The Biologic Relevance of PIM Kinases in the Context of Multiple Myeloma and Their Potential As Therapeutic Targets in Combination Drug Therapy

The proto-oncogene proviral integration sites for moloney murine leukemia virus (PIM) are serine/threonine kinases currently under investigation as therapeutic targets in hematologic cancers, where they have important functional roles as mediators of apoptosis, cell migration and homing. Thus far in Multiple Myeloma (MM), the PIM Kinases have been shown to mediate cap-dependent and cap-independent translation through the PI3K/AKT pathway and the activation of mTOR signaling (Lu J et al. 2013). In the context of the bone marrow microenvironment, PIM kinase expression in MM cells is known to be elevated in the presence of bone marrow stromal cells, and is mediated by the IL-6/STAT3 pathway, as well as the TNFα/NFΚB pathway (activated by Osteoclast secretion) (Hiasa M et al. 2014). Here, we have further studied the role of PIM kinases in MM. We observed elevated expression of PIM 1, 2 and 3 in patient derived myeloma cells (CD138+) as opposed to the stromal compartment (CD138-), confirming that PIM expression is predominantly hematologic lineage-specific. Inhibition of all three kinases by a pan PIM inhibitor results in reduced cell viability in tested human-derived MM cell lines, reinforcing the importance of the kinases as targets in drug therapy, as shown previously. Because the expression of Pim2 is consistently higher in primary tumor cells as well as in human MM lines, compared to Pim1 and Pim3, we sought to identify their distinct biologic significance.

Single knock down (KD) of each kinase resulted in varying effects on cell viability, suggesting, together with the PIM expression profile, that the three kinases play different roles in the biology of MM. As the Pim2 KD resulted in the most profound decrease in cell viability, we focused our efforts to dissect the mechanistic importance of Pim2. Since Pim1 and Pim2 regulate the DNA damage response (DDR) via checkpoint kinase 1 (Chk1) in other hematologic malignancies, we investigated the effect of Pim2 KD on major signaling factors involved in the DDR. Transient KD resulted in phosphorylation of DDR pathway markers including ATR, CHK1/2, P21 and H2AX, and mimicked the effects of Doxorubicin treatment (a known DNA Damage causing agent). Furthermore, Doxorubicin treatment downregulated Pim2 expression, suggesting that Pim2 functions as an upstream regulator of the DDR pathway in MM.

Pim2 appears to be the most relevant target in MM; however, because of a lack of a specific PIM2 inhibitor we used the pan-PIM inhibitor as a tool compound. Although the pan PIM kinase inhibitor showed single agent activity, combination approaches were more efficacious. Combining the pan-PIM inhibitor with bortezomib shows a significant synergistic effect on cell viability in multiple MM cell lines (MM1S, U266, KMS-12BM). Based on our KD experiments Pim2 predominantly mediates cell viability, suggesting that specifically targeting Pim2 in combination with Bortezomib will have a more direct effect on MM cell survival. To confirm the role of Pim2, ongoing experiments aim to focus on the effects of ectopically expressing Pim2 in the context of myeloma. We propose that Pim2 overexpression will not only serve anti-apoptotic purposes, but will further protect against DNA Damage in human MM cell lines.