Gerlinde Wernig

All Publications

• Selective hematopoietic stem cell ablation using CD117-antibody-drug-conjugates enables safe and effective transplantation with immunity preservation. Nature communications Czechowicz, A., Palchaudhuri, R., Scheck, A., Hu, Y., Hoggatt, J., Saez, B., Pang, W. W., Mansour, M. K., Tate, T. A., Chan, Y. Y., Walck, E., Wernig, G., Shizuru, J. A., Winau, F., Scadden, D. T., Rossi, D. J. 2019; 10 (1): 617

  Abstract

  Hematopoietic stem cell transplantation (HSCT) is a curative therapy for blood and immune diseases with potential for many settings beyond current standard-of-care. Broad HSCT application is currently precluded largely due to morbidity and mortality associated with genotoxic irradiation or chemotherapy conditioning. Here we show that a single dose of a CD117-antibody-drug-conjugate (CD117-ADC) to saporin leads to>99% depletion of host HSCs, enabling rapid and efficient donor hematopoietic cell engraftment. Importantly, CD117-ADC selectively targets hematopoietic stem cells yet does not cause clinically significant side-effects. Blood counts and immune cell function are preserved following CD117-ADC treatment, with effective responses by recipients to both viral and fungal challenges. These results suggest that CD117-ADC-mediated HSCT pre-treatment could serve as a non-myeloablative conditioning strategy for the treatment of a wide range of non-malignant and malignant diseases, and might be especially suited to gene therapy and gene editing settings in which preservation of immunity is desired.

  View details for PubMedID 30728354

• Doxycycline Reduces Scar Thickness and Improves Collagen Architecture. Annals of surgery Moore, A. L., desJardins-Park, H. E., Duoto, B. A., Mascharak, S., Murphy, M. P., Irizarry, D. M., Foster, D. S., Jones, R. E., Barnes, L. A., Marshall, C. D., Ransom, R. C., Wernig, G., Longaker, M. T. 2018

  Abstract

  OBJECTIVE: To investigate the effects of local doxycycline administration on skin scarring.BACKGROUND: Skin scarring represents a major source of morbidity for surgical patients. Doxycycline, a tetracycline antibiotic with off-target effects on the extracellular matrix, has demonstrated antifibrotic effects in multiple organs. However, doxycycline's potential effects on skin scarring have not been explored in vivo.METHODS: Female C57BL/6J mice underwent dorsal wounding following an established splinted excisional skin wounding model. Doxycycline was administered by local injection into the wound base following injury. Wounds were harvested upon complete wound closure (postoperative day 15) for histological examination and biomechanical testing of scar tissue.RESULTS: A one-time dose of 3.90 mM doxycycline (2 mg/mL) within 12 hours of injury was found to significantly reduce scar thickness by 24.8% (P < 0.0001) without compromising tensile strength. The same effect could not be achieved by oral dosing. In doxycycline-treated scar matrices, collagen I content was significantly reduced (P = 0.0317) and fibers were favorably arranged with significantly increased fiber randomness (P = 0.0115). Common culprits of altered wound healing mechanics, including angiogenesis and inflammation, were not impacted by doxycycline treatment. However, engrailed1 profibrotic fibroblasts, responsible for scar extracellular matrix deposition, were significantly reduced with doxycycline treatment (P = 0.0005).CONCLUSIONS: Due to the substantial improvement in skin scarring and well-established clinical safety profile, locally administered doxycycline represents a promising vulnerary agent. As such, we favor rapid translation to human patients as an antiscarring therapy.

  View details for PubMedID 30585822

• Surgical adhesions in mice are derived from mesothelial cells and can be targeted by antibodies against mesothelial markers. Science translational medicine Tsai, J. M., Sinha, R., Seita, J., Fernhoff, N., Christ, S., Koopmans, T., Krampitz, G. W., McKenna, K. M., Xing, L., Sandholzer, M., Sales, J. H., Shoham, M., McCracken, M., Joubert, L., Gordon, S. R., Poux, N., Wernig, G., Norton, J. A., Weissman, I. L., Rinkevich, Y. 2018; 10 (469)

  Abstract

  Peritoneal adhesions are fibrous tissues that tether organs to one another or to the peritoneal wall and are a major cause of postsurgical and infectious morbidity. The primary molecular chain of events leading to the initiation of adhesions has been elusive, chiefly due to the lack of an identifiable cell of origin. Using clonal analysis and lineage tracing, we have identified injured surface mesothelium expressing podoplanin (PDPN) and mesothelin (MSLN) as a primary instigator of peritoneal adhesions after surgery in mice. We demonstrate that an anti-MSLN antibody diminished adhesion formation in a mouse model where adhesions were induced by surgical ligation to form ischemic buttons and subsequent surgical abrasion of the peritoneum. RNA sequencing and bioinformatics analyses of mouse mesothelial cells from injured mesothelium revealed aspects of the pathological mechanism of adhesion development and yielded several potential regulators of this process. Specifically, we show that PDPN+MSLN+ mesothelium responded to hypoxia by early up-regulation of hypoxia-inducible factor 1 alpha (HIF1alpha) that preceded adhesion development. Inhibition of HIF1alpha with small molecules ameliorated the injury program in damaged mesothelium and was sufficient to diminish adhesion severity in a mouse model. Analyses of human adhesion tissue suggested that similar surface markers and signaling pathways may contribute to surgical adhesions in human patients.

