- Anatomic Pathology
Board Certification: American Board of Pathology, Anatomic Pathology (2013)
Board Certification: American Board of Pathology, Hematology (2013)
Residency: Stanford Hospital and Clinics (2013) CA
Fellowship: Stanford Hospital and Clinics (2012) CA
Residency: Stanford Hospital and Clinics (2011) CA
Fellowship, Friedrich-Wilhelms-University Bonn, Germany, Hematology/Oncology (2004)
Residency, Friedrich-Wilhlems-Uniersity Bonn, Germany, Internal Medicine (2001)
Medical Education: Medical University of Vienna (1999) Austria
Current Research and Scholarly Interests
Fibrotic diseases is a cover term coined by our laboratory to address complications of the excessive scarring of fibrous tissue. They occur when fibroblasts – a critical component of the structural tissue of the body – proliferate and include, but are not limited to lung fibrosis, kidney and liver fibrosis, scleroderma, bone marrow fibrosis, wound healing and surgical adhesions. Despite fibrotic diseases being life-threatening-- the mortality rate of some are higher than that of cancer-- current treatments are ineffective and/or entirely nonexistent.
Our mission is to identify new targets for treatment through uncovering the underlying mechanisms of inflammation and fibrosis. We seek to understand how fibroblasts crosstalk with one another, with the immune system, and with epithelial and mesenchymal cells. By integrating single-cell transcriptional profiling, next-generation shotgun proteomics with mass cytometry and chromatin studies of patient-derived primary tissues in combination with in vivo modeling of fibrotic disease in mice, we gain insight into the pathophysiology of fibrotic diseases. We employ immunotherapy combined with small molecules in order to manipulate signaling pathways at the transcriptional level to disrupt pro-fibrotic cell function and fate. The transcriptional networks we study play key roles in fibrotic disease, metabolism, bone physiology, cancer, and immunology. Understanding them will provide the critical foundation to translate our findings into immunotherapies and clinical practice for fibrotic diseases.
Independent Studies (7)
- Directed Reading in Pathology
PATH 299 (Win, Spr)
- Early Clinical Experience in Pathology
PATH 280 (Win, Spr)
- Graduate Research
PATH 399 (Win, Spr)
- Graduate Research
STEMREM 399 (Aut)
- Medical Scholars Research
PATH 370 (Win, Spr)
- Undergraduate Research
PATH 199 (Win, Spr)
- Undergraduate Research
STEMREM 199 (Aut, Win, Spr, Sum)
- Directed Reading in Pathology
Graduate and Fellowship Programs
Hematopathology (Fellowship Program)
CD47 prevents the elimination of diseased fibroblasts in scleroderma.
2020; 5 (16)
Scleroderma is a devastating fibrotic autoimmune disease. Current treatments are partly effective in preventing disease progression but do not remove fibrotic tissue. Here, we evaluated whether scleroderma fibroblasts take advantage of the "don't-eat-me-signal" CD47 and whether blocking CD47 enables the body's immune system to get rid of diseased fibroblasts. To test this approach, we used a Jun-inducible scleroderma model. We first demonstrated in patient samples that scleroderma upregulated transcription factor JUN and increased promoter accessibilities of both JUN and CD47. Next, we established our scleroderma model, demonstrating that Jun mediated skin fibrosis through the hedgehog-dependent expansion of CD26+Sca1- fibroblasts in mice. In a niche-independent adaptive transfer model, JUN steered graft survival and conferred increased self-renewal to fibroblasts. In vivo, JUN enhanced the expression of CD47, and inhibiting CD47 eliminated an ectopic fibroblast graft and increased in vitro phagocytosis. In the syngeneic mouse, depleting macrophages ameliorated skin fibrosis. Therapeutically, combined CD47 and IL-6 blockade reversed skin fibrosis in mice and led to the rapid elimination of ectopically transplanted scleroderma cells. Altogether, our study demonstrates the efficiency of combining different immunotherapies in treating scleroderma and provides a rationale for combining CD47 and IL-6 inhibition in clinical trials.
View details for DOI 10.1172/jci.insight.140458
View details for PubMedID 32814713
Expansion of Bone Precursors through Jun as a Novel Treatment for Osteoporosis-Associated Fractures.
Stem cell reports
Osteoporosis and osteoporotic fractures lead to decreased life quality and high healthcare costs. Current treatments prevent losses in bone mass and fractures to some extent but have side effects. Therefore, better therapies are needed. This study investigated whether the transcriptionfactor Jun has a specific pro-osteogenic potency and whether modulating Jun could serve as a novel treatment for osteoporosis-associated fractures. We demonstrate that ectopically transplanted whole bones and distinct osteoprogenitors increase bone formation. Perinatal Jun induction disturbs growth plate architecture, causing a striking phenotype with shortened and thickened bones. Molecularly, Jun induces hedgehog signaling in skeletal stem cells. Therapeutically, Jun accelerates bone growth and healing in a drilling-defect model. Altogether, these results demonstrate that Jun drives bone formation by expanding osteoprogenitor populations and forcing them into the bone fate, providing a rationale for future clinical applications.
View details for DOI 10.1016/j.stemcr.2020.02.009
View details for PubMedID 32197115
Activation of JUN in fibroblasts promotes pro-fibrotic programme and modulates protective immunity.
