Abstract 1006: Understanding the resistance mechanisms of MTC to RTK inhibitors

Abstract

Abstract Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor caused by activating mutations in the RET proto-oncogene. Vandetanib is an FDA approved systemic therapy targeting RET, EGFR, VEGFR, BRK, TIE2, members of EPH receptors kinase family, and members of Src family of tyrosine kinases. Treatment of MTC patients with metastatic disease leads to partial response accompanied by adverse side effects, poor tolerability, and the development of resistance. The mechanisms of MTC resistance to Vandetanib are not completely understood. Our overall goal is to understand the genetic and epigenetic mechanisms of resistance to Vandetanib, which would lead to novel combination therapies to prevent resistance or utilized to treat MTC patients. TT cell line, a MTC cell line with activating mutation C634W in RET, was used to generate resistant cell lines at 600 nM concentration compared to 270 nM IC50 in the parental cell line. To study the resistance in MTC, exome sequencing was done to identify genetic alterations such as novel mutations and copy number alterations. RNA-seq was performed to identify altered pathways, alternation in drug targets expression, and splicing events of some mRNAs. CRISPR KO screening was performed to validate our finding and identify genes that are synthetic lethal or conforming resistance to vandetanib. Also, we are analyzing the chromosomal changes by SKY analysis to validate copy number variations identified by exome sequencing and investigate tumor heterogeneity and epigenetic changes will be studied by DNA methylation. Preliminary results from exome sequencing confirmed that there is an enrichment in variant allele fraction in resistant cell line of C634W mutation from 0.72 to 0.99 in drug resistant cells. Also, RNA-seq data showed that there is an increased an expression in mutant RET transcript from log2(TPM) of 8.58 to 9.37 in resistance cell lines. Moreover, SKY analysis demonstrated four copies of chromosome 10 where RET gene is located indicating that there three mutant copies and one wild type copy. Western blot data confirmed the activation and an increased in RET expression in resistant cell line. CRISPR Knock-Out data showed that Cdk5, NF-ĸB, and IGF-1 were enriched in resistant cell line; whereas STAT, drug resistance, and mTOR pathway genes found to be depleted, suggest these genes may be synthetic lethal with vandetanib. Overall, our results suggest that the resistance is a result of an increase in mutated RET copies leading to increased RET transcripts. We will explore this hypothesis by performing CRISPR activation screening to identify genes confirms resistance upon activation. Also, we will perform SKY analysis in resistant cell line to determine the copy number of chromosome10. Additionally, simple western and phosphoproteomics will be conducted to identify other signaling alongside RET that may be altered as part of the resistance mechanism. Citation Format: Arwa Fallatah. Understanding the resistance mechanisms of MTC to RTK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1006.