Synergistic Activity of Combinations of JAK2 Kinase Inhibitor with PI3K/mTOR, MEK or PIM Kinase Inhibitor Against Human Myeloproliferative Neoplasm Cells Expressing JAK2V617F

Abstract 798

The mutant JAK2-V617F tyrosine kinase (TK) is present in the majority of patients with BCR-ABL negative myeloproliferative neoplasms (MPNs). JAK2-V617F activates downstream signaling through the STAT, RAS/MAPK and PI3/AKT pathways, conferring proliferative and survival advantages in the MPN hematopoietic progenitor cells (HPCs). We have previously reported that pan-histone deacetylase (HDAC) inhibitors e.g. panobinostat (PS) (Novartis Pharmaceuticals), depleted mRNA expression of JAK2-V617F, and disrupted the chaperone association of with hsp90 with JAK2-V617F, thereby promoting the degradation of JAK2-V617F by the proteasome. This led to attenuation of the levels and downstream transcriptional activity of STAT3 and STAT5, resulting in growth arrest and apoptosis of MPN HPCs. Additionally, co-treatment with PS and a JAK2 kinase inhibitor, TG101209, further depleted JAK/STAT signaling and synergistically induced apoptosis of JAK2-V617F expressing HEL92.1.7 and Ba/F3-JAK2V617F cells, as well as exerted greater lethality against primary CD34+CD38-Lin- MPN versus normal CD34+ HPCs. In the present studies, we determined the cytotoxic effects of inhibiting JAK2-STAT3/5 in conjunction with pharmacologic targeting of the collateral, pro-growth and pro-survival signaling through PI3K/AKT, RAF-MEK or PIM1 kinases in MPN cells. For this, the cytotoxic effects of co-treatment with TG101209 and the MEK inhibitor (AZD6244, AstraZenaca), dual PI3K/mTOR inhibitor (BEZ235, Novartis) or the PIM1 kinase inhibitor (SGI-1776, SuperGen) were evaluated in HEL92.1.7, Ba/F3-JAK2V617 and primary human MPN cells. Treatment with BEZ235 dose-dependently attenuated the levels of p-JAK2, p-STAT5, p-STAT3, p-AKT, p-ERK1/2 and p-4EBP1. Co-treatment with BEZ235 and TG101209 was synergistically lethal against the cultured MPN and primary CD34+ MF-MPN cells (combination indices < 1.0). Co-treatment with AZD6244 and TG101209 also induced synergistic apoptosis of cultured MPN cells (combination indices of < 1.0). This was associated with greater attenuation of the levels of p-AKT and p-ERK1/2. PIM1 is a cytoplasmic serine/threonine kinase that serves as a downstream effector of several cytokine signaling pathways promoting cell survival and proliferation. PIM1 collaborates in Myc-induced transformation and known to phosphorylate 4EBP1 and eIF4B, thereby promoting protein translation. Co-treatment with TG101209 and the PIM1 kinase inhibitor, SGI-1776 also induced synergistic apoptosis of HEL92.1.7 cells and Ba/F3-JAK2V617F cells (combination indices < 1.0) but not of Ba/F3-hEpoR cells. PIM kinase mediates PRAS40 phosphorylation and induces mTORC1 activity in phosphorylating 4EBP1. Consistent with this, co-treatment with SGI-1776 and TG-101209 inhibited p-PRAS40 and p-4EBP1 levels in cultured MPN but not in normal progenitor cells. These findings demonstrate for the first time that combined treatment with a MEK inhibitor, PIM1 kinase inhibitor or dual PI3K/mTOR inhibitor enhances the anti-JAK2-V617F activity of TG101209 in cultured and primary human MPN cells. Our findings support the rationale to determine the in vivo activity of TG101209 in combination with inhibitors of MEK, PIM1 or PI3K/mTOR kinase against human MPN cells.