Menin regulates the serine biosynthetic pathway in Ewing sarcoma.

Developmental transcription programs are epigenetically regulated by multi-protein complexes, including the menin- and MLL-containing trithorax (TrxG) complexes, which promote gene transcription by depositing the H3K4me3 activating mark at target gene promoters. We recently reported that in Ewing sarcoma, MLL1 (lysine methyltransferase 2A, KMT2A) and menin are overexpressed and function as oncogenes. Small molecule inhibition of the menin-MLL interaction leads to loss of menin and MLL1 protein expression, and to inhibition of growth and tumorigenicity. Here, we have investigated the mechanistic basis of menin-MLL-mediated oncogenic activity in Ewing sarcoma. Bromouridine sequencing (Bru-seq) was performed to identify changes in nascent gene transcription in Ewing sarcoma cells, following exposure to the menin-MLL interaction inhibitor MI-503. Menin-MLL inhibition resulted in early and widespread reprogramming of metabolic processes. In particular, the serine biosynthetic pathway (SSP) was the pathway most significantly affected by MI-503 treatment. Baseline expression of SSP genes and proteins (PHGDH, PSAT1, and PSPH), and metabolic flux through the SSP were confirmed to be high in Ewing sarcoma. In addition, inhibition of PHGDH resulted in reduced cell proliferation, viability, and tumor growth in vivo, revealing a key dependency of Ewing sarcoma on the SSP. Loss of function studies validated a mechanistic link between menin and the SSP. Specifically, inhibition of menin resulted in diminished expression of SSP genes, reduced H3K4me3 enrichment at the PHGDH promoter, and complete abrogation of de novo serine and glycine biosynthesis, as demonstrated by metabolic tracing studies with 13 C-labeled glucose. These data demonstrate that the SSP is highly active in Ewing sarcoma and that its oncogenic activation is maintained, at least in part, by menin-dependent epigenetic mechanisms involving trithorax complexes. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Svoboda LK ，Teh SSK ，Sud S ，Kerk S ，Zebolsky A ，Treichel S ，Thomas D ，Halbrook CJ ，Lee HJ ，Kremer D ，Zhang L ，Klossowski S ，Bankhead AR ，Magnuson B ，Ljungman M ，Cierpicki T ，Grembecka J ，Lyssiotis CA ，Lawlor ER ... -  《-》

Tumorigenicity of Ewing sarcoma is critically dependent on the trithorax proteins MLL1 and menin.

Svoboda LK ，Bailey N ，Van Noord RA ，Krook MA ，Harris A ，Cramer C ，Jasman B ，Patel RM ，Thomas D ，Borkin D ，Cierpicki T ，Grembecka J ，Lawlor ER ... -  《Oncotarget》

EWS-FLI1 and Menin Converge to Regulate ATF4 Activity in Ewing Sarcoma.

Ewing sarcomas are driven by EWS-ETS fusions, most commonly EWS-FLI1, which promotes widespread metabolic reprogramming, including activation of serine biosynthesis. We previously reported that serine biosynthesis is also activated in Ewing sarcoma by the scaffolding protein menin through as yet undefined mechanisms. Here, we investigated whether EWS-FLI1 and/or menin orchestrate serine biosynthesis via modulation of ATF4, a stress-response gene that acts as a master transcriptional regulator of serine biosynthesis in other tumors. Our results show that in Ewing sarcoma, ATF4 levels are high and that ATF4 modulates transcription of core serine synthesis pathway (SSP) genes. Inhibition of either EWS-FLI1 or menin leads to loss of ATF4, and this is associated with diminished expression of SSP transcripts and proteins. We identified and validated an EWS-FLI1 binding site at the ATF4 promoter, indicating that the fusion can directly activate ATF4 transcription. In contrast, our results suggest that menin-dependent regulation of ATF4 is mediated by transcriptional and post-transcriptional mechanisms. Importantly, our data also reveal that the downregulation of SSP genes that occurs in the context of EWS-FLI1 or menin loss is indicative of broader inhibition of ATF4-dependent transcription. Moreover, we find that menin inhibition similarly leads to loss of ATF4 and the ATF4-dependent transcriptional signature in MLL-rearranged B-cell acute lymphoblastic leukemia, extending our findings to another cancer in which menin serves an oncogenic role. IMPLICATIONS: These studies provide new insights into metabolic reprogramming in Ewing sarcoma and also uncover a previously undescribed role for menin in the regulation of ATF4.

