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Germ-line JAK2 mutations in the kinase domain are responsible for hereditary thrombocytosis and are resistant to JAK2 and HSP90 inhibitors by Caroline.

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Presentation on theme: "Germ-line JAK2 mutations in the kinase domain are responsible for hereditary thrombocytosis and are resistant to JAK2 and HSP90 inhibitors by Caroline."— Presentation transcript:

1 Germ-line JAK2 mutations in the kinase domain are responsible for hereditary thrombocytosis and are resistant to JAK2 and HSP90 inhibitors by Caroline Marty, Cécile Saint-Martin, Christian Pecquet, Sarah Grosjean, Joseph Saliba, Céline Mouton, Emilie Leroy, Ashot S. Harutyunyan, Jean-François Abgrall, Rémi Favier, Aurélie Toussaint, Eric Solary, Robert Kralovics, Stefan N. Constantinescu, Albert Najman, William Vainchenker, Isabelle Plo, and Christine Bellanné-Chantelot Blood Volume 123(9): February 27, 2014 ©2014 by American Society of Hematology

2 Clinical and molecular features of 2 pedigrees with hereditary thrombocytosis.
Clinical and molecular features of 2 pedigrees with hereditary thrombocytosis. (A) Filled symbols represent individuals with JAK2 germ-line mutations. Below each symbol is reported the JAK2 genotype with “N” indicating a normal allele, the age (years) at diagnosis for affected individuals or the age at last examination for asymptomatic relatives, and the platelet level (×109/L). (B) Sequence electropherograms of the germ-line JAK2 mutations. Caroline Marty et al. Blood 2014;123: ©2014 by American Society of Hematology

3 Scheme and in silico modeling of the JAK2 mutants.
Scheme and in silico modeling of the JAK2 mutants. (A) Scheme and location of germ-line JAK2 mutations. (B) Superimposition of the crystal structures of the active JH1 kinase and JH2 pseudokinase domains of JAK2 wild type (WT) based on 2 radiograph crystal structure studies.20,21 On the left in blue: coordinate of active JH1 (protein data bank [PDB] code: 2B7A). On the right in green: coordinate of active JH2 (PDB code: 4FVQ). Two of the mutations, R938Q and R867Q, are located in the JH1, whereas the S755R mutation is located in the JH2 (pink residues). The germ-line JAK2 S755R/R938Q mutant therefore contains mutations in both JH2 and JH1. The activation loop is represented in red in both JH1 and JH2. The glycine-rich loop is in yellow, and the catalytic loop is in orange. The salt bridge between D869 and R867 is only formed in the inactive JH1, which is the coordinate represented on the small square on the left. The coordinate of inactive JH1 is PDB code 3UGC. The salt bridge between K752 and D635 is formed in active JH2 (WT and V617F). The coordinate shown here in the small square on the right is the active JH2 WT (PDB code: 4FVQ). Caroline Marty et al. Blood 2014;123: ©2014 by American Society of Hematology

4 Sensitivities to EPO and TPO of Ba/F3-EPOR and Ba/F3-MPL cells.
Sensitivities to EPO and TPO of Ba/F3-EPOR and Ba/F3-MPL cells. (A) Ba/F3-EPOR cells expressing each of the JAK2 forms were cultured for 48 hours either in the absence of cytokine (black arrow, x-axis) or in the presence of increasing doses of EPO (0.01, 0.02, 0.03, 0.05, 0.1, 0.3, and 1 U/mL). Viable cells were quantified by WST-1 proliferation assay. Dose-response curves are means expressed in percentages of maximum growth value ± standard error of the mean (SEM; n = 4 in triplicate). (B) Proliferation was assayed 48 hours after culturing Ba/F3-MPL cells expressing each of the JAK2 forms in the absence of cytokine (black arrow, x-axis) or in the presence of increasing doses of TPO (0.005, 0.015, 0.05, 0.15, 0.5, 1.5, and 5 ng/mL). The asterisk indicates 0.05 ng/mL TPO. Data are means ± SEM (n = 4 independent experiments performed in triplicate). Two-tailed Student t test: **P < .01; ****P < (C) Ba/F3-MPL cell lines were plated at 30 000 cells and cultured over a period of 72 hours in the absence or presence of either 0.05 ng/mL or 5 ng/mL TPO and numbered. Curves show cumulative cell number ± standard deviation of a typical experiment out of 3 performed in triplicate. Insert represents data at 5 ng/mL TPO with an adapted y-axis scale. Caroline Marty et al. Blood 2014;123: ©2014 by American Society of Hematology

