Ribosomal proteins (RP) regulate specific gene expression by selectively translating subsets of mRNAs. Indeed, in Diamond–Blackfan anaemia and 5q- syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anaemia. Little is known about the molecular mechanisms of selective mRNA translation and involvement of ribosomal-associated factors in this process. Ribonuclease inhibitor (RNH1) is an ubiquitously expressed protein that binds to and inhibits pancreatic-type ribonucleases. Here we report that RNH1 binds to ribosomes and regulates erythropoiesis by controlling translation of the erythroid transcription factor GATA1. Rnh1-deficient mice die between embryonic days E8.5 to E10 due to impaired production of mature erythroid cells from progenitor cells. In Rnh1-deficient embryos, mRNA levels of Gata1 are normal, but GATA1 protein levels are decreased. At the molecular level, we found that RNH1 binds to the 40S subunit of ribosomes and facilitates polysome formation on Gata1 mRNA to confer transcript-specific translation. Further, RNH1 knock down in human CD34+ progenitor cells decreased erythroid differentiation without affecting myelopoiesis. Our results reveal an unsuspected role for RNH1 in the control of GATA1 mRNA translation and erythropoiesis.
Vijaykumar Chennupati, Diogo F.T. Veiga, Kendle M. Maslowski, Nicola Andina, Aubry Tardivel, Eric Chi-Wang Yu, Martina Stilinovic, Cedric Simillion, Michel A. Duchosal, Manfredo Quadroni, Irene Roberts, Vijay G. Sankaran, H. Robson MacDonald, Nicolas Fasel, Anne Angelillo-Scherrer, Pascal Schneider, Trang Hoang, Ramanjaneyulu Allam
Disordered coagulation contributes to death in sepsis and lacks effective treatments. Existing markers of disseminated intravascular coagulation (DIC) reflect its sequelae rather than its causes, delaying diagnosis and treatment. Here we show that disruption of the endothelial Tie2 axis is a sentinel event in septic DIC. Proteomics in septic DIC patients revealed a network involving inflammation and coagulation with the Tie2 antagonist, Angiopoietin-2 (Angpt-2), occupying a central node. Angpt-2 was strongly associated with traditional DIC markers including platelet counts, yet more accurately predicted mortality in two large independent cohorts (combined N = 1077). In endotoxemic mice, reduced Tie2 signaling preceded signs of overt DIC. During this early phase, intravital imaging of microvascular injury revealed excessive fibrin accumulation, a pattern remarkably mimicked by Tie2 deficiency even without inflammation. Conversely, Tie2 activation normalized pro-thrombotic responses by inhibiting endothelial tissue factor and phosphatidylserine exposure. Critically, Tie2 activation had no adverse effects on bleeding. These results mechanistically implicate Tie2 signaling as a central regulator of microvascular thrombus formation in septic DIC and indicate that circulating markers of the Tie2 axis could facilitate earlier diagnosis. Finally, interventions targeting Tie2 may normalize coagulation in inflammatory states while averting the bleeding risks of current DIC therapies.
Sarah J. Higgins, Karen De Ceunynck, John Kellum, Xiuying Chen, Xuesong Gu, Sharjeel A. Chaudhry, Sol Schulman, Towia A. Libermann, Shulin Lu, Nathan I. Shapiro, David C. Christiani, Robert Flaumenhaft, Samir M. Parikh
Patients with myeloproliferative neoplasms (MPNs) frequently progress to bone marrow failure or acute myeloid leukemia (AML), and mutations in epigenetic regulators such as the metabolic enzyme isocitrate dehydrogenase (IDH) are associated with poor outcomes. Here, we showed that combined expression of Jak2V617F and mutant IDH1R132H or Idh2R140Q induces MPN progression, alters stem/progenitor cell function, and impairs differentiation in mice. Jak2V617F Idh2R140Q–mutant MPNs were sensitive to small-molecule inhibition of IDH. Combined inhibition of JAK2 and IDH2 normalized the stem and progenitor cell compartments in the murine model and reduced disease burden to a greater extent than was seen with JAK inhibition alone. In addition, combined JAK2 and IDH2 inhibitor treatment also reversed aberrant gene expression in MPN stem cells and reversed the metabolite perturbations induced by concurrent JAK2 and IDH2 mutations. Combined JAK2 and IDH2 inhibitor therapy also showed cooperative efficacy in cells from MPN patients with both JAK2mut and IDH2mut mutations. Taken together, these data suggest that combined JAK and IDH inhibition may offer a therapeutic advantage in this high-risk MPN subtype.
