Systems level characterizations of single and combination drug mechanisms of action in vitro and in vivo

Abstract

Small molecule characterization is a critical limiting step in cancer drug development. At the present time, high throughput screens of natural products and combinatorial synthesis libraries generate more pharmaceutical leads than can be characterized in detail. Lead optimization further generates many derivatives of these cytotoxic hits in an attempt to generate optimized compounds with better physical or chemical properties. This leaves many promising agents stranded in drug development and poorly characterized. In addition, most small molecules interact biochemically with a diverse set of proteins. While characterizing the diversity of biochemical interactions that can occur is important to understanding function, only a subset are likely to be necessary or sufficient for therapeutic efficacy. In light of this diversity, the functional characterization of the mechanisms of cell death by cytotoxic agents should improve drug discovery by allowing for the early prioritization of cytotoxic leads, derivatized compounds, and targeted inhibitors on the basis of the mechanisms by which they cause death in intact cells. Using RNAi mediated suppression of key mediators of apoptosis; we found that we could predict the functional mechanisms of drug action in lymphoma cells across many categories of cytotoxic therapeutics with as few as 8 shRNAs. Beyond single drug mechanisms, most drugs used in cancer are used as drug combinations. These combinations were largely formulated on two principles: compounds must have a unique mechanism of action so that more cumulative drug can be dosed with non-overlapping toxicity, and they must have statistically independent mechanisms of drug resistance. However, beyond clinical efficacy, the basic mechanisms of combination therapy have never been examined. Thus, in light of the central role of apoptosis in guiding mammalian cell death to cancer therapy, we sought to examine the functional signatures of cell death in the face of combination therapy. Surprisingly we find that RNAi mediated suppression of cell death mediators in response to common cytotoxic regimens, averages both sensitivity and resistance to therapy and neutralizes the effects of genetic variation. This suggests that common cytotoxic regimens are intrinsically depersonalized and difficult to genetically stratify.