DNAR-04. Variance in Methyl-guanine methyl transferase (MGMT) expression as a defining feature of intra-tumoral heterogeneity that contribute to acquired temozolomide resistance in glioblastoma

Abstract BACKGROUND The concepts of mean and variance serve complementary roles in biological research. While the former captures the central tendencies of a trait in populations, the latter defines the degree of spread or dispersion of the data points relative to that central tendency. Most biological studies have focused on the differences in means. Here, we explore the relevance of variance in protein expression as a mechanism of therapeutic resistance for glioblastomas. METHODS Preclinical studies. RESULTS Temozolomide treatment induced polyribonucleotide nucleotidyltransferase 1 (PNPT1) dependent degradation of miR-181d, a methyl-guanine methyl transferase (MGMT) regulating miRNA, resulting in de-repression of MGMT expression and acquired temozolomide resistance. In addition to MGMT, miR-181d additionally downregulates PNPT1. Degradation of miR-181d, therefore, creates a feed-forward cycle, expediting an increase in the steady-state MGMT level and acquired temozolomide resistance. The degradation requires the activation of the DNA damage response through Ataxia Telangiectasia and Rad3-related (ATR) kinase. The degradation of miR-181d in glioblastoma cells increased both the mean and the variance of MGMT expression in the cell population. Subclone reconstituted glioblastoma populations with similar populational mean MGMT levels but with differences in the variance of MGMT expression exhibited differential temozolomide sensitivity, with the higher MGMT variance population showing increased resistance. This variance is one defining feature of intra-tumoral heterogeneity, and the variance-mediated temozolomide resistance is suppressed by exogenous administration of miR-181d. CONCLUSIONS Our study reveals a novel mechanism of temozolomide resistance in glioblastoma, wherein chemotherapy-induced miR-181d degradation increases MGMT variance. This finding offers a mechanistic basis for understanding the contribution of intra-tumoral heterogeneity to acquired temozolomide resistance and highlights the potential for miRNA delivery as a therapeutic strategy.