Fig. 1: A chemical mutagenesis screen identifies of ruxolitinib resistant JAK2 mutations L902Q and L983F. | Nature Communications

Fig. 1: A chemical mutagenesis screen identifies of ruxolitinib resistant JAK2 mutations L902Q and L983F.

From: Efficacy of JAK1/2 inhibition in murine myeloproliferative neoplasms is not mediated by targeting oncogenic signaling

Fig. 1

A Immunoblot analysis of JAK2-V617F and JAK2V617F + L902Q expressing Ba/F3 cells in presence of indicated concentrations of ruxolitinib. The samples derive from the same experiment but different gels for pJAK2, total JAK2, HSP90, another for pSTAT5, total STAT5 were processed in parallel. Uncropped images are provided as a source data file. B MTT incorporation assay of JAK2-V617F and JAK2V617F + L902Q expressing Ba/F3 cells in presence of indicated ruxolitinib concentrations. Data represent mean ± SEM. Three independent experiments were performed. One representative experiment (n = 3) was shown. C MTT incorporation assay of JAK2-V617F, JAK2V617F + L902Q, and JAK2-V617F + L983F expressing Ba/F3 cells in presence of indicated ruxolitinib concentrations. Data represent mean ± SEM. Three independent experiments were performed. One representative (n = 3 technical replicates) experiment was shown. D Immunoblot analysis (n = 2) of JAK2-V617F, JAK2V617F + L902Q and JAK2-V617F + L983F expressing Ba/F3 cells in presence of indicated concentrations of ruxolitinib. The samples derive from the same experiment but different gels for pSTAT5, total STAT5, another for JAK” and β-actin were processed in parallel. Uncropped images are provided as a Source Data file. E The binding of ruxolitinib to the JH1 domain of JAK2 kinase was modeled using the SwissDock tool to understand the structural consequences of mutations conferring resistance to ruxolitinib. Ruxolitinib fits well into the ATP-binding pocket of JAK2 WT. 91% of its solvent accessible surface area is buried in the complex. The drug is held by numerous hydrophobic interactions with residues Leu 855, Val 863, Ala 880, Val 911, Met 929, Leu 932 and Leu 983 that line the binding pocket. Leu 902 does not directly interact with ruxolitinib, however it is close to the binding pocket and its mutation to Gln with a polar side chain significantly disturbs the binding of the inhibitor. While it is still almost completely buried, the propanenitrile and cyclopentyl moieties essentially exchange their positions leading to unfavorable interactions between the Asp 994 side chain and the cyclopentyl ring.

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