Primary atopic disorders are a group of inborn errors of immunity that skew the immune system toward severe allergic disease. Defining the biology underlying these extreme monogenic phenotypes reveals shared mechanisms underlying common polygenic allergic disease and identifies potential drug targets. Germline gain-of-function (GOF) variants in JAK1 are a cause of severe atopy and eosinophilia. Modeling the JAK1GOF (p.A634D) variant in both zebrafish and human induced pluripotent stem cells (iPSCs) revealed enhanced myelopoiesis. RNA-Seq of JAK1GOF human whole blood, iPSCs, and transgenic zebrafish revealed a shared core set of dysregulated genes involved in IL-4, IL-13, and IFN signaling. Immunophenotypic and transcriptomic analysis of patients carrying a JAK1GOF variant revealed marked Th cell skewing. Moreover, long-term ruxolitinib treatment of 2 children carrying the JAK1GOF (p.A634D) variant remarkably improved their growth, eosinophilia, and clinical features of allergic inflammation. This work highlights the role of JAK1 signaling in atopic immune dysregulation and the clinical impact of JAK1/2 inhibition in treating eosinophilic and allergic disease.
Catherine M. Biggs, Anna Cordeiro-Santanach, Sergey V. Prykhozhij, Adam P. Deveau, Yi Lin, Kate L. Del Bel, Felix Orben, Robert J. Ragotte, Aabida Saferali, Sara Mostafavi, Louie Dinh, Darlene Dai, Katja G. Weinacht, Kerry Dobbs, Lisa Ott de Bruin, Mehul Sharma, Kevin Tsai, John J. Priatel, Richard A. Schreiber, Jacob Rozmus, Martin C.K. Hosking, Kevin E. Shopsowitz, Margaret L. McKinnon, Suzanne Vercauteren, Michael Seear, Luigi D. Notarangelo, Francis C. Lynn, Jason N. Berman, Stuart E. Turvey
BACKGROUND. Chronotherapy is a drug intervention at specific times of the day to optimize efficacy and minimize adverse effects. Its value in hematologic malignancy remains to be explored, in particular in adult patients. METHODS. We performed chronotherapeutic analysis using two cohorts of diffuse large B cell lymphoma (DLBCL) patients undergoing chemotherapy with a dichotomized schedule (morning or afternoon). The effect of a morning or afternoon schedule of rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) on survival and drug tolerability were evaluated in a survival cohort (n = 210) and an adverse event cohort (n = 129), respectively. Analysis of ~14,000 healthy subjects was followed to identify the circadian variation in hematologic parameters. RESULTS. Both progression-free survival (PFS) and overall survival (OS) of female, but not male, patients were significantly shorter when patients received chemotherapy mostly in the morning (PFS hazard ratio [HR] 0.357; P = 0.033 and OS HR 0.141; P = 0.032). The dose intensity was reduced in female patients treated in the morning (cyclophosphamide 10%; P = 0.002, doxorubicin 8%; P = 0.002 and rituximab 7%; P = 0.003). This was mainly attributable to infection and neutropenic fever: female patients treated in the morning suffered from a higher incidence of infections (16.7% vs 2.4%) and febrile neutropenia (20.8% vs 9.8%) as compared to those treated in the afternoon. The sex-specific chronotherapeutic effects can be explained by the larger daily fluctuation of circulating leukocytes and neutrophils in females than in males. CONCLUSIONS. In female DLBCL patients, R-CHOP treatment in the afternoon can reduce the toxicity while it improves the efficacy and the survival outcome.
Dae Wook Kim, Ja Min Byun, Jeong-Ok Lee, Jae Kyoung Kim, Youngil Koh
Precision medicine can significantly improve outcomes for cancer patients, but implementation requires comprehensive characterization of tumor cells to identify therapeutically exploitable vulnerabilities. Here we describe somatic biallelic TET2 mutations in an elderly patient with acute myeloid leukemia (AML) that was chemoresistant to anthracycline and cytarabine (Ara-C), but acutely sensitive to 5’-azacitidine (5’-Aza) hypomethylating monotherapy resulting in long-term morphological remission. Given the role of TET2 as a regulator of genomic methylation, we hypothesized that mutant TET2 allele dosage affects response to 5’-Aza. Using an isogenic cell model system and an orthotopic mouse xenograft, we demonstrate that biallelic TET2 mutations confer sensitivity to 5’-Aza compared to cells with monoallelic mutation. Our data argue in favor of using hypomethylating agents for chemoresistant disease or as first line therapy in patients with biallelic TET2-mutated AML and demonstrate the importance of considering mutant allele dosage in the implementation of precision medicine for cancer patients.
