Abstract 4609: High-grade TP53-mutated endometrial carcinomas have decreased NRF2 antioxidant activity
Authors: Beinse Guillaume , Just Pierre-Alexandre , Rance Bastien , Izac Brigitte , Letourneur Franck , Saidu Nathaniel Edward , Chouzenoux Sandrine , Goldwasser François , Pasmant Eric , Batteux Frederic , Borghese Bruno , Leroy Karen , Alexandre Jérome 
Published: August 2018

Abstract
Background
NRF2 is a major transcription factor regulating the expression of antioxidative/detoxifying enzymes. Its activation, due to mutations or activation of signaling pathways, has been associated with chemoresistance and poor prognosis in several tumors. NRF2 status remains unclear in endometrial carcinoma (EC). We aimed to identify molecular alterations associated with NRF2 activation in the four molecular subgroups of EC described by the Cancer Genome Atlas (TCGA), and explored its impact on EC prognosis.
Methods
Ninety patients treated in Cochin Hospital (2012 to 2017) for EC were included. Formalin-fixed paraffin-embedded tissue samples were processed for histopathological analysis and DNA and RNA extractions. Next generation sequencing (NGS) of NFE2L2 (encoding NRF2), KEAP1 and CUL3 genes and a panel of 15 genes significantly mutated in EC was performed using AmpliseqTM panels on Ion TorrentTM PGM (Thermo Fisher). The nuclear expression of mismatch repair (MMR) and NRF2 proteins were analyzed by immunochemistry (IHC). NRF2 activity was assessed by NQO1, GCLC, and AKR1C3 mRNA expressions, normalized to MRPL19 and TBP housekeeping genes, using TaqManTM assays and quantitative RT-PCR. Clinical event of interest was event-free survival (EFS) (progression, relapse, or death).
Results
Tumors were classified according to NGS and IHC data as POLE exonuclease domain mutated (N=3, 3%), MMR-deficient (dMMR) (N=28, 31%), TP53 mutated (N=23, 25%), and MMR-proficient tumors (N=32, 36%). NRF2 nuclear immunostaining was not correlated to NRF2 activity. The 3 tumors with highest NRF2 target genes expression harbored known NRF2 pathway activating mutations (NFE2L2 p.W24G, KEAP1 p.R336*, KEAP1 p.D422N). In addition, 2 dMMR tumors showed intermediate/high (1st quartile) NRF2 target genes expression and low allele ratio NFE2L2 mutations, suggesting NRF2 subclonal activation. No correlation was observed between NRF2 activity and PI3K or KRAS pathways mutations. The TP53 subgroup showed a strikingly lower NQO1 expression compared to dMMR or pMMR tumors (ANOVA p&lt;.05). This observation was confirmed by in silico analyses of publicly available TCGA data. NQO1 low expression was significantly associated with poor EFS, independently of tumor stage (Cox p&lt;.01).
Conclusion
In contrast with previous reports based on IHC, NRF2 activation is a rare event in EC, associated with NFE2L2 or KEAP1 mutations. NQO1 downregulation in the TP53-mutated subgroup might be explained by the interaction of NRF2 with TP53 missense mutants, which has been shown to impair its anti-oxidant transcriptional activity while enhancing proteasome expression (Walerych et al. Nat Cell Biol 2016). Very low levels of NQO1 expression identifies a subset of EC with a poor prognosis, which might be sensitive to specific combination therapies (Liu et al. Nat Commun. 2017).
Citation Format: Guillaume Beinse, Pierre-Alexandre Just, Bastien Rance, Brigitte Izac, Franck Letourneur, Nathaniel Edward Saidu, Sandrine Chouzenoux, François Goldwasser, Eric Pasmant, Frederic Batteux, Bruno Borghese, Karen Leroy, Jérome Alexandre. High-grade TP53-mutated endometrial carcinomas have decreased NRF2 antioxidant activity [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 4609.