  View details for PubMedID 30487249

• Direct targeting of the mouse optic nerve for therapeutic delivery. Journal of neuroscience methods Mesentier-Louro, L. A., Dodd, R., Domizi, P., Nobuta, H., Wernig, M., Wernig, G., Liao, Y. J. 2018

  Abstract

  BACKGROUND: Animal models of optic nerve injury are often used to study central nervous system (CNS) degeneration and regeneration, and targeting the optic nerve is a powerful approach for axon-protective or remyelination therapy. However, the experimental delivery of drugs or cells to the optic nerve is rarely performed because injections into this structure are difficult in small animals, especially in mice.NEW METHOD: We investigated and developed methods to deliver drugs or cells to the mouse optic nerve through 3 different routes: a) intraorbital, b) through the optic foramen and c) transcranial.RESULTS: The methods targeted different parts of the mouse optic nerve: intraorbital proximal (intraorbital), intracranial middle (optic-foramen) or intracranial distal (transcranial) portion.COMPARISON WITH EXISTING METHODS: Most existing methods target the optic nerve indirectly. For instance, intravitreally delivered cells often cannot cross the inner limiting membrane to reach retinal neurons and optic nerve axons. Systemic delivery, eye drops and intraventricular injections do not always successfully target the optic nerve. Intraorbital and transcranial injections into the optic nerve or chiasm have been performed but these methods have not been well described. We approached the optic nerve with more selective and precise targeting than existing methods.CONCLUSIONS: We successfully targeted the murine optic nerve intraorbitally, through the optic foramen, and transcranially. Of all methods, the injection through the optic foramen is likely the most innovative and fastest. These methods offer additional approaches for therapeutic intervention to be used by those studying white matter damage and axonal regeneration in the CNS.

  View details for PubMedID 30389488

• Reduced Scar Thickness Achieved by Topical Doxycycline Is Mediated by Specific Skin Fibroblast Populations and Not Immune Cell Infiltrate Moore, A. L., Murphy, M. P., Irizarry, D. M., Des Jardins-Park, H. E., Duoto, B. A., Mascharak, S., Foster, D. S., Jones, R., Wernig, G., Longaker, M. T. ELSEVIER SCIENCE INC. 2018: S210–S211

  View details for DOI 10.1016/j.jamcollsurg.2018.07.462

  View details for Web of Science ID 000447760600413

• Mouse Model with cJUN Over-Expression Eludes to Deep Dermal Fibroblast Expansion and Immune Cell Recruitment as the Biologic Mechanism of Hypertrophic Scarring Moore, A. L., Duoto, B. A., Des Jardins-Park, H. E., Mascharak, S., Wernig, G., Longaker, M. T. ELSEVIER SCIENCE INC. 2018: S208

  View details for DOI 10.1016/j.jamcollsurg.2018.07.456

  View details for Web of Science ID 000447760600409

• Stem cell therapy for treatment of ischemic optic neuropathy Mesentier-Louro, L., Yang, N., Shariati, A., Domizi, P., Dodd, R., Wernig, G., Wernig, M., Liao, Y. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2018

  View details for Web of Science ID 000442912501176

• Unifying mechanism for different fibrotic diseases PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wernig, G., Chen, S., Cui, L., Van Neste, C., Tsai, J. M., Kambham, N., Vogel, H., Natkunam, Y., Gilliland, D. G., Nolan, G., Weissman, I. L. 2017; 114 (18): 4757-4762

  Abstract

  Fibrotic diseases are not well-understood. They represent a number of different diseases that are characterized by the development of severe organ fibrosis without any obvious cause, such as the devastating diseases idiopathic pulmonary fibrosis (IPF) and scleroderma. These diseases have a poor prognosis comparable with endstage cancer and are uncurable. Given the phenotypic differences, it was assumed that the different fibrotic diseases also have different pathomechanisms. Here, we demonstrate that many endstage fibrotic diseases, including IPF; scleroderma; myelofibrosis; kidney-, pancreas-, and heart-fibrosis; and nonalcoholic steatohepatosis converge in the activation of the AP1 transcription factor c-JUN in the pathologic fibroblasts. Expression of the related AP1 transcription factor FRA2 was restricted to pulmonary artery hypertension. Induction of c-Jun in mice was sufficient to induce severe fibrosis in multiple organs and steatohepatosis, which was dependent on sustained c-Jun expression. Single cell mass cytometry revealed that c-Jun activates multiple signaling pathways in mice, including pAkt and CD47, which were also induced in human disease. αCD47 antibody treatment and VEGF or PI3K inhibition reversed various organ c-Jun-mediated fibroses in vivo. These data suggest that c-JUN is a central molecular mediator of most fibrotic conditions.