Anna Sophia McKenney, Allison N. Lau, Amritha Varshini Hanasoge Somasundara, Barbara Spitzer, Andrew M. Intlekofer, Jihae Ahn, Kaitlyn Shank, Franck T. Rapaport, Minal A. Patel, Efthymia Papalexi, Alan H. Shih, April Chiu, Elizaveta Freinkman, Esra A. Akbay, Mya Steadman, Raj Nagaraja, Katharine Yen, Julie Teruya-Feldstein, Kwok-Kin Wong, Raajit Rampal, Matthew G. Vander Heiden, Craig B. Thompson, Ross L. Levine
Oncogenic addiction to the Fms-like tyrosine kinase 3 (FLT3) is a hallmark of acute myeloid leukemia (AML) that harbors the FLT3–internal tandem duplication (FLT3-ITD) mutation. While FLT3 inhibitors like sorafenib show initial therapeutic efficacy, resistance rapidly develops through mechanisms that are incompletely understood. Here, we used RNA-Seq–based analysis of patient leukemic cells and found that upregulation of the Tec family kinase BMX occurs during sorafenib resistance. This upregulation was recapitulated in an in vivo murine FLT3-ITD–positive (FLT3-ITD+) model of sorafenib resistance. Mechanistically, the antiangiogenic effects of sorafenib led to increased bone marrow hypoxia, which contributed to HIF-dependent BMX upregulation. In in vitro experiments, hypoxia-dependent BMX upregulation was observed in both AML and non-AML cell lines. Functional studies in human FLT3-ITD+ cell lines showed that BMX is part of a compensatory signaling mechanism that promotes AML cell survival during FLT3 inhibition. Taken together, our results demonstrate that hypoxia-dependent upregulation of BMX contributes to therapeutic resistance through a compensatory prosurvival signaling mechanism. These results also reveal the role of off-target drug effects on tumor microenvironment and development of acquired drug resistance. We propose that the bone marrow niche can be altered by anticancer therapeutics, resulting in drug resistance through cell-nonautonomous microenvironment-dependent effects.
Jolieke G. van Oosterwijk, Daelynn R. Buelow, Christina D. Drenberg, Aksana Vasilyeva, Lie Li, Lei Shi, Yong-Dong Wang, David Finkelstein, Sheila A. Shurtleff, Laura J. Janke, Stanley Pounds, Jeffrey E. Rubnitz, Hiroto Inaba, Navjotsingh Pabla, Sharyn D. Baker
As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non–BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.
