Abstract 3133: Identification and functional validation of novel genetically-linked breast cancer targets through pooled gain-of-function screening.

Abstract

Abstract Breast cancer is one of the most common cancer types, with greater than 450,000 deaths reported per year worldwide. Through genome wide sequencing efforts, multiple genetic alterations have been identified, including mutations and amplifications in genes such as v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), GATA binding protein 3 (GATA3), phosphatidylinositol 3-kinase alpha catalytic subunit (PIK3CA) as well as novel genomic rearrangements such as the recently identified MAGI3-AKT3 fusion. Now that breast cancer can be characterized to an unprecedented level, one of the key challenges remaining is to identify and distinguish critical ‘driver’ events responsible for tumor progression, from neutral ‘passenger’ lesions. In order to achieve this, we utilized high resolution aCGH analysis of 50 purified breast cancer samples (made up of Her2+, estrogen receptor positive (ER+) and triple negative tumors with variable responses to SOC regimens), in combination with a Gain-of-Function transformation screen to identify and validate novel breast targets. 158 genomic regions were found to be recurrently amplified, consisting of 759 genes in total. The top 32 focally amplified genes, along with 12 cancer-relevant mutant alleles were prioritized and a library generated utilizing the pTRIPZ-tetracycline regulated inducible lentiviral vector system. These 44 genes were subsequently combined into 16 different target pools (5-13 targets per pool, co-expressing genes that were co-amplified in the same clinical specimen) and evaluated for their ability to transform immortalized breast epithelial MCF10A cells (both wild-type and p53 -/- cells). Through this screening approach, p21-activated kinase 1 (PAK1) was identified, whose kinase activity was required to robustly transform MCF10A cells through regulating multiple signalling pathways including MAPK. Several other putative oncogenes were also identified and will be presented here, including the glycosyltransferse asparagine-linked glycosylation 8 (ALG8). Interestingly, PAK1 and ALG8 are co-amplified in both breast (8%) and ovarian cancers (11%). Our target validation studies have suggested that ALG8 can support PAK1-induced transformation, as dramatic suppression of soft-agar colony growth was seen in co-amplified breast cancer cell lines upon combined siRNA treatment to both targets. Thus, this combined high resolution aCGH profiling and functional screening approach has enabled the successful identification of novel oncogenic targets in breast cancer. Citation Format: Krishna Vasudevan, Axel Hernandez, Zhongwu Lai, Yonghong Xiao, Nin Guan, Carolyn Hardy, Robert Godin, Christopher Denz, Minwei Ye, Elizabeth Lenkiewicz, Stephanie Savage, Michael T. Barrett, Donna Prunkard, Peter Rabinovitch, Mark Basik, Ewa Przybytkowski, Kevin Webster, Michael Zinda, Emma-Louise Jenkins. Identification and functional validation of novel genetically-linked breast cancer targets through pooled gain-of-function screening. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3133. doi:10.1158/1538-7445.AM2013-3133