Myeloproliferative disorders (MPDs) are a group of conditions characterized by chronic increases in some or all of the blood cells. Recently, we and others have identified a mutation in the JAK2 tyrosine kinases in classic MPDs including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The mutant enzyme designated JAK2V617F possesses deregulated and enhanced kinase activity and induces constitutive activation of downstream signaling events in transfected cells. We have now generated transgenic mice expressing the mutated enzyme in the hematopoietic system driven by a vav gene promoter. The transgenic mice displayed marked increases in blood counts and developed phenotypes that closely resembled human ET and PV in a transgene dose- dependent manner. The mice also developed PMF-like symptoms as they aged. Our data provides unequivocal evidence that JAK2V617F can cause MPDs and that the phenotypes are determined by the expression level of the mutant enzyme. Furthermore, by generating JAK2 transgenic mice and crossing them with JAK2V617F transgenic mice, we have found that over- expression of wild type JAK2 is not only non-pathogenic but also totally suppresses the pathogenic function of JAK2V617F, suggesting that wild type JAK2 antagonizes mutant JAK2V617F. These transgenic mouse models provide an invaluable tool to study MPDs and normal hematopoiesis. In this study, we will utilize these tools to reveal the pathological role of JAK2V617F, the molecular and cellular mechanism on which MPDs develop, and the interplay of mutant JAK2V617F with wild type JAK2. Specifically, we will fully characterize the phenotypes of JAK2V617F transgenic mice and reveal the dose-dependent nature of JAK2V617F-induced MPD phenotypes. We will thoroughly investigate the effects of JAK2V617F on the proliferation, differentiation, and survival of hematopoietic stem/progenitor cells by using various in vitro cell culture methods and in vivo stem cell transplant techniques and analyze the activation of various signaling pathways in these cells. This study will provide novel insight into the mechanism by which abnormal hematopoiesis arises in MPD patients and normal hematopoiesis is regulated under healthy conditions. It addresses how JAK2V617F causes MPDs and answers the question why a single point mutation causes so many phenotypes. This study should have major implications in development of therapeutic drugs and procedures to treat MPDs in the future.
This study concerns myeloproliferative disorders (MPDs) which represent a heterogeneous group of diseases characterized by increased proliferation of erythroid, megakaryocytic and/or myeloid lineages. We have identified an acquired mutation of tyrosine kinase JAK2 in these diseases and demonstrated the pathogenic role of the mutant enzyme by using transgenic mouse models. This study is intended to reveal how the mutant enzyme causes the disease. Understanding the pathogenic mechanism of the mutant enzyme will eventually allow us to develop therapeutic drugs and procedures to treat the diseases. PHS 398/2590 (Rev. 11/07) Page 2 REVISED BUDGET Program Director/Principal Investigator (Last, First, Middle): Zhao, Zhizhuang, Joe DETAILED BUDGET FOR INITIAL BUDGET PERIOD DIRECT COSTS ONLY FROM 09/30/2009 THROUGH 09/29/2010 PERSONNEL (Applicant organization only) Months Devoted to Project NAME ROLE ON PROJECT Zhizhuang Joe Zhao PD/PI Tina Ho Research Associate Shaofeng Wang Research Scholar Wanming Zhao Research Scholar Cal. Mnths Acad. Mnths Summer Mnths 4.2 12 12 12 INST.BASE SALARY DOLLAR AMOUNT REQUESTED (omit cents) SALARY REQUESTED FRINGE BENEFITS TOTAL 160,000 56,000 21,566 77,566 56,222 56,222 21,651 77,873 27,300 27,300 273 27,573 23,980 23,980 240 24,220 SUBTOTALS 163,502 43,730 207,232 CONSULTANT COSTS 0 EQUIPMENT (Itemize) 0 SUPPLIES (Itemize by category) General lab supplies 25,000 Chemicals 4,000 29,000 TRAVEL Travel for three members to attend domestic scientific meetings4,500 PATIENT CARE COSTS INPATIENT 0 OUTPATIENT 0 ALTERATIONS AND RENOVATIONS (Itemize by category) 0 OTHER EXPENSES (Itemize by category) Lab analysis and service 8,000 Publication 2,000 Animal per diem 22,000 32,000 CONSORTIUM/CONTRACTUAL COSTS DIRECT COSTS 0 SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD (Item 7a, Face Page) $ 272,732 CONSORTIUM/CONTRACTUAL COSTS FACILITIES AND ADMINISTRATIVE COSTS 0 TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD $ 272,732 PHS 398 (Rev. 11/07) Page Form Page 4 REVISED BUDGET Program Director/Principal Investigator (Last, First, Middle): Zhao, Zhizhuang, Joe BUDGET FOR ENTIRE PROPOSED PROJECT PERIOD DIRECT COSTS ONLY BUDGET CATEGORY TOTALS INITIAL BUDGET PERIOD (from Form Page 4) ADDITIONAL YEARS OF SUPPORT REQUESTED 2nd 3rd 4th 5th PERSONNEL: Salary and fringe benefits. Applicant organization only. 207,232 211,122 CONSULTANT COSTS 0 0 EQUIPMENT 0 0 SUPPLIES 29,000 29,000 TRAVEL 4,500 4,500 PATIENT CARE COSTS INPATIENT 0 0 OUTPATIENT 0 0 ALTERATIONS AND RENOVATIONS 0 0 OTHER EXPENSES 32,000 32,000 CONSORTIUM/ CONTRACTUAL COSTS DIRECT 0 0 SUBTOTAL DIRECT COSTS (Sum = Item 8a, Face Page) 272,732 