Jiménez JA ，Apfelbaum AA ，Hawkins AG ，Svoboda LK ，Kumar A ，Ruiz RO ，Garcia AX ，Haarer E ，Nwosu ZC ，Bradin J ，Purohit T ，Chen D ，Cierpicki T ，Grembecka J ，Lyssiotis CA ，Lawlor ER ... -  《-》

EWS-FLI1 reprograms the metabolism of Ewing sarcoma cells via positive regulation of glutamine import and serine-glycine biosynthesis.

Ewing sarcoma (EWS) is a soft tissue and bone tumor that occurs primarily in adolescents and young adults. In most cases of EWS, the chimeric transcription factor, EWS-FLI1 is the primary oncogenic driver. The epigenome of EWS cells reflects EWS-FLI1 binding and activation or repression of transcription. Here, we demonstrate that EWS-FLI1 positively regulates the expression of proteins required for serine-glycine biosynthesis and uptake of the alternative nutrient source glutamine. Specifically, we show that EWS-FLI1 activates expression of PHGDH, PSAT1, PSPH, and SHMT2. Using cell-based studies, we also establish that EWS cells are dependent on glutamine for cell survival and that EWS-FLI1 positively regulates expression of the glutamine transporter, SLC1A5 and two enzymes involved in the one-carbon cycle, MTHFD2 and MTHFD1L. Inhibition of serine-glycine biosynthesis in EWS cells impacts their redox state leading to an accumulation of reactive oxygen species, DNA damage, and apoptosis. Importantly, analysis of EWS primary tumor transcriptome data confirmed that the aforementioned genes we identified as regulated by EWS-FLI1 exhibit increased expression compared with normal tissues. Furthermore, retrospective analysis of an independent data set generated a significant stratification of the overall survival of EWS patients into low- and high-risk groups based on the expression of PHGDH, PSAT1, PSPH, SHMT2, SLC1A5, MTHFD2, and MTHFD1L. In summary, our study demonstrates that EWS-FLI1 reprograms the metabolism of EWS cells and that serine-glycine metabolism or glutamine uptake are potential targetable vulnerabilities in this tumor type.

Sen N ，Cross AM ，Lorenzi PL ，Khan J ，Gryder BE ，Kim S ，Caplen NJ ... -  《-》

EWS-FLI1-regulated Serine Synthesis and Exogenous Serine are Necessary for Ewing Sarcoma Cellular Proliferation and Tumor Growth.

Despite a growing body of knowledge about the genomic landscape of Ewing sarcoma, translation of basic discoveries into targeted therapies and significant clinical gains has remained elusive. Recent insights have revealed that the oncogenic transcription factor EWS-FLI1 can impact Ewing sarcoma cellular metabolism, regulating expression of 3-phosphoglycerate dehydrogenase (PHGDH), the first enzyme in de novo serine synthesis. Here, we have examined the importance of serine metabolism in Ewing sarcoma tumorigenesis and evaluated the therapeutic potential of targeting serine metabolism in preclinical models of Ewing sarcoma. We show that PHGDH knockdown resulted in decreased Ewing sarcoma cell proliferation, especially under serine limitation, and significantly inhibited xenograft tumorigenesis in preclinical orthotopic models of Ewing sarcoma. In addition, the PHGDH inhibitor NCT-503 caused a dose-dependent decrease in cellular proliferation. Moreover, we report a novel drug combination in which nicotinamide phosphoribosyltransferase (NAMPT) inhibition, which blocks production of the PHGDH substrate NAD+, synergized with NCT-503 to abolish Ewing sarcoma cell proliferation and tumor growth. Furthermore, we show that serine deprivation inhibited Ewing sarcoma cell proliferation and tumorigenesis, indicating that Ewing sarcoma cells depend on exogenous serine in addition to de novo serine synthesis. Our findings suggest that serine metabolism is critical for Ewing sarcoma tumorigenesis, and that targeting metabolic dependencies should be further investigated as a potential therapeutic strategy for Ewing sarcoma. In addition, the combination strategy presented herein may have broader clinical applications in other PHGDH-overexpressing cancers as well.

Issaq SH ，Mendoza A ，Kidner R ，Rosales TI ，Duveau DY ，Heske CM ，Rohde JM ，Boxer MB ，Thomas CJ ，DeBerardinis RJ ，Helman LJ ... -  《-》