2020; 11 (1): 2795
The transcription factor JUN is highly expressed in pulmonary fibrosis. Its induction in mice drives lung fibrosis, which is abrogated by administration of anti-CD47. Here, we use high-dimensional mass cytometry to profile protein expression and secretome of cells from patients with pulmonary fibrosis. We show that JUN is activated in fibrotic fibroblasts that expressed increased CD47 and PD-L1. Using ATAC-seq and ChIP-seq, we found that activation of JUN rendered promoters and enhancers of CD47 and PD-L1 accessible. We further detect increased IL-6 that amplified JUN-mediated CD47 enhancer activity and protein expression. Using an in vivo mouse model of fibrosis, we found two distinct mechanisms by which blocking IL-6, CD47 and PD-L1 reversed fibrosis, by increasing phagocytosis of profibrotic fibroblasts and by eliminating suppressive effects on adaptive immunity. Our results identify specific immune mechanisms that promote fibrosis and suggest a therapeutic approach that could be used alongside conventional anti-fibrotics for pulmonary fibrosis.
View details for DOI 10.1038/s41467-020-16466-4
View details for PubMedID 32493933
Elucidating the fundamental fibrotic processes driving abdominal adhesion formation.
2020; 11 (1): 4061
Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti-JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients.
View details for DOI 10.1038/s41467-020-17883-1
View details for PubMedID 32792541
JUN Drives Pathologic Scarring by Activating Key Fibroproliferative Pathways in Fibroblast Subpopulations
ELSEVIER SCIENCE INC. 2019: E215–E216
View details for Web of Science ID 000492749600516
- Selective hematopoietic stem cell ablation using CD117-antibody-drug-conjugates enables safe and effective transplantation with immunity preservation NATURE COMMUNICATIONS 2019; 10
- Direct targeting of the mouse optic nerve for therapeutic delivery JOURNAL OF NEUROSCIENCE METHODS 2019; 313: 1–5
- DOXYCYCLINE REDUCES SCARRING BY MODULATING COLLAGEN ARCHITECTURE BMJ PUBLISHING GROUP. 2019: 157–58
Modeling chronic Graft-vs-Host disease in MHC-matched mouse strains: genetics, graft composition and tissue targets.
Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation
Graft-vs-host disease (GVHD) remains a major complication of allogeneic hematopoietic cell transplantation. Acute GVHD results from direct damage by donor T cells, while the biology of chronic GVHD (cGVHD) with its autoimmune-like manifestations remains poorly understood - mainly due to the paucity of representative preclinical models. We examined over an extended time period seven MHC-matched, minor antigen mismatched mouse models for development of cGVHD. Development and manifestations of cGVHD were determined by a combination of MHC-allele type and recipient strain, with BALB recipients being the most susceptible. The C57BL/6 into BALB.B combination most closely modeled the human syndrome. In this strain combination moderate acute GVHD was observed and BALB.B survivors developed overt cGVHD at 6-12 months affecting eyes, skin and liver. Naïve CD4+ cells caused this syndrome as no significant pathology was induced by grafts composed of purified hematopoietic stem cells (HSC) or HSC plus memory CD4+ or CD8+ cells. Furthermore, co-transferred naïve and memory CD4+ T cells demonstrated differential homing patterns and locations of persistence. No clear association with donor TH17 cells and the phenotype of acute or chronic GVHD was observed in this model. Donor CD4+ cells caused injury to medullary thymic epithelial cells, a key population responsible for negative T-cell selection, suggesting that impaired thymic selection was an underlying cause of the cGVHD syndrome. In conclusion, we report for the first time that the C57BL/6 into BALB.B combination is a representative model of cGVHD that evolves from immunological events during the early post-transplant period.
View details for DOI 10.1016/j.bbmt.2019.08.001
View details for PubMedID 31415899
Doxycycline Reduces Scar Thickness and Improves Collagen Architecture.
Annals of surgery
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
2018; 10 (469)
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
- Reduced Scar Thickness Achieved by Topical Doxycycline Is Mediated by Specific Skin Fibroblast Populations and Not Immune Cell Infiltrate ELSEVIER SCIENCE INC. 2018: S210–S211
- Mouse Model with cJUN Over-Expression Eludes to Deep Dermal Fibroblast Expansion and Immune Cell Recruitment as the Biologic Mechanism of Hypertrophic Scarring ELSEVIER SCIENCE INC. 2018: S208
Stem cell therapy for treatment of ischemic optic neuropathy
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2018
View details for Web of Science ID 000442912501176
- Doxycyline Improves Wound Healing via Nonantibiotic Associated Mechanisms ELSEVIER SCIENCE INC. 2017: S162–S163
Unifying mechanism for different fibrotic diseases
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2017; 114 (18): 4757-4762
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
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
2016; 166 (2): 451-467
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
2015; 160 (6): 1196-1208
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
2013; 73 (1): 373-384
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
2012; 26 (12): 2538-2545
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
2012; 26 (4): 720-727
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
Physiological Jak2V617F Expression Causes a Lethal Myeloproliferative Neoplasm with Differential Effects on Hematopoietic Stem and Progenitor Cells
2010; 17 (6): 584-596
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
2008; 111 (9): 4788-4796
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
2008; 111 (7): 3751-3759
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
View details for PubMedCentralID PMC2275031
Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced polycythemia vera
2008; 13 (4): 311-320
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
2006; 108 (8): 2770-2779
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
2006; 3 (7): 1140-1151
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
2006; 107 (11): 4274-4281
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
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
2005; 102 (52): 18962-18967
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
2005; 102 (33): 11852-11857
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
2005; 7 (4): 387-397
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
2002; 35 (1): 95-104
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