Friedrich Stölzel, Sarah E. Fordham, Devi Nandana, Wei-Yu Lin, Helen J. Blair, Claire J. Elstob, Hayden L. Bell, Brigitte Mohr, Leo Ruhnke, Desiree Kunadt, Claudia Dill, Daniel A. Allsop, Rachel E. Piddock, Emmanouela-Niki Soura, Catherine Vida Park, Mohd Fadly, Thahira Rahman, Abrar A. Alharbi, Manja Wobus, Heidi Altmann, Christoph Röllig, Lisa Wagenführ, Gail L. Jones, Tobias Menne, Graham H. Jackson, Helen J. Marr, Jude Fitzgibbon, Kenan Onel, Manja Meggendorfer, Amber Robinson, Zuzanna Bziuk, Emily Bowes, Olaf Heidenreich, Torsten Haferlach, Sara Villar, Beñat Ariceta, Rosa Ayala Diaz, Steven J. Altschuler, Lani Wu, Felipe Prosper, Pau Montesinos, Joaquin Martinez-Lopez, Martin Bornhäuser, James M. Allan
Despite the efficacy of tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML), malignant long-term hematopoietic stem cells (LT-HSC) persist as a source of relapse. However, LT-HSC are heterogenous and the most primitive, drug-resistant LT-HSC subpopulations are not well characterized. In normal hematopoiesis, self-renewal and long-term reconstitution capacity is enriched within LT-HSCs with low c-Kit expression (c-KitLow). Here, using a transgenic CML mouse model, we found that long-term engraftment and leukemogenic capacity were restricted to c-KitLow CML LT-HSC. CML LT-HSC demonstrated enhanced differentiation with expansion of mature progeny following exposure to the c-Kit ligand, stem cell factor (SCF). Conversely, SCF deletion led to depletion of normal LT-HSC but increase in c-KitLow and total CML LT-HSC with reduced generation of mature myeloid cells. CML c-KitLow LT-HSC showed reduced cell cycling, and expressed enhanced quiescence and inflammatory gene signatures. SCF administration led to enhanced depletion of CML primitive progenitors but not LT-HSC after TKI treatment. Human CML LT-HSC with low or absent c-Kit expression were markedly enriched after TKI treatment. We conclude that CML LT-HSC expressing low c-Kit levels are enriched for primitive, quiescent, drug-resistant leukemia initiating cells and represent a critical target for eliminating disease persistence.
Mansi Shah, Harish Kumar, Shaowei Qiu, Hui Li, Mason Harris, Jianbo He, Ajay Abraham, David K. Crossman, Andrew Paterson, Robert S. Welner, Ravi Bhatia
Diamond–Blackfan anemia (DBA) is a genetic blood disease caused by heterozygous loss-of-function mutations in ribosomal protein (RP) genes, most commonly RPS19. The signature feature of DBA is hypoplastic anemia occurring in infants, although some older patients develop multi-lineage cytopenias with bone marrow hypocellularity. The mechanism of anemia in DBA is not fully understood and even less is known about the pancytopenia that occurs later in life, in part because patient hematopoietic stem and progenitor cells (HSPCs) are difficult to obtain, and the current experimental models are suboptimal. We modeled DBA by editing healthy human donor CD34+ HSPCs with CRISPR/Cas9 to create RPS19 haploinsufficiency. In vitro differentiation revealed normal myelopoiesis and impaired erythropoiesis, as observed in DBA. After transplantation into immunodeficient mice, bone marrow repopulation by RPS19+/− HSPCs was profoundly reduced, indicating hematopoietic stem cell (HSC) impairment. The erythroid and HSC defects resulting from RPS19 haploinsufficiency were partially corrected by transduction with an RPS19-expressing lentiviral vector or by Cas9 disruption of TP53. Our results define a tractable, biologically relevant experimental model of DBA based on genome-editing of primary human HSPCs and they identify an associated HSC defect that emulates the pan-hematopoietic defect of DBA.
