Pharmacologic loss of mutated JAKs can overcome persistence to ruxolitinib. (A) Proliferation of JAK-inhibitor persistent BaF/3 cell lines (JAK1V658F and JAK2R683G) with ruxolitinib. Cell growth relative to DMSO-treated control is shown. Data from wells plated in triplicates (s.d.) are shown and are representative of 3 independent experiments. (B) Western blot analysis revealing retained phosphorylation of JAK downstream targets Stat3 and Stat5 in persistent cells (BaF/3 JAK2R683G) treated with increasing concentrations of ruxolitinib. (C) PU-H71 inhibits potently the proliferation of ruxolitinib-persistent cells. Cell growth relative to DMSO-treated control is shown. Data from wells plated in triplicates (s.d.) are shown and are representative of 3 independent experiments. In each case, IC₅₀ values are displayed. (D) Treatment with HSP90 inhibitor PU-H71 strongly reduces constitutive activation of signaling pathways downstream of mutant JAKs and causes degradation of total and phosphorylated JAK2 in ruxolitinib-persistent cells. Protein analysis of whole-cell lysates from BaF/3 JAK1V658F and CRLF2 wt/JAK2R683S cell lines is shown. Tubulin is shown as loading control. (E) Western blot analysis revealing that activation of Tyk2 is restricted to JAK-inhibitor persistent BaF/3 cell lines. (F) Immune precipitation of Jak1 and Tyk2 from JAK2R683G-expressing BaF/3 cells demonstrated that both tyrosine kinases only directly interact with phosphorylated Jak2 in persistent cells. (G) Immune precipitation of Hsp90 and Jak1 from ruxolitinib-persistent JAK1V658F-mutant cell lines (MOHITO, left panel; BaF/3, right panel) resulted in coprecipitation of the kinase and the chaperone protein with PU-H71–coated beads. Ctrl, control; DMSO, dimethylsulfoxide; IB, immunoblot; IP, immunoprecipitation; log, logarithmic; p, phosphorylated form; pers, ruxolitinib-persistent cells; s.d., standard deviation; sens, ruxolitinib-naive cells; wt, wild type.