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The epidermal growth factor receptors, EGFR (erbB-1) and HER2 (erbB-2), and their down stream signaling events promote tumor growth and survival in a variety of epithelial tumors. Current treatment strategies used to target these receptors include mAbs directed against their extracellular domains and small molecule inhibitors of their tyrosine kinase activities. The efficacy of these types of therapeutics is increasingly being found to be contingent on the genotype of the targeted cancer. In vitro evaluation of mAbs is reliant on selection of tumor cell lines driven by the specific antigen proteins of interest and their associated pathways. We selected a panel of eight human tumor cell lines, six breast cancer (BT474, SK-BR-3, MDA-MB-453, MDA-MB-231, MDA-MB-468 and MCF-7), one colon (SW48) and one skin (A431) with varying levels of HER2, EGFR and EGFR/HER2 expression to evaluate the mAbs cetuximab and trastuzumab for antiproliferative effects, ability to induce apoptosis and the ability to block cell cycle. These findings were compared to results obtained after treatment with lapatinib, erlotinib, Ly294002 and Tamoxifen. The BT474 and SK-BR-3 breast cancer cell lines with HER2 amplified expression demonstrated cytostatic antiproliferative and G1/S cell cycle block sensitivity to the anti-HER2 antibody trastuzumab. The more prominent inhibitory effects observed in the BT474 cell line were further confirmed by measurable induced decreased levels of phospho-Akt and increased levels of the CDK2 inhibitor p27Kip1 following treatment with trastuzumab, lapatinib and Ly294002. The SW48 colon and A431 skin cancer cell lines with EGFR mutations and EGFR over expression, respectively, demonstrated cytostatic antiproliferative sensitivity to the anti-EGFR antibody cetuximab. This in vitro cell-based multiplexed approach for assessment of oncology therapeutics based on genetically directed cell line selection, demarcation of cytostatic and cytotoxic cell proliferation inhibition and assessment of receptor down stream signaling biomarkers offers greater predictive efficacious value and can be used to identify optimal candidates for combination therapies. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-14-17.

Targeting Receptor Tyrosine Kinases (RTKs) for therapeutic efficacy has been a promising approach for modern oncological agents. However, not all cancers respond to these agents and understanding of the feedback and resistance mechanisms is not complete. We have used an In vitro cellular profiling of 240 genetically characterized tumor human cell lines to help understand genotypes that convey sensitivity and resistance to these agents. While most of the sensitive cell lines were unique to a given RTK inhibitor, many of the resistant cell lines had similar genotypes. We have investigated downstream genes for mutations, over-expression and phosphorylation status. Phosphorylation levels pAKT, pERK, and pJNK, and as well as basal levels of p27KIP, EGFR and ERBB2 protein levels were analyzed. Many of the downstream mutations or activated genes confer resistance to the RTK inhibitors such as AKT, PTEN and PI3K mutations. In addition to downstream activation, we have found that overexpressed RTKs often seem to confer resistance to other RTK inhibitors. For example, EGFR over-expressed cell lines disproportionately populated the group of cell lines resistant to an IGFR-1 receptor agonist. We have investigated cross talk between these various RTKs. Both EGFR and IGFR-1 inhibitors were tested in a combinatorial approach through 60 cell lines for synergy and antagonism. We evaluate synergy calculations through combination index and bliss analysis across 60 cell lines. Other combinations are also in the process of being tested for synergy. 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 239. doi:10.1158/1538-7445.AM2011-239

In vitro cellular response profiling of tumor human cell lines has become a widely used approach for the targeted cancer therapeutics development. Correlation of the drug sensitivity and resistance cellular response with genomic data offers a robust and sensitive system for predicting clinical efficacy and identifying more efficacious patient populations. We have developed a high throughput cellular approach to evaluate the relationship between tumor genotypes and drug sensitivity over 240 human tumor cell lines. A panel of the cancer therapeutic agents was tested for proliferative, apoptotic and cell cycle arrest responses using multiplexed high content screening with automated fluorescence microscopy and image analysis based technology. Growth index was measured using nuclear dye. Activated caspase 3 antibodies were used for the apoptosis induction detection. Phospho-histone H3 antibodies were used to measure the cell cycle block. The eighteen cancer therapeutics were used in this study: BMS-387032 (SNS-032), PD0325901, PD173074, API-2 (Triciribine), Lapatinib (Tykerb), BMS-536924, Geldanamycin, Sorafenib, Dasatinib, Erlotinib, VX-680, Sunitinib, Everolimus, Tandutinib, Cl 1040, Doxorubicin, Paclitaxel and Staurosporine. We generated cell line profiles to reveal drug sensitivity and resistance patterns and identified markers associated with a specific response. It was found that Raf/Ras mutations confer sensitivity to MEK inhibitors, PD0325901 and Cl 1040, while PIK3CA and RB mutations were associated with resistance. Resistance to IGF-1R, FGFR, and EGFR inhibitors correlated with PI3K/PTEN/Akt or Raf/Ras pathway activation. EGFR mRNA overexpression was associated with resistance to FGFR and IGF-1R inhibitors. Resistance to AKT inhibitor, API-2 (Triciribine) was associated with PTEN mutations and amplification of AKT. APC mutations were associated with resistance to Aurora kinase inhibitor, VX-680, while mutations in the beta-catenin gene were linked with a sensitive phenotype. This approach could provide insight into the mechanisms of enhanced susceptibility or resistance which in turn could be used for the optimization of the targeted cancer therapeutics. 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 4845. doi:10.1158/1538-7445.AM2011-4845