  View details for DOI 10.1073/pnas.1621375114

  View details for PubMedID 28424250

• Unifying Mechanism of fibrotic diseases Proceedings of the National Academy of Science Wernig, G., Weissman, I. L. 2017; 114 (18): 4757-4762

  View details for DOI 10.1073/pnas.1621375114

• Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types CELL Loh, K. M., Chen, A., Koh, P. W., Deng, T. Z., Sinha, R., Tsai, J. M., Barkal, A. A., Shen, K. Y., Jain, R., Morganti, R. M., Shyh-Chang, N., Fernhoff, N. B., George, B. M., Wernig, G., Salomon, R. E., Chen, Z., Vogel, H., Epstein, J. A., Kundaje, A., Talbot, W. S., Beachy, P. A., Ang, L. T., Weissman, I. L. 2016; 166 (2): 451-467

  Abstract

  Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence, comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end, here, we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%-99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively, this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. VIDEO ABSTRACT.

  View details for DOI 10.1016/j.cell.2016.06.011

  View details for PubMedID 27419872

• Tuning Cytokine Receptor Signaling by Re-orienting Dimer Geometry with Surrogate Ligands CELL Moraga, I., Wernig, G., Wilmes, S., Gryshkova, V., Richter, C. P., Hong, W., Sinha, R., Guo, F., Fabionar, H., Wehrman, T. S., Krutzik, P., Demharter, S., Plo, I., Weissman, I. L., Minary, P., Majeti, R., Constantinescu, S. N., Piehler, J., Garcia, K. C. 2015; 160 (6): 1196-1208

  Abstract

  Most cell-surface receptors for cytokines and growth factors signal as dimers, but it is unclear whether remodeling receptor dimer topology is a viable strategy to "tune" signaling output. We utilized diabodies (DA) as surrogate ligands in a prototypical dimeric receptor-ligand system, the cytokine Erythropoietin (EPO) and its receptor (EpoR), to dimerize EpoR ectodomains in non-native architectures. Diabody-induced signaling amplitudes varied from full to minimal agonism, and structures of these DA/EpoR complexes differed in EpoR dimer orientation and proximity. Diabodies also elicited biased or differential activation of signaling pathways and gene expression profiles compared to EPO. Non-signaling diabodies inhibited proliferation of erythroid precursors from patients with a myeloproliferative neoplasm due to a constitutively active JAK2V617F mutation. Thus, intracellular oncogenic mutations causing ligand-independent receptor activation can be counteracted by extracellular ligands that re-orient receptors into inactive dimer topologies. This approach has broad applications for tuning signaling output for many dimeric receptor systems.

  View details for DOI 10.1016/j.cell.2015.02.011

  View details for PubMedID 25728669

• STAT5 Is Crucial to Maintain Leukemic Stem Cells in Acute Myelogenous Leukemias Induced by MOZ-TIF2 CANCER RESEARCH Tam, W. F., Haehnel, P. S., Schueler, A., Lee, B. H., Okabe, R., Zhu, N., Pante, S. V., Raffel, G., Mercher, T., Wernig, G., Bockamp, E., Sasca, D., Kreft, A., Robinson, G. W., Hennighausen, L., Gilliland, D. G., Kindler, T. 2013; 73 (1): 373-384

  Abstract

  MOZ-TIF2 is a leukemogenic fusion oncoprotein that confers self-renewal capability to hematopoietic progenitor cells and induces acute myelogenous leukemia (AML) with long latency in bone marrow transplantation assays. Here, we report that FLT3-ITD transforms hematopoietic cells in cooperation with MOZ-TIF2 in vitro and in vivo. Coexpression of FLT3-ITD confers growth factor independent survival/proliferation, shortens disease latency, and results in an increase in the number of leukemic stem cells (LSC). We show that STAT5, a major effector of aberrant FLT3-ITD signal transduction, is both necessary and sufficient for this cooperative effect. In addition, STAT5 signaling is essential for MOZ-TIF2-induced leukemic transformation itself. Lack of STAT5 in fetal liver cells caused rapid differentiation and loss of replating capacity of MOZ-TIF2-transduced cells enriched for LSCs. Furthermore, mice serially transplanted with Stat5(-/-) MOZ-TIF2 leukemic cells develop AML with longer disease latency and finally incomplete penetrance when compared with mice transplanted with Stat5(+/+) MOZ-TIF2 leukemic cells. These data suggest that STAT5AB is required for the self-renewal of LSCs and represents a combined signaling node of FLT3-ITD and MOZ-TIF2 driven leukemogenesis. Therefore, targeting aberrantly activated STAT5 or rewired downstream signaling pathways may be a promising therapeutic option.

