Since their discovery in 1986, Ral (Ras-like) GTPases have emerged as critical regulators of diverse cellular functions. Ral-selective guanine nucleotide exchange factors (RalGEFs) function as downstream effectors of the Ras oncoprotein, and the RalGEF-Ral signaling network comprises the third best characterized effector of Ras-dependent human oncogenesis. Because of this, Ral GTPases as well as their effectors are being explored as possible therapeutic targets in the treatment of RAS mutant cancer. The two Ral isoforms, RalA and RalB, interact with a variety of downstream effectors and have been found to play key and distinct roles in both normal and neoplastic cell physiology including regulation of vesicular trafficking, migration and invasion, tumor formation, metastasis, and gene expression. In this review we provide an overview of Ral biochemistry and biology, and we highlight recent discoveries.

Background:The highly related small GTPases RalA and RalB exhibit distinct functions in cancer cell processes. Results: Posttranslational modifications signaled by the C-terminal CAAX motif contribute to Ral isoform differences in subcellular localization, activation, and protein stability. Conclusion: Modifications catalyzed by RCE1, ICMT, and protein palmitoyl acyltransferase cause Ral isoform-specific functional consequences. Significance: Inhibitors of CAAX motif modifications will have complex consequences on Ral GTPase regulation.

Summary Pharmacologic inhibitors of protein kinases comprise the vast majority of approved signal transduction inhibitors for cancer treatment. An important facet of their clinical development is the identification of the key substrates critical for their driver role in cancer. One approach for substrate identification involves evaluating the phosphorylation events associated with stable expression of an activated protein kinase. Another involves genetic or pharmacologic inhibition of protein kinase expression or activity. However, both approaches are limited by the dynamic nature of signaling, complicating whether phosphorylation changes are primary or secondary activities of kinase function. We have developed rapamycin-regulated (RapR) protein kinases as molecular tools that allow for the study of spatiotemporal regulation of signaling. Here we describe the application of this technology to the Src tyrosine kinase and oncoprotein (RapR-Src). We describe how to achieve stable expression of this tool in cell lines and how to subsequently activate the tool and determine its function in signaling and morphology.

The high frequency (95%) of KRAS mutations in pancreatic ductal adenocarcinomas (PDAC), together with strong evidence for the KRAS dependency of PDAC growth, makes the K-Ras oncoprotein an attractive therapeutic target. However, to date the development of direct inhibitors of K-Ras has not been successful, with most efforts focused on targeting components of K-Ras downstream effector signaling. While the pharmaceutical industry has centered on inhibitors of the Raf or PI3K effector signaling pathways, there is substantial evidence that K-Ras-dependent cancer growth must rely on the activities of other effector pathways. Of those pathways, the Ral guanine nucleotide exchange factor RalGEF) effector pathway, leading to activation of the Ras-like (Ral) small GTPases is well-validated to drive PDAC growth. Interestingly, while the two Ral isoforms share significant sequence identity (82%) as well as the same set of effectors, our studies found that they have strikingly different roles in PDAC growth. While RalA but not RalB is necessary for PDAC anchorage-independent growth in vitro and tumorigenic growth in vivo, RalB is the critical driver of PDAC invasion in vitro and metastasis in vivo. The mechanistic basis for their different biological roles in cancer remains to be elucidated. We hypothesize that their divergent C-terminal sequences and the posttranslational modifications that they signal for result in distinct subcellular localizations that then contribute to Ral isoform-selective effector interactions that promote distinct biological consequences in PDAC. To evaluate our hypothesis, we initiated studies to determine the effector(s) that mediates RalA-dependent anchorage-independent growth and tumorigenesis in vivo, the effector(s) that mediates RalB-dependent invasion and metastasis in vivo; and the role of the C-terminal hypervariable domain in causing distinct RalA- and RalB-dependent phenotypes and effector utilization. We found that both RalA and RalB subcellular localization is dependent on the C-terminal CAAX tetrapeptide signaled modifications by Rce1 and ICMT. We also determined that RalB preferentially activated mTORC1 to regulate pancreatic tumor cell Matrigel migration, through the Sec5 effector and regulation of exocyst function. Citation Format: Leanna R. Gentry, Timothy D. Martin, David J. Reiner, Channing J. Der. Mechanistic dissection of Ral GTPase signaling in driving KRAS-dependent pancreatic cancer growth. [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 A08. doi: 10.1158/1557-3125.RASONC14-A08

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The effectiveness of cancer therapeutics targeting signal transduction pathwaysis compromised by a diversity of mechanisms that drive de novo or acquired resistance. Two recent studies identify mTOR activation as a point of convergence of mechanisms that cause resistance to inhibitors of the Raf-MEK-ERK and PI3K signaling.

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