The project addresses the identification and validation of new aims for a combinatorial therapy of Mcl-1-dependent types of cancer
Final Report Abstract
Bcl-2 pro-survival proteins are widely considered to be the guardian of cell survival in the face of both extrinsic and intrinsic stress stimuli. Many cancer cells, for example, depend on Bcl-2 prosurvival proteins to maintain cell viability in the face of oncogenic signaling. Mcl-1, a pro-survival member of the Bcl-2 family, is involved in tissue development, malignancy, and response to anticancer therapeutics. The project’s aim was to elucidate new synthetic lethality opportunities in cancer therapy, targeting Mcl-1. In the cancer context, synthetic lethality is defined as identifying and inhibiting two (or more) cancer cell pathways to exploit their synergies, resulting in a significant clinical development opportunity. Cancer cell lines, whose survival depends on Mcl-1, often show a different threshold for Mcl-1 knockdown to elicit cell death. It was expected that related signal transduction pathways act in synergy with Mcl-1 to enhance cancer cell survival and therefore represent new therapeutic opportunities for drug development. The project identified genes that maintain high expression of Mcl-1 in multiple myeloma cells (MM). MM is a malignant monoclonal plasma cell disease of the bone marrow. 10% of all hematological malignancies are MM incidents. However, despite the improved survival in MM patients and the impact of novel therapeutics, nearly all patients relapse marking MM as an incurable disease. Therefore there is an urgent need for new approaches and novel therapeutics in MM treatment. We could demonstrate that the KMS-11 MM cell line is highly addicted to Mcl-1, siRNA knockdown resulted in rapid cell lethality which could be rescued by over-expression of murine Mcl-1. This lethality could be entirely rescued by Bcl-2. Of around 400 single lethal hits in a KMS-11 genome-wide siRNA screen the loss of only a few proteins - among them eIF1A - was partially rescued by Bcl-2 in a secondary screen. The prediction was that some of these siRNA hits were lethal because they might be involved in selective regulation of Mcl-1 levels and provoke the loss of Mcl-1. As expected, limiting eIF1A expression resulted in rapid loss of Mcl-1 due to the labile nature of this rapidly-turned over protein, accounting for cell lethality via the Bcl-2 family pathway. Thus, the vast majority of cell deaths in response to a brief loss of the expression of individual proteins occurred by pathways unable to be rescued by Bcl-2. Validation studies included de-convoluted siRNA knockdown assays demonstrating knockdown of the intended target by Western blot. Additionally, retrovirus infected multiple myeloma cells expressing the target shRNA confirmed the results showing target protein as well as Mcl-1 knockdown. Further validation assays were performed in which the hit gene was over-expressed under knockdown conditions to rescue the phenotype. These results are even more intriguing, since Bcl-2 could not rescue the lethality and loss of Mcl-1 due to knocking down another member of the protein synthesis machinery, RPL19. The results suggest that eIF1A is particularly relevant to the synthesis of proteins such as Mcl-1 that are constituents of pathways linked to the Bcl-2 regulated death machinery. Mcl-1 perhaps more than any other pro-survival member of the Bcl-2 family is subject to extensive levels of regulation. Mcl- 1 mRNA and protein levels are highly regulated within a cell allowing for a rapid induction or loss of Mcl-1 protein expression. This study helps to understand the multiple regulatory pathways with which a cancer cell maintains high levels of Mcl-1 and therefore its pro-cancer activity and discovered novel targets that could be developed into new therapeutic opportunities for drug development.
Publications
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Programming cancer cells for high expression levels of Mcl1. EMBO Rep. 2013 Apr;14 (4):328-36
Ertel F, Nguyen M, Roulston A, Shore GC
