Hematology
Abstract
Anemia is a common and disabling complication of chronic kidney disease (CKD). Current therapies can be burdensome, and full correction of anemia is limited by cardiovascular side effects. New approaches that may offer additional therapeutic options are needed. We explored the anti-anemic effects of erythroferrone, an erythroid hormone that induces iron mobilization by suppressing the master iron-regulatory hormone hepcidin. In a preclinical murine model of adenine-induced CKD, transgenic augmentation of erythroferrone mobilized iron, increased hemoglobin concentrations by approximately 2 g/dl, and modestly improved renal function without affecting systemic or renal inflammation, fibrosis, or markers of mineral metabolism. This study supports the concept that therapeutic augmentation of erythroferrone is a promising approach for alleviating CKD-associated anemia.
Authors
Brian Czaya, Joseph D. Olivera, Moya Zhang, Amber Lundin, Christian D. Castro Andrade, Grace Jung, Mark R. Hanudel, Elizabeta Nemeth, Tomas Ganz
Abstract
RNA splicing factor SF3B1 is one of the most recurrently mutated genes in chronic lymphocytic leukemia (CLL) and frequently co-occurs with chromosome 13q deletion (del(13q)). This combination is associated with poor prognosis in CLL, suggesting these lesions increase CLL aggressiveness. While del(13q) in murine B cells (Mdr mice), but not expression of Sf3b1-K700E, drives the initiation of CLL, we hypothesize that SF3B1 mutation accelerates CLL progression. In this study, we crossed mice with a B-cell-specific Sf3b1-K700E allele with Mdr mice to determine the impact of Sf3b1 mutation on CLL progression. We found that the co-occurrence of these two lesions in murine B cells caused acceleration of CLL. We showed that Sf3b1-K700E impacted alternative RNA splicing of Nfatc1 and activated mTOR signaling and the MYC pathway, contributing to CLL acceleration. Moreover, concurrent inhibition of RNA splicing and mTOR pathways led to cell death in vitro and in vivo in murine CLL cells with SF3B1 mutation and del(13q). Our results thus suggest that SF3B1 mutation contributes to the aggressiveness of CLL by activating the mTOR pathway through alternative splicing of Nfatc1, providing a rationale for targeting mTOR and RNA splicing in the subset of CLL patients with both SF3B1 mutations and del(13q).
Authors
Bo Zhang, Prajish Iyer, Meiling Jin, Elisa ten Hacken, Zachary Cartun, Kevyn L. Hart, Mike Fernandez, Kristen Stevenson, Laura Rassenti, Emanuela M. Ghia, Thomas J. Kipps, Donna Neuberg, Ruben Carrasco, Wing Chan, Joo Y. Song, Yu Hu, Catherine Wu, Lili Wang
Abstract
β-Thalassemia is a genetic disorder arising from mutations in the β-globin gene, leading to ineffective erythropoiesis and iron overload. Ineffective erythropoiesis, a hallmark of β-thalassemia, is an important driver of iron overload, which contributes to liver fibrosis, diabetes, and cardiac disease. Iron homeostasis is regulated by the hormone hepcidin; BMP6/hemojuvelin–mediated (BMP6/HJV-mediated) signaling induces hepatic hepcidin expression via SMAD1/5, with transmembrane serine protease 6 (TMPRSS6) being a negative regulator of HJV. Individuals with loss-of-function mutations in the TMPRSS6 gene show increased circulating hepcidin and iron-refractory iron-deficiency anemia, suggesting that blocking TMPRSS6 may be a viable strategy to elevate hepcidin levels in β-thalassemia. We generated a human mAb (REGN7999) that inhibits TMPRSS6. In an Hbbth3/+ mouse model of β-thalassemia, REGN7999 treatment led to significant reductions in liver iron, reduced ineffective erythropoiesis, and showed improvements in RBC health, running distance during forced exercise, and bone density. In a phase I, doubleblind, randomized, placebo-controlled study in healthy human volunteers (NCT05481333), REGN7999 increased serum hepcidin and reduced serum iron with an acceptable tolerability profile. Our results suggest that, by both reducing iron and improving RBC function, inhibition of TMPRSS6 by REGN7999 may offer a therapy for iron overload and impaired erythropoiesis in β-thalassemia.
