Abstract 1956: miR-181d degradation mediated genetic heterogeneity and acquired resistance
Authors: Ramakrishnan Valya , Akers Johnny , Nguyen Thien , Wang Aaron , Adhikari Bandita , Hirshman Brian , Li Jie , Sarkaria Jann , Hua Wei , Ying Mao , Nitta Masayuki , Jiang Tao , Carter Bob , Chen Clark C. 
Published: August 2018

Abstract
When unrepaired, alkylated DNA can induce cell death or trigger mutagenesis. Cellular capacity for repair of these lesions by O6-methylguanine methyltransferase (MGMT) dictates the equilibrium between cell viability and genetic diversity. Treatment of glioblastoma cells with temozolomide (TMZ) induced ATM- and Rad3-related (ATR) kinase dependent polyribonucleotide nucleotidyltransferase 1 (PNPT1) degradation of miR-181d. miR-181d suppresses MGMT expression; its degradation increases the mean MGMT expression of the cell population. miR-181d degradation also magnifies the cell-to-cell variability in MGMT expression, expanding the genetic heterogeneity of the population. This expanded heterogeneity enhances the “fitness” of the population and constitutes a novel form of chemotherapeutic resistance. These effects can be suppressed by overexpression of miR-181d, suggesting miRNA delivery as a strategy for glioblastoma therapy. To characterize the mechanism of acquired resistance, we profiled the expression of 2400 miRNAs before and after TMZ treatment. In independent patient-derived neurosphere lines, the majority of miRNAs remained unchanged after treatment. However, miR-181d was consistently suppressed after TMZ treatment. Our previous work demonstrated miR-181d as the master regulator of MGMT. We confirmed TMZ-induced suppression of miR-181d using independent in vitro and in vivo models as well as matched pre- and post-TMZ treated clinical specimens. TMZ-induced miR-181d suppression persisted after transcriptional inhibition, suggesting degradation as the primary mechanism. We performed an siRNA screen and identified polyribonucleotide nucleotidyltransferase 1 (PNPT1) as the gene responsible for miR-181d degradation. CRISPR inactivation of PNPT1 eliminated TMZ-induced suppression of miR-181d; this was rescued by wild-type PNPT1 but not by PNPT1 harboring RNAse-inactivating mutations. TMZ-induced degradation of miR-181d requires ATR kinase. Silencing or inhibition of ATR eliminated binding of PNPT1 to miR-181d and prevented degradation of miR-181d. TMZ-sensitizing effects of ATR inhibition were reversed by anti-miR-181d, suggesting miR-181d is essential in this process. In addition to elevating the mean MGMT expression of the population, single-cell analysis revealed that miR-181d degradation broadened the cell-to-cell variability in MGMT expression in vitro. In matched clinical pre- and post-TMZ treated specimens, variability in MGMT expression was significantly elevated in post-TMZ samples. This was recapitulated using The Cancer Genome Atlas (TCGA) database. We propose that miR-181d degradation-mediated expansion of genetic heterogeneity enhances the “fitness” of the population, constituting a novel form of chemotherapeutic resistance. These effects are suppressed by miR-181d overexpression, suggesting miRNA delivery as a strategy for glioblastoma therapy.
Citation Format: Valya Ramakrishnan, Johnny Akers, Thien Nguyen, Aaron Wang, Bandita Adhikari, Brian Hirshman, Jie Li, Jann Sarkaria, Wei Hua, Mao Ying, Masayuki Nitta, Tao Jiang, Bob Carter, Clark C. Chen. miR-181d degradation mediated genetic heterogeneity and acquired resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1956.