Abstract 2916: Protein arginine methyltransferase 5 as a therapeutic target for KRAS mutated colorectal cancer
Authors: Shifteh David , Sapir Tzuriel , Goel Sanjay , Maitra Radhashree 
Published: September 2020

Introduction

Protein Arginine Methyl Transferase 5 (PRMT5) is a transcription regulator for multiple cellular processes. It is being tested as a potential target/biomarker in several cancer types. Herein we analyzed the therapeutic efficacy of PRMT5 in KRAS mutated and wildtype (WT) colorectal cancer (CRC) cells. This cohort of patients (40-45%) have a worse prognosis when treated with monoclonal antibodies that have added significant therapeutic benefit for KRAS wild type CRC. Currently, there is no specific FDA approved alternate therapy guidelines for this subgroup of patients.

Methodology

Four CRC cell lines [HCT116 and SW620 (KRAS Mutant); and HKE3, LIM 2405 (KRAS Wildtype)] were analyzed for basal level PRMT5 expression by western blot and qPCR analysis. Next, cells were treated with PRMT5 inhibitor (EPZ015666) at 1µM and 10µM concentration for 72 hours and High-Throughput Presto Blue HS Cell Viability Assay was performed in a 96 well plate reading emission at 595 nm. Cell viability was confirmed by flow cytometry with Via Count reagent. Finally, Annexin V/7AAD Apoptosis assay and propidium Iodide cell cycle analysis were performed.

ResultsBasal level of PRMT5 expression is significantly higher in KRAS mutated cells as compared to KRAS WT cells at both transcriptional and translational levels, as determined by qPCR (2.5 Fold; p=0.01) and Western Blot analysis (2.5-Fold; p=0.011).PRMT5 inhibitor treatment showed a significant reduction in cell viability in KRAS mutated cells compared to KRAS WT cells after 72 hours of treatment by High-Throughput Presto Blue HS Cell Viability Assay (at 1 µM 2.8-fold inhibition; p=0.00003 & at 10µM 2.1-fold; p=0.0025).A 5.3-fold increase in apoptosis was observed in KRAS mutated CRC cells when compared to KRAS WT cells by Annexin V/7AAD Apoptosis Assay (p=0.05) post 72 hours of 10µM inhibitor treatment.Cell cycle analysis of KRAS mutant and wild type CRC cells by flow cytometry using propidium iodide indicated a significant (p= 0.026) G2 phase arrest (7.7% increase) of the mutant cells compared to the wildtype cells when treated with 10µM PRMT5 inhibitor for 72 hours.

Conclusion

PRMT5 expression is significantly upregulated in KRAS mutated CRC cells when compared to KRAS WT CRC cells. While PRMT5 inhibitor treatment showed efficacy in both wildtype and mutant cells, cell cycle arrest and apoptosis are significantly upregulated for the KRAS mutant cells. In addition, significant reduction in cell viability was observed in KRAS mutant cells. PRMT5 functions by symmetrical di-methylation of several proteins including the histones. The mechanism of crosstalk between KRAS mutation and PRMT5 methylation will provide valuable insight into the disease characteristics of KRAS mutated CRC. Further analysis is in progress to delineate the molecular mechanism.

Citation Format: David Shifteh, Tzuriel Sapir, Sanjay Goel, Radhashree Maitra. Protein arginine methyltransferase 5 as a therapeutic target for KRAS mutated colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2916.