Authors
Heinrich E. Lob, Nikhil Singh, Kusha Mohammadi, Larisa Ivanova, Beth Crowell, Hyon J. Kim, Leah Kravets, Nanditha M. Das, Yonaton Ray, Jee Hae Kim, Sylvie Rottey, Emily Labriola-Tompkins, Hazem E. Hassan, Lorna Farrelly, Harvey F. Chin, Marilena Preda, Leigh Spencer Noakes, Kei Saotome, Matthew Franklin, Marc W. Retter, Elif Karayusuf, John J. Flanagan, William Olson, Kalyan C. Nannuru, Vincent Idone, Michael E. Burczynski, Olivier A. Harari, Lorah Perlee, Griet Van Lancker, Andrew J. Murphy, Aris N. Economides, Sarah J. Hatsell
Abstract
Mitochondrial DNA (mtDNA) shares characteristics with bacterial DNA and activates immune cells via Toll like receptor (TLR)-9. Extracellular vesicles (EVs) and mtDNA have been found in blood products and can activate immune cells; we sought to characterize their evolution in stored blood products. From a previous study of hemolysis in 13,403 blood donors, a second blood unit was drawn from 651 donors and sampled at days 10, 21, and 42. EV counts and RBC-EVs increased with storage time, and EV levels were higher in males and in RBC units processed in AS-1 compared to AS-3. mtDNA levels were higher in females and RBC units processed in AS-3. EV populations and mtDNA levels were highly correlated within donors for 98 donations obtained 2-12 months apart. Quantitative trait locus analysis revealed several genetic associations, most notably linking mtDNA levels with polymorphisms in ANKLE1, which encodes an erythroid-specific protein that preferentially cleaves mtDNA. These data suggest that donor-intrinsic factors may influence mtDNA and EV levels found in RBC units. This finding lends impetus to determining if genetic or environmental factors control levels of these immune mediators in blood donors.
Authors
Xutao Deng, Clara Di Germanio, Erika G. Marques de Menezes, Pamela Milani, Mars Stone, Heather Tanner, Sonia Coco Bakkour, Daniel M. Chafets, Sarah E. Reese, Nareg H. Roubinian, Steven Kleinman, Tamir Kanias, Michael P. Busch, Eric J. Earley, Grier P. Page, Travis Nemkov, Angelo D’Alessandro, Philip J. Norris
Abstract
Sickle cell disease (SCD) causes severe morbidity and early mortality, yet it varies phenotypically. Both air pollution and SCD affect the cardiorespiratory, inflammatory, and endothelial systems; however, limited evidence exists on the effect of long-term air pollution exposure in SCD. We hypothesized that annual ambient (outdoor) concentrations of fine particulate matter (PM2.5), particles with a diameter of 2.5 μm or less, at a child’s home would be significantly associated with worse clinical, laboratory, and stroke-risk imaging outcomes. Patient data for this retrospective study were obtained from a cohort of children with SCD (from 2010 to 2019). Annual PM2.5 exposure was estimated using remote-sensing air pollution datasets. Statistical analyses employed fixed effects multivariable models, offering a robust approach to isolate the effect of PM2.5 exposure. The final cohort included 1,089 children with SCD. Higher annual PM2.5 concentrations were significantly associated with more annual hospital days, higher likelihood of hospitalization and abnormal stroke-risk screening, and elevated inflammatory markers. Of note, hydroxyurea use mitigated the inflammatory response to PM2.5 but did not mitigate the effect of PM2.5 on clinical outcomes. Importantly, the elevated stroke risk associated with PM2.5 exposure persisted, even among children receiving hydroxyurea therapy, highlighting a critical concern in pediatric SCD management. These results underscore the clinical importance of addressing environmental factors for comprehensive SCD care.
