Uncovering a player responsible for boosting RIT1 protein abundance and cancer promoting activity

Inappropriate activation of the Ras signaling pathway is a major cancer driver in many tissues. In lung cancer, activating KRAS mutations are common among patients and a lot of research has aimed at finding ways to disarm this pathway. Research from the lab of Dr. Alice Berger has identified another important gene in lung cancer, the Ras-like gene, RIT1, that they have found to be a rare oncogene (diagnosed in less than 5% of cases). RIT1 is capable of activating Ras signaling in tumors that do not harbor KRAS mutations and curiously, many of these cancers do not have mutations in the RIT1 gene either. Instead, the Berger lab has uncovered that RIT1 protein abundance is a major factor promoting oncogenic activity. Therefore, the Berger research team has focused on understanding the players regulating RIT1 protein abundance to identify potential therapeutic opportunities to treat RIT1-driven diseases.

Previously, the Berger lab published a genome-wide CRISPR screen in RIT1-mutant cells to identify genetic vulnerabilities of RIT1-driven lung cancer. “In other words, what genes do RIT1-mutant cells rely on in order to drive cancer growth?” shares Dr. Amanda Riley, a former graduate student in the Berger lab. Currently there are no targeted therapies for RIT1-driven diseases and since RIT1 itself is difficult to target due to its protein structure, this approach could identify genes that are important for RIT1 function. Thus, this method could identify opportunities to block RIT1 by inhibiting those genes, by either using existing inhibitors or provide rationale for new inhibitor development. “This could offer more specific and efficient treatment options for patients,” Dr. Riley explains. This genome-wide screen identified USP9X as a top essential gene. In their recently published iScience study led by Riley, the Berger research team asked “why USP9X was essential and what role USP9X plays in promoting RIT1-driven cell growth,” states Dr. Riley. While the exact mechanisms by which RIT1 drives lung cancer are still being worked out, elevated RIT1 activity has been shown to affect tumor-promoting processes including cell cycle regulation, chromosomal integrity and epithelial to mesenchymal transition (EMT). However, what is clear is that RIT1 protein abundance is important for its cancer promoting function.

The Berger lab’s previous work identified USP9X, which is a deubiquitinase—an enzyme that removes ubiquitin marks from other proteins, as an important RIT1 regulator. “Ubiquitin chains can mark proteins for degradation, and USP9X removes these ubiquitin chains to prevent degradation,” and as a result, this would lead to increased RIT1 abundance, Dr. Riley explains. Following up on their prior work, the researchers sought to validate that USP9X was indeed responsible for regulating RIT1 protein abundance by removing the ubiquitin marks that would normally signal for RIT1 degradation. To do so, they performed functional tests to understand if USP9X was essential for regulating RIT1 abundance and could help promote its oncogenic phenotypes. They found that depletion of USP9X reduced RIT1 protein levels in multiple lung cancer cell lines, which led to reduced cell proliferation and a reversal of RIT1-driven resistance to targeted therapies. To understand whether depletion of USP9X had the same effects in vivo, Dr. Riley describes that they “injected USP9X knockout cells in mice, [and] we found that USP9X knockout tumors grew slower. This was an exciting addition to the paper because it built upon our in vitro findings and further supported the notion that USP9X could be a promising new drug target for RIT1-driven cancers.” Consistent with their hypothesis, the researchers found that USP9X does indeed remove polyubiquitin chains from RIT1, which regulates RIT1 abundance and stability. “Our model suggests that USP9X increases the abundance of RIT1, which is important for RIT1-driven tumor growth. The exact mechanisms underlying RIT1 in lung cancer are still being explored and is an exciting area of ongoing research,” Dr. Riley states. However, she acknowledges that they “have yet to identify the E3 ligase(s) responsible for ubiquitinating mutant RIT1. Our CRISPR screen results identified several candidate E3 ligases, and future work will focus on characterizing these ligases. Additionally, our work found that USP9X targets both mutant and wild-type RIT1. RIT1 amplifications are also linked to cancer, but future work is needed to understand the oncogenic mechanisms underlying RIT1 amplifications in lung cancer.”

Since “USP9X depletion abrogates RIT1-driven cell growth, USP9X inhibition could be a promising new therapeutic avenue for treating RIT1-driven cancers,” shares Dr. Riley. “Although our work focused on lung cancer, RIT1 is implicated in other cancer types. There are some USP9X [targeting drugs] currently available, and it will be interesting to test these compounds and work on developing more specific and efficient USP9X inhibitors.” She concludes by acknowledging that this work was possible thanks to “some incredible collaborators on this project. Dr. Lixin Wan’s group at the Moffitt Cancer Center in Florida are experts in ubiquitination and performed many of the ubiquitination experiments presented in the paper. Dr. Pau Castel–another RIT1 expert–contributed mass spec data to help support our findings. It was fantastic to work with all of these talented scientists around the country!”

This work was supported by the National Institutes of Health.

Fred Hutch/UW/Seattle Children’s Cancer Consortium member Dr. Alice Berger contributed to this work.

Riley AK, Grant M, Snell A, Cromwell E, Vichas A, Moorthi S, Rominger C, Modukuri SP, Urisman A, Castel P, Wan L, Berger AH. The deubiquitinase USP9X regulates RIT1 protein abundance and oncogenic phenotypes. iScience. 2024.