We also investigated the effects of PU H71 in MUTZ five cells, a human acute lymphoblas tic leukemia cell line a short while ago described to get a JAK2R683G mutation, and identified that this JAK2 mutant lymphoid cell line was also sensitive to PU H71. These data demonstrate that JAK2V617F/MPLW515L mutant cells are uniformly sensitive to PU H71 and suggest HSP90 inhibition may perhaps inhibit the proliferation of JAK2 mutant/dependent cells in additional malignancies. We following investigated the effects of HSP90 inhibition on sig nal transduction pathways in JAK2/MPL mutant and wild style hematopoietic cell lines. Treatment with PU H71 markedly decreased phosphorylation of JAK2 in Ba/F3 EPOR JAK2V617F and Ba/F3 MPLW515L cells. We also observed dose depen dent inhibition of downstream signaling pathways, which includes phos phorylation of STAT3, STAT5, and MAP kinase, at physiologically achievable concentrations.
We observed potent inhi bition of downstream signaling pathways in JAK2V617F good UKE one cells but not in JAK2V617F unfavorable THP one cells. Equivalent effects on signaling in Ba/F3 cells expressing JAK2/MPL mutations and in JAK2V617F mutant human leukemia cell lines were observed with 17 DMAG. JAK2 can be a HSP90 client protein and associates selleck chemical with PU H71/HSP90. Given that PU H71 potently inhibited development and signaling with the distinct JAK2 dependent cell lines, we subsequent evaluated wheth er PU H71 mediated HSP90 inhibition led to JAK2 degradation. Western blot analysis showed that PU H71 or 17 DMAG deal with ment led to dose dependent degradation of total JAK2 in the two isogenic and leukemic cell lines at con centrations associated with inhibition of growth and signaling. Of note, degradation of the two JAK2 and Raf1, a recognized HSP90 client protein, was observed at similar concentrations of PU H71.
We mentioned comparable final results in cells ectopically expressing MPLW515L alone or with overexpression of JAK2, demonstrating PU H71 treatment results in JAK2 degrada tion and inhibition of signaling in cells expressing endogenous selleck chemical GX15-070 or improved amounts of JAK2. We subsequent established irrespective of whether JAK2 is usually a bona fide HSP90 chaperone client protein. Immunoprecipitation experiments in Ba/F3 cells expressing JAK2/MPL mutants and in JAK2V617F mutant and wild kind leukemia cells demonstrated that JAK2 particularly associates with HSP90. Addi tionally, we demonstrated precipitation of JAK2 and HSP90 by PU H71 coated agarose beads, confirming direct engagement of your JAK2 HSP90 complicated by PU H71. Of note, PU H71 treatment resulted in JAK2 degradation in JAK2 mutant, MPL mutant, and in JAK2 wild kind cells.
This suggested to us that unphosphory lated, wild form JAK2 is also an HSP90 client protein; in help of this, we observed the association of JAK2, HSP90, and PU H71 in JAK2 wild type THP 1 cells.