Synergistic Induction of Cell Death By Combined Inhibition of PIM and AKT Kinases in Cytogenetically Defined Standard and High-Risk Multiple Myeloma

The PIM and PI3K/Akt pathways have been studied extensively for their effects on cell growth, proliferation and survival and shown to have both distinct and overlapping functions. The PIM proteins (PIM1, 2 and 3) are serine/threonine kinases that are constitutively active, with control of PIM signaling largely at the level of transcriptional regulation. Recent studies have shown PIM2 mRNA to be highly expressed in multiple myeloma (MM) and Akt has previously been shown to be constitutively active in primary MM cells in at least 50% of samples. As results of targeting single signaling pathways can be compromised by compensatory mechanisms, we have investigated the effects of the pan-PIM inhibitor AZD1897 and the Akt inhibitor AZD5363 alone and in combination in MM, utilising a panel of cell lines representing the major MM translocation subtypes and primary patient CD138+ samples.

Western blotting of a panel of human myeloma cell lines (HMCL) and primary MM samples showed that PIM2 is the major PIM isoform expressed. PIM2 protein levels were highest in HMCL with the (4;14) translocation. This finding was confirmed in primary samples where levels were quantified by densitometry (corrected for GAPDH expression) – relative levels in cases with t(4;14) 3.6±1, t(11;14) 1±0.4 and hyperdiploidy 0.7±0.3 (n=5, 6 and 4 respectively; p=0.02 by ANOVA).

PIM and Akt inhibitors reduced proliferation in HMCL but had modest effects on cell death when used on their own. Combined PIM+Akt inhibition led to a more marked reduction in proliferation and produced synergistic cytotoxicity with induction of >50% cell death at 72 hours in 4 of 4 cell lines with t(4;14), 2 of 3 with t(14;16) and 2 of 4 with t(11;14). Chou-Talalay analysis confirmed synergistic effects with combination indices ranging from 0.1 to 0.8. Direct comparison of inhibitors of Akt, PI3K (GDC0941) and mTORC1/2 (KU-0063794) in combination with AZD1897 showed that the PIM+Akt inhibitor combination was most effective at inducing cell death in 8 out of 12 HMCL. As Akt is downstream of PI3K, this result may appear surprising – however, AZD5363 inhibits both Akt and has additional direct activity against p70S6K/S6K1. As previous studies have shown a PI3K-independent component to S6K signaling in some MM cells, this may at least partly explain this phenomenon.

Investigation of downstream signaling showed that PIM inhibition alone led to decreased BAD S112 phosphorylation and of Akt alone to reduced FOXO3 and PRAS40 phosphorylation, confirming blockade of these validated targets at the concentrations employed. Combined PIM+Akt inhibition had enhanced effects compared with either agent alone on mTORC1 outputs, including phosphorylation of 4EBP1 and S6. Importantly, combined inhibition synergised to significantly reduce MCL1 levels in cells that showed marked cell death in response to AZD1897+AZD5363, but not in those that did not, suggesting a mechanistic basis for the cytotoxicity observed.

Next we investigated the effect of PIM and Akt inhibition in 23 primary MM samples, subdivided by FISH analysis into standard risk [t(11;14), hyperdiploidy, normal] or adverse risk [t(4;14), t(14;16), gain of 1q, TP53 deleted] groups. Viable CD138+ cell survival after 72 hours in the standard risk group (n=13) was 74±4% of control for AZD1897(PIM inhibitor), 65±4% for AZD5363(Akt inhibitor) and 50±4% for the combination. The equivalent figures for the adverse risk group (n=10) were 70±4%, 57±8% and 38±6%, indicating that dual PIM and Akt targeting is effective across all clinical risk categories.

We also assessed the effect of the combination of individual PIM or Akt inhibitors with other anti-MM agents. In HMCL treated with dexamethasone, enhanced effects were seen particularly for the AZD5363+Dex combination eg H929 cells, AZD5363 54% survival, Dex (10nM) 95%, both 15%; KMS28BM 59%, 90%, 28% respectively. Dual PIM+Akt inhibition could also enhance the effects of bortezomib in some HMCL eg H929 PIM+Akt 81% survival, BTZ (25nM) 73%, combination 28%.

In conclusion, combined inhibition of PIM (AZD1897) and Akt (AZD5363) kinases resulted in significant anti-MM activity across all cytogenetically defined clinical risk groups, with synergistic induction of cell death. Biomarkers of activity included mTORC1 targets such as p4EBP1 and pS6 and also MCL1 levels. These results provide a rationale for clinical studies of combined PIM and Akt inhibition in MM.