Dual Inhibition Of Mcl-1 By The Combination Of Carfilzomib and TG02 In Multiple Myeloma

Carfilzomib (Kyprolis^TM) is a proteasome inhibitor that is FDA approved for single-agent use in relapsed and refractory multiple myeloma (MM). In order to enhance the therapeutic efficacy of carfilzomib, we sought to combine carfilzomib in a rational approach with other novel agents. TG02 is a multi-kinase inhibitor known to target JAK2 and CDK9. The rationale for co-treatment with carfilzomib and TG02 is that both independently target Mcl-1 and multiple myeloma cells are dependent on this anti-apoptotic protein for survival.

Four different human myeloma cell lines (HMCL), MM1.s, RPMI 8226, H929 and U266, were initially treated for 24 hours with carfilzomib, TG02 or the combination. MM1.s were the most sensitive to carfilzomib treatment (IC50 8.4 nM) and H929 were the most resistant (IC50 25.5 nM). MM1.s was also the most sensitive to TG02 treatment (IC50 197.6 nM). Co-treatment with carfilzomib and TG02 resulted in at least additive cell death in all four lines (additive in RPMI 8226 and U266, greater then additive in MM1.s and H929). Pulse dosing of carfilzomib was also preformed to better mimic the pharmacokinetics of carfilzomib in the clinic. When cells were pulsed for 1 hour with carfilzomib, then treated with TG02 for 23 hours, or pulsed with both carfilzomib and TG02, then treated with TG02 for 23 hours, similar patterns of activity were observed with continuous dosing. However, higher concentrations of carfilzomib were required to reach an IC50. Activity of the combination was also observed in freshly isolated samples from relapsed/refractory MM patients. Co-culture of HMCL with Hs-5 stromal cells or the conditioned medium from these cells had mixed results. The combination did not overcome protection by co-culture or addition of conditioned medium in RPMI 8226 or KMS18 cells, but did in MM1.s and H929 cells. Interestingly the latter two lines are where synergistic activity of the combination is observed.

We next determined the molecular basis for the increased apoptosis. HMCL were treated with carfilzomib, TG02 or the combination for 24 hours. After 6 hours cells were isolated for western blot analysis and RT-qPCR to determine protein and mRNA levels of the Bcl-2 family of proteins, respectively. Consistent with our hypothesis, treatment with carfilzomib caused an increase in NOXA, an inhibitor of Mcl-1, at the mRNA level in all cell lines. TG02 treatment resulted in a decrease in Mcl-1 protein expression. However, unexpectedly we also observed an increase in NOXA mRNA with TG02 treatment alone. More surprisingly the decrease in Mcl-1 protein did not appear to be due to the predicted decrease in Mcl-1 transcription. Mcl-1 mRNA levels did not change following TG02 treatment. Since the loss of Mcl-1 at the protein level occurs in the presence of carfilzomib, the effect of TG02 is unlikely to be due to increased degradation. Therefore we reasoned that regulation of translation of Mcl-1 is the most likely mechanism and data will be presented on this. Consistent with this possibility over expression of Mcl-1 in RPMI 8226 cells confers significantly less protection than over expression of Bcl-2 or Bcl-x_L. Taken together these data suggest that dual inhibition of Mcl-1 through decreased expression and induction of 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.