  View details for DOI 10.1158/0008-5472.CAN-12-0255

  View details for Web of Science ID 000313019800037

  View details for PubMedID 23149921

• Anti-CD47 antibodies promote phagocytosis and inhibit the growth of human myeloma cells LEUKEMIA Kim, D., Wang, J., Willingham, S. B., Martin, R., Wernig, G., Weissman, I. L. 2012; 26 (12): 2538-2545

  Abstract

  Multiple myeloma is a plasma cell neoplasm residing in bone marrow. Despite advances in myeloma therapies, novel therapies are required to improve patient outcomes. CD47 is highly expressed on myeloma cells and a potential therapeutic candidate for myeloma therapies. Flow cytometric analysis of patient bone marrow cells revealed that myeloma cells overexpress CD47 when compared with non-myeloma cells in 73% of patients (27/37). CD47 expression protects cells from phagocytosis by transmitting an inhibitory signal to macrophages. Here we show that blocking CD47 with an anti-CD47 monoclonal antibody increased phagocytosis of myeloma cells in vitro. In xenotransplantation models, anti-CD47 antibodies inhibited the growth of RPMI 8226 myeloma cells and led to tumor regression (42/57 mice), implicating the eradication of myeloma-initiating cells. Moreover, anti-CD47 antibodies retarded the growth of patient myeloma cells and alleviated bone resorption in human bone-bearing mice. Irradiation of mice before myeloma cell xenotransplantation abolished the therapeutic efficacy of anti-CD47 antibodies delivered 2 weeks after radiation, and coincided with a reduction of myelomonocytic cells in spleen, bone marrow and liver. These results are consistent with the hypothesis that anti-CD47 blocking antibodies inhibit myeloma growth, in part, by increasing phagocytosis of myeloma cells.

  View details for DOI 10.1038/leu.2012.141

  View details for Web of Science ID 000312186000013

  View details for PubMedID 22648449

• EXEL-8232, a small-molecule JAK2 inhibitor, effectively treats thrombocytosis and extramedullary hematopoiesis in a murine model of myeloproliferative neoplasm induced by MPLW515L LEUKEMIA Wernig, G., Kharas, M. G., Mullally, A., Leeman, D. S., Okabe, R., George, T., Clary, D. O., Gilliland, D. G. 2012; 26 (4): 720-727

  Abstract

  About 10% of patients with essential thrombocythemia (ET) or myelofibrosis (MF) that lack mutations in JAK2 harbor an activating mutation in the thrombopoietin receptor, MPLW515L. Distinct from the JAK2V617F retroviral transplant model, the MPLW515L model recapitulates many features of ET and MF, including severe fibrosis and thrombocytosis. We have tested EXEL-8232, an experimental potent JAK2 inhibitor, for efficacy in suppression of thrombocytosis in vivo and for its ability to attenuate MPLW515L myeloproliferative disease. EXEL-8232 was administered for 28 days q12 h by oral gavage at doses of 30 or 100 mg/kg, prospectively. Animals treated with EXEL-8232 at 100 mg/kg had normalized high platelet counts, eliminated extramedullary hematopoiesis in the spleen and eliminated bone marrow fibrosis, whereas the wild-type controls did not develop thrombocytopenia. Consistent with a clinical response in this model, we validated surrogate end points for response to treatment, including a reduction of endogenous colony growth and signaling inhibition in immature erythroid and myeloid primary cells both in vitro and upon treatment in vivo. We conclude that EXEL-8232 has efficacy in treatment of thrombocytosis in vivo in a murine model of ET and MF, and may be of therapeutic benefit for patients with MPL-mutant MPN.

  View details for DOI 10.1038/leu.2011.261

  View details for PubMedID 22005786

• EXEL-8232, a Small Molecule JAK2 inhibitor, Effectively Treats Thrombocytosis and Extramedullary Hematopoiesis in a Murine Model of Myeloproliferative Disease Induced by MPLW515L Wernig, G., Kharas, M., Clary, D., Gilliland, G., George, T. NATURE PUBLISHING GROUP. 2011: 329A

  View details for Web of Science ID 000291285001051

• Physiological Jak2V617F Expression Causes a Lethal Myeloproliferative Neoplasm with Differential Effects on Hematopoietic Stem and Progenitor Cells CANCER CELL Mullally, A., Lane, S. W., Ball, B., Megerdichian, C., Okabe, R., Al-Shahrour, F., Paktinat, M., Haydu, J. E., Housman, E., Lord, A. M., Wernig, G., Kharas, M. G., Mercher, T., Kutok, J. L., Gilliland, D. G., Ebert, B. L. 2010; 17 (6): 584-596

  Abstract

  We report a Jak2V617F knockin mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem cell (HSC) compartment has the unique capacity for disease initiation but does not have a significant selective competitive advantage over wild-type HSCs. In contrast, myeloid progenitor populations are expanded and skewed toward the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to JAK2V617F-positive MPN.