Senthil Velan Bhoopalan, Jonathan S. Yen, Thiyagaraj Mayuranathan, Kalin D. Mayberry, Yu Yao, Maria Angeles Lillo Osuna, Yoonjeong Jang, Janaka S.S. Liyange, Lionel Blanc, Steven R. Ellis, Marcin W. Wlodarski, Mitchell J. Weiss
Mutations in the BRCA1 tumor suppressor gene, such as 5382insC (BRCA15382insC), give carriers an increased risk for breast, ovarian, prostate and pancreatic cancers. We have previously reported that, in mice, Brca1 deficiency in the hematopoietic system leads to pancytopenia and, as a result, early lethality. Here we explore the cellular consequences of Brca1 null and BRCA1 5382insC alleles in combination with Tp53 deficiency in the murine hematopoietic system. We find that Brca1 and Tp53 co-deficiency leads to a highly penetrant erythroproliferative disorder that is characterized by hepatosplenomegaly and expanded megakaryocyte erythroid progenitor (MEP) and immature erythroid blast populations. The expanded erythroid progenitor populations in both bone marrow and spleen have the capacity to transmit the disease into secondary mouse recipients, suggesting Brca1 and Tp53 co-deficiency provides a new murine model of hematopoietic neoplasia. This Brca1/Tp53 model replicates Poly (ADP-ribose) polymerase (PARP) inhibitor olaparib sensitivity seen in existing Brca1/Tp53 breast cancer models and has the benefits of monitoring disease progression and drug responses via peripheral blood analyses without sacrificing experimental animals. In addition, this erythroid neoplasia develops much faster than murine breast cancer, allowing for increased efficiency of future preclinical studies.
Gerardo Lopez-Perez, Ranjula Wijayatunge, Kelly B. McCrum, Sam R. Holmstrom, Victoria E. Mgbemena, Theodora S. Ross
Acute graft-versus-host disease (aGvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT) inflicted by alloreactive T cells primed in secondary lymphoid organs (SLOs) and subsequent damage to aGvHD target tissues. In recent years, regulatory T cell (Treg) transfer and/or expansion has emerged as a promising therapy to modulate aGvHD. However, cellular niches essential for fostering Tregs to prevent aGvHD have not been explored, yet. Here, we tested whether and to what extent MHC class II (MHCII) expressed on Ccl19+ fibroblastic reticular cells (FRCs) shape the donor CD4+ T cell response during aGvHD. Animals lacking MHCII expression on Ccl19-Cre-expressing FRCs (MHCIIΔCcl19) showed aberrant CD4+ T cells activation in the effector phase resulting in exacerbated aGvHD that was associated with significantly reduced expansion of Foxp3+ Tregs and invariant natural killer T (iNKT) cells. Skewed Treg maintenance in MHCIIΔCcl19 mice resulted in loss of protection from aGvHD provided by adoptively transferred donor Tregs. In contrast, although FRCs upregulated co-stimulatory surface receptors, degraded and processed exogenous antigens after myeloablative irradiation, FRCs were dispensable to activate alloreactive CD4+ T cells in two mouse models of aGvHD. In sum, these data reveal an immunoprotective, MHCII-mediated function of FRC niches in secondary lymphoid organs (SLOs) after allo-HCT and highlights a hitherto unknown framework of cellular and molecular interactions that regulate CD4+ T cell alloimmunity.
Haroon Shaikh, Joern Pezoldt, Zeinab Mokhtari, Juan Gamboa Vargas, Duc-Dung Le, Josefina Peña Mosca, Estibaliz Arellano-Viera, Michael A.G. Kern, Caroline Graf, Niklas Beyersdorf, Manfred B. Lutz, Angela Riedel, Maike Büttner-Herold, Alma Zernecke, Hermann Einsele, Antoine-Emmanuel Saliba, Burkhard Ludewig, Jochen Huehn, Andreas Beilhack
Acquired aplastic anemia (AA) is caused by autoreactive T-cell-mediated destruction of early hematopoietic cells. Somatic loss of human leukocyte antigen (HLA) Class I alleles was identified as a mechanism of immune escape in surviving hematopoietic cells of some AA patients. However, pathogenicity, structural characteristics and clinical impact of specific HLA alleles in AA remain poorly understood. Here, we evaluated somatic HLA loss in 505 AA patients from two multi-institutional cohorts. Using a combination of HLA mutation frequencies, peptide-binding structures, and association with AA in an independent cohort of 6,323 patients from the National Marrow Donor Program, we identified 19 AA risk alleles and 12 non-risk alleles and established a novel AA HLA pathogenicity stratification. Our results define pathogenicity for the majority of common HLA-A/B alleles across diverse populations. Our study demonstrates that HLA alleles confer different risks of developing AA, but once AA develops, specific alleles are not associated with response to immunosuppression or trans-plant outcomes. However, higher pathogenicity alleles, particularly HLA-B*14:02, are associated with higher rates of clonal evolution in adult AA patients. Our study provides novel insights into the immune pathogenesis of AA, opening the door to future autoantigen identification and improved under-standing of clonal evolution in AA.
