Impact of isolated germline JAK2V617I mutation on human hematopoiesis by Adam J. Mead, Onima Chowdhury, Christian Pecquet, Alexandra Dusa, Petter Woll, Deborah Atkinson, Adam Burns, Joannah Score, Michelle Rugless, Ruth Clifford, Simon Moule, Nicola Bienz, Paresh Vyas, Nick Cross, Rosemary E. Gale, Shirley Henderson, Stefan N. Constantinescu, Anna Schuh, and Sten Eirik W. Jacobsen Blood Volume 121(20):4156-4165 May 16, 2013 ©2013 by American Society of Hematology

PB and BM hematopoietic phenotype of germline JAKV617I. PB and BM hematopoietic phenotype of germline JAKV617I. (A) Quantification of Lin-CD34+, phenotypic HSCs (CD45+Lin–CD34+CD38–CD90+CD45RA–), multipotent progenitors (MPPs; CD45+Lin–CD34+CD38–CD90–CD45RA–), common myeloid progenitors (CMPs; CD45+Lin–CD34+CD38+CD123+CD45RA–), granulocyte macrophage progenitors (GMPs; CD45+Lin–CD34+CD38+CD123+CD45RA+), and megakaryocyte erythroid progenitors (MEPs; CD45+Lin–CD34+CD38+CD123–CD45RA–) in the PB of normal controls (n = 4); JAK2V617I-positive samples (n = 4); patients with JAK2V617F-positive PV (n = 3); ET (n = 3), and myelofibrosis (n = 3). (B) Quantification of phenotypic HSCs, MPPs, CMPs, GMPs, and MEPs in the BM of normal controls (n = 8) and JAK2V617I-positive samples. (C) Numbers of BFU-E, CFU-GM, and CFU-GEMM in the BM of NC (n = 8) and JAK2V617I-positive (n = 4) cases. (D) Numbers and sizes of BM CFU-Mk in NC (n = 3) versus JAK2V617I (n = 4) patients. Error bars represent SEM. P values are shown if < .05. Adam J. Mead et al. Blood 2013;121:4156-4165 ©2013 by American Society of Hematology

Long-term engraftment kinetics of germline JAK2V617I CD34+ cells after xenograft transplantation. Long-term engraftment kinetics of germline JAK2V617I CD34+ cells after xenograft transplantation. Human CD45 engraftment kinetics for age-matched NBM cases: (A) controls, n = 2 subjects; 2 recipient mice per sample and (B) JAK2V617I, n = 4, cases C1-C4; 1-3 recipient mice per samples after injection of 100 000 CD34+ cells into NOD/LtSz-scidIL2Rgnull (NSG) mice. P values in (B) represent a comparison of JAK2V617I and NBM engraftment levels at each respective time. (C) Engraftment kinetics of CD19+ B cell, CD15/33/66b+ myeloid cells, and CD34+ progenitor cells expressed as a percentage of total hCD45+ cells. (D) Quantification of HSCs, MEPs, GMPs, and CMPs in the BM at 18 weeks after transplantation. Error bars represent SEM. P values are indicated if < .1. Adam J. Mead et al. Blood 2013;121:4156-4165 ©2013 by American Society of Hematology

JAK2V617I induces only weak constitutive activation but marked cytokine hyperresponsiveness. JAK2V617I induces only weak constitutive activation but marked cytokine hyperresponsiveness. (A) A representative analysis (1/2 independent experiments with similar results) of Ba/F3 EpoR cells that were engineered to overexpress equal levels of wild-type JAK2, JAK2V617F, or JAK2V617I by bicistronic retroviral transduction and cell sorting. Cells growing in medium supplemented with IL3 (left panel) or cells that acquired autonomous growth, namely cells expressing JAK2V617F or JAK2V617I (right panel, cells are denoted “Sel.”) were starved for 5 hours without serum and cytokines and then stimulated with EPO (20 U/mL) as indicated for 15 minutes, and then lysed in 1% NP40 buffer and assessed by Western blotting for specific phosphorylation at sites that reflect activation of JAK2, STAT1, STAT3, STAT5, and Erk1/2, and for total level of JAK2, STAT3, Erk1/2, HA, and β-actin protein expression. (B) A representative analysis (1/2 independent experiments with similar results) of Ba/F3 TpoR cells that were engineered to overexpress equal levels of wild-type JAK2, JAK2V617F, or JAK2V617I by bicistronic retroviral transduction and cell sorting. Cells growing in medium supplemented with IL3 were starved for 5 hours without serum and cytokines and then stimulated with Tpo (20 ng/mL) as indicated, and then lysed after 15 minutes in 1% NP40 buffer and assessed by Western blotting for specific phosphorylation at sites that reflect activation of JAK2, STAT1, STAT3, STAT5, and Erk1/2, and for total level of JAK2, STAT3, Erk1/2, HA, and β-actin protein expression. (C-D) Results of luciferase assays in γ2A fibrosarcoma JAK2-deficient cells transfected with cDNAs coding for TpoR (C) or EpoR (D) along with wild-type JAK2, JAK2V617F, or JAK2V617I, along with STAT5 and STAT-dependent firefly luciferase and pRLTK-driven renilla luciferase (rlu), for normalization. Dual luciferase was measured 24 hours after transfection. Results of 1 representative experiment of 3 independent experiments are shown. Data are expressed as means of triplicates, and error bars indicate SD. *P < .05, **P < .01, ***P < .001. Adam J. Mead et al. Blood 2013;121:4156-4165 ©2013 by American Society of Hematology

Cytokine response assays. Cytokine response assays. (A-B) Colony formation with rhSCF at 50 ng/mL and (A) rhG-CSF (Amgen) or (B) rhTPO, each at 100 ng/mL or 0.8 ng/mL. Results are from normal subjects (n = 6), JAK2V617I cases (n = 4), and JAK2V617F-positive MPNs (n = 6), expressed as numbers of colonies formed with 0.8 ng/mL rhG-CSF or rhTPO as a percentage of colony numbers formed with 100 ng/mL rhG-CSF or rhTPO. (C) The numbers of BFU-E generated with conditioned methylcellulose (see Methods) in the presence of rhEPO at 5 U/mL, 0.05 U/mL, or 0.005 U/mL, or in the absence of EPO. Results are expressed as a percentage relative to total numbers of BFU-Es generated at 5 U/mL for normal controls (n = 4), JAK2V617I cases (n = 4), and JAK2V717F cases (n = 7). P values are shown if < .1. Error bars represent SEM. Adam J. Mead et al. Blood 2013;121:4156-4165 ©2013 by American Society of Hematology