eric Perkey, Frederick Allen, Valerie M. Tesmer, Jennifer Chase, Rami Khoriaty, Catherine E. Keegan, Ivan Maillard, Jayakrishnan Nandakumar
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