Project description:Fifty percent of cutaneous melanomas are driven by activated BRAFV600E, but tumors treated with RAF inhibitors, even when they respond dramatically, rapidly adapt and develop resistance. Thus, there is a pressing need to identify the major mechanisms of intrinsic and adaptive resistance and develop drug combinations that target these resistance mechanisms. In a combinatorial drug screen on a panel of 12 treatment-naïve BRAFV600E mutant melanoma cell lines of varying levels of resistance to MAPK pathway inhibition we identified the combination PLX4720, a targeted inhibitor of mutated BRaf, and lapatinib, an inhibitor of the ERBB family of receptor tyrosine kinases, as synergistically cytotoxic in the subset of cell lines that displayed the most resistance to PLX4720. To identify potential mechanisms of resistance to PLX4720 treatment and synergy with lapatinib treatment we performed a multi-platform functional genomics analysis to profile the genome as well as the transcriptional and proteomic responses of these cell lines to treatment with PLX4720. We found modest levels of resistance correlated with the zygosity of the BRAF V600E allele and RTK mutational status. Layered over base-line resistance was substantial upregulation of many ERBB pathway genes in response to BRaf inhibition, thus generating the vulnerability to combination with lapatinib. The transcriptional responses of ERBB pathway genes are associated with a number of transcription factors, including ETS2 and its associated cofactors that represent a convergent regulatory mechanism conferring synergistic drug susceptibility in the context of diverse mutational landscapes. 12 BRAF mutant melanomas and 4 melanomas with WT BRAF were exposed plx4720 treatment to evaluate their responses after 8 hours of treatment. 5 of the 12 BRAF mutant melanomas responses were also evaluated in response to the treatment of lapatinib alone, masitinib alone, the combination of lapatinib with plx4720, or the combination of masitinib with plx4720. All samples were run in at least triplicate.
Project description:These data provide scientific information to understand the mechanism of action of lapatinib resistance in HER2-positive patients and to test the combination of HER2-targeted agents and GSK1363089 (foretinib) in the clinic by using an acquired lapatinib-resistant cell line. Cell lines (BT474, an HER2-positive and lapatinib-sensitive cell line; BT474-J4, an acquired lapatinib-resistant cell line) were treated with lapatinib alone (1uM), foretinib (GSK1363089) alone (0.1 uM), a combination of lapatinib (1 uM) and foretinib (0.1 uM), or DMSO control for 24 hours. Triplicates were performed.
Project description:Acquired drug resistance prevents targeted cancer therapy from achieving stable and complete responses. Emerging evidence implicates a key role for nonmutational mechanisms including changes in cell state during early stages of acquired drug resistance. Targeting nonmutational resistance may therefore present a therapeutic opportunity to eliminate residual surviving tumor cells and impede relapse. A variety of cancer cell lines harbor quiescent, reversibly drug-tolerant âpersisterâ cells which survive cytotoxic drugs including targeted therapies and chemotherapies. These persister cells survive drug through nonmutational mechanisms which are poorly understood. Specifically targeting persister cells is a promising strategy to prevent tumor relapse. We sought to identify therapeutically exploitable vulnerabilities in persister cells using the HER2-amplified breast cancer line BT474 as an experimental model. Similar to other persister cell models, upon treatment with the HER2 inhibitor lapatinib (2uM concentration) for nine or more days, the majority of BT474 cells die, revealing a small population of quiescent surviving persister cells. Removal of lapatinib allows the persister cells to regrow and to re-acquire sensitivity to lapatinib. Subsequent lapatinib treatment re-derives persister cells. The reversibility of persister cell drug resistance indicates a nonmutational resistance mechanism. Here we provide RNAseq gene expression profiling data generated from parental BT474 cells compared to BT474 persister cells generated from nine days of treatment with 2 uM lapatinib. These data can be used to identify genes and pathways which are upregulated in persister cells, revealing potential therapeutic targets. 3 biological replicates of BT474 persister cells, two biological replicates of BT474 parental cells
Project description:ChIP-seq for PPARGC1A was performed in OE19 cells to identify genomic binding sites. Cells were treated with 500 nM lapatinib for 48 hours.
Project description:ChIP-seq for HNF4A, PPARGC1A and H3K27ac was performed in OE19 cells to identify genomic binding sites. Cells were treated with DMSO or 500 nM lapatinib for 48 hours.
Project description:To understand the gene network that controls plant tolerance to cold stress, we carried out a near full genome transcript expression profiling in Arabidopsis using Affymetrix GeneChips that contain approximately 24,000 genes. For microarray analysis, Arabidopsis seedlings were cold treated at 0 C for 0 h, 3 h, 6 h, and 24 h. A total of 939 genes were statistically determined to be cold-regulated with 655 being up-regulated and 284 down-regulated. A large number of the early cold-responsive genes encode transcription factors that likely control late-responsive genes, which implies a multitude of transcriptional cascades. In addition, many genes involved in post-transcriptional and chromatin level regulation were also cold regulated suggesting their involvement in cold responsive gene regulation. A number of genes important for the biosynthesis or signaling of plant hormones, such as abscisic acid, gibberellic acid and auxin, are regulated by cold stress, which is of potential importance in coordinating cold tolerance with growth and development. We compared the cold-responsive transcriptomes of wild type and ice1, a mutant defective in an upstream transcription factor required for chilling and freezing tolerance. The transcript levels of many cold-responsive genes were altered in the ice1 mutant not only during cold stress conditions, but also before cold treatments. Our study provides a global picture of the Arabidopsis cold-responsive transcriptome and its control by ICE1, and thus will be valuable for understanding gene regulation under cold stress and the molecular mechanisms of cold tolerance. Experiment Overall Design: Two replicates for each time point of 0 hours, 3 hours, 6 hours and 24 hours of cold treatment for the wildtype (control) and ice1 (mutant).
