SH2B3 mutation as a potential resistance mechanism to oncolytic virus therapy.

Philippos Apolinario Costa Yale New Haven Hospital, New Haven, CT info_outline Philippos Apolinario Costa, Jeffrey Joseph Ishizuka, Kelly Olino, Alexander Frey, Daniel Lee

Authors Philippos Apolinario Costa Yale New Haven Hospital, New Haven, CT info_outline Philippos Apolinario Costa, Jeffrey Joseph Ishizuka, Kelly Olino, Alexander Frey, Daniel Lee Organizations Yale New Haven Hospital, New Haven, CT, Yale School of Medicine, New Haven, CT Abstract Disclosures Research Funding No funding sources reported Background: Oncolytic viruses (OV) are viruses that preferentially destroy cancer cells while sparing healthy cells. Currently, talimogene laherparepvec (TVEC) is the sole FDA-approved OV product; however, resistance to therapy often emerges. To further understand OV resistance mechanisms, we performed transcriptome and mutational analysis on patient samples using a discordant lesion approach. Methods: An 80-year-old woman was diagnosed with Merkel cell carcinoma of the right lower extremity and underwent wide local excision. Her disease recurred, and she developed multiple metastatic lesions over three years. During that time, she was further treated with radiation, immunotherapy, and intralesional TVEC. Despite the different treatments, her disease eventually progressed, leading to her death. We analyzed the primary lesion and four discordant lesions that either persisted or recurred following treatment. Treatment data were extracted from medical records, and lesions were classified based on their resistance to therapies. DNA and RNA were extracted from tissue, and whole exome sequencing and bulk RNA sequencing were performed. Whole exome data was compared between specimens to determine mutation enrichment over time. Gene Set Enrichment Analysis was used to interpret bulk RNA sequencing data and compare transcriptional states of lesions. Results: Lesions four and five recurred following TVEC treatment and were, therefore, classified as TVEC resistant. Whole exome sequencing of all five lesions identified a heterozygous point mutation (P521L) in the SH2B3 gene in lesions four and five only. Additionally, the variant allelic fraction of this point mutation increased from 33% in lesion four to 44% in lesion five, where it was the highest frequency variant. When comparing lesion five to lesion one, Gene Set Enrichment Analysis showed an increase in both the hallmark inflammatory response gene signature and the hallmark interferon alpha response gene signature. SH2B3 is a gene that encodes the LNK protein, which functions as a negative regulator of the JAK-STAT signaling pathway and is, therefore, a negative regulator of interferon signaling. A mutation leading to a loss of function in SH2B3 would conceptually lead to upregulation of interferon signaling, which has previously been shown to promote an antiviral state within the tumor microenvironment and limit the effectiveness of OV treatment. Conclusions: Sequencing of discordant MCC lesions revealed enrichment in the mutational fraction of SH2B3 associated with an increase in interferon alpha and inflammatory signaling among lesions that were resistant to TVEC treatment. This is the first study to report a possible genetic driver for OV resistance. We introduced using CRISPR-HDR P521L SH2B3 in MCC cell lines to investigate its impact on LNK expression and its role as a mechanism of OV treatment resistance, with results to be presented.