  View details for DOI 10.1016/j.ccr.2010.05.015

  View details for Web of Science ID 000278952300010

  View details for PubMedID 20541703

  View details for PubMedCentralID PMC2909585

• High-throughput sequence analysis of the tyrosine kinome in acute myeloid leukemia BLOOD Loriaux, M. M., Levine, R. L., Tyner, J. W., Froehling, S., Scholl, C., Stoffregen, E. P., Wernig, G., Erickson, H., Eide, C. A., Berger, R., Bernard, O. A., Griffin, J. D., Stone, R. M., Lee, B., Meyerson, M., Heinrich, M. C., Deininger, M. W., Gilliland, D. G., Druker, B. J. 2008; 111 (9): 4788-4796

  Abstract

  To determine whether aberrantly activated tyrosine kinases other than FLT3 and c-KIT contribute to acute myeloid leukemia (AML) pathogenesis, we used high-throughput (HT) DNA sequence ana-lysis to screen exons encoding the activation loop and juxtamembrane domains of 85 tyrosine kinase genes in 188 AML patients without FLT3 or c-KIT mutations. The screen identified 30 nonsynonymous sequence variations in 22 different kinases not previously reported in single-nucleotide polymorphism (SNP) databases. These included a novel FLT3 activating allele and a previously described activating mutation in MET (METT1010I). The majority of novel sequence variants were stably expressed in factor-dependent Ba/F3 cells. Apart from one FLT3 allele, none of the novel variants showed constitutive phosphorylation by immunoblot analysis and none transformed Ba/F3 cells to factor-independent growth. These findings indicate the majority of these alleles are not potent tyrosine kinase activators in this cellular context and that a significant proportion of nonsynonymous sequence variants identified in HT DNA sequencing screens may not have functional significance. Although some sequence variants may represent SNPs, these data are consistent with recent reports that a significant fraction of such sequence variants are "passenger" rather than "driver" alleles and underscore the importance of functional assessment of candidate disease alleles.

  View details for DOI 10.1182/blood-2007-07-101394

  View details for PubMedID 18252861

• The Jak2V617F oncogene associated with myeloproliferative diseases requires a functional FERM domain for transformation and for expression of the Myc and Pim proto-oncogenes BLOOD Wernig, G., Gonneville, J. R., Crowley, B. J., Rodrigues, M. S., Reddy, M. M., Hudon, H. E., Walz, C., Reiter, A., Podar, K., Royer, Y., Constantinescu, S. N., Tomasson, M. H., Griffin, J. D., Gilliland, D. G., Sattler, M. 2008; 111 (7): 3751-3759

  Abstract

  The V617F activating point mutation in Jak2 is associated with a proportion of myeloproliferative disorders. In normal hematopoietic cells, Jak2 signals only when associated with a growth factor receptor, such as the erythropoietin receptor (EpoR). We sought to identify the molecular requirements for activation of Jak2V617F by introducing a point mutation in the FERM domain (Y114A), required for receptor binding. Whereas BaF3.EpoR cells are readily transformed by Jak2V617F to Epo independence, we found that the addition of the FERM domain mutation blocked transformation and the induction of reactive oxygen species. Further, while cells expressing Jak2V617F had constitutive activation of STAT5, cells expressing Jak2V617F/Y114A did not, suggesting that signaling is defective at a very proximal level. In addition, expression of the Myc and Pim proto-oncogenes by Jak2V617F was found to be FERM domain dependent. An inducible constitutively active STAT5 mutant expressed in BaF3 cells was sufficient to induce Myc and Pim. Finally, the FERM domain in Jak2V617F was also required for abnormal hematopoiesis in transduced primary murine fetal liver cells. Overall, our results suggest that constitutive activation of Jak2 requires an intact FERM domain for a transforming phenotype, and is necessary for activation of the major target of Jak2, STAT5.

