The use of molecularly targeted drugs as single agents has shown limited utility in many tumor types, largely due to the complex and redundant nature of oncogenic signaling networks. Targeting of the PI3K/AKT/mTOR pathway through inhibition of mTOR in combination with aromatase inhibitors has seen success in particular sub-types of breast cancer and there is a need to identify additional synergistic combinations to maximize the clinical potential of mTOR inhibitors. We have used loss-of-function RNAi screens of the mTOR inhibitor rapamycin to identify sensitizers of mTOR inhibition. RNAi screens conducted in combination with rapamycin in multiple breast cancer cell lines identified six genes, AURKB, PLK1, PIK3R1, MAPK12, PRKD2, and PTK6 that when silenced, each enhanced the sensitivity of multiple breast cancer lines to rapamycin. Using selective pharmacological agents we confirmed that inhibition of AURKB or PLK1 synergizes with rapamycin. Compound-associated gene expression data suggested histone deacetylation (HDAC) inhibition as a strategy for reducing the expression of several of the rapamycin-sensitizing genes, and we tested and validated this using the HDAC inhibitor entinostat in vitro and in vivo. Our findings indicate new approaches for enhancing the efficacy of rapamycin including the use of combining its application with HDAC inhibition.

MicroRNAs (miRNAs) regulate the expression of hundreds of genes. However, identifying the critical targets within a miRNA-regulated gene network is challenging. One approach is to identify miRNAs that exert a context-dependent effect, followed by expression profiling to determine how specific targets contribute to this selective effect. In this study, we performed miRNA mimic screens in isogenic KRAS-Wild-type (WT) and KRAS-Mutant colorectal cancer (CRC) cell lines to identify miRNAs selectively targeting KRAS-Mutant cells. One of the miRNAs we identified as a selective inhibitor of the survival of multiple KRAS-Mutant CRC lines was miR-126. In KRAS-Mutant cells, miR-126 over-expression increased the G1 compartment, inhibited clonogenicity and tumorigenicity, while exerting no effect on KRAS-WT cells. Unexpectedly, the miR-126-regulated transcriptome of KRAS-WT and KRAS-Mutant cells showed no significant differences. However, by analyzing the overlap between miR-126 targets with the synthetic lethal genes identified by RNAi in KRAS-Mutant cells, we identified and validated a subset of miR-126-regulated genes selectively required for the survival and clonogenicity of KRAS-Mutant cells. Our strategy therefore identified critical target genes within the miR-126-regulated gene network. We propose that the selective effect of miR-126 on KRAS-Mutant cells could be utilized for the development of targeted therapy for KRAS mutant tumors.

To identify vulnerable genes in breast cancer cells that may be therapeutic candidates for treatment of breast cancer, we conducted an unbiased, large-scale, synthetic siRNA-mediated RNAi screen. Using cell viability as the read-out, we first screened a whole human kinome library plus 350 additional genes (four siRNAs per gene) in a cell line representative of triple negative (ER -ve, PR -ve, Her2/Neu -ve) breast cancer, MDA-MB-468. We identified approximately 40 kinases whose loss-of-function reduced the viability of MDA-MB-468 cells based on the criteria of at least two of four siRNAs per gene generating a Z score of ≤ −1.5. To validate the top candidates from our screen, PLK1, AURKB and PCTK3/CDK18), we tested additional siRNAs (from multiple vendors) in different breast cancer cell lines including ER positive cell lines, CAMA-1 and MCF-7, HER-2 amplified cell lines, HCC-1954 and AU565, triple-Negative (Basal A) cell lines HCC-1937 and MDA-MB-468, and triple-Negative (Basal B) cell lines BT549 and MDA-MB-231. We found silencing of PLK1, AURKB or PCTK3/CDK18 reduced the cell viability in majority of these cell lines to a certain degree and also induced cell cycle arrest and apoptosis. PLK1 and AURKB are being actively pursued as molecular targets in cancer but little is known of the function of the PCTK3/CDK18 protein, with most of the limited functional studies of PCTK3/CDK18 having focused on a role in neuronal cell signaling. One study has though reported an increase in the expression of PCTK3/CDK18 in breast cancer (Valladares et al., Cancer Genet Cytogenet. 2006 170:147). We found silencing of PCTK3/CDK18 is lethal to most breast cancer cells, but it is well tolerated in the untransformed breast epithelial cell line MCF10A. The PCTK3/CDK18 gene encodes a member of the PCTAIRE protein kinase subfamily of CDC2-related serine/threonine-specific protein kinases. We observed that with multiple siRNAs, we induced an 80% decrease in the viability of MDA-MB-468 cells 96 hours following siRNA transfection. To ensure that the observed inhibitory effect is indeed due to silencing of PCTK3/CDK18 we performed an RNAi rescue experiment, by creating a silent third-codon point mutation within the region targeted by one of the PCTK3 siRNAs. We found the RNAi-induced inhibition and cell cycle arrest is countered by expression of a functional version of the target gene that is resistant to the silencing siRNA. No specific inhibitor of PCTK3/CDK18 is available, but a proteome-wide CDK/CRK-specific kinase inhibitor RGB-286147 does decrease cell viability, and induce cell cycle arrest and apoptosis of MDA-MB-468 cells. We are currently further investigating the normal and cancer-related functional roles of PCTK3/CDK18 and are pursuing strategies for the identification of PCTK3/CDK18 specific inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 267. doi:1538-7445.AM2012-267