Abstract A03: Modeling the therapeutic benefit of Ras-family inhibition in vivo
Authors: Burkhart Deborah L. , Gamper Ivonne , Rebocho Ana P. , Yan Haixi , Littlewood Trevor D. , Evan Gerard I. 
Published: May 2015

Cancers depend upon co-opting the core machinery that allows a normal cell to convert extrinsic signals into a cellular response such as proliferation, arrest, or apoptosis. Inhibition of the protein families at the core of this machinery may cause the regression of tumors; however, this inhibition could disrupt normal tissues that depend upon continuous proliferation. One such protein family that holds a key place in this regulatory machinery is the Ras family of small GTPases, comprised of N-Ras, K-Ras and H-Ras. The Ras proteins integrate cell surface signals from Receptor Tyrosine Kinases (RTKs) and transduce their consolidated signal via a cascade of downstream intracellular kinases that regulate transcription factors, cell architecture, cell cycle and metabolism.

In order to model the potential therapeutic index of Ras-family inhibition in vivo, we have generated a novel transgenic mouse in which the expression of a pan-Ras dominant negative protein (H-RasN17) is regulated by doxycycline. These mice enable us to model inhibition of the Ras pathway in vivo and thus determine the likely therapeutic efficacy of Ras inhibition.

Expression of H-RasN17 reduces the expression of phospho-Erk in mouse embryonic fibroblasts (MEFs). Furthermore, persistent expression delays entry into the cell cycle from G0 and slows asynchronously proliferating cells. These effects are completely reversible when expression of H-RasN17 is switched off, through removal of doxycycline. H-RasN17 sequesters Ras-specific guanine exchange factors (GEFs) and therefore may not inhibit active, oncogenic Ras mutants that are constitutively associated with GTP. However, we find that H-RasN17 also reduces proliferation of MEFs that express oncogenic forms of K-Ras. Further studies will explore the ability of H-RasN17 to inhibit transformation induced by oncogenic forms of K-Ras, and determine the mechanism of inhibition.

Preliminary in vivo data suggest that the effects of systemic H-RasN17 expression depend upon its level of expression in different tissues. High levels of H-RasN17 generally result in increased apoptosis in several tissues, particularly the pancreas, within a week of expression, whereas lower levels are well tolerated by adult mice for up to two months.

To complement our in vitro analysis of Ras inhibition in cells expressing oncogenic K-ras, we have expressed H-RasN17 in KrasG12D driven non-small cell lung tumors. Early results suggest that expression of H-RasN17 in these tumors reduces tumor burden, most likely by slowing tumor growth rather than by inducing cell death. Currently, we are further investigating the effects of both high and low levels of H-RasN17 on normal tissues and also in additional tumor models such as LSL-BrafV619E melanoma, MMTV-Her2 mammary gland tumors, and Eμ-Myc lymphomas.

Citation Format: Deborah L. Burkhart, Ivonne Gamper, Ana P. Rebocho, Haixi Yan, Trevor D. Littlewood, Gerard I. Evan. Modeling the therapeutic benefit of Ras-family inhibition in vivo. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A03. doi: 10.1158/1557-3125.RASONC14-A03