Functional understanding of Ras-Ral signaling in development and cancer

Ras is a small GTPase frequently mutated in various types of cancer. All knowledge of how Ras signaling influences our cells can potentially be exploited to fight cancer. Several of the signaling pathways downstream of Ras are well-known, whereas others are less understood, such as signaling through Ral, which is itself a small GTPase. The main goal of the project is to describe Ras-Ral signaling at the molecular level. Our strategy is to perform a screen in fruit flies for genes that modify a phenotype induced by active Ral. In the first part of the project, we have expressed active Ral in different fly tissues to characterize the phenotypes. I have also expressed dominant negative Ral or removed Ral by RNAi and analyzed the phenotypes. Furthermore, the dependence of these phenotypes on Ral and known Ral-interacting proteins have been verified. In addition to genetic tools, I have also used biochemical approaches with Drosophila S2 cell culture to answer specific questions of Ral activation. Through this screening effort I have discovered a role for Ral in the development of the larval hematopoietic system, which brings insight on how development of specific cell types occurs. These results have been presented at conferences and received excellent feedback, and are now being prepared for publication. A subgoal of the project was to characterize the proteins CNK, Rap1 and Ral in the development of the fly eye. This system allows testing the effect of these proteins on cell shape, polarity and development. In June 2014 we published results from a screen for genes that modify a fly eye phenotype induced by CNK. The gene coding for Rap1 was one of the candidates from the screen, and we could chow that Rap1 activity is required to maintain the apical domain of photoreceptor cells during development. These and other related results will be pursued in future projects.

Ras mutations are found in more than 30 % of human cancers. Therefore an urgent need persists for identification and functional understanding of modulators of Ras signaling, since they are highly relevant as therapeutic targets. Ras signals through sever al downstream pathways that have already been delineated, including the Raf-MAPK, the PI3K-AKT and the Ral axis. Of these, the Ral pathway is the least studied, but emerging evidence for a role of Ral in Ras-induced oncogenesis has spurred a recent intere st in this pathway. To understand how the Ras-Ral signaling axis contributes to normal and malignant growth it is important to understand the spatiotemporal regulation of this pathway. This can be achieved by identification of genetic interactors of Ral t hat will bring new functional knowledge of the pathway and uncover potential therapeutic targets. Drosophila is a powerful system to identify conserved genes that functionally interact with specific human oncogenes. As a homologue of Ral is also found in flies, we anticipate that expression of Ral during development affects some of the same protein networks that are perturbed in human cancers. Therefore we will use the phenotype induced by expression of constitutively active Ral during eye development to isolate genetic modifiers of this phenotype. We expect the identification and characterization of these modifiers will lead to an enhanced understanding of the spatiotemporal regulation of the Ras-Ral signaling axis, potentially uncovering new therapeutic targets to the benefit of cancer patients.