Brincidofovir induces potent anti‐tumor activity in MYC‐driven Natural Killer/T‐cell Lymphoma with loss of transcriptional repressor TLE1

Abstract

Introduction: Natural killer/T-cell lymphoma (NKTCL) is an aggressive Epstein-Barr virus (EBV)-associated malignancy which is prevalent in Asian countries. Brincidofovir (BCV), a lipid conjugated form of cidofovir, is a biologically-active acyclic nucleoside phosphonate recently shown to exhibit both antiviral and antitumor properties. Methods: We investigated BCV in NKTCL cell-lines (n = 11) and mouse xenograft models. Whole exome sequencing, bulk RNAseq and single cell RNAseq (scRNAseq) were employed to examine mechanisms of action, and validated using orthogonal assays. Results: BCV inhibited viability in all NKTCL cell-lines in a dose- and time-dependent manner. Highest sensitivity was demonstrated in four cell-lines KAI-3, NK-S1, NK-92 and KHYG-1 (IC50 36.0 to 303.6 ng/ml). Intraperitoneal BCV (40 mg/kg, 2X per week) inhibited tumor growth in NOD/SCID mice NK-S1 xenografts, compared with vehicle alone (p = 0.0005, two-tailed t-test). Whole exome sequencing of the cell-lines revealed widespread mutations in the JAK/STAT and DNA repair pathways, with no clear correlation observed with BCV sensitivity. RNAseq and Hallmark gene set enrichment analysis showed that MYC target pathways (FDR q < 0.001) were prominently upregulated in the four sensitive cell-lines compared to the rest. RNAseq of BCV-treated cell-lines (KAI-3 and NK-S1) revealed striking downregulation of MYC target pathways. scRNAseq revealed distinct temporal cell states evoked by BCV, including appearance of cell clusters enriched for p53 and apoptosis pathways, cell cycle and DNA repair pathways, as well as type I/II interferon and cytokine signaling. Western blot and quantitative PCR showed that markers of replication stress, DNA damage and STING signaling were potently upregulated, implicating an immunogenic form of cell death. Notably, TLE1, a known transcriptional repressor of the MYC oncogene, was the topmost downregulated gene (log2FoldChange -7.39, adjusted p = 3.51E-31) in the four BCV-sensitive cell-lines on RNAseq. This finding was verified on qPCR (p = 0.0061, Mann-Whitney test) and Western blot. In keeping with this result, RNAseq on NKTCL patient samples (n = 36) showed that TLE1-low tumors were enriched for MYC target pathways, which conferred worse progression-free survival (HR 3.44, 95% CI: 1.23–10.49, p = 0.0301). Conclusions: Taken together, these results suggest that BCV may play a novel role in the treatment of MYC-driven NKTCL. Loss of TLE1, a putative predictive biomarker, implies that BCV may favour patients in this poor prognostic subgroup. The research was funded by: Symbio Pharmaceuticals, Tanoto Foundation Professorship in Medical Oncology, New Century Foundation Limited, Ling Foundation, Singapore Ministry of Health’s National Medical Research Council Transition Award (TA21jun-0005), Research Training Fellowship Seed Fund (SEEDFD21jun-0002), Large Collaborative Grant (OFLCG18May-0028), and Collaborative Centre Grant (TETRAD II). Keywords: Diagnostic and Prognostic Biomarkers, Extranodal non-Hodgkin lymphoma, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. J. Y. Chan Consultant or advisory role: Symbio Pharmaceuticals Research funding: Symbio Pharmaceuticals M. Hazama Employment or leadership position: SymBio Pharmaceuticals Limited K. Fukushima Employment or leadership position: SymBio Pharmaceuticals Limited C. K. Ong Research funding: SymBio Pharmaceuticals Limited