Synergistic Activity of Co-Treatment with PIM1 Kinase Inhibitor SGI-1776 and Histone Deacetylase Inhibitor Panobinostat or Heat Shock Protein 90 Inhibitor AUY922 against Human CML and Myeloproliferative Neoplasm (MPN) Cells.

Abstract 2651

Poster Board II-627

The oncoprotein PIM1 is a cytoplasmic serine/threonine kinase, which is overexpressed and implicated in the pathogenesis of hematologic malignancies. PIM1 activity promotes cell survival and proliferation, and is a downstream mediator of cytokine receptor signaling involving the JAK/STATs. PIM1 phosphorylates and inactivates Bad, which enhances the anti-apoptotic functions of Bcl-2. PIM1 also mediates the phosphorylation and inactivation of cell cycle inhibitors p21 and p27, thereby promoting progression through G1 and G2/M phases of the cell cycle. PIM1 phosphorylates serine 10 on histone H3, and has been shown to phosphorylate and stabilize Myc, thereby collaborating in Myc-induced transformation. Further, PIM1 is known to phosphorylate 4EBP1 on the same residues (threonine 37 and 46) as mTOR kinase. It can also phosphorylate eIF4B, thereby maintaining high levels of protein translation. PIM1 is known to be chaperoned as a heat shock protein 90 client protein. In the present studies, we demonstrate that treatment with the PIM kinase inhibitor SGI-1776 (SuperGen Inc.) dose-dependently (500 nM to 2.0 μM) depleted p-BAD (Ser112), p-p70S6K, p-4EBP1, p-Ser10 Histone H3 and c-Myc levels, while inducing p27 and p21 expression, in the cultured human CML-BC K562 and erythroleukemia HEL92.1.7 (with homozygous JAK2-V617F mutation). Treatment with SGI-1776 induced apoptosis of K562 and HEL92.1.7 (HEL) cells as well as Ba/F3-hEpoR-JAK2-V617F but not Ba/F3-hEpoR cells. Similar effects of SGI-1776 were observed in primary CML-BC and MPN (myeloproliferative neoplasm) cells. Treatment with the non-geldanamycin analogue hsp90 inhibitor AUY922 (Novartis Pharma) or with the pan-HDAC inhibitor panobinostat (PS) (Novartis Pharma) disrupted the chaperone association of PIM1 with hsp90 and increased binding to hsp70, thereby promoting the proteasomal degradation of PIM1 in K562 and HEL cells. Treatment with PS (20–100 nM) also depleted PIM1 mRNA as measured by qPCR. Co-treatment with SGI-1776 (0.5 to 2.0 μM) and clinically achievable concentrations of AUY922 (e.g., 25 nM) caused greater depletion of pBAD (Ser112), pp70S6K, p4EBP1, cyclin B1, c-Raf, c-Myc, and AKT and synergistically induced apoptosis of K562 and HEL92.1.7 cells (combination indices < 1.0). As compared to treatment with either agent alone, co-treatment with SGI-1776 and AUY922 or PS also exerted greater cytotoxicity against primary CML-BC and MF-MPN cells. Importantly, co-treatment with PS significantly enhanced SGI-1776-induced apoptosis of purified primary CD34+CD38-Lin- hematopoietic stem cells expressing JAK2-V617F derived from a patient with primary myelofibrosis. Taken together, these pre-clinical findings indicate that simultaneously depleting PIM kinase levels by AUY922 or PS and inhibiting PIM1 kinase activity with SGI-1776 induces synergistic apoptosis against cultured and primary CML-BC and MPN cells. Additionally, our findings suggest that co-treatment with PS and SGI-1776 may be an effective treatment strategy against hematopoietic stem cells expressing JAK2-V617F in patients with MF-MPN.