Authors
Paul E. George, Grace Kalmus, Joseph Lipscomb, David H. Howard, Benjamin Kopp, Wilbur A. Lam, Stefanie Ebelt
Abstract
Heme iron (HI), derived principally from hemoglobin (Hb) in animal foods, is a highly bioavailable source of dietary iron for humans. Despite several decades of focused research, however, molecular mechanisms governing HI absorption remain undefined. Previous studies in mice and rats have not produced a consensus, definitive model of efficient HI absorption/utilization. We hypothesized that a nutritional approach, using semipurified, HI-containing diets, could be utilized to establish a tractable rodent model of HI absorption that could ultimately be employed to test the roles of receptors, transporters, and enzymes using genetic engineering technology. Experiments were designed to assess HI utilization by feeding animals AIN-93G–based, HI-enriched experimental diets formulated with lyophilized porcine RBCs, containing approximately 85% HI and 15% nonheme iron (NHI). Total iron was within the physiological range (50–75 ppm) and precisely matched NHI control diets containing ferrous sulfate were utilized as comparators. Notably, in Sprague-Dawley (S-D) rats and C57BL/6 (B6) mice, dietary HI effectively (a) resolved iron-deficiency anemia; (b) supported normal pregnancy, lactation, and neonatal development; and (c) contributed to iron loading in Hamp-KO mice and rats (modeling hereditary hemochromatosis). A nutritional paradigm has thus been established that facilitates investigation into mechanisms of HI absorption by S-D rats and B6 mice.
Authors
Jennifer K. Lee, Yue He, Shireen R.L. Flores, Regina R. Woloshun, Xiaoyu Wang, Jacob S. Shine, Pearl O. Ebea-Ugwuanyi, Sitara Sriram, Melissa Fraga, Sean Zhu, Yang Yu, Iqbal Hamza, James F. Collins
Abstract
Overall survival (OS) in multiple myeloma (MM) varies between a couple of months to more than 20 years, influenced by tumor characteristics, the tumor microenvironment (TME), and patient factors such as age and frailty. We analyzed sequential BM samples from 45 MM patients with OS < 3 years versus > 8 years using mass cytometry and bulk TCRβ sequencing. Patients with long OS demonstrated stability in the TME and T cell environments, while those with short OS had significant changes at relapse, including fewer T cells, increased Treg cells, and more activated and exhausted CD8 T cells. Notably, higher PD-1 expression in CD8 T cells at diagnosis correlated with short OS. Additionally, short-OS patients exhibited a more monoclonal T cell environment at relapse, with abundance of hyperexpanded clones. These findings reveal distinct immune cell differences between patients with short and long OS.
Authors
Alenka Djarmila Behsen, Esten Nymoen Vandsemb, Tobias Schmidt Slørdahl, Karen Dybkær, Maja Zimmer Jakobsen, Muhammad Kashif, Johan Lund, Vincent Luong, Astrid Marta Olsnes, Anders Waage, Anne Marit Sponaas, Kristine Misund
Abstract
Bronchiolitis obliterans syndrome (BOS) is a progressive, fatal obstructive lung that occurs following lung transplant, where it is termed chronic lung allograft dysfunction BOS (CLAD-BOS), or as the primary manifestation of pulmonary chronic graft versus host disease (cGVHD-BOS) following allogeneic hematopoietic stem cell transplant. Disease pathogenesis is poorly understood, however chronic alloreactivity is common to both conditions, suggesting a shared pathophysiology. We performed single-cell RNA-Seq (scRNA-Seq) on explanted human lungs from 4 CLAD-BOS patients, 3 cGVHD-BOS patients, and 3 deceased controls to identify cell types, genes, and pathways enriched in BOS to better understand disease mechanisms. In both forms of BOS, we found an expanded population of CD8+ tissue resident memory T-cells (TRM), which was distinct to BOS compared to other chronic lung diseases. In addition, BOS samples expressed genes and pathways associated with macrophage chemotaxis and proliferation, including in non-immune cell populations. We also identify dysfunctional stromal cells in BOS, characterized by pro and anti-fibrotic gene programs. These data suggest substantial cellular and molecular overlap between CLAD- and cGVHD-BOS and therefore, common pathways for possible therapeutic intervention.
