Targeting the Ras-Ral effector pathway for cancer treatment

Gentry, Leanna

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March 19, 2019

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Gentry, Leanna
- Affiliation: School of Medicine, Department of Pharmacology

Abstract

The RAS oncogene is the most frequently mutated gene in human cancers, and this activated Ras oncoprotein has been shown to be required for both cancer initiation and maintenance. Great strides have been made in understanding Ras signaling in cancer since the discovery of its involvement in human cancers in 1982, with numerous Ras effector pathways and modes of Ras regulation having been identified as contributing to Ras-driven oncogenesis. However, there has been limited success in developing strategies for therapeutically targeting Ras-driven oncogenesis. One effort that has gained popularity in recent years is the inhibition of Ras effector signaling. The Ral (Ras-like) small GTPases, discovered shortly after Ras in an attempt to identify RAS-related genes, are activated downstream of Ras by Ral guanine nucleotide exchange factors (RalGEFs). The Ral family members have since emerged as critical regulators of key cellular processes and, importantly, have been characterized as playing a role in tumorigenesis and invasion of multiple cancer types. Interestingly, divergent roles for RalA and RalB are often observed in within a cancer. Due to the high affinity of Ral for GTP, which activates Ral upon binding, the Ral GTPase family cannot be targeted directly. Therefore, indirect inhibition of Ral must be considered for targeting Ral-dependent phenotypes in Ras-driven cancers. This could be achieved through inhibition of Ral association with the plasma membrane, which is thought to be required for its activation and subsequent signaling. Alternatively, downstream effectors of Ral with validated roles in cancer could be inhibited. Posttranslational processing of the CAAX motif located on the C-termini of Ral GTPases, among other proteins, has been considered essential for their proper subcellular localization, activation, and function. The first and essential step of this process is prenylation by GGTase. Prenylation signals for further CAAX processing by the enzymes RCE1 and ICMT, which are under consideration as therapeutic targets. We determined that the modifications regulated by these enzymes have distinct roles and consequences for Ral GTPases. We found that both RalA and RalB require RCE1 for association with the plasma membrane, and that the absence of RCE1 caused a sustained activation of both RalA and RalB. In contrast, ICMT deficiency disrupted plasma membrane localization of RalB but not RalA, whereas RalA depended on ICMT for efficient localization to recycling endosomes. Furthermore, ICMT deficiency caused increased stability of RalB protein but not RalA. Lastly, we found that palmitoylation was critical for proper subcellular localization of RalB but not RalA. In summary, we identified isoform-specific consequences of CAAX modifications that could be contributing to the divergent localization and activities of the Ral proteins. In order to address inhibiting Ral effectors, we sought to determine the effect of inhibiting TBK1, a kinase that is a validated effector of RalB, in pancreatic ductal adenocarcinoma, a disease characterized by greater than 90% of cases containing a K-Ras mutation. We found that a novel small molecule inhibitor of TBK1, while effective at inhibiting signaling, had a minimal effect on pancreatic cancer cell proliferation in vitro and in vivo. However, when combined with inhibition of ERK1/2, we found a synergistic proliferation defect and induction of apoptosis. This suggests combination approaches with TBK1 inhibitors may provide therapeutic benefit in the treatment of K-Ras-driven pancreatic cancer. Overall, this work provides further insight into strategies for targeting Ral for the treatment of cancer.

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