  View details for DOI 10.1182/blood-2007-07-102186

  View details for Web of Science ID 000254569300064

  View details for PubMedID 18216297

• Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced polycythemia vera CANCER CELL Werning, G., Kharas, M. G., Okabe, R., Moore, S. A., Leeman, D. S., Cullen, D. E., Gozo, M., McDowell, E. P., Levine, R. L., Doukas, J., Mak, C. C., Noronha, G., Martin, M., Ko, Y. D., Lee, B. H., Soll, R. M., Tefferi, A., Hood, J. D., Gilliland, D. G. 2008; 13 (4): 311-320

  Abstract

  We report that TG101348, a selective small-molecule inhibitor of JAK2 with an in vitro IC50 of approximately 3 nM, shows therapeutic efficacy in a murine model of myeloproliferative disease induced by the JAK2V617F mutation. In treated animals, there was a statistically significant reduction in hematocrit and leukocyte count, a dose-dependent reduction/elimination of extramedullary hematopoiesis, and, at least in some instances, evidence for attenuation of myelofibrosis. There were no apparent toxicities and no effect on T cell number. In vivo responses were correlated with surrogate endpoints, including reduction/elimination of JAK2V617F disease burden assessed by quantitative genomic PCR, suppression of endogenous erythroid colony formation, and in vivo inhibition of JAK-STAT signal transduction as assessed by flow cytometric measurement of phosphorylated Stat5.

  View details for DOI 10.1016/j.ccr.2008.02.009

  View details for Web of Science ID 000254817400006

  View details for PubMedID 18394554

• JAK2T875N is a novel activating mutation that results in myeloproliferative disease with features of megakaryoblastic leukemia in a murine bone marrow transplantation model BLOOD Mercher, T., Wernig, G., Moore, S. A., Levine, R. L., Gu, T., Frohling, S., Cullen, D., Polakiewicz, R. D., Bernard, O. A., Boggon, T. J., Lee, B. H., Gilliland, D. G. 2006; 108 (8): 2770-2779

  Abstract

  Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia associated with a poor prognosis. However, there are relatively few insights into the genetic etiology of AMKL. We developed a screening assay for mutations that cause AMKL, based on the hypothesis that constitutive activation of STAT5 would be a biochemical indicator of mutation in an upstream effector tyrosine kinase. We screened human AMKL cell lines for constitutive STAT5 activation, and then used an approach combining mass spectrometry identification of tyrosine phosphorylated proteins and growth inhibition in the presence of selective small molecule tyrosine kinase inhibitors that would inform DNA sequence analysis of candidate tyrosine kinases. Using this strategy, we identified a new JAK2T875N mutation in the AMKL cell line CHRF-288-11. JAK2T875N is a constitutively activated tyrosine kinase that activates downstream effectors including STAT5 in hematopoietic cells in vitro. In a murine transplant model, JAK2T875N induced a myeloproliferative disease characterized by features of AMKL, including megakaryocytic hyperplasia in the spleen; impaired megakaryocyte polyploidization; and increased reticulin fibrosis of the bone marrow and spleen. These findings provide new insights into pathways and therapeutic targets that contribute to the pathogenesis of AMKL.

  View details for DOI 10.1182/blood-2006-04-014712

  View details for Web of Science ID 000241131700046

  View details for PubMedID 16804112

• MPLW515L is anovel somatic activating mutation in myelofibrosis with myeloid metaplasia PLOS MEDICINE Pikman, Y., Lee, B. H., Mercher, T., McDowell, E., Ebert, B. L., Gozo, M., Cuker, A., Wernig, G., Moore, S., Galinsky, I., DeAngelo, D. J., Clark, J. J., Lee, S. J., Golub, T. R., Wadleigh, M., Gilliland, D. G., Levine, R. L. 2006; 3 (7): 1140-1151

  Abstract

  The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR).DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9-4.0 x 10(12)/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis.Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits certain features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD.

  View details for DOI 10.1371/journal.pmed.0030270

  View details for Web of Science ID 000239493300031

  View details for PubMedID 16834459

• Expression of Jak2V617F causes a polycythemia vera-like disease with associated myelofibrosis in a murine bone marrow transplant model BLOOD Wernig, G., Mercher, T., Okabe, R., Levine, R. L., Lee, B. H., Gilliland, D. G. 2006; 107 (11): 4274-4281

  Abstract

  An acquired somatic mutation, Jak2V617F, was recently discovered in most patients with polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), and essential thrombocythemia (ET). To investigate the role of this mutation in vivo, we transplanted bone marrow (BM) transduced with a retrovirus expressing either Jak2 wild-type (wt) or Jak2V617F into lethally irradiated syngeneic recipient mice. Expression of Jak2V617F, but not Jak2wt, resulted in clinicopathologic features that closely resembled PV in humans. These included striking elevation in hemoglobin level/hematocrit, leukocytosis, megakaryocyte hyperplasia, extramedullary hematopoiesis resulting in splenomegaly, and reticulin fibrosis in the bone marrow. Histopathologic and flow cytometric analyses showed an increase in maturing myeloid lineage progenitors, although megakaryocytes showed decreased polyploidization and staining for acetylcholinesterase. In vitro analysis of primary cells showed constitutive activation of Stat5 and cytokine-independent growth of erythroid colony-forming unit (CFU-E) and erythropoietin hypersensitivity, and Southern blot analysis for retroviral integration indicated that the disease was oligoclonal. Furthermore, we observed strain-specific differences in phenotype, with Balb/c mice demonstrating markedly elevated leukocyte counts, splenomegaly, and reticulin fibrosis compared with C57Bl/6 mice. We conclude that Jak2V617F expression in bone marrow progenitors results in a PV-like syndrome with myelofibrosis and that there are strain-specific modifiers that may in part explain phenotypic pleiotropy of Jak2V617F-associated myeloproliferative disease in humans.

