Title: FOLR1 Upregulation- A Mechanism Behind Minimal Residual Disease in Tepotinib-Treated MET Amplified NSCLC

1Soumya Malhotra, 2 Simon Baldacci, 2 Francesco Facchinetti, 2 William Feng, 2 Pasi A. Janne 1Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, Fort Lauderdale, Florida 2Department of Medical Oncology, Dana Farber Cancer Institute, Boston, Massachusetts

Background: MET proto-oncogene encodes a receptor tyrosine kinase involved in cell proliferation and survival. Therefore, MET dysregulation can lead to unchecked tumor growth, invasion, and metastases. MET tyrosine kinase inhibitors (TKIs), such as tepotinib, have changed the landscape of MET Amplified Non-Small Cell Lung Cancer (NSCLC); however, patients relapse frequently due to the incomplete removal of tumor cells, resulting in the establishment of minimal residual disease (MRD). MRD, described as persister cancer cells remaining in the body after treatment, forms the basis for increased resistance development in MET Amplified NSCLC. In recent years, through systematic evaluation, Folate Receptor Alpha (FOLR1), a cell surface folate binding protein, has been implicated in cell growth regulation, DNA biosynthesis, repair, and methylation, contributing to cancer growth and metastases in solid tumors. This study investigates FOLR1 upregulation as a resistance mechanism in tepotinib-treated MET Amplified NSCLC.

Methods: Cell populations were divided into control and treated. The control population was subdivided into two to account for growth time and plate crowding. Drug toxicity assays were conducted using the Pierce BCA Protein Assay protocol to determine optimal treatment dosage. The control population was treated with low concentrations of dimethyl sulfoxide (DMSO), while the treated population received 5nM tepotinib. Both populations were treated the day following cell plating, with drug renewal performed on day five. All live cells and their culture media were harvested separately on day ten. FOLR1 expression was assessed in the harvested cells utilizing Reverse Transcription- Quantitative Polymerase Chain Reaction (RT-qPCR), Flow Cytometry, Western Blot, and in the culture media using enzyme-linked immunosorbent assay (ELISA) assays.

Results: The results were analyzed using the GraphPad Prism program. Both populations were confirmed to express FOLR1. Control populations showed a baseline FOLR1 expression, thus confirming its presence in MET Amplified cancer cells. Moreover, the persister cells in the treated population showed a significant upregulation consistent across qPCR, ELISA, Western Blot, and Flow Cytometry assays. These results indicate that FOLR1 changes start at the transcriptional level, as shown by the increased mRNA expression through RT-qPCR. mRNA changes lead to increased cell surface and total FOLR1 protein, evidenced by the Flow Cytometry and Western Blot. Furthermore, increased FOLR1 concentrations in the culture media, measured through ELISA, affirms high FOLR1 cleavage in persister cells. These results are consistent with previous studies confirming increased FOLR1 serum concentrations in NSCLC patients.

Conclusion: Our findings provide a rationale for targeting FOLR1 in MET Amplified NSCLC. Ongoing studies to elucidate tepotinib response on FOLR1 knockout cells would strengthen our results. Furthermore, simultaneous in-vivo testing is helping progress our understanding of adverse effects related to targeting FOLR1. Antibody-drug conjugates and Chimeric Antigen Receptor T-cells are at the forefront of cancer research as targeted agents that link a cytotoxic drug to a monoclonal antibody or re-engineer T cells to specifically recognize a cellular surface antigen, improving the efficacy of chemotherapy and reducing systemic exposure and toxicity. Using this technology for FOLR1 targeted treatments could provide a new advent for managing MET Amplified NSCLC with decreased resistance-related failures.