Obesity promotes a chronic inflammatory and hypercoagulable state that drives cardiovascular disease, type 2 diabetes, fatty liver disease, and several cancers. Elevated thrombin activity underlies obesity-linked thromboembolic events, but the mechanistic links between the thrombin/fibrin(ogen) axis and obesity-associated pathologies are incompletely understood. In this work, immunohistochemical studies identified extravascular fibrin deposits within white adipose tissue and liver as distinct features of mice fed a high-fat diet (HFD) as well as obese patients. Fibγ390–396A mice carrying a mutant form of fibrinogen incapable of binding leukocyte αMβ2-integrin were protected from HFD-induced weight gain and elevated adiposity. Fibγ390–396A mice had markedly diminished systemic, adipose, and hepatic inflammation with reduced macrophage counts within white adipose tissue, as well as near-complete protection from development of fatty liver disease and glucose dysmetabolism. Homozygous thrombomodulin-mutant ThbdPro mice, which have elevated thrombin procoagulant function, gained more weight and developed exacerbated fatty liver disease when fed a HFD compared with WT mice. In contrast, treatment with dabigatran, a direct thrombin inhibitor, limited HFD-induced obesity development and suppressed progression of sequelae in mice with established obesity. Collectively, these data provide proof of concept that targeting thrombin or fibrin(ogen) may limit pathologies in obese patients.
Anna K. Kopec, Sara R. Abrahams, Sherry Thornton, Joseph S. Palumbo, Eric S. Mullins, Senad Divanovic, Hartmut Weiler, A. Phillip Owens III, Nigel Mackman, Ashley Goss, Joanne van Ryn, James P. Luyendyk, Matthew J. Flick
BACKGROUND. Ibrutinib has been shown to have immunomodulatory effects by inhibiting Bruton’s tyrosine kinase (BTK) and IL-2–inducible T cell kinase (ITK). The relative importance of inhibiting these 2 kinases has not been examined despite its relevance to immune-based therapies. METHODS. Peripheral blood mononuclear cells from chronic lymphocytic leukemia (CLL) patients on clinical trials of ibrutinib (BTK/ITK inhibitor; n = 19) or acalabrutinib (selective BTK inhibitor; n = 13) were collected serially. T cell phenotype, immune function, and CLL cell immunosuppressive capacity were evaluated. RESULTS. Ibrutinib markedly increased CD4+ and CD8+ T cell numbers in CLL patients. This effect was more prominent in effector/effector memory subsets and was not observed with acalabrutinib. Ex vivo studies demonstrated that this may be due to diminished activation-induced cell death through ITK inhibition. PD-1 and CTLA-4 expression was significantly markedly reduced in T cells by both agents. While the number of Treg cells remained unchanged, the ratio of these to conventional CD4+ T cells was reduced with ibrutinib, but not acalabrutinib. Both agents reduced expression of the immunosuppressive molecules CD200 and BTLA as well as IL-10 production by CLL cells. CONCLUSIONS. Ibrutinib treatment increased the in vivo persistence of activated T cells, decreased the Treg/CD4+ T cell ratio, and diminished the immune-suppressive properties of CLL cells through BTK-dependent and -independent mechanisms. These features provide a strong rationale for combination immunotherapy approaches with ibrutinib in CLL and other cancers. TRIAL REGISTRATION. ClinicalTrials.gov NCT01589302 and NCT02029443. Samples described here were collected per OSU-0025. FUNDING. The National Cancer Institute.