  View details for Web of Science ID 000237877300020

  View details for PubMedID 16478879

  View details for PubMedCentralID PMC1895786

• Role of JAK-STAT signaling in the pathogenesis of myeloproliferative disorders. Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program Levine, R. L., Wernig, G. 2006: 233-?

  Abstract

  The identification of JAK2V617F mutations in polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis (MF) represents an important advance in our understanding of these myeloproliferative disorders (MPD). Most, if not all, patients with PV and a significant number of patients with ET and MF are JAK2V617F positive, and the mutation likely arises in the hematopoietic stem cell compartment. JAK2V617F is a constitutively active tyrosine kinase that is able to activate JAK-STAT signaling most efficiently when co-expressed with the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte colony-stimulating factor receptor (GCSFR). Data from murine models supports the central role of JAK2V617F in the pathogenesis of MPD, as expression of JAK2V617F in a bone marrow transplantation assay results in polycythemia and myelofibrosis in recipient mice. Activation of JAK-STAT signaling by JAK2V617F in some, but not all MPD patients with ET and MF led to the identification of the constitutively active MPLW515L allele in ET and MF. Small molecule inhibitors of JAK-STAT signaling are currently being developed, which offer potential for molecularly targeted therapy for patients with PV, ET, and MF. Despite these advances, many questions remain regarding the role of a single disease allele in three phenotypically distinct MPD, the potential clinical efficacy of JAK2 inhibitors, and the identity of oncogenic alleles in JAK2V617F/MPLW515-negative MPD.

  View details for PubMedID 17124066

• Expression of a homodimeric type I cytokine receptor is required for JAK2V617F-mediated transformation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lu, X. H., Levine, R., Tong, W., Wernig, G., Pikman, Y., Zarnegar, S., Gilliland, D. G., Lodish, H. 2005; 102 (52): 18962-18967

  Abstract

  A recurrent somatic activating mutation in the nonreceptor tyrosine kinase JAK2 (JAK2V617F) occurs in the majority of patients with the myeloproliferative disorders polycythemia vera, essential thrombocythemia, myelofibrosis with myeloid metaplasia, and, less commonly, chronic myelomonocytic leukemia. We do not understand the basis for the specificity of the JAK2V617F mutation in clonal disorders of the myeloid, but not lymphoid, lineage, nor has the basis for the pleiotropic phenotype of JAK2V617F-associated myeloproliferative disorders been delineated. However, the presence of the identical mutation in patients with related, but clinicopathologically distinct, myeloid disorders suggests that interactions between the JAK2V617F kinase and other signaling molecules may influence the phenotype of hematopoietic progenitors expressing JAK2V617F. Here, we show that coexpression of the JAK2V617F mutant kinase with a homodimeric Type I cytokine receptor, the erythropoietin receptor (EpoR), the thrombopoietin receptor, or the granulocyte colony-stimulating-factor receptor, is necessary for transformation of hematopoietic cells to growth-factor independence and for hormone-independent activation of JAK-STAT signaling. Furthermore, EpoR mutations that impair erythropoietin-mediated JAK2 or STAT5 activation also impair transformation mediated by the JAK2V617F kinase, indicating that JAK2V617F requires a cytokine receptor scaffold for its transforming and signaling activities. Our results reveal the molecular basis for the prevalence of JAK2V617F in diseases of myeloid lineage cells that express these Type I cytokine receptors but not in lymphoid lineage cells that do not.

