Abstract
Abstract 2940
Background:
The ubiquitin-proteasome pathway has been validated as a therapeutic target for multiple myeloma by the incorporation of proteasome inhibitors such as bortezomib and carfilzomib into our standards of care against this disease. A minority of patients achieve complete remission with single-agent therapy, however, and proteasome inhibitors may induce on- and off-target anti-apoptotic effects and clinical toxicities that limit their utility. Unlike proteasome inhibitors, which have a broad impact on intracellular proteolysis, E3 ligases are responsible for ubiquitination of only a small subset of proteins. Our hypothesis proposed that inhibition of the Human double minute (HDM)-2 E3 ligase, which in part is responsible for p53 ubiquitination, could be a more rational therapeutic target for myeloma, where p53 mutations are less common than is the case in solid tumors.
Methods:
We used Nutlin-3a and MI-63, which bind to the p53 binding pocket of HDM-2, as prototypic agents, and studied their activity against both wild-type (wt) and mutant (mut) p53 cell line models of multiple myeloma. These studies were supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma.
Results:
Inhibition of HDM-2 in wt p53 (MM1.S, H929, MOLP-8) cells with Nutlin-3a or MI-63 reduced viability, with a median inhibitory concentration (IC50) in the 0.5–2 μM range. In wt cells, genomic and proteomic studies showed that MI-63 induced transcription of downstream gene targets including p21, PUMA and Bax, and promoted accumulation of p53, p21, p27, and Bax. This was accompanied by cell cycle arrest at G1, and induction of apoptosis as demonstrated by Annexin V staining and caspase activation. Interestingly, these agents also showed activity against mut p53 cells such as RPMI 8226 and U266, though with higher IC50's of 30–40 μM. Knockdown of HDM-2 sensitized mut p53 myeloma cells to MI-63, suggesting that this agent was inducing cell death in an on-target manner. In p53 mutant cells, MI-63 induced both type I cell death and type II, autophagic cell death, as determined by conversion of microtubule-associated protein 1 light chain 3, and activation of transcription of autophagy (ATG) genes ATG3 and 5. Since autophagy can be a mechanism of cell survival or cell death, we tested the impact of the autophagy inhibitors chloroquine and 3-methyladenine, and found that they reduced the impact of MI-63 on viability. Moreover, knockdown of ATG5 helped rescue cells as well, supporting the possibility that autophagic activation was a mechanism of cell death in these myeloma models. Synergistic anti-myeloma effects were seen when MI-63 was combined with the BH3 mimetic ABT-737, with decreased viability and enhanced apoptosis, accompanied by increased Bax activation and caspase cleavage. Notably, ABT-737 also enhanced the effects of MI-63 on mut p53 myeloma models, where increased levels of both type I and II programmed cell death were seen.
Conclusions:
Together, our findings support the possibility that HDM-2 is a promising therapeutic target for multiple myeloma, and also provide a rationale for translation of HDM-2 inhibitors alone, and in combination with ABT-737, to the clinic.
Disclosures:
Leopold: Ascenta Therapeutics: Employment, Equity Ownership.
Inhibition of the MDM2 E3 Ligase Induces Apoptosis and Autophagy in Wild-Type and Mutant p53 Models of Multiple Myeloma, and Acts Synergistically with ABT-737
