Efficacy Of ABT-199 In Multiple Myeloma

Great strides have been made in the last 15 years in the treatment of multiple myeloma, with FDA approval of both IMIDs and proteasome inhibitors, 10-year survival rates are now achievable in around 25% of patients. However, for the remaining 75%, disease relapse due to drug resistance remains a significant clinical concern. Therefore novel therapeutic approaches to be used in combination with the current active agents are required.

The Bcl-2 family of proteins regulates apoptosis and presents an exciting target for therapy. We have demonstrated that myeloma cell lines can be dependent on the anti-apoptotic protein Mcl-1 or can be co-dependent on Mcl-1 and Bcl-2/x_L for survival. The distinction between Mcl-1 dependence and co-dependence is the distribution of the pro-apoptotic BH3-only protein Bim. In Mcl-1-dependent lines, Bim is primarily associated with Mcl-1. In contrast in the co-dependent lines, Bim is either predominately associated with Bcl-2/x_L or when it is released from Bcl-2/x_L it cannot bind to Mcl-1 because of the presence of the Mcl-1 inhibitor, Noxa. This renders these cells sensitive to the Bcl-2/x_L inhibitor ABT-737. We have confirmed these findings in freshly isolated patient samples and demonstrated that among the 5 patient samples tested, Bim was associated with both Mcl-1 and Bcl-x_L and the cells were sensitive to ABT-737. This suggests that co-dependence on Mcl-1 and Bcl-2/x_L may be a common phenomena in myeloma. However, given the adverse effects seen with Bcl-x_L inhibition, ABT-737 or the related compound Navitoclax may be difficult to use for the treatment of multiple myeloma. However, ABT-199, a Bcl-2-specific inhibitor, has been developed and we report on preclinical testing in multiple myeloma.

Freshly isolated plasma cells from 3 myeloma patients were treated with either ABT-737 or ABT-199 for 24 h and IC50s for each drug were compared. In each patient sample, the plasma cells were less sensitive to ABT-199 than to ABT-737 (MM49: 199 IC50 1.4 μM vs. 737 IC50 0.9 μM; MM51: 199 IC50 0.34 μM vs. 737 IC50 0.07 μM; MM52: 199 IC50 1.3 μM vs. 737 IC50 0.25 μM), and only MM51 was truly sensitive to ABT-199. CoIP studies in MM51 revealed very little Bim bound to Mcl-1 and none bound to Bcl-x_L, suggesting the majority of the Bim present in the cell must be bound to Bcl-2. These data suggest that myeloma cells are more likely to be co-dependent on Mcl-1 and Bcl-x_L for survival than Mcl-1 and Bcl-2. However Bcl-2 dependence can exist in myeloma.

We also performed dose curves in 4 multiple myeloma cells lines, representing 4 different Bim binding patterns and sensitivity to ABT-737. 8226, MM.1s, and KMS18 are all co-dependent cell lines (sensitive to ABT-737), while KMS11 is Mcl-1 dependent. Of these cell lines, KMS18 is the only one with the majority of Bim bound to Bcl-2, therefore should be sensitive to ABT-199. Surprisingly this was not the case. 8226 was the only cell line that was sensitive to ABT-199 (IC50 2.2 μM), while MM1.s, KMS18, and KMS11 were insensitive with IC50s of 6.2 μM, 6.8 μM, and 24.7 μM respectively.

In order to gain a mechanistic understanding of these data, we performed CoIP studies to determine the pattern of Bim binding to Mcl-1, Bcl-x_L, and Bcl-2 following treatment with ABT-199. We found in KMS18 cells, upon treatment, Bim is released from Bcl-2 and bound by Mcl-1 thereby preventing apoptosis. We have already demonstrated that very little Bim is bound to Bcl-2 in KMS11 and MM.1s, which is consistent with the lack of sensitivity in these cell lines. However in 8226 cells, the high expression of Noxa prevents the Bim released from Bcl-2 from binding to Mcl-1, thereby promoting apoptosis. Silencing of Noxa in this cell line raises the IC50 of ABT-199 2-fold.

Next we investigated the effectiveness of combining ABT-199 with the proteasome inhibitor carfilzomib, which has been shown to be a potent inducer of Noxa. A synergistic response to this drug combination was seen in KMS18 cells. CoIP studies revealed that in the presence of Cz, the Bim released from Bcl-2 by ABT-199 could no longer bound to Mcl-1, and was free to activate Bak and Bax. Only additive responses were seen in 8226, MM.1s and KMS11 cell lines.

Taken together these data suggest ABT-199 alone may only be an effective treatment for multiple myeloma in a small subset of patients. However, combining it with either Noxa inducers or Mcl-1 inhibitors could be a promising approach for the treatment of this disease.