  View details for DOI 10.1073/pnas.0509714102

  View details for Web of Science ID 000234350000034

  View details for PubMedID 16365288

• The vast majority of bone-marrow-derived cells integrated into mdx muscle fibers are silent despite long-term engraftment PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wernig, G., Janzen, V., SCHAFER, R., Zweyer, M., Knauf, U., Hoegemeier, O., Mundegar, R. R., Garbe, S., Stier, S., Franz, T., Wernig, M., Wernig, A. 2005; 102 (33): 11852-11857

  Abstract

  Bone-marrow-derived cells can contribute nuclei to skeletal muscle fibers. However, serial sectioning of muscle in mdx mice implanted with GFP-labeled bone marrow reveals that only 20% of the donor nuclei chronically incorporated in muscle fibers show dystrophin (or GFP) expression, which is still higher than the expected frequency of "revertant" fibers, but there is no overall increase above controls over time. Obviously, the vast majority of incorporated nuclei either never or only temporarily turn on myogenic genes; also, incorporated nuclei eventually loose the activation of the beta-actin::GFP transgene. Consequently, we attempted to enhance the expression of dystrophin. In vivo application of the chromatin-modifying agents 5-azadeoxycytidine and phenylbutyrate as well as local damage by cardiotoxin injections caused a small increase in dystrophin-positive fibers without abolishing the appearance of "silent" nuclei. The results thus confirm that endogenous repair processes and epigenetic modifications on a small-scale lead to dystrophin expression from donor nuclei. Both effects, however, remain below functionally significant levels.

  View details for DOI 10.1073/pnas.0502507102

  View details for Web of Science ID 000231317000051

  View details for PubMedID 16085712

  View details for PubMedCentralID PMC1187970

• Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis CANCER CELL Levine, R. L., Wadleigh, M., Cools, J., Ebert, B. L., Wernig, G., Huntly, B. J., Boggon, T. J., Wlodarska, L., Clark, J. J., Moore, S., Adelsperger, J., Koo, S., Lee, J. C., Gabriel, S., Mercher, T., D'Andrea, A., Frohling, S., Dohner, K., Marynen, P., Vandenberghe, P., Mesa, R. A., Tefferi, A., GRIFFIN, J. D., Eck, M. J., Sellers, W. R., Meyerson, M., Golub, T. R., Lee, S. J., Gilliland, D. G. 2005; 7 (4): 387-397

  Abstract

  Polycythemia vera (PV), essential thrombocythemia (ET), and myeloid metaplasia with myelofibrosis (MMM) are clonal disorders arising from hematopoietic progenitors. An internet-based protocol was used to collect clinical information and biological specimens from patients with these diseases. High-throughput DNA resequencing identified a recurrent somatic missense mutation JAK2V617F in granulocyte DNA samples of 121 of 164 PV patients, of which 41 had homozygous and 80 had heterozygous mutations. Molecular and cytogenetic analyses demonstrated that homozygous mutations were due to duplication of the mutant allele. JAK2V617F was also identified in granulocyte DNA samples from 37 of 115 ET and 16 of 46 MMM patients, but was not observed in 269 normal individuals. In vitro analysis demonstrated that JAK2V617F is a constitutively active tyrosine kinase.

  View details for DOI 10.1016/j.ccr.2005.03.023

  View details for Web of Science ID 000228741700013

  View details for PubMedID 15837627

• Correction of CFTR malfunction and stimulation of Ca2+- activated Cl- channels restore HCO3- secretion in cystic fibrosis bile ductular cells HEPATOLOGY Zsembery, A., Jessner, W., Sitter, G., Spirli, C., Strazzabosco, M., Graf, J. 2002; 35 (1): 95-104

  Abstract

  In view of the occurrence of hepatobiliary disorders in cystic fibrosis (CF) this study addresses the role of the cystic fibrosis transmembrane conductance regulator (CFTR) and of Ca(2+)-activated Cl(-) channels in promoting HCO3- secretion in bile ductular cells. Human cholangiocytes were isolated from control livers and from 1 patient with CF (DeltaF508/G542X mutations). Single channel and whole cell currents were analyzed by patch clamp techniques, and HCO3- secretion was determined by fluorometric analysis of the rate of recovery of intracellular pH following alkaline loading. In control cholangiocytes, both cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) catalytic subunit, activated CFTR Cl(-) channels that exhibited a nonrectifying conductance of 8 pS and appeared in clusters. Activation of Cl(-) current by cAMP was associated with an increase in the rate of HCO3- secretion. The basal rate of HCO3- secretion was lower in CF than in control cholangiocytes. In both control and CF cholangiocytes, raising intracellular Ca(2+) concentrations with ionomycin led to a parallel activation of Cl(-) current and HCO3- secretion. Consistent with reports that premature stop codon mutations (class I; e.g., G542X) can be read over by treatment with aminoglycoside antibiotics, exposure of CF cholangiocytes to gentamicin restored activation by cAMP of Cl(-) current and HCO3- secretion. The observation that activation of Ca(2+)-dependent Cl(-) channels can substitute for cystic fibrosis transmembrane conductance regulator (CFTR) in supporting HCO3- secretion and the efficacy of gentamicin in restoring CFTR function and HCO3- secretion in class I mutations are of potential clinical interest.

  View details for DOI 10.1053/jhep.2002.30423

  View details for Web of Science ID 000172983300013

  View details for PubMedID 11786964