Alvocidib Enhances Venetoclax Sensitivity in Unfavorable-Risk Multiple Myeloma Cells In Vitro and In Vivo

Abstract:

Background: Multiple myeloma (MM) is a lymphoproliferative neoplasm involving mature plasma cells and remains incurable. New and more effective treatment options are clearly needed for higher-risk MM groups. The BCL-2-specific BH3-mimetic venetoclax (ABT-199) has been reported to be active in patients with the t(11;14) translocation, an intermediate risk group in response to immunomodulatory drugs (IMiDS) and proteasome inhibitors (PIs), prompting efforts to extend venetoclax activity to include more resistant, higher-risk MM subsets.

Methods:

Effects of the CDK9 inhibitor alvocidib (flavopiridol; FP) on responses to the BCL-2-specific BH3-mimetic venetoclax (ABT-199) were examined in multiple myeloma (MM) cells, including higher-risk sub-types exhibiting low BCL-2/MCL-1 expression profiles and limited sensitivity to venetoclax alone. Cell death and protein expression were evaluated by western blot, and immunofluorescence. Xenograft models, including an orthotopic murine model (NOD/SCID-γ mice) and an immune-competent model (C57BL/KaLwR1 mice) were used to characterize combination effects in vivo.

Results:

Alvocidib (100-200 nM; 24 h) synergistically increased ABT-199 (250-750 nM) lethality in both ABT-199-sensitive and insensitive MM cells, and these interactions were synergistic by Median Dose Effect analysis with CI values < 1.0 in both t(4;14) H929 and OPM-2 cells. Synergistic interactions or markedly enhanced lethality were also observed in multiple other intermediate or high-risk models (e.g., KMS11(t(4;14)), OPM2(t(4;14)), RPMI8226 (t(14;16) and t(8;22)), and KMS28-PE (t(4;14))). The alvocidib/venetoclax regimen was effective in various drug-resistant MM lines e.g., bortezomib, melphalan, and revlimid. Alvocidib blocked CDK9 activation and P-TEFb phosphorylation, down-regulated MCL-1, increased BCL-2/MCL-1 ratios, and up-regulated BIM. MCL-1 ectopic expression or knock-down in MM cells significantly diminished or increased ABT-199 sensitivity respectively. CDK9 shRNA knock-down triggered MCL-1 down-regulation and increased ABT-199 activity, while BIM knock-down significantly reduced alvocidib/venetoclax lethality. HS-5 cell co-culture failed to protect MM cells from the FP/ABT-199 regimen, suggesting circumvention of microenvironmental signals. The effects of venetoclax +/- alvocidib were investigated in a larger number of primary specimens (N =23). Combined treatment very significantly reduced the survival of cells compared to single agent treatment (24 h; P < 0.0001 and 0.0016). In the sub-set of specimens in which primitive progenitor cells could be identified (CD138-/CD19+/CD20+/CD127+; N=18), significant reductions in survival following combined treatment were also observed (P < 0.0074 and 0.0001). In contrast, alvocidib did not increase ABT199 lethality in normal CD34+ cord blood cells. To assess the in vivo relevance of these findings, NOD/SCID-γ mice were inoculated with fluorescently-labled bortezomib-resistant PS-R cells. Combined treatment sharply reduced tumor burden and significantly prolonged survival compared to single-agent treatment (P < 0.0001). Median survival was 79 days for animals in the combined treatment group, versus 62, 63, and 71 days for those in vehicle, venetoclax, or alvocidib groups, respectively. In addition, combined treatment was not associated with significant increases in weight loss or other forms of toxicity e.g., behavioral changes, fur loss etc. Finally, parallel studies were performed in an immune-competent model. C57BL/KaLwR1 mice were injected intravenously with murine MM 5TGM1 cells stably expressing luciferase. Combined administration suppressed tumor growth, manifested by clearly diminished intensity and significantly prolonged survival compared to single agents (P < 0.001), but did not lead to increases in toxicity e.g., weight loss. Specifically, median survival was 69 days for animals in the combined treatment group, versus 39, 49, and 53 days for those in vehicle, venetoclax, or alvocidib groups, respectively.

Conclusions:

These findings argue that CDK9 inhibitors e.g., FP may increase the anti-myeloma activity of ABT-199, including in unfavorable-risk MM minimally responsive to ABT-199 alone.