Effect of the Pan-PIM Kinase Inhibitor, AZD1208, Alone and in Combination with Bortezomib in Multiple Myeloma
Authors: Santo Loredana , Ramachandran Janani , Cirstea Diana D , Eda Homare , Nemani Neeharika , Hideshima Teru , Dharminder Chauhan , Huszar Dennis , Anderson Kenneth C. , Raje Noopur 
Published: October 2019

Abstract
The proto-oncogene proviral integration site for moloney murine leukemia virus (PIM) kinases (PIM-1, PIM-2, and PIM-3) are serine/threonine kinases upregulated in multiple cancer cells including multiple myeloma (MM). Our preliminary data show higher expression of PIM 1/2/3 in patient derived tumor cells (CD138+) compared to the stromal compartment (CD138-), confirming that PIM expression is predominant in MM cell compartment. Although PIM2 expression is significantly higher than PIM1 or PIM3 in patient MM cells and in human MM cell lines, transient knockout of PIM1, PIM2 and PIM3 individually showed a similar cytotoxicity in U266 MM cells 24 hours post transfection, suggesting that all three isoforms play a role in MM cell survival and that an inhibitor of all three isoforms would have a therapeutic advantage in MM. AZD1208 is a potent, highly selective PIM kinase inhibitor that effectively inhibits all three isoforms. Here we investigated the preclinical activity of AZD1208 in MM. Maximal cytotoxicity was observed in 72h culture, with IC50 of 1µM in KMS-12BM.
AZD1208 1µM inhibited the phosphorylation of BAD at Ser-112 (a direct PIM kinase substrate) and the phosphorylation of P70S6K T389 (downstream of the mTOR-C1 complex) in KMS12-BM, MM1S and MM.1R MM cells, confirming that PIM kinases regulate mTOR activity. The effect on downstream signaling pathways correlated with the inhibitory effect on KMS-12BM, but not MM1S and MM1R, cell survival, suggesting alternative feedback mechanisms of resistance. AZD1208 (2µM) induced increased PARP cleavage after 24 hr in KMS-12BM cells; however, only high doses of AZD1208 (10µM) induced apoptotic signaling cascade in MM1S and MM1R cells. Since Checkpoint kinase 1 (Chk1) is a critical component of the DNA damage response and is regulated by PIM1 and PIM2 in other hematologic malignancies, we further investigated the effect of AZD1208 on major signaling checkpoints involved in the DNA damage response. Increasing doses of AZD1208 induced phosphorylation of Chk1 (Ser 345), Chk2 (Thr68), histone H2A family member H2A.X (Ser 139), and p53 (Ser15) after 4h of treatment in KMS-12BM cell lines, suggesting that cell death induced by AZD1208 is a consequence of the DNA damage response.
We next examined the combination treatment of AZD1208 with bortezomib, which is not known to induce DNA damage. A synergistic cytotoxic effect was observed in MM1S, U266 and KMS-12BM. Importantly, PIM1 and PIM2 expression was increased by bortezomib, suggesting that PIM1 and PIM2 are substrate of proteasome. On going studies aim at understanding the mechanism underlying this synergistic combination and in validating its efficacy in vivo in order to provide the rationale for clinical protocols of PIM kinase inhibitors in MM.

Disclosures
Hideshima: Acetylon Pharmaceuticals: Consultancy. Huszar:AstraZeneca: Employment, Equity Ownership. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership. Raje:novartis, Amgen, Celgene, Millenium, Onyx: Consultancy; Eli Lilly, Acetylon: Research Funding.