5 Signaling studies in Ba/F3-EPOR and Ba/F3-MPL cells and STAT activities in γ2A cells.
Signaling studies in Ba/F3-EPOR and Ba/F3-MPL cells and STAT activities in γ2A cells. (A) Ba/F3-EPOR or (B) Ba/F3-MPL cells expressing the different JAK2 constructs were serum- and cytokine-starved for 6 hours prior to a 15-minute stimulation with (A) 1 U/mL EPO and (B) 0.05 and 5 ng/mL TPO at 37°C, as indicated. Cells were lysed, and the phosphorylation status of STAT1, STAT3, STAT5, AKT, and ERK1/2 was examined by western blotting with the respective anti-phospho specific antibodies, as indicated. Expression of HSC70 in the samples was used as loading control and was consistent with expression of total AKT, ERK1/2, and the individual STAT isoforms. Blots shown were reproduced in 2 independent experiments. The quantification of the phospho-STAT5 (P-STAT5) blots for unstimulated Ba/F3-EPOR and Ba/F3-MPL were shown as P-STAT5/HSC70 ratios (arbitrary units [AU]) and displayed below the figures. (C) JAK2-deficient γ2A cells were transfected to express the various JAK2 mutants in the presence of equal amounts of JAK2 WT and MPL. STAT1-, STAT3-, or STAT5-dependent transcriptional activity was measured 24 hours after transfection by the dual firefly (pGRR5 reporter responding to STATs) and renilla (pRL-TK reporter with constitutive expression) luciferase system. Shown are averages ± SEM of 9 experiments in triplicate. Two-tailed Student t test: *P < .05; **P < .01; ***P < .001. Caroline Marty et al. Blood 2014;123: ©2014 by American Society of Hematology

6 Sensitivity of JAK2 R867Q and JAK2 S755R/R938Q mutants to JAK2 and HSP90 inhibitors.
Sensitivity of JAK2 R867Q and JAK2 S755R/R938Q mutants to JAK2 and HSP90 inhibitors. (A) Ba/F3-MPL cells expressing either JAK2 R867Q, JAK2 S755R/R938Q, or JAK2 V617F could be maintained in WEHI-supplemented medium but also exhibited cytokine-independent growth (Autonomous). Autonomous or WEHI-maintained cells were serum-starved for 6 hours and stimulated, or not (no TPO), with 5 ng/mL TPO for 15 minutes (+TPO). The constitutive phosphorylation level of each of the JAK2 constructs was analyzed by western blotting. The level of P-STAT5 was also detected, and expression of HSC70 served as a loading control. Blots are representative of a typical experiment. (B) Growth of autonomous Ba/F3-MPL cells expressing JAK2 V617F (×), R867Q (▽), and S755R/R938Q mutants (□), as well as WEHI-dependent Ba/F3-MPL expressing JAK2 WT (gray circle), was determined in response to treatment with various concentrations of INCB018424, TG101348, CYT-387, AZ960, (C) the PI3K inhibitor LY , or (D) AUY922. A WST-1 proliferation assay was performed after 72 hours of exposure to the inhibitors, in the presence of 5 ng/mL TPO. Data (means ± SEM) were calculated as percentages of vehicle-treated cells and were conducted in duplicate in 4 independent experiments. Significant differences for R867Q and S755R/R938Q mutants compared with JAK2 WT were found for all inhibitors except for LY (P < .05 using the 2-tailed Student t test). (E) The 50% inhibitory concentration (IC50) values of cytokine-independent Ba/F3-MPL cells exposed to inhibitors for 72 hours were calculated using GraphPad PRISM software. (F) Coimmunoprecipitation (IP) was performed in Ba/F3-MPL cells using an anti-JAK2 antibody and blotted for the presence of HSP90 and MPL. Cell homogenates (H) show the amount of proteins before the IP. A quantification of both HSP90 and JAK2 in the IP fractions using ImageJ software (National Institutes of Health) was performed, and the histogram represents the HSP90/JAK2 ratios in AU. Blots shown were reproduced in 2 independent experiments. Caroline Marty et al. Blood 2014;123: ©2014 by American Society of Hematology

