A Newly Developed IAP Inhibitor (IAPi) Induces Apoptosis of Human Myeloma Cells and Synergises with Conventional and Novel Anti-Myeloma Therapeutics

The IAPs (inhibitor of apoptosis proteins) are a family of 8 structurally related caspase inhibitors that efficiently block the execution phase of apoptosis induced by various stimuli including death receptor activation. Based on reports of elevated expression of IAPs in multiple myeloma (MM) we evaluated the therapeutic potential of a novel IAP (IAPi) in MM. A panel of 9 genetically heterogeneous human myeloma cell lines (HMCL) were screened and found to be sensitive to IAPi with an IC₅₀ of 25 to 50μM in a dose and time dependent manner. Induction of cell death was confirmed by Annexin V and propidium iodide staining of treated HMCL. Mechanistic studies were then undertaken utilising 3 HMCL with high (NCI H929), intermediate (LP-1) or low (OPM2) sensitivity to IAPi with QRT-PCR demonstrating the highest expression of XIAP in the most IAPi sensitive HMCL NCI H929. In all 3 HMCL IAPi exposure was associated with rapid cleavage of caspase 3, caspase 7, ROCK-1, cytosolic degradation of ICAD, nuclear translocation of CAD and PARP cleavage confirming cell death via apoptosis. Replicate experiments utilising caspase specific inhibitors and siRNA knock-down of caspases 3 and 7 confirmed this to be both caspase 3 and caspase 7 dependent. Co-culture of HMCL with conditioned media from the human stromal cell line HS-5 or primary MM bone marrow mononuclear cells reproducibly demonstrated enhanced IAPi-induced HMCL apoptosis with a median increase in HMCL killing of 1.2 fold compared to HMCL treated in standard culture media. Similarly, IAPi activity was not abrogated but enhanced in the presence of either IL-6 or IGF-1 (1.1 and 1.3 fold, respectively) when compared to HMCL treated without the addition of exogenous growth factors. Primary MM cells from 9 multiply relapsed MM patients were treated with IAPi in an autologous bone marrow co-culture assay with concentrations of IAPi ranging from 10–100μM inducing a median of 23% (range, 8–56% as determined by Apo2.7 staining) MM cell killing at 48 hours. Finally the anti-MM activity of IAPi in combination with other conventional (etoposide, adriamycin, velcade) and novel (HSP90 inhibitor, agonistic TRAIL DR5 monoclonal antibody) therapeutics was investigated against both HMCL and primary MM tumour samples. All combinations showed synergistic cell killing with Combination Indices of <1 (Calcusyn) when compared to either agent alone. We conclude that IAPi induces apoptosis of MM cells via a caspase 3 and caspase 7 dependent process. Furthermore, IAPi retains anti-MM activity in the context of pro-survival cytokine exposure and induces synergistic killing of MM cells when combined with both conventional and novel anti-MM therapeutics. Based on these preliminary observations further investigation of the role of IAPi as a potential tumour sensitising agent for the therapy of MM is justified.