Caspase-3 is a cysteine–aspartic acid protease that cleaves cellular targets and executes cell death. Our current understanding is caspase-3 is activated by the cleavage of the interdomain linker and then subsequent cleavage of the N-terminal prodomain. However, previous reports have suggested that removal of the prodomain can result in the constitutive activation of caspase-3, although other studies have not observed this. To address this question in a more physiological setting, we developed an inducible doxycycline system to express a mutant form of caspase-3 that lacks the prodomain (∆28). We found that the removal of the prodomain renders the cells more susceptible to death signals, but the caspase is not constitutively active. To elucidate the regions of the prodomain that regulate activity, we created deletion constructs that remove 10 and 19 N-terminal amino acids. Surprisingly, removal of the first 10 amino acids renders caspase-3 inactive. Following serum withdrawal, the interdomain linker is cleaved, however, the remaining prodomain is not removed. Therefore, there is a specific amino acid or stretch of amino acids within the first 10 that are important for prodomain removal and caspase-3 function. We created different point mutations within the prodomain and found amino acid D9 is vital for caspase-3 function. We hypothesize that an initial cleavage event at D9 is required to allow cleavage at D28 that causes the complete removal of the prodomain allowing for full caspase activation. Together these findings demonstrate a previously unknown role of the prodomain in caspase activation.

Carfilzomib (Kyprolis Ò ), a second generation proteasome inhibitor, is FDA approved for single-agent use among relapsed/refractory multiple myeloma (MM). To enhance the therapeutic efficacy of carfilzomib, we sought to combine carfilzomib with other novel agents. TG02, a multi-kinase inhibitor, targets JAK2 and CDK9. The rationale for co-treatment with carfilzomib and TG02 is that both independently target Mcl-1 and most myeloma cells are dependent on this anti-apoptotic protein for survival. We observed at least additive effects using the combination treatment in MM cell lines and patient samples. To determine how the bone marrow environment affects the efficacy of the combination we conducted co-culture experiments with Hs-5 stromal cells. We also examined the mechanism of increased apoptosis by determining the affect on expression of the Bcl-2 family of proteins. We found that carfilzomib increases NOXA mRNA expression, as expected, and TG02 treatment caused a decrease in Mcl-1 protein but not mRNA levels. Consistent with this possibility, we find silencing CDK9 does not change carfilzomib sensitivity in the same manner as addition of TG02. Since changes in Mcl-1 protein occur in the presence of a proteasome inhibitor we hypothesize that regulation of Mcl-1 translation is the most likely mechanism. Taken together our data suggest that dual inhibition of Mcl-1 via decreased expression and the induction of its antagonist NOXA by the combination of carfilzomib and TG02 is active in myeloma and warrants further testing preclinically and in clinical trials. Moreover, regulation of Mcl-1 by TG02 is more complex than initially appreciated.