276,622 CONSORTIUM/ CONTRACTUAL COSTS F&A TOTAL DIRECT COSTS 272,732 276,622 TOTAL DIRECT COSTS FOR ENTIRE PROPOSED PROJECT PERIOD $ 549,354 JUSTIFICATION. Follow the budget justification instructions exactly. Use continuation pages as needed. The original proposal has three aims. In this revised application, we deleted Aim 3 and left Aim 1 and 2 intact. Aim 1 and Aim 2 represent more than 60% of the work. In order to accomplish these aims in two years, we increased the effort of Tina Ho, Research Associate, from original 50% to 100%. This results in an increase in annual budget by nearly $25,000. A 3% increase in salary was budgeted for staff members (not including PI) in the second year. Travel will be reimbursed at actual and reasonable costs. Fringe benefit rates are with DHHS for approval, only approved rates will be charged to grant at time of award. PHS 398 (Rev. 11/07) Page Form Page 5 REVISED CHECKLIST Program Director/Principal Investigator (Last, First, Middle): Zhao, Zhizhuang, Joe CHECKLIST TYPE OF APPLICATION (Check all that apply.) NEW application. (This application is being submitted to the PHS for the first time.) RESUBMISSION of application number: Resubmission of requested forms for 1R01HL094591-01A1 (This application replaces a prior unfunded version of a new, renewal, or revision application.) RENEWAL of grant number: (This application is to extend a funded grant beyond its current project period.) REVISION to grant number: (This application is for additional funds to supplement a currently funded grant.) CHANGE of program director/principal investigator. Name of former program director/principal investigator: CHANGE of Grantee Institution. Name of former institution: List Country(ies) FOREIGN application Domestic Grant with foreign involvement Involved: INVENTIONS AND PATENTS (Renewal appl. only) No Yes If Yes,Previously reported Not previously reported 1. PROGRAM INCOME (See instructions.) All applications must indicate whether program income is anticipated during the period(s) for which grant support is request. If program income is anticipated, use the format below to reflect the amount and source(s). Budget Period Anticipated Amount Source(s) 2. ASSURANCES/CERTIFICATIONS (See instructions.) In signing the application Face Page, the authorized organizational representative agrees to comply with the policies, assurances and/or certifications listed in the application instructions when applicable. Descriptions of individual assurances/certifications are provided in Part III and listed in Part I, 4.1 under Item 14. If unable to certify compliance, where applicable, provide an explanation and place it after this page. 3. FACILITIES AND ADMINSTRATIVE COSTS (F&A)/ INDIRECT COSTS. See specific instructions. DHHS Agreement dated: 06/23/2008 No Facilities And Administrative Costs Requested. DHHS Agreement being negotiated with Regional Office. No DHHS Agreement, but rate established with Date CALCULATION* (The entire grant application, including the Checklist, will be reproduced and provided to peer reviewers as confidential information.) a. Initial budget period: Amount of base $ 272,732 x Rate applied 46.50 % = F&A costs $ 126,820 b. 02 year Amount of base $ 276,622 x Rate applied 46.50 % = F&A costs $ 128,629 c. 03 year Amount of base $ x Rate applied % = F&A costs $ d. 04 year Amount of base $ x Rate applied % = F&A costs $ e. 05 year Amount of base $ x Rate applied % = F&A costs $ TOTAL F&A Costs $ 255,449 *Check appropriate box(es): Salary and wages base Modified total direct cost base Off-site, other special rate, or more than one rate involved (Explain) Explanation (Attach separate sheet, if necessary.): Other base (Explain) 4. DISCLOSURE PERMISSION STATEMENT: If this application does not result in an award, is the Government permitted to disclose the title of your proposed project, and the name, address, telephone number and e-mail address of the official signing for the applicant organization, to organizations that may be interested in contacting you for further information (e.g., possible collaborations, investment)? Yes No PHS 398 (Rev. 11/07) Page Checklist Form Page
| Shi, Kaiyao; Zhao, Wanke; Chen, Yun et al. (2014) Cardiac hypertrophy associated with myeloproliferative neoplasms in JAK2V617F transgenic mice. J Hematol Oncol 7:25 |
| Chen, Yun; Guo, Yao; Han, Jiayu et al. (2012) Generation and characterization of a highly effective protein substrate for analysis of FLT3 activity. J Hematol Oncol 5:39 |
| Guo, Yao; Chen, Yun; Xu, Xuesong et al. (2012) SU11652 Inhibits tyrosine kinase activity of FLT3 and growth of MV-4-11 cells. J Hematol Oncol 5:72 |
| Zhao, Anna H; Gao, Rufei; Zhao, Zhizhuang J (2011) Development of a highly sensitive method for detection of JAK2V617F. J Hematol Oncol 4:40 |
| Zhao, Wanming; Gao, Rufei; Lee, Jiyun et al. (2011) Relevance of JAK2V617F positivity to hematological diseases--survey of samples from a clinical genetics laboratory. J Hematol Oncol 4:4 |
| Wang, Shaofeng; Dong, Hongbo; Han, Jiayu et al. (2010) Identification of a variant form of tyrosine phosphatase LYP. BMC Mol Biol 11:78 |
