Hakim Djaballah

Abstract LB-273: Preclinical assesment of a novel CDC7 inhibitor: Genomewide RNAi screening identifies unique synergetic and resistance genes

Despite extensive drug discovery efforts, drug-candidate failure and patients relapsing in the clinic remain as persistent problems. While insufficient drug-gene engagement leads to drug failure, de novo escape mutations give rise to patients relapsing, calling the need for systemic studies on how genes influence drug responsiveness. Towards this end, we have explored a functional short hairpin RNA (shRNA) based genomic screening platform aimed at interrogating drug-gene engagement and assessing its consequences on signaling pathways. We propose this concept as a novel way to evaluate drug candidates prior to clinical trials enabling liability assessment and predicting clinical outcome. We took advantage of the arrayed shRNA library produced in lentiviral particles and characterized by several obvious advantageous features including shRNA targeting one hairpin at a time and on the fly high content whole well microscopy imaging analysis. We carried out three parallel genomewide shRNA screens in the absence or presence of the novel CDC7 kinase inhibitor (MSK-777) at its IC20 and IC50 and have identified several gene candidates that influence MSK-777 sensitivity and resistance. These include synergizers that enhance MSK-777 sensitivity and rescuers that confer MSK-777 resistance. IPA analysis mapped clusters of these hits to multiple major pathways among them were the NF-kB pathway, the ubiquitin-proteasome pathway, DNA replication, and several epigenetic regulatory genes. We will present and discuss this concept together with the emerging pathways as a means to identify both key therapeutic targets and biomarkers of sensitivity and resistance. Thus, allowing for not only a broader applicability of assessing candidate genes that modulate specific drug agents, but also for the identification of a tailored and more efficacious therapeutic regimen to treat cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-273. doi:10.1158/1538-7445.AM2011-LB-273

Abstract 5503: Dual fluorescence high throughput screen detects MUC16 selective therapeutic candidates

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC BACKGROUND: The MUC16 gene encodes the CA125 ovarian cancer antigen. This glycoprotein is expressed in the majority of Epithelial Ovarian Cancers (EOC) but has limited expression elsewhere in the adult human. Expression of the carboxy-portion of MUC16 results in increased invasion, soft agar growth and in vivo growth. OBJECTIVES: We sought to develop a suitable microscopy based assay system to screen for and identify small molecules which have a differential effects against MUC16+ cells compared to the isogenic MUC16 negative ovarian cancer cell lines. Using this assay we seek to identify MUC16 specific small molecules for development of potential chemotherapy drug candidates against ovarian cancer. METHODS: Carboxy-terminus portions of the Human MUC16 gene were introduced into SK-Ov-3 and A2780 EOC cell lines and labeled with the red fluorescent protein (mCherry) vector. Control lines were labeled with green fluorescent protein (GFP) vector. Relative toxicity of tested compounds on MUC16+ (mCherry) and MUC16- (GFP) cell populations was assessed by cell viability using ratiometric fluorescence intensities of both co-cultured isogenic cells. Compounds with differential activity were selected, resupplied and tested in a 96 well plate viability assay for confirmation. RESULTS: Both A2780 and SK-Ov-3 isogenic pairs had similar in vitro growth characteristics. A small scale pilot screen of 3,119 compounds was completed for each pair of isogenic cells. Six compounds, including Quinacrine, Ellipticine and Acriflavinium were relatively selective for MUC16+ A2780 cells and these three compounds were among the hits identified in the secondary SK-Ov-3 dual fluorescence screen. Caspase3 apoptosis assay was used to confirm Quinacrine as the most selective inhibitor in the MUC16+ cells. CONCLUSIONS: MUC16 +/− dual fluorescence screen is an example of a high throughput screening strategy to identify potentially specific modulators of key oncogenic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5503.