Dual inhibition of Mcl-1 by the combination of carfilzomib and TG02 in multiple myeloma

Abstract

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.

TG02 inhibits proteasome inhibitor–induced HSF1 serine 326 phosphorylation and heat shock response in multiple myeloma

“…This is consistent with our previous findings with carfilzomib. 10 Taken together, our data show that TG02 inhibits pS326 in MM cell lines and patient samples.…”

“…Consistent with previous findings, the combination of TG02 and bortezomib results in a strong additive effect on apoptosis, and we confirm our previous findings that the combination of TG02 and carfilzomib results in an additive effect in MM.1s cells ( Figure 2B). 10,13 We observe that TG02 strongly inhibits PI-pS326, PI-induced HSP27 upregulation, and PI-induced HSP40 upregulation in H929 cells ( Figure 2C, left). TG02 also inhibits PI-pS326 in U266 cells but does not lead to HSP inhibition ( Figure 2C, right).…”

Positive transcription elongation factor b (P-TEFb) is a therapeutic target in human multiple myeloma

“…Such findings, along with the observation that Mcl-1 knock-down analogously increased proteasome inhibitor lethality, argue that these actions reflect disabling of Mcl-1 cytoprotective activities. However, there are alternative mechanisms by which CDK inhibition may promote cell death in multiple myeloma and other cell types including a) enhanced DNA damage [ 41 ]; b) disruption of cytoprotective autophagy [ 42 ]; and c) disabling of cytoprotective ER stress responses [ 43 ], both of which can be triggered by BH3-mimetics and proteasome inhibitors [ 23 , 44 ]. It is therefore possible that these actions may contribute to the activity of these combinations.…”

“…Although limited trials of CDK9 inhibitors have not yet established clear single-agent activity in MM [ 17 , 45 ], it is possible that sub-optimal scheduling or pharmacokinetics may have limited the efficacy of such agents to date [ 46 ]. However, it is likely that the ultimate value of such agents may lie in rational combinations e.g., with BH3-mimetics or proteasome inhibitors, particularly in the setting of resistant disease [ 23 , 44 ] or disease characterized by pronounced P-TEFb activation. In this regard, clinical studies suggest that dinaciclib may have some, albeit limited single-agent activity in MM [ 17 ], and initial trials combining dinaciclib with bortezomib show some promise [ 30 , 45 ].…”

Exploiting the pro-apoptotic function of NOXA as a therapeutic modality in cancer

“…Indeed, some cell lines have undetectable Noxa, either at mRNA or protein level, while other myeloma cell lines express very high levels of endogenous NOXA [120]. Since the vast majority of MM cells are known to be strongly MCL1 dependent for survival [121,122], NOXA is expected to play a major role in the induction of cell death through the neutralization of MCL1 [77, 123,124]. In accordance, S63845 is effective in MM cells that depend on MCL1 [108].…”