Timothy S. Olson, Benjamin F. Frost, Jamie L. Duke, Marian Dribus, Hongbo M. Xie, Zachary D. Prudowsky, Elissa Furutani, Jonas Gudera, Yash B. Shah, Deborah Ferriola, Amalia Dinou, Ioanna Pagkrati, Soyoung Kim, Yixi Xu, Meilun He, Shannon Zheng, Sally Nijim, Ping Lin, Chong Xu, Taizo Nakano, Joseph H. Oved, Beatriz M. Carreno, Yung-Tsi Bolon, Shahinaz M. Gadalla, Steven G.E. Marsh, Sophie Paczesny, Stephanie J. Lee, Dimitrios S. Monos, Akiko Shimamura, Alison A. Bertuch, Loren Gragert, Stephen Spellman, Daria V. Babushok
Antithrombin, a major endogenous anticoagulant, is a serine protease inhibitor (serpin). We characterized the biological and clinical impact of variants involving C-terminal antithrombin. We performed comprehensive molecular, cellular, and clinical characterization of patients with C-terminal antithrombin variants from a cohort of 444 unrelated individuals with confirmed antithrombin deficiency. We identified 17 patients carrying 12 C-terminal variants, 5 of whom had the p.Arg445Serfs*17 deletion. Five missense variants caused qualitative deficiency, and 7, including 4 insertion-deletion variants, induced severe quantitative deficiency, particularly p.Arg445Serfs*17 (antithrombin <40%). This +1 frameshift variant had a molecular size similar to that of WT antithrombin but possessed a different C-terminus. Morphologic and cotransfection experiments showed that recombinant p.Arg445Serfs*17 was retained at the endoplasmic reticulum and had a dominant-negative effect on WT antithrombin. Characterization of different 1+ frameshift, aberrant C-terminal variants revealed that protein secretion was determined by frameshift site. The introduction of Pro441 in the aberrant C-terminus, shared by 5 efficiently secreted variants, partially rescued p.Arg445Serfs*17 secretion. C-terminal antithrombin mutants have notable heterogeneity, related to variant type and localization. Aberrant C-terminal variants caused by 1+ frameshift, with similar size as WT antithrombin, may be secreted or not, depending on frameshift site. The severe clinical phenotypes of these genetic changes are consistent with their dominant-negative effects.
Carlos Bravo-Pérez, Mara Toderici, Joseph E. Chambers, José A. Martínez-Menárguez, Pedro Garrido-Rodriguez, Horacio Pérez-Sanchez, Belén de la Morena-Barrio, José Padilla, Antonia Miñano, Rosa Cifuentes-Riquelme, Vicente Vicente, Maria L. Lozano, Stefan J. Marciniak, Maria Eugenia de la Morena-Barrio, Javier Corral
POEMS syndrome is a rare monoclonal plasma cell disorder with unique symptoms distinct from other plasma cell neoplasms, including high serum VEGF levels. Since the prospective isolation of POEMS clones has not yet been successful, their real nature remains unclear. We herein performed the single-cell RNA sequencing of bone marrow plasma cells from patients with POEMS syndrome and identified POEMS clones that had immunoglobulin λ light chain (IGL) sequences (IGLV1-36, 40, 44, and 47) with amino acid changes specific to POEMS syndrome. The proportions of POEMS clones in plasma cells were markedly smaller (median: 12.9%) than in multiple myeloma (MM) (96–100%) and monoclonal gammopathy of undetermined significance (MGUS) patients (57–81%). Single-cell transcriptomes revealed that POEMS clones were CD19-negative, CD138-positive, and MHC class II-low, which allowed for their prospective isolation. POEMS clones expressed significantly lower levels of c-MYC and CCND1 than MM, accounting for their small size. VEGF mRNA was not up-regulated in POEMS clones, directly indicating that VEGF is not produced by POEMS clones. These results reveal unique features of POEMS clones and enhance our understanding of the pathogenesis of POEMS syndrome.
Yusuke Isshiki, Motohiko Oshima, Naoya Mimura, Kensuke Kayamori, Yurie Miyamoto-Nagai, Masahide Seki, Yaeko Nakajima-Takagi, Takashi Kanamori, Eisuke Iwamoto, Tomoya Muto, Shokichi Tsukamoto, Yusuke Takeda, Chikako Ohwada, Sonoko Misawa, Jun-ichiro Ikeda, Masashi Sanada, Satoshi Kuwabara, Yutaka Suzuki, Emiko Sakaida, Chiaki Nakaseko, Atsushi Iwama
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