Abstract 1252: TIMELESS is a KSR1-like effector of Ras-driven colon tumorigenesis

Abstract

Multiple studies have revealed that Ras-driven tumors acquire unique vulnerabilities by adapting cellular mechanisms that promote uncontrolled proliferation and suppress apoptosis. Targeting these vulnerabilities provide opportunities to develop novel, efficacious cancer therapeutics that lack the harmful side effects accompanying current therapies. RNA interference (RNAi) of the molecular scaffold Kinase Suppressor of Ras 1 (KSR1), which modulates ERK activation downstream of oncogenic Ras, selectively kills malignant, Ras-driven cancer cells, but does not kill immortalized, non-transformed human colon epithelial cells (HCECs). With the exception of a minor hair follicle defect, KSR1-/- mice are fertile and phenotypically normal, suggesting that KSR1 is not required for normal cell survival and that Ras-driven and KSR1-dependent pathways may yield valuable new targets for therapeutic development. To identify targets, like KSR1, that are required for cancer cell survival but not normal cell survival, we used a gene expression-based signature screening approach termed Functional Signature Ontology (FUSION, Potts et al. Sci. Signaling 2013) to screen 15,172 genes in the K-RasD13-bearing human colorectal cancer cell line HCT116. We quantified the functional similarity between KSR1 and each individual gene screened using Euclidean Distance and Pearson Correlation similarity metrics. Additional metrics were added to identify the best targets for biological validation including off-target analysis (siRNA seed sequences), cell viability evaluation, expression analysis, and enrichment analysis. Initial biological validation is completed by assessing cell viability following transient depletion of a screen hit in anchorage-independent and normal culture conditions in HCECs and HCT116s. Due to the similarity between the gene expression signatures, Timeless Circadian Clock (TIMELESS) was identified as being KSR1-like and a potential target. We found that transient TIMELESS depletion decreases cell viability in HCT116 cells under anchorage-independent conditions (47% decrease, p < 0.0001, N = 4). In normal culture conditions, TIMELESS depletion decreases cell viability in HCT116 cells, but not HCECs (HCEC 14% decrease, p > 0.05; HCT116 49% decrease, p < 0.0001, N = 6). TIMELESS is upregulated at the RNA level in colon tumors compared to normal colon tissue (∼2.2 fold, p < 0.0001) (TCGA) and is upregulated at the protein level in three human colon cancer cell lines (HCT116, SW480, SW620) bearing activated Ras compared to HCECs (4-7 fold). Our data indicate that the FUSION screen provides a platform for identifying novel therapeutic targets and demonstrates the potential to identify oncogene-specific vulnerabilities in an unbiased manner. TIMELESS overexpression represents a vulnerability in Ras-driven tumors that will reveal novel and selective targets found in Ras-driven cancers that can be used in the development of selective therapeutics. Citation Format: Beth K. Clymer, Kurt W. Fisher, David L. Kelly, Michael A. White, Robert E. Lewis. TIMELESS is a KSR1-like effector of Ras-driven colon tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1252.

Molecular Signal Integration of Aging and Diabetes Mellitus

“…The Timeless protein can be responsible for circadian rhythms in pancreatic β-cells [55]. It is believed that the Timeless protein can be identified as a kinase suppressor with Ras-1-like activity [56].…”