Sascha Dietrich, Małgorzata Oleś, Junyan Lu, Leopold Sellner, Simon Anders, Britta Velten, Bian Wu, Jennifer Hüllein, Michelle da Silva Liberio, Tatjana Walther, Lena Wagner, Sophie Rabe, Sonja Ghidelli-Disse, Marcus Bantscheff, Andrzej K. Oleś, Mikołaj Słabicki, Andreas Mock, Christopher C. Oakes, Shihui Wang, Sina Oppermann, Marina Lukas, Vladislav Kim, Martin Sill, Axel Benner, Anna Jauch, Lesley Ann Sutton, Emma Young, Richard Rosenquist, Xiyang Liu, Alexander Jethwa, Kwang Seok Lee, Joe Lewis, Kerstin Putzker, Christoph Lutz, Davide Rossi, Andriy Mokhir, Thomas Oellerich, Katja Zirlik, Marco Herling, Florence Nguyen-Khac, Christoph Plass, Emma Andersson, Satu Mustjoki, Christof von Kalle, Anthony D. Ho, Manfred Hensel, Jan Dürig, Ingo Ringshausen, Marc Zapatka, Wolfgang Huber, Thorsten Zenz
Nervous system injury is a frequent result of cancer therapy involving cranial irradiation, leaving patients with marked memory and other neurobehavioral disabilities. Here, we report an unanticipated link between bone marrow and brain in the setting of radiation injury. Specifically, we demonstrate that bone marrow–derived monocytes and macrophages are essential for structural and functional repair mechanisms, including regeneration of cerebral white matter and improvement in neurocognitive function. Using a granulocyte-colony stimulating factor (G-CSF) receptor knockout mouse model in combination with bone marrow cell transplantation, MRI, and neurocognitive functional assessments, we demonstrate that bone marrow–derived G-CSF–responsive cells home to the injured brain and are critical for altering neural progenitor cells and brain repair. Additionally, compared with untreated animals, animals that received G-CSF following radiation injury exhibited enhanced functional brain repair. Together, these results demonstrate that, in addition to its known role in defense and debris removal, the hematopoietic system provides critical regenerative drive to the brain that can be modulated by clinically available agents.
Jorg Dietrich, Ninib Baryawno, Naema Nayyar, Yannis K. Valtis, Betty Yang, Ina Ly, Antoine Besnard, Nicolas Severe, Karin U. Gustafsson, Ovidiu C. Andronesi, Tracy T. Batchelor, Amar Sahay, David T. Scadden
STAT5B is often mutated in hematopoietic malignancies. The most frequent STAT5B mutation, Asp642His (N642H), has been found in over 90 leukemia and lymphoma patients. Here, we used the Vav1 promoter to generate transgenic mouse models that expressed either human STAT5B or STAT5BN642H in the hematopoietic compartment. While STAT5B-expressing mice lacked a hematopoietic phenotype, the STAT5BN642H-expressing mice rapidly developed T cell neoplasms. Neoplasia manifested as transplantable CD8+ lymphoma or leukemia, indicating that the STAT5BN642H mutation drives cancer development. Persistent and enhanced levels of STAT5BN642H tyrosine phosphorylation in transformed CD8+ T cells led to profound changes in gene expression that were accompanied by alterations in DNA methylation at potential histone methyltransferase EZH2-binding sites. Aurora kinase genes were enriched in STAT5BN642H-expressing CD8+ T cells, which were exquisitely sensitive to JAK and Aurora kinase inhibitors. Together, our data suggest that JAK and Aurora kinase inhibitors should be further explored as potential therapeutics for lymphoma and leukemia patients with the STAT5BN642H mutation who respond poorly to conventional chemotherapy.
Ha Thi Thanh Pham, Barbara Maurer, Michaela Prchal-Murphy, Reinhard Grausenburger, Eva Grundschober, Tahereh Javaheri, Harini Nivarthi, Auke Boersma, Thomas Kolbe, Mohamed Elabd, Florian Halbritter, Jan Pencik, Zahra Kazemi, Florian Grebien, Markus Hengstschläger, Lukas Kenner, Stefan Kubicek, Matthias Farlik, Christoph Bock, Peter Valent, Mathias Müller, Thomas Rülicke, Veronika Sexl, Richard Moriggl
V617F driver mutation of JAK2 is the leading cause of the Philadelphia-chromosome-negative myeloproliferative neoplasms (MPNs). Although thrombosis is a leading cause of mortality and morbidity in MPNs, the mechanisms underlying their pathogenesis are unclear. Here, we identified pleckstrin-2 (Plek2) as a downstream target of the JAK2/STAT5 pathway in erythroid and myeloid cells, and showed that it is upregulated in a JAK2V617F-positive MPN mouse model and in patients with MPNs. Loss of Plek2 ameliorated JAK2V617F-induced myeloproliferative phenotypes including erythrocytosis, neutrophilia, thrombocytosis, and splenomegaly, thereby reverting the widespread vascular occlusions and lethality in JAK2V617F-knockin mice. Additionally, we demonstrated that a reduction in red blood cell mass was the main contributing factor in the reversion of vascular occlusions. Thus, our study identifies Plek2 as an effector of the JAK2/STAT5 pathway and a key factor in the pathogenesis of JAK2V617F-induced MPNs, pointing to Plek2 as a viable target for the treatment of MPNs.
