Abstract 5835: Oncogenic activation of the serine synthesis pathway by the scaffolding protein menin

Abstract

Abstract Developmental transcription programs are epigenetically regulated by multi-protein complexes including the MLL-containing trithorax (TrxG) complexes, which methylate H3K4 to promote gene transcription. We recently reported that the TrxG protein MLL1 and its binding partner menin are overexpressed by and function as oncogenes in Ewing sarcoma. Moreover, small molecule inhibition of the menin-MLL1 protein-protein interaction leads to loss of menin and MLL1 protein expression and to inhibition of growth and tumorigenicity. For the current study we investigated the mechanistic basis of menin-MLL1 mediated oncogenic activity in Ewing sarcoma. Bru-seq analysis was performed to identify changes in nascent gene transcription in Ewing sarcoma cells following exposure to the menin-MLL interaction inhibitor MI-503. Identified targets were validated by qRT-PCR, western blot, immunocytochemistry and by interrogation of published databases. Loss of function was achieved by lentiviral shRNAs and by inhibitor treatment. Exposure of three independent Ewing sarcoma cell lines to MI-503 resulted in significant and reproducible modulation of nearly 100 genes. Bioinformatics analysis of MI-503-regulated genes revealed that the serine synthesis pathway (SSP) was the most significantly impacted pathway downstream of MI-503 treatment. The SSP has recently emerged as a major mediator of tumorigenicity in multiple human cancers. qRT-PCR confirmed reduced expression of SSP enzyme-encoding genes PHGDH, PSAT1 and PSPH in MI-503 treated cells. To determine if the SSP contributes to Ewing sarcoma pathogenesis we evaluated expression of SSP genes and proteins in tumors and cell lines. Our findings showed that Ewing sarcomas express high levels of PHGDH, PSAT1, and PSPH and interrogation of the Cancer Cell Line Encyclopedia revealed that PHGDH expression is higher in Ewing sarcoma than in any other cancer in the database. Studies of Ewing sarcoma cells in serine and glycine free media showed that cells retain viability but that exposure to the PHGDH inhibitor, CBR-5884, inhibits survival in both standard and serine/glycine-free conditions, confirming the importance of the pathway to tumor pathogenesis. To determine how menin-MLL1 promote SSP activation we performed loss of function studies and discovered that knockdown of menin, but not of MLL1, leads to a significant reduction in SSP gene expression, implicating menin as the primary mediator of SSP gene activation. Transcriptional up regulation the SSP pathway has been linked to ATF4 in non-small cell lung cancer. Significantly, ATF4 protein expression is high in Ewing sarcoma cells and ATF4 transcript and protein expression are down regulated following MI-503 treatment and menin knockdown. Together these data demonstrate that the SSP is highly active in Ewing sarcoma and that its activation is induced, at least in part, by a novel menin-ATF4 transcriptional axis. Citation Format: Laurie Kathleen Svoboda, Selina Shiqing Teh, Sudha Sud, Armand Bankhead, Brian Magnuson, Mats Ljungman, Costas Lyssiotis, Tomasz Cierpicki, Jolanta Grembecka, Elizabeth R. Lawlor. Oncogenic activation of the serine synthesis pathway by the scaffolding protein menin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5835. doi:10.1158/1538-7445.AM2017-5835