Meixiao Long, Kyle Beckwith, Priscilla Do, Bethany L. Mundy, Amber Gordon, Amy M. Lehman, Kami J. Maddocks, Carolyn Cheney, Jeffrey A. Jones, Joseph M. Flynn, Leslie A. Andritsos, Farrukh Awan, Joseph A. Fraietta, Carl H. June, Marcela V. Maus, Jennifer A. Woyach, Michael A. Caligiuri, Amy J. Johnson, Natarajan Muthusamy, John C. Byrd
The lack of mechanistic explanations for many genotype-phenotype associations identified by GWAS precludes thorough assessment of their impact on human health. Here, we conducted an expression quantitative trait locus (eQTL) mapping analysis in erythroblasts and found erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of red blood cells (rbc). The same SNPs were previously associated with mean corpuscular hemoglobin concentration (MCHC) and susceptibility to severe malaria infection. We showed that Atp2b4–/– mice demonstrate increased MCHC, confirming ATP2B4 as the causal gene at this GWAS locus. Using CRISPR-Cas9, we fine mapped the genetic signal to an erythroid-specific enhancer of ATP2B4. Erythroid cells with a deletion of the ATP2B4 enhancer had abnormally high intracellular calcium levels. These results illustrate the power of combined transcriptomic, epigenomic, and genome-editing approaches in characterizing noncoding regulatory elements in phenotype-relevant cells. Our study supports ATP2B4 as a potential target for modulating rbc hydration in erythroid disorders and malaria infection.
Samuel Lessard, Emily Stern Gatof, Mélissa Beaudoin, Patrick G. Schupp, Falak Sher, Adnan Ali, Sukhpal Prehar, Ryo Kurita, Yukio Nakamura, Esther Baena, Jonathan Ledoux, Delvac Oceandy, Daniel E. Bauer, Guillaume Lettre
Angioimmunoblastic T cell lymphoma (AITL) represents a distinct, aggressive form of peripheral T cell lymphoma with a dismal prognosis. Recent exome sequencing in patients with AITL has revealed the frequent coexistence of somatic mutations in the Rho GTPase RhoA (RhoAG17V) and loss-of-function mutations in the 5-methylcytosine oxidase TET2. Here, we have demonstrated that TET2 loss and RhoAG17V expression in mature murine T cells cooperatively cause abnormal CD4+ T cell proliferation and differentiation by perturbing FoxO1 gene expression, phosphorylation, and subcellular localization, an abnormality that is also detected in human primary AITL tumor samples. Reexpression of FoxO1 attenuated aberrant immune responses induced in mouse models adoptively transferred with T cells and bearing genetic lesions in both TET2 and RhoA. Our findings suggest a mutational cooperativity between epigenetic factors and GTPases in adult CD4+ T cells that may account for immunoinflammatory responses associated with AITL patients.
Shengbing Zang, Jia Li, Haiyan Yang, Hongxiang Zeng, Wei Han, Jixiang Zhang, Minjung Lee, Margie Moczygemba, Sevinj Isgandarova, Yaling Yang, Yubin Zhou, Anjana Rao, M. James You, Deqiang Sun, Yun Huang
Runt-related transcription factor 1 (RUNX1) is generally considered to function as a tumor suppressor in the development of leukemia, but a growing body of evidence suggests that it has pro-oncogenic properties in acute myeloid leukemia (AML). Here we have demonstrated that the antileukemic effect mediated by RUNX1 depletion is highly dependent on a functional p53-mediated cell death pathway. Increased expression of other RUNX family members, including RUNX2 and RUNX3, compensated for the antitumor effect elicited by RUNX1 silencing, and simultaneous attenuation of all RUNX family members as a cluster led to a much stronger antitumor effect relative to suppression of individual RUNX members. Switching off the RUNX cluster using alkylating agent–conjugated pyrrole-imidazole (PI) polyamides, which were designed to specifically bind to consensus RUNX-binding sequences, was highly effective against AML cells and against several poor-prognosis solid tumors in a xenograft mouse model of AML without notable adverse events. Taken together, these results identify a crucial role for the RUNX cluster in the maintenance and progression of cancer cells and suggest that modulation of the RUNX cluster using the PI polyamide gene-switch technology is a potential strategy to control malignancies.