Baobing Zhao, Yang Mei, Lan Cao, Jingxin Zhang, Ronen Sumagin, Jing Yang, Juehua Gao, Matthew J. Schipma, Yanfeng Wang, Chelsea Thorsheim, Liang Zhao, Timothy Stalker, Brady Stein, Qiang Jeremy Wen, John D. Crispino, Charles S. Abrams, Peng Ji
The transcription factor PU.1 is often impaired in patients with acute myeloid leukemia (AML). Here, we used AML cells that already had low PU.1 levels and further inhibited PU.1 using either RNA interference or, to our knowledge, first-in-class small-molecule inhibitors of PU.1 that we developed specifically to allosterically interfere with PU.1-chromatin binding through interaction with the DNA minor groove that flanks PU.1-binding motifs. These small molecules of the heterocyclic diamidine family disrupted the interaction of PU.1 with target gene promoters and led to downregulation of canonical PU.1 transcriptional targets. shRNA or small-molecule inhibition of PU.1 in AML cells from either PU.1lo mutant mice or human patients with AML-inhibited cell growth and clonogenicity and induced apoptosis. In murine and human AML (xeno)transplantation models, treatment with our PU.1 inhibitors decreased tumor burden and resulted in increased survival. Thus, our study provides proof of concept that PU.1 inhibition has potential as a therapeutic strategy for the treatment of AML and for the development of small-molecule inhibitors of PU.1.
Iléana Antony-Debré, Ananya Paul, Joana Leite, Kelly Mitchell, Hye Mi Kim, Luis A. Carvajal, Tihomira I. Todorova, Kenneth Huang, Arvind Kumar, Abdelbasset A. Farahat, Boris Bartholdy, Swathi-Rao Narayanagari, Jiahao Chen, Alberto Ambesi-Impiombato, Adolfo A. Ferrando, Ioannis Mantzaris, Evripidis Gavathiotis, Amit Verma, Britta Will, David W. Boykin, W. David Wilson, Gregory M.K. Poon, Ulrich Steidl
Apoptosis delimits platelet life span in the circulation and leads to storage lesion, which severely limits the shelf life of stored platelets. Moreover, accumulating evidence indicates that platelet apoptosis provoked by various pathological stimuli results in thrombocytopenia in many common diseases. However, little is known about how platelet apoptosis is initiated or regulated. Here, we show that PKA activity is markedly reduced in platelets aged in vitro, stored platelets, and platelets from patients with immune thrombocytopenia (ITP), diabetes, and bacterial infections. Inhibition or genetic ablation of PKA provoked intrinsic programmed platelet apoptosis in vitro and rapid platelet clearance in vivo. PKA inhibition resulted in dephosphorylation of the proapoptotic protein BAD at Ser155, resulting in sequestration of prosurvival protein BCL-XL in mitochondria and subsequent apoptosis. Notably, PKA activation protected platelets from apoptosis induced by storage or pathological stimuli and elevated peripheral platelet levels in normal mice and in a murine model of ITP. Therefore, these findings identify PKA as a homeostatic regulator of platelet apoptosis that determines platelet life span and survival. Furthermore, these results suggest that regulation of PKA activity represents a promising strategy for extending platelet shelf life and has profound implications for the treatment of platelet number-related diseases and disorders.
Lili Zhao, Jun Liu, Chunyan He, Rong Yan, Kangxi Zhou, Qingya Cui, Xingjun Meng, Xiaodong Li, Yang Zhang, Yumei Nie, Yang Zhang, Renping Hu, Yancai Liu, Lian Zhao, Mengxing Chen, Weiling Xiao, Jingluan Tian, Yunxiao Zhao, Lijuan Cao, Ling Zhou, Anning Lin, Changgeng Ruan, Kesheng Dai