Lung cancers in never- and light-smokers often harbor targetable oncogenic mutations in Ras pathway genes. Here, a novel OCLN-RASGRF1 fusion is identified in an otherwise Ras wild-type lung tumor. Studying this and other RASGRF1 fusions, the authors show that these fusions lead to malignant phenotypes that can be reversed by MEK inhibition.
In this issue of Clinical Cancer Research, Hunihan and colleagues describe the discovery and tumorigenic role of a RASGRF1 fusion in lung adenocarcinoma (1). Lung cancer is the deadliest cancer and historically has been difficult to treat. Over recent decades, studies of cancer genomes resulted in the identification of oncogenic mutational “drivers'' which in turn fueled the development of targeted therapies for lung cancer. These therapeutic advances, coupled with smoking cessation efforts, lung cancer screening, and improved diagnostics, are starting to lower the lung cancer mortality rate (2). Almost all the targetable oncogenic drivers of lung adenocarcinoma are part of the Ras/MAPK signal transduction pathway. Previous cancer genome studies have identified mutations in this pathway in upwards of 75% of lung adenocarcinoma samples (3).
Traditionally, lung cancer was considered to be a disease restricted to individuals with a history of smoking. However, a troubling recent increase in the incidence of lung cancer, particularly lung adenocarcinoma, in never-smokers has been reported (4, 5). Lung cancer in patients with minimal or no history of smoking (never-/light-smokers) is strikingly different from those with a history of heavy smoking. Never-/light-smokers tend to have a high frequency of actionable mutations that drive carcinogenesis, are more likely to be women, and the tumors that occur are predominantly adenocarcinomas (6). While lung cancers in such patients have actionable oncogenic drivers, some tumors lack known oncogenic mutations and therefore lack targeted therapy opportunities. One can postulate that these patients harbor novel mutations that go undetected by current diagnosis methods.
To identify rare oncogenic drivers in never-/light-smokers, Hunihan and colleagues evaluated a cohort of 103 lung adenocarcinomas from individuals with a less-than-10 pack-year smoking history using existing clinical molecular diagnostic data or targeted next-generation sequencing. Ninety-five percent of the individuals harbored alterations in established driver genes such as EGFR or KRAS. In five samples, routine clinical molecular profiling did not identify any known drivers. The authors performed whole-exome sequencing (WES) and RNA sequencing (RNA-seq) on these samples to identify any missed driver mutations or novel alterations. In one sample, they identified a novel in-frame fusion of the Ras guanine nucleotide exchange factor gene RASGRF1 and the tight junction gene OCLN (Fig. 1A).
RASGRF1 encodes a guanine nucleotide exchange factor that catalyzes the release of GDP from Ras proteins allowing its return to a GTP-bound active state and thus promoting Ras signaling. The rearrangement resulted in the fusion of the transmembrane domain of OCLN to the catalytically active C-terminal Ras-GEF domain of RASGRF1 (OCLN-RASGRF1). Querying publicly available data, the authors identified two similar RASGRF1 fusions involving SLC4A4 or IQGAP1 in pancreatic ductal adenocarcinoma and giant cell carcinoma, respectively. The authors suggest that the identification of RASGRF1 fusions in other cancers, particularly those that are Ras wild-type, could help identify additional Ras-driven cancers and thus open novel opportunities for therapeutic intervention. Similar RASGRF1 fusions have also been identified involving TMEM154 (7), TMEM87A (8), CD63, EHBP1, and ABCC2 (9) in different cancer models validating the recurrent nature of such fusions and the potentially pan-cancer nature of RASGRF1 fusions.
The authors went on to functionally evaluate OCLN-RASGRF1, SLC4A4-RASGRF1, and IQGAP1-RASGRF1 fusions in a series of in vitro and in vivo assays. Each fusion was found to stimulate increased levels of GTP-bound Ras and activation of the RAF-MEK-ERK pathway (Fig. 1B). OCLN-RASGRF1 appeared more active compared with the other two fusions. Membrane localization studies of OCLN-RASGRF1 showed that the fusion protein localized to the plasma membrane. The precise implication of this localization remains to be elucidated, but authors suggested that the localization might be required to promote activation of Ras, which also requires membrane association.
Further supporting the role of OCLN-RASGRF1 as an oncogenic driver in lung cancer, expression of the fusion promoted anchorage-independent growth of NIH3T3 cells and IL3-independent proliferation of Ba/F3 cells. Expression of OCLN-RASGRF1 in NIH3T3 cells also promoted robust tumor formation in immune deficient mice, demonstrating the oncogenic potential of this fusion and which could provide a model system to interrogate the drug sensitivities of the fusion protein in the future. Currently there are no known inhibitors of RASGRF1 and cells expressing the RASGRF1 fusion were not sensitive to inhibition of another Ras-GEF protein, SOS1. The authors therefore screened 1,728 small molecule inhibitors to find compounds with selective activity against OCLN-RASGRF1. Sixteen inhibitors specifically impaired viability of OCLN-RASGRF1-expressing Ba/F3 cells. Four classes of compounds showed a significant impact on cell viability: RAF/MEK/ERK inhibitors, PI3K inhibitors, HMG-CoA reductase inhibitors, and antiestrogens. Among these, the MEK inhibitor trametinib appeared particularly attractive for further study. Trametinib induced potent inhibition of ERK signaling and induced apoptosis in PaCaDD13 cells, a pancreatic cell line which harbors a SLC4A4-RASGRF1 fusion.
Overall, this work highlights the need for deeper genetic interrogation of otherwise oncogene-negative lung tumors to identify novel therapeutic vulnerabilities. RASGRF1 fusions have been found in other cancer types, and the current study expands the oncogenic role of RASGRF1 fusions to include lung cancer, a disease strongly connected to Ras activation. One limitation of this study is the use of non-lung cancer cells (NIH3T3) for the demonstration of the oncogenic potential of OCLN-RASGRF1. However, NIH3T3 cells are a classic model of Ras activation that has correlated well with lung cancer potential for other Ras pathway genetic variants. In addition, the precise mechanism of fusion activation remains to be elucidated. Both upregulation of RASGRF1 via the OCLN promoter or alteration of cellular localization of RASGRF1 may play a role.
The identification of OCLN-RASGRF1 as a novel driver in a subset of lung adenocarcinomas adds to the list of Ras pathway drivers in lung cancer and suggests other as yet unknown genomic alterations may activate this pathway in lung cancer. Hunihan and colleagues’ identification of RASGRF1 fusions as oncogenic drivers of lung cancer raises the possibility of therapeutic targeting of RASGRF1 fusions in lung and other tumors in the future. If successful, RASGRF1 fusions may be the latest in a long list of critical biomarkers in lung cancer.
A.H. Berger reports grants from NIH, American Cancer Society, and Pardee Foundation during the conduct of the study as well as personal fees from Puma Biotechnology outside the submitted work. No disclosures were reported by the other author.
S. Moorthi was supported in part by a postdoctoral research fellowship from the Fred Hutch Translational Data Science Integrated Research Center. A.H. Berger was supported in part by NIH/NCI R37CA252050 and the Fred Hutch Innovators Network Endowed Chair.