Functional genomic analysis in cancer personalized therapy

Abstract

Cancer personalized therapy targets molecular pathways that drive the behavior of cancer cells. Emerging technologies, in particular the “next generation” sequencing, have rapidly increased the discovery of genomic aberrations including gene mutations, copy number variations and chromosomal rearrangements. Further, there are large numbers of targeted therapies in the clinic representing a toolkit of drugs. Development of approaches to rapidly determine whether new aberrations are drivers or passengers and to identify drugs that will benefit patients with these aberrations becomes a challenge. Success of such development will escalate extensive sequencing of patient tumors at the level of kinome, exome and whole genome. High throughput technologies will allow functional interrogation of the genomic aberrations to the emerging toolkit of targeted drugs. We report a moderate throughput functional genomic approach that can rapidly and efficiently: 1) determine oncogenic activity of specific genomic aberrations, 2) identify targeted therapies likely to be active against the specific genomic aberration, and 3) distinguish on-target effects from non-specific toxicity of targeted reagents. In our study, we expressed independently a wide series of abnormal genes identified in patient tumors into IL-3 dependent Ba/F3 cells using lentiviral vectors. These genes include BCR-ABL, KRAS, HRAS, wild-type PIK3CA and its mutants (H1047R, E545K, R88Q), wild-type PIK3R1 and its mutants (K567E, N453del), wild-type AKT1, 2, 3 and their mutants E17K, GNAQ Q209L and LPA 1, 2, 3. Cells were removed from IL3 to allow manifest of the construct effects. RT-PCR using primers containing the exogenous vector sequence confirmed gene expression. Two different functions were readily assessed. In the first 10 days after transfection, expression of the gene constructs protected cells from death induced by IL3 deprivation. 4 to 6 weeks after transfection, the ability of the constructs allowed Ba/F3 cells to proliferate in the absence of IL3. These two assays provide a rapid assessment of gain of function due to overexpression of wild type or mutant constructs. Stable cell lines were established with a wide variety of constructs demonstrating the applicability of the approach. In a proof of concept study, the stable cell lines were then assessed for the effect of the constructs on sensitivity to a variety of drugs targeting MEK, PI3K, AKT, and LPA receptors in the presence or absence of IL3. As expected, expression of KRAS or HRAS rendered cells exquisitely sensitive to inhibition of MEK in the absence of IL3. Exogenous IL3 protected cells from MEK inhibition-induced cell death, indicating the on-target effect of cellular response, thus providing a ready counter screen for toxicity. In summary, our results support the notion that integration of functional genomic analysis into patient management and drug selection has the opportunity to rapidly improve patient outcomes.