Authors
Patrick W. Mellors, Ana N. Nottingham, Bruno Casino Remondo, Maksim Shestov, Joseph D. Planer, Andrew R. Peterson, Yun Ying, Su Zhou, Jason D. Christie, Joshua M. Diamond, Edward Cantu, Maria C. Basil, Saar Gill
Abstract
BACKGROUND. The graft-vs-leukemia (GVL) effect contributes to the efficacy of allogeneic stem cell transplantation (alloSCT). However, relapse, indicative of GVL failure, is the greatest single cause of treatment failure. Based on preclinical data showing that IFN-γ is important to sensitize myeloblasts to alloreactive T cells, we performed a phase I trial of IFN-γ combined with donor leukocyte infusions (DLI) in myeloblastic malignancies that relapsed post-HLA-matched alloSCT. METHODS. Patients with relapsed acute myeloid leukemia or myelodysplastic syndrome after alloSCT were eligible. Patients self-administered IFN-γ for 4 weeks (cohort 1) or 1 week (cohort 2), followed by DLI and concurrent IFN-γ for a total of 12 weeks. Bone marrow samples were analyzed by single-cell RNA sequencing (scRNAseq) to assess in vivo responses to IFN-γ by malignant myeloblasts. RESULTS. IFN-γ monotherapy was well tolerated by all subjects (n=7). Treatment-related toxicities after DLI included: grade I-II graft-versus-host disease (n=5), immune effector cell-associated neurotoxicity syndrome (n=2), and idiopathic pulmonary syndrome (n=1), all of which resolved with corticosteroids. Four of 6 DLI recipients achieved minimal residual disease-negative complete remissions and full donor hematopoietic recovery. Median overall survival was 579 days (range, 97-906) in responders. ScRNAseq confirmed in vivo activation of IFN-γ response pathway in hematopoietic stem cell-like or myeloid progenitor cells after IFN-γ in analyzed samples. CONCLUSIONS. IFN-γ was safe and well tolerated in this phase I study of IFN-γ for relapsed AML/MDS post-alloSCT, with a promising efficacy signal when combined with DLI. Larger studies are needed to formally test the efficacy of this approach. TRIAL RESGISTRATION. ClinicalTrials.gov NCT04628338. FUNDING. The research was supported by The UPMC Hillman Cancer Center Cancer Immunology and Immunotherapy Program (CIIP) Pilot Award and Cure Within Reach: Drug Repurposing Clinical Trials to Impact Blood Cancers. Recombinant IFN-gamma (Actimmune®) was donated by Horizon Therapeutics.
Authors
Sawa Ito, Emily Geramita, Kedwin Ventura, Biswas Neupane, Shruti Bhise, Erika M. Moore, Scott Furlan, Warren D. Shlomchik
Abstract
Thrombin promotes the proliferation and function of CD8+ T cells. To test if thrombin prevents exhaustion and sustains antiviral T cell activity during chronic viral infection, we depleted the thrombin-precursor prothrombin to 10% of normal levels in mice prior to infection with the clone 13 strain of lymphocytic choriomeningitis virus. Unexpectedly, prothrombin insufficiency resulted in 100% mortality after infection that was prevented by depletion of CD8+ T cells, suggesting that reduced availability of prothrombin enhances virus-induced immunopathology. Yet, the number, function, and apparent exhaustion of virus-specific T cells were measurably unaffected by prothrombin depletion. Histological analysis of the lung, heart, liver, kidney, spleen, intestine, and brain did not reveal any evidence of hemorrhage or increased tissue damage in low prothrombin mice that could explain mortality. Viral loads were also similar in infected mice regardless of prothrombin levels. Instead, infection of prothrombin-depleted mice resulted in a severe, T cell-dependent anemia associated with increased hemolysis. Thus, thrombin plays an unexpected protective role in preventing hemolytic anemia during virus infection, with potential implications for patients who are using direct thrombin inhibitors as an anticoagulant therapy.
Authors
Rachel Cantrell, H. Alex Feldman, Leah Rosenfeldt, Ayad Ali, Benjamin Gourley, Cassandra Sprague, Daniel Leino, Jeff Crosby, Alexey Revenko, Brett Monia, Stephen N. Waggoner, Joseph S. Palumbo
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