An ex vivo Familial Genetic Strategy for Determining Mechanism of Action

One of the greatest challenges in anticancer drug development is the discovery of molecular targets and biochemical interactions required for drug action. Lapses in drug efficacy and unanticipated toxicity, the two biggest causes of drug failure in clinical trials, are often attributed to our limited understanding of drug mechanism and cost the pharmaceutical industry millions. Genomics is rapidly emerging as tool for mechanism elucidation. Our approach is one of the latest to link drugs to the genes which influence their activity. This ex vivo familial genetics strategy uses a collection of extensively genotyped, normal, healthy, human cell lines from multigenerational families. Cell lines are phenotyped for cytotoxic response to anticancer agents, heritability analysis gives a measure of the degree to which genetic influences response, and linkage analysis suggests regions of the genome which are associated with the observed variation in response. To evaluate this strategy as method for mechanism elucidation, we first asked whether the system could produce pharmacological and genomic profiles related to a shared mechanism for a class of structurally related compounds. The in vitro sensitivity of CEPH cell lines the camptothecin, Topoisomerase 1 inhibitors (Top1), was studied. Heritability analysis estimates that genetics accounts for as much 20% of the observed variation in cytotoxic response to these drugs. Linkage analysis revealed a pattern of six quantitative trait loci (QTLs) that were shared by all of the camptothecins and independently replicated with a second of camptothecin analogues. The pattern of QTLs observed with the camptothecins was compared to those of the indenisoquinolones, a structurally distinct class of Top1 inhibitors. The objective was to identify which if any QTLs are related to the general mechanism of Top1 inhibition or should be considered class-specific. Finally, the model was assessed for its ability to stratify compounds by mechanism based on their biological and genomic profiles. Cell lines were phenotyped for response to approximately 30 drugs belonging to 8 mechanistic classes. Intraclass biological and genomic profiles were more similar to each other than to compounds belonging to distinct mechanistic classes. This work could have a significant impact on drug discovery and development as it provides a strategy for not only making predictions about mechanism of action for novel therapies, but for identifying genes involved in variable response to chemotherapeutic agents as well

Identification and Replication of Loci Involved in Camptothecin-Induced Cytotoxicity Using CEPH Pedigrees

To date, the Centre d'Etude Polymorphism Humain (CEPH) cell line model has only been used as a pharmacogenomic tool to evaluate which genes are responsible for the disparity in response to a single drug. The purpose of this study was demonstrate the model's ability to establish a specific pattern of quantitative trait loci (QTL) related to a shared mechanism for multiple structurally related drugs, the camptothecins, which are Topoisomerase 1 inhibitors. A simultaneous screen of six camptothecin analogues for in vitro sensitivity in the CEPH cell lines resulted in cytotoxicity profiles and orders of potency which were in agreement with the literature. For all camptothecins studied, heritability estimates for cytotoxic response averaged 23.1±2.6%. Nonparametric linkage analysis was used to identify a relationship between genetic markers and response to the camptothecins. Ten QTLs on chromosomes 1, 3, 5, 6, 11, 12, 16 and 20 were identified as shared by all six camptothecin analogues. In a separate validation experiment, nine of the ten QTLs were replicated at the significant and suggestive levels using three additional camptothecin analogues. To further refine this list of QTLs, another validation study was undertaken and seven of the nine QTLs were independently replicated for all nine camptothecin analogues. This is the first study using the CEPH cell lines that demonstrates that a specific pattern of QTLs could be established for a class of drugs which share a mechanism of action. Moreover, it is the first study to report replication of linkage results for drug-induced cytotoxicity using this model. The QTLs, which have been identified as shared by all camptothecins and replicated across multiple datasets, are of considerable interest; they harbor genes related to the shared mechanism of action for the camptothecins, which are responsible for variation in response

Targeted drug discovery for pediatric leukemia

Despite dramatic advances in the treatment of pediatric leukemia over the past 50 years, there remain subsets of patients who respond poorly to treatment. Many of the high-risk cases of childhood leukemia with the poorest prognosis have been found to harbor specific genetic signatures, often resulting from chromosomal rearrangements. With increased understanding of the genetic and epigenetic makeup of high-risk pediatric leukemia has come the opportunity to develop targeted therapies that promise to be both more effective and less toxic than current chemotherapy. Of particular importance is an understanding of the interconnections between different targets within the same cancer, and observations of synergy between two different targeted therapies or between a targeted drug and conventional chemotherapy. It has become clear that many cancers are able to circumvent a single specific blockade, and pediatric leukemias are no exception in this regard. This review highlights the most promising approaches to new drugs and drug combinations for high-risk pediatric leukemia. Key biological evidence supporting selection of molecular targets is presented, together with a critical survey of recent progress towards the discovery, pre-clinical development, and clinical study of novel molecular therapeutics