A chink in lung cancer’s armor

From the Berger Lab, Human Biology Division

The study of cancer is all about classification.  Tumors of the pancreas, breast or lung are further subdivided in classes, subclasses and sub-subclasses in order to understand their individual characteristics and devise specific therapies based on their unique sensitivities.  Lung cancer is broadly divided into two categories, small cell (SCLC) and non-small cell lung cancer (NSCLC) based on the appearance of cells under the microscope and response to chemotherapies.  NSCLC, the most common form of lung cancer, can be further subdivided into three additional categories: adenocarcinoma, squamous cell carcinoma and large cell carcinoma.  Adenocarcinoma is the most common carcinoma accounting for 40% of lung cancer diagnoses.  Again, lung adenocarcinoma subtypes can be grouped by the genetic alterations that drive tumor development.  Changes in activity or expression level of different member of the epidermal growth factor receptor (EGFR)/RAS pathway led to aberrant activation of downstream pathways that drive proliferation are a common feature in a majority of lung adenocarcinomas. Mutation or amplification of EGFR or KRAS as well as other members of this pathway are well documented.  The laboratory of Dr. Alice Berger in the Human Biology Division recently added a new member to the list of oncogenes in this pathway, the small RAS-family GTPase RIT1.  As with other members of the EGFR/RAS pathway, mutations or amplification of RIT1 are frequent, and, interestingly, mutually exclusive of EGFR or KRAS changes.  Dr. Berger explained the need for additional research on this new oncogene: “RIT1 is mutated or amplified in up to 14% of lung adenocarcinomas, but very little is known about the function of this gene. RIT1 mutations often occur as the only RAS pathway mutation in a lung tumor – so patients with RIT1 mutant lung cancer don’t have the effective targeted therapy options that patients with EGFR mutant lung cancer can access.”

Dr. Berger’s laboratory in collaboration with Cancer Consortium members Robert Bradley, John Lee and Emily Hatch recently published a report detailing the unique functional profile of RIT1 mutated lung adenocarcinoma in the journal Nature Communications.  Since RIT1, EGFR and KRAS mutations are mutually exclusive, it is likely that all three activate the same pathway. However, since they occur at different nodes in the pathway, the investigators sought to determine whether genetic or pharmacological sensitivities were similar or distinct among lung cancer cell lines harboring each mutation.  To interrogate the entire genome for potential drug targets, a CRISPR/Cas9 gene editing approach was used to disrupt nearly 20,000 genes and identify which disruptions led to specific growth inhibition or lethality in the context of EGFR, KRAS or RIT1 mutations.  To this end, the researchers constructed a PC9-derived lung cancer cell line, which was first engineered to express the bacterial Cas9 protein, a critical component of the CRISPR gene editing complex.  The mutant protein (EGFR, KRAS or RIT1) was introduced through lentiviral transduction.  Finally, the three cell lines were individually transduced with libraries of guide RNA constructs targeting 19,114 human genes.  The cells were grown in culture approximately 12 population doublings allowing sufficient time for targeted gene disruption to manifest a robust phenotypic impact on cell growth and viability.  To determine which disruptions impacted cell growth, genomic DNA was extracted and guide RNAs from the cells were PCR amplified and sequenced.  The viability of a given gene disruption was proportional to the abundance of the guide RNA in the sequencing output.  Computational analysis of the sequencing data identified genetic dependencies unique to each of the three EGFR/RAS pathway oncogenes as well as dependencies common to most or all of them. 

In addition to previously identified interactions among EGFR/RAS pathway members, the investigators found a new and unexpected interaction.  RIT1 mutant cells are uniquely sensitive to disruption of mitotic spindle assembly checkpoint functions.  This critical checkpoint monitors sister chromatid attachment during the metaphase portion of mitosis to ensure proper chromosome segregation during anaphase.  The checkpoint assures that both daughter cells receive the proper complement of chromosomes.  Thus, pharmacological modulation of chromosome segregation may have clinical efficacy in RIT1 mutant lung cancer.  The authors demonstrate that inhibition of Aurora A kinase, a component of the spindle assembly checkpoint, leads to an increase in mitotic errors to a greater extent in RIT1 mutant cells when compared to RIT1 wildtype cells.  This unanticipated discovery may open the way to new therapies for lung adenocarcinoma. 

Another new interaction was uncovered between the transcriptional activator YAP1 and RIT1.  YAP1 is part of the Hippo pathway that regulates cell growth and proliferation and activation of YAP1 synergizes with RIT1 mutation to transform normal small airway lung epithelial cells.  Reexamination of existing data on human lung cancer confirmed that the majority of RIT1 mutant lung cancer occurs in the context of Hippo pathway mutations expected to lead to increased YAP1 activity.  Interestingly, the RIT1/YAP1 interaction is unique to RIT1 and is not found with EGFR or KRAS.  This intriguing finding is likely to lead to a better understanding of the genetic basis of lung cancer.  Dr. Berger summarizes: “By taking a genome-wide approach, we discovered two totally new mechanisms of RIT1 function that may be targetable in lung cancer. The first mechanism was a role for RIT1 in the spindle assembly checkpoint which leaves RIT1 mutant cells vulnerable to anti-mitotic therapies. The second was an unexpected synergy between mutant RIT1 and the YAP oncoprotein – something that we observed directly in human lung tumors as well as in our cell line screens. We hope that these early-stage discoveries will lead to new therapies for RIT1-mutant disease in the future.”


Graphical Representation from Heffner Lab
Image from Heffner Lab

This research was supported by grants from the National Cancer Institute, National Institute of General Medical Sciences, National Science Foundation, Lung Cancer Research Foundation, Hutch United as well as donations from the Smith family to A.H.B.

Fred Hutch/UW Cancer Consortium members Robert K. Bradley, John K. Lee, Emily M. Hatch and Alice H. Berger contributed to this study.

Vichas A., Riley A.K., Nkinsi N.T., Kamlapurkar S., Parrish P.C.R., Lo A., Duke F., Chen J., Fung I., Watson J., Rees M., Gabel A.M., Thomas J.D., Bradley R.K., Lee J.K., Hatch E.M., Baine M.K., Rekhtman N., Ladanyi M., Piccioni F., Berger A.H. (2021). Integrative oncogene-dependency mapping identifies RIT1 vulnerabilities and synergies in lung cancer. Nat Commun. 12(1), 4789.