7 Effect of JAK2 mutations on protein stability and chaperone function for MPL cell-surface expression. Effect of JAK2 mutations on protein stability and chaperone function for MPL cell-surface expression. (A) Ba/F3-MPL cells expressing either JAK2 WT, V617F, R867Q, or S755R/R938Q and maintained in WEHI-supplemented medium were treated with CHX (50 μg/mL) for 0, 0.5, 1, 2, 5, 8, and 24 hours. Levels of both total JAK2 and MPL proteins were examined by western blotting, and β-actin serves as loading control. Table shows means ± SEM of the half-lives (T1/2) of JAK2 WT and mutants and mature cell-surface MPL interpolated from y = 0.5 on the curves corresponding to half of the protein remaining in CHX-treated cells compared with CHX-untreated cells (n = 3). Significance compared with JAK2 WT T1/2 or cell-surface MPL T1/2 in presence of JAK2 WT was assessed using the 2-tailed Student t test. *P < .05; **P < .01; ***P < (B) Ba/F3 cells expressing the FLAG-tagged MPL and transduced with the bicistronic retroviral pMIGR-IRES-GFP vector encoding either JAK2 WT, V617F, R867Q, S755R/R938Q, S755R, or R938Q were sorted for equal GFP levels and maintained in IL-3–supplemented medium. GFP expression allowed monitoring of JAK2 level in the various cell lines, and MPL cell-surface expression was assessed by flow cytometry using PE fluorescence labeling of the extracellular FLAG tag. Histogram shows the mean fluorescence intensities (MFIs) of PE-labeled cell-surface MPL. (C) Histogram shows means ± SEM of PE MFI in fold related to Ba/F3-MPL cells with no overexpression of JAK2 (Endog.) (n = 3). Significance compared with the Ba/F3–MPL–JAK2 WT condition was calculated using the 2-tailed Student t test. *P < .05. Caroline Marty et al. Blood 2014;123: ©2014 by American Society of Hematology

8 Assessment of myeloid progenitor amplification and signaling in platelets from JAK2-mutated patients from families 1 and 2. Assessment of myeloid progenitor amplification and signaling in platelets from JAK2-mutated patients from families 1 and 2. Mononuclear cells from R867Q (n = 2) patients, S755R/R938Q (n = 4) patients, or control donors (n = 5) were purified and plated (A-B) in methylcellulose with SCF/IL-3/EPO to study BFU-E progenitors or (C-D) in serum-free fibrin clot with SCF and increasing doses of TPO, (E) with 10 ng/mL TPO to study CFU-MK progenitors. Data represent means ± standard deviation for each patient plated in triplicate. (F-G) Histograms represent the means ± SEM of CFU-MK progenitors in the presence of SCF and TPO (10 ng/mL) for patients (n = 5 for S755R/938Q, n = 3 for R867Q) and controls (n = 4). Two-tailed Student t test: **P < .01. (H-I) The numbers of MKs per cluster were counted. (J-K) Platelets were isolated from R867Q (n = 2) patients, S755R/R938Q (n = 4) patients, or control donors (n = 3); washed in phosphate-buffered saline; and stimulated or not with TPO (5 ng/mL). Platelets were lysed, and the phosphorylation status of JAK2, STAT1, STAT3, STAT5, AKT, and ERK1/2 was examined by western blotting with the respective anti-phospho specific antibodies, as indicated. Expressions of HSC70 and of total STAT proteins, AKT, and ERK in the samples were used as loading controls. Blots show representative results in 2 S755R/R938Q patients (P1, P3) (J) and 2 R867Q patients (P′2, P′3) (K). Caroline Marty et al. Blood 2014;123: ©2014 by American Society of Hematology


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