Abstract 1434: Loss of PTEN confers resistance to neddylation inhibition through TOP2A in glioblastoma
Authors: Taylor Brett , Ferdosi Shayesteh , Tang Nanyun , Bybee Rita , Reid George , Hartman Lauren , Finlay Darren , Vuori Kristiina , Lee Matthew , Peng Sen , Furnari Frank , Dhruv Harshil , Berens Michael 
Published: September 2020

Abstract
As a disease that has not seen lasting advances in its standard of care for decades, glioblastoma (GBM) calls for novel therapeutic approaches, and given GBM's heterogeneous and aggressive nature, precision medicine stands out as an appealing treatment modality for this tragic disease. Neddylation is a post-translational modification process that regulates protein function and turnover. MLN4924, or Pevonedistat, inhibits the E1 ligase Nedd8 activating enzyme (NAE) and, in doing so, the E1, E2, and E3 neddylation cascade. In its most researched role, NEDD-8 is conjugated to the cullin subunit of cullin-RING ligases (CRLs), a family of E3 ligases that facilitate the ubiquitination of different substrates. Thus, inhibition of neddylation decreases ubiquitination and degradation of particular substrates: e.g., p21, p27, wee-1, and other cell cycle regulatory and pro-apoptotic proteins. We and others have shown that neddylation stands out a promising therapeutic approach to cancer and GBM, given the vulnerability to MLN4924 in subset of different in vitro and in vivo models. In this work, we establish the selective vulnerability to MLN4924 in GBM and explore the biological mechanisms that underlie this differential response. Using the Cancer Cell Line Encyclopedia (CCLE) and the Genomics of Drug Sensitivity in Cancer (GDSC) databases, we found that mutations and deletions in phosphatase and tensin homolog (PTEN) are associated with resistance to MLN4924 in both low-grade gliomas and GBM. Our established models validated these findings, as our sensitive models – GB1 (IC50= 0.28 μM) and LN18 (IC50 = 0.19 μM), – expressed PTEN, while our resistant models – M059K (IC50 = 5.5 μM) and SNU1105 (IC50 = 20.9 μM) – did not. Knocking down PTEN with siRNA in our sensitive models increased their IC50 values three- to four-fold. This relationship was further validated in multiple isogenic systems. We sought to uncover whether this PTEN-status dependent resistance was contingent on its well-studied lipid phosphatase activity and PI3K/AKT signaling. While the resistant models had higher baseline levels of phosphorylated AKT (p-AKT), we found that MLN4924 treatment did not consistently alter p-AKT levels in our sensitive and/or resistant models. We also used a PTEN null isogenic glioma stem cell system (GSC23), in which wild-type PTEN and lipid phosphatase dead (G129A) PTEN was re-expressed, and found that both PTEN models sensitized GSC23 to MLN4924 treatment equally. While independent of the PI3K/AKT axis, we did find that there were significant increases in topoisomerase II alpha (TOP2A) levels in the post-treatment lysate of our resistant (PTEN-null) models. Targeting this increase in TOP2A expression, we found strong synergy with two different TOP2 poisons – etoposide and doxorubicin – and MLN4924 in a combination DDR. For the first time, we show that PTEN status not only serves as a novel biomarker for MLN4924 response but also reveals a vulnerability to TOP2A inhibitors when used in combination with MLN4924.
Citation Format: Brett Taylor, Shayesteh Ferdosi, Nanyun Tang, Rita Bybee, George Reid, Lauren Hartman, Darren Finlay, Kristiina Vuori, Matthew Lee, Sen Peng, Frank Furnari, Harshil Dhruv, Michael Berens. Loss of PTEN confers resistance to neddylation inhibition through TOP2A in glioblastoma [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 1434.