Ken Morita, Kensho Suzuki, Shintaro Maeda, Akihiko Matsuo, Yoshihide Mitsuda, Chieko Tokushige, Gengo Kashiwazaki, Junichi Taniguchi, Rina Maeda, Mina Noura, Masahiro Hirata, Tatsuki Kataoka, Ayaka Yano, Yoshimi Yamada, Hiroki Kiyose, Mayu Tokumasu, Hidemasa Matsuo, Sunao Tanaka, Yasushi Okuno, Manabu Muto, Kazuhito Naka, Kosei Ito, Toshio Kitamura, Yasufumi Kaneda, Paul P. Liu, Toshikazu Bando, Souichi Adachi, Hiroshi Sugiyama, Yasuhiko Kamikubo
Hematopoietic transitions that accompany fetal development, such as erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors do not execute the adult morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects may also contribute to inferior platelet recovery after cord blood stem cell transplantation and may underlie inefficient platelet production by megakaryocytes derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors has identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism that is known to drive adult megakaryocyte morphogenesis. This blockade resulted from neonatal-specific expression of an oncofetal RNA-binding protein, IGF2BP3, which prevented the destabilization of the nuclear RNA 7SK, a process normally associated with adult megakaryocytic P-TEFb activation. Knockdown of IGF2BP3 sufficed to confer both phenotypic and molecular features of adult-type cells on neonatal megakaryocytes. Pharmacologic inhibition of IGF2BP3 expression via bromodomain and extraterminal domain (BET) inhibition also elicited adult features in neonatal megakaryocytes. These results identify IGF2BP3 as a human ontogenic master switch that restricts megakaryocyte development by modulating a lineage-specific P-TEFb activation mechanism, revealing potential strategies toward enhancing platelet production.
Kamaleldin E. Elagib, Chih-Huan Lu, Goar Mosoyan, Shadi Khalil, Ewelina Zasadzińska, Daniel R. Foltz, Peter Balogh, Alejandro A. Gru, Deborah A. Fuchs, Lisa M. Rimsza, Els Verhoeyen, Miriam Sansó, Robert P. Fisher, Camelia Iancu-Rubin, Adam N. Goldfarb
Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase–mediated (DNA-PK–mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK–deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK–deficient quiescent leukemia cells and BRCA/DNA-PK–deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients.
Margaret Nieborowska-Skorska, Katherine Sullivan, Yashodhara Dasgupta, Paulina Podszywalow-Bartnicka, Grazyna Hoser, Silvia Maifrede, Esteban Martinez, Daniela Di Marcantonio, Elisabeth Bolton-Gillespie, Kimberly Cramer-Morales, Jaewong Lee, Min Li, Artur Slupianek, Daniel Gritsyuk, Sabine Cerny-Reiterer, Ilona Seferynska, Tomasz Stoklosa, Lars Bullinger, Huaqing Zhao, Vera Gorbunova, Katarzyna Piwocka, Peter Valent, Curt I. Civin, Markus Muschen, John E. Dick, Jean C.Y. Wang, Smita Bhatia, Ravi Bhatia, Kolia Eppert, Mark D. Minden, Stephen M. Sykes, Tomasz Skorski
Mutations of the splicing factor–encoding gene
Bon Ham Yip, Violetta Steeples, Emmanouela Repapi, Richard N. Armstrong, Miriam Llorian, Swagata Roy, Jacqueline Shaw, Hamid Dolatshad, Stephen Taylor, Amit Verma, Matthias Bartenstein, Paresh Vyas, Nicholas C.P. Cross, Luca Malcovati, Mario Cazzola, Eva Hellström-Lindberg, Seishi Ogawa, Christopher W.J. Smith, Andrea Pellagatti, Jacqueline Boultwood
The eleven-nineteen leukemia (ENL) protein family, composed of ENL and AF9, is a common component of 3 transcriptional modulators: AF4–ENL–P-TEFb complex (AEP), DOT1L-AF10-ENL complex (referred to as the DOT1L complex) and polycomb-repressive complex 1 (PRC1). Each complex associates with chromatin via distinct mechanisms, conferring different transcriptional properties including activation, maintenance, and repression. The mixed-lineage leukemia (
Hiroshi Okuda, Boban Stanojevic, Akinori Kanai, Takeshi Kawamura, Satoshi Takahashi, Hirotaka Matsui, Akifumi Takaori-Kondo, Akihiko Yokoyama
Developing erythrocytes take up exceptionally large amounts of iron, which must be transferred to mitochondria for incorporation into heme. This massive iron flux must be precisely controlled to permit the coordinated synthesis of heme and hemoglobin while avoiding the toxic effects of chemically reactive iron. In cultured animal cells, iron chaperones
Moon-Suhn Ryu, Deliang Zhang, Olga Protchenko, Minoo Shakoury-Elizeh, Caroline C. Philpott