Abstract 2002: MAX mutations in endometrial cancer are associated with poor patient outcome, altered E-box binding, and increased tumor vascularity
Authors: Walker Christopher J. , Rush Craig , Dama Paola , Stein Maggie , Shakya Reena , O’Hern Matthew , Mutch David G. , Cohn David E. , Goodfellow Paul J. 
Published: July 2016

Abstract
In contrast to aberrant MYC-signaling, MYC associated factor X (MAX) mutation is not common in many cancers but occurs in a limited number of specific tissue types, where it may be important. We sequenced MAX in 540 endometrioid endometrial cancer (EEC) DNAs. Mutations were seen 5.37% of samples with two hotspots, H28R (n = 12) and R60Q/W (n = 4 R60Q and n = 1 R60W), which accounted for ∼60% of the mutations seen. MAX mutation was associated with reduced recurrence-free survival (RFS) (P = 0.03; HR [95% CI] = 3.798 [1.10-13.0]) and with advanced stage (P = 0.024) and high tumor grade (P = 0.048). The association with reduced RFS remained significant in multivariable analysis (P = 0.05 HR = 2.40 [1.00-5.74]), strongly suggesting MAX mutation contributes to clinical aggressiveness in EEC.
Focusing on the H28R hotspot mutation, we performed whole transcriptome expression profiling using AN3CA endometrial cancer cell lines stably expressing exogenous long and short isoforms of flag-tagged MAX-WT and MAX-H28R. Unsupervised hierarchical clustering and principle component analysis proved that the expression profiles for the MAX-H28R long cells were strikingly different than the MAX-H28R short, MAX-WT and unmodified cells.
The MAX:DNA interface contains a predicted hydrogen bond between his28 and a guanine residue in the E-box motif. We hypothesized that the his-arg mutation seen in EECs would alter DNA binding, and assessed altered binding via chromatin immunoprecipitation/qPCR (ChIP). MAX-H28R had higher affinity for the NPM1 and CDK4 promoter regions, both of which include E-boxes that are well-established MYC/MAX targets. EMSAs for the same regions confirmed ChIP results, and showed that the supershifted MAX-WT and MAX-H28R complexes had different motilities, consistent with altered confirmation. We showed that an anti-MYC antibody was not able to supershift the EMSA fragment, indicating MYC is not part of the MAX:DNA complex. Co-IP, however, showed MAX-H28R retains MYC binding ability. Based on these findings, we conclude the differences seen in EMSA were with MAX homodimers. RNA-seq revealed differential gene expression in MAX-H28R mutated tumors, and ChIP showed enhanced MAX-H28R affinity for E-boxes in several of the differentially expressed genes.
In vivo, MAX-H28R AN3CA xenografts were markedly hemorrhagic compared to MAX-WT. MECA-32 (pan-endothelial marker) staining proved that the MAX-mutant tumors had significantly more vessels per field (2.4-fold increase, P = 0.002). Work is ongoing to test for paracrine factors elaborated by the H28R-mutant AN3CA cells that might contribute to the vascular phenotype.
Taken together these data implicate MAX mutation as a novel driver of endometrial tumorigenesis, contributing to tumor aggressiveness through enhanced tumor vascularity and potentially other mechanisms.
Citation Format: Christopher J. Walker, Craig Rush, Paola Dama, Maggie Stein, Reena Shakya, Matthew O’Hern, David G. Mutch, David E. Cohn, Paul J. Goodfellow. MAX mutations in endometrial cancer are associated with poor patient outcome, altered E-box binding, and increased tumor vascularity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2002.