Abstract
Abstract The molecular pathogenesis of many cancer types, including multiple myeloma (MM), involves alterations in the PI3K/Akt/mTOR and cyclin/CDK/CDKI/Rb (Rb) pathways. Targeting these pathways individually has shown limited efficacy. Here, however, we show the combination of an HDAC inhibitor with rapamycin synergistically inhibits proliferation in 88% of human myeloma cell lines tested (p<0.01), as well as effectively controlling tumor growth in long-term preclinical studies. To understand the synergistic molecular mechanism of this combination, candidate pathway analysis and a systems-level approach were taken. We found the combination antagonized the oncogenic activation of the AKT pathway associated with single-agent rapamycin treatment, along with inhibiting the ERK/MAPK pathway to a much greater extent than either single agent alone. For a more unbiased approach, gene expression profiling (GEP) was coupled with a systems-level gene co-expression network analysis. This analysis delineated the contribution of each inhibitor to the overall gene expression change of the combination by considering not only measures of fold-change and significance testing, but also the degree of gene expression inter-connectedness. With these findings, a network of five gene modules was constructed, where each module represents a particular gene expression effect of the combination. Each module of genes was then individually tested for functional and clinical enrichment. Of particular interest, the module containing genes cooperatively affected by both compounds was highly enriched (p<0.001) for genes involved in cell cycle (especially mitotic processes), immune recognition, and DNA damage/repair, which we have investigated further. Specifically, we confirmed the down-regulation of RRM2, a gene involved in DNA synthesis and repair, by western blot and validated an increase in DNA damage markers with combination treatment. Additionally, we determined that specific RRM2 inhibition decreased MM cell viability, which decreased further when combined with rapamycin. Gene Set Enrichment Analysis of drug-induced gene expression profiles demonstrated that all gene expression modules associated with the drug combination were significantly enriched (p<0.01) when comparing healthy donors to MM patients in a large, publicly available GEP dataset. Finally, interrogation of the cooperative drug signature in publicly available patient GEP datasets with survival annotation found it predictive of increased survival (p<0.01), thus linking the drug combination-induced transcriptional changes to predictions for enhanced survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C111.