Specific inhibition of JAK2 V617F signaling by modulation of hTpoR conformation. (A) Illustration of 2 strategies used to specifically inhibit JAK2 V617F-driven activation of hTpoR. Left: Helical wheel diagram positioned at interface V. Asparagine and glutamine residues facing each other in an α-helix have the property to form hydrogen bonds to stabilize the interface in which they lie. Mutation of Q516 to another residue (except asparagine) is predicted to disrupt the JAK2-V617F–specific active interface. Right: Glycines positioned at regular positions on the same face of an α-helix form the so-called glycine zipper, which can stabilize a specific interface. (B) STAT5 transcriptional activity measured by luciferase assay of HEK293T transiently transfected with hJAK2 WT or V617F and indicated hTpoR WT or mutants. Values represent the mean (±SD) of 3 independent experiments performed in triplicate. Each dot represents a replicate. (C-H) Left: STAT1/3/5 phosphorylation in Ba/F3 cells expressing either hJAK2 V617F (C-E) or hJAK2 WT (F-H) together with the indicated hTpoR mutants. Values represent the mean fluorescence intensities (MFI) of 3 to 4 independent experiments. Right: Representative flow cytometry measurement of STAT1/3/5 phosphorylation in Ba/F3 cells expressing hJAK2 V617F (C-E) or hJAK2 WT (F-H) and hTpoR mutants. (I-J) Proliferation assay of Ba/F3 cells stably transduced with hJAK2 V617F (I) or hJAK2 WT (J) and indicated as hTpoR WT or mutants. Values represent the mean (±SD) of 3 independent experiments performed in triplicate (N = 3, n = 9). (K) Flow cytometry analysis of cell surface expression of HA-tagged hTpoR WT and indicated mutants on Ba/F3 stably expressing hJAK2 WT or V617F and indicated hTpoR WT and mutants. (L) Representative western blot of HA-hTpoR WT and mutants stably expressed in Ba/F3 cells. (M-N) Luminescent viability assay (CellTiter-Glo) used to measure proliferation of TF-1 cells stably transduced with hJAK2 V617F (M) or hJAK2 WT (N) with the indicated hTpoR mutants. Values represent mean (±SD) of 8 biological replicates (n = 8). (B-K, M-N) Two-way analysis of variance followed by SIDAK multiple comparison test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Specific inhibition of JAK2 V617F signaling by modulation of hTpoR conformation. (A) Illustration of 2 strategies used to specifically inhibit JAK2 V617F-driven activation of hTpoR. Left: Helical wheel diagram positioned at interface V. Asparagine and glutamine residues facing each other in an α-helix have the property to form hydrogen bonds to stabilize the interface in which they lie. Mutation of Q516 to another residue (except asparagine) is predicted to disrupt the JAK2-V617F–specific active interface. Right: Glycines positioned at regular positions on the same face of an α-helix form the so-called glycine zipper, which can stabilize a specific interface. (B) STAT5 transcriptional activity measured by luciferase assay of HEK293T transiently transfected with hJAK2 WT or V617F and indicated hTpoR WT or mutants. Values represent the mean (±SD) of 3 independent experiments performed in triplicate. Each dot represents a replicate. (C-H) Left: STAT1/3/5 phosphorylation in Ba/F3 cells expressing either hJAK2 V617F (C-E) or hJAK2 WT (F-H) together with the indicated hTpoR mutants. Values represent the mean fluorescence intensities (MFI) of 3 to 4 independent experiments. Right: Representative flow cytometry measurement of STAT1/3/5 phosphorylation in Ba/F3 cells expressing hJAK2 V617F (C-E) or hJAK2 WT (F-H) and hTpoR mutants. (I-J) Proliferation assay of Ba/F3 cells stably transduced with hJAK2 V617F (I) or hJAK2 WT (J) and indicated as hTpoR WT or mutants. Values represent the mean (±SD) of 3 independent experiments performed in triplicate (N = 3, n = 9). (K) Flow cytometry analysis of cell surface expression of HA-tagged hTpoR WT and indicated mutants on Ba/F3 stably expressing hJAK2 WT or V617F and indicated hTpoR WT and mutants. (L) Representative western blot of HA-hTpoR WT and mutants stably expressed in Ba/F3 cells. (M-N) Luminescent viability assay (CellTiter-Glo) used to measure proliferation of TF-1 cells stably transduced with hJAK2 V617F (M) or hJAK2 WT (N) with the indicated hTpoR mutants. Values represent mean (±SD) of 8 biological replicates (n = 8). (B-K, M-N) Two-way analysis of variance followed by SIDAK multiple comparison test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

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