Co-Treatment with JAK2 Inhibitor TG101209 and Panobinostat or hsp90 Inhibitor AUY922 Attenuates Mutant JAK2-V617F Levels and Activity in Human Myeloproliferative Disorder Cells

The mutant JAK2-V617F is present and constitutively active in myeloproferative disorders (MPD), including polycythemia vera (PV), essential thrombocytosis and primary myelofibrosis (MF). JAK2-V617F activates the signal transduction through the STAT, RAS/MAPK and PI3K/AKT pathways, conferring proliferative and survival advantage in the MPD hematopoietic progenitor cells (HPCs). We have previously reported that the panhistone deacetylase (HDAC) inhibitors e.g., panobinostat (Novartis Pharmaceuticals Inc), induces growth arrest and apoptosis of neoplastic HPCs. Panobinostat (PS), by inhibiting HDAC6, induces hyperacetylation of heat shock protein (hsp) 90, which attenuates the ATP binding and chaperone function of hsp90. This is associated with polyubiquitylation and degradation of hsp90 client protein signaling kinases, e.g, FLT-3, Bcr-Abl, AKT and c-RAF. Additionally, the mutant versions of signaling oncoprotein kinases, e.g., FLT-3, Bcr-Abl, B-RAF and c-KIT, may be even more dependent on hsp90 chaperone function for maintaining their proper folding and active conformation. This makes the mutant oncoprotein kinase even more susceptible to misfolding and proteasomal degradation upon inhibition of hsp90 function. In the present studies we determined the effects of PS or the non-geldanamycin analogue hsp90 inhibitor (AUY922, Novartis) and/or the JAK-2 inhibitor TG101209 (TG) (TargeGen, San Diego, CA) in the cultured (human erythroleukemia HEL and mouse BaF3 cells expressing JAK2-V617F) and primary human MPD cells (from two patients with PV and MF. Treatment with PS (10 to 30 nM) dose-dependently increased % of cells in G1 and decreased % of cells in S phase of cell cycle, as well as induced apoptosis of HEL and BaF3/JAK2-V617F cells. This was associated with attenuation in the levels of JAK2-V617F, p-JAK2, p-STAT3, p-STAT5, p-AKT (serine 473), AKT, p-ERK1/2, p-GATA1 (serine 310), GATA1 and Bcl-xL in HEL cells. PS induced lysine acetylation of hsp90 but not of JAK2. Similar effects were observed, following treatment with AUY922 (AUY). Both PS and AUY induced significantly less apoptosis of BaF3/hEpoR versus BaF3/JAK2-V617F cells. PS and AUY also reduced the chaperone association of JAK2-V617F with hsp90. Treatment with TG (0.2 to 2.0 uM) also dose-dependently attenuated the levels of p-STAT3, p-STAT5, p-AKT (serine 473), AKT, p-ERK1/2, p-GATA1 (serine 310) and Bcl-xL, which was associated with apoptosis of HEL and BaF3/JAK2-V617F cells. Co-treatment with PS and TG induced more apoptosis of the cultured and primary MPD cells than treatment with either agent alone. AUY also increased apoptosis of HEL cells induced by TG. Co-treatment with PS and AUY synergistically induced apoptosis of HEL cells (combination indices < 1.0). Co-treatment with 20 nM of PS or 15 nM of AUY augmented TG mediated attenuation of p-STAT3, p-STAT5 and Bcl-xL levels in HEL and BaF3/JAK2-V617F cells. These findings demonstrate for the first time that pan-HDAC inhibitor PS and hsp90 inhibitor AUY attenuate the levels and signaling of JAK2-V617F and enhance the anti-JAK2-V617F activity of TG in human MPD cells. Our findings also support the rationale to determine the in vivo activity of PS or AUY in combination with JAK2 inhibitors against human MPD.