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

Abstract

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.

Trial registration: ClinicalTrials.gov NCT04067336 NCT04065399.

Conflict of interest statement

Conflict of interest: Dr. Grembecka and Dr. Cierpicki receive research support from Kura Oncology, Inc. and have equity ownership in the company.

©2021 American Association for Cancer Research.

Figures

Figure 1.. ATF4 Modulates Serine Biosynthesis Pathway Genes in Ewing sarcoma.

A,ATF4 gene expression in patient tumors from three independently published Ewing sarcoma datasets [–34]. B,ATF4 mRNA and protein levels in Ewing sarcoma cell lines and non-Ewing mesenchymal cell lines (N=3). C, Trypan blue exclusion proliferation assay after shRNA knockdown of ATF4 (N=2). D, qRT-PCR and E, representative western blot of SSP (PHGDH, PSAT1, and PSPH) mRNA and protein (A673 & A4573– 30 µg, TC32– 50 µg) in Ewing sarcoma cell lines after 96 hours of shATF4 knockdown (N=3). F, Chromatin immunoprecipitation qRT-PCR (ChIP-qPCR) for ATF4 at PHGDH and PSAT1 gene promoters. Negative control is a promoter region in chr2 without ATF4 binding (N=3). G, Representative western blots and qRT-PCR for SSP expression in ATF4 overexpressing (OE) Ewing sarcoma cells (A4573– 30 µg, TC32– 50 µg) (N=2, N=3). Error bars represent SEM from independent biological replicates. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; Two-tailed t-test.

Figure 2.. EWS-FLI1 Directly Binds to and Activates ATF4.

A, qRT-PCR and B, representative western blot (30 µg) for FLI1 (EWS-FLI1), and SSP (PHGDH, PSAT1, and PSPH) mRNA and protein levels after 96 hours of EWS-FLI1 knockdown (N=3, N=4). C, qRT-PCR for ATF4 and PSAT1 mRNA at 48 (N=2) and 96 (N=3) hours post-shFLI1 knockdown in A673, A4573, and TC32 cells. D, Representative western blot (30 µg) for FLI1 (EWS-FLI1) and ATF4 protein levels after 48 and 96 hours of FLI1 (EWS-FLI1) knockdown (N=3). Western blots depicted for FLI1 and GAPDH are the same as those presented in panel B and are reproduced here for ease of comparison. E, Integrative Genomics Viewer (IGV) screenshot of publicly available ChIP-seq tracks for FLI1 (EWS-FLI1), H3K27Ac, and H3K4me3 at the ATF4 and PHGDH gene promoters in Ewing sarcoma cell lines and primary tumors [8]. F, ChIP-qPCR for FLI1 (EWS-FLI1) at the PHGDH and ATF4 gene promoters (N=3). Negative control is a region in chr2 without EWS-FLI1 binding. Error bars represent SEM from independent biological replicates. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; Two-tailed t-test.

Figure 3.. Menin Inhibition Leads to Loss of ATF4 in Ewing sarcoma.

A, Representative western blot (50 µg) of MEN1 in a panel of Ewing sarcoma and non-Ewing sarcoma cell lines. B, Representative western blot (A673 & A4573– 30 µg, TC32– 50 µg) for ATF4 and SSP (PHGDH, PSAT1, and PSPH) protein after 96 hours of treatment with 3 µM MI-503 or DMSO control (N=3). C, qRT-PCR for ATF4 and D, SSP (PHGDH, PSAT1, and PSPH) mRNA in three Ewing sarcoma cell lines after 96 hours of treatment with 3 µM MI-503 or DMSO (N=3). E, ChIP-qPCR for ATF4 at PHGDH and PSAT1 gene promoters after 96 hours of 3 µM MI-503 treatment compared to DMSO control (N=4). F, qRT-PCR for MEN1 and ATF4 mRNA after 72 hours of doxycycline (dox)-inducible MEN1 knockdown (N=3). G, Representative western blot (30 µg) of MEN1 and ATF4 in A673 dox-inducible shNS or shMEN1 cells after 72 hours treatment with dox. H, qRT-PCR for SSP mRNA after MEN1 knockdown (N=3). Error bars represent SEM from independent biological replicates. * p<0.05; **p<0.01; *** p<0.001; **** p<0.0001; Two-tailed t-test.

Figure 4.. Menin Inhibition Downregulates ATF4 Expression in an H3K4me3-Independent Manner.

A, Representative western blot (A673 & A4573– 30 µg, TC32– 50 µg) of MEN1, ATF4, H3K4me3 and total H3 levels after 96 hours of treatment with 3 µM MI-503 or DMSO control. B, ChIP-qPCR for H3K4me3 enrichment at the ATF4 gene promoter after 96 hours of MI-503 treatment compared to DMSO control. Negative control is a gene desert region in chr2 without H3K4me3 enrichment (N=3). C, Representative western blot of MLL2 (KMT2B), MEN1, and ATF4 protein after knockdown of MLL2, and qRT-PCR for ATF4 and SSP (PHGDH, PSAT1, PSPH) mRNA after KMT2B knockdown (N=3). D, Representative western blot for MEN1, ATF4, phosphorylated-eIF2a (Ser51) and total eIF2a after 96 hours of 3 µM MI-503 treatment in A673, A4573, and TC32 cells (N=3). E, Representative western blot for P-eIF2a (Ser51) and total eIF2a after 24 and 48 hours of 3 µM MI-503 treatment in A673 cells (N=2). Error bars represent SEM from independent biological replicates. * p<0.05; Two-tailed t-test.

Figure 5.. EWS-FLI1 Inhibition in Ewing sarcoma Broadly Inhibits an ATF4-dependent Gene Expression Program.

A, Heatmap of Log2Fold Change in expression from publicly available RNA-seq data of shRNA knockdown of EWS-FLI1 (iEF), luciferase-targeting control (iLuc), and wildtype EWS-FLI1 cDNA (WT-EF) rescue in A673 cells [35]. B, qRT-PCR validation of ATF4 and select ATF4 target genes after 96 hours of FLI1 (EWS-FLI1) knockdown (N=3, N=4). Error bars represent SEM from independent biological replicates. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; Two-tailed t-test.

Figure 6.. Menin Inhibition Identifies an ATF4-dependent Gene Expression Signature.

A, Volcano plot of global changes in mRNA levels from previously published RNA-seq of MI-503-treated A673, A4573, and TC71 Ewing sarcoma cells [18]. Cells were treated with 3 µM vehicle (MI-NC) or MI-503 for 72 hours and analyzed by bromouridine sequencing (Bru-seq). Genes marked in red show <−2-fold change in gene expression and adjusted p<0.05. B, Top downregulated genes from combined Bru-seq data of all three cell lines after 3 µM MI-503 treatment for 72 hours [18]. C, Top transcription factor motifs identified using the HOMER software by motif analysis of promoters of genes downregulated in (A) from three independent cell lines (A673, A4573. TC71). D, qRT-PCR of ATF4 and select ATF4 target genes after 96 hours of treatment with 3 µM MI-503 or DMSO control (N=3). Error bars represent SEM from independent biological replicates. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; Two-tailed t-test.

Figure 7.. Menin Inhibition Downregulates ATF4 Activity in MLLr Leukemia Cell Lines.

A, qRT-PCR of ATF4 and select ATF4 target genes in MV4;11 B-ALL and B, MOLM13 AML cell lines after 7 days of treatment with 25 and 50 nM MI-3454, a next-generation menin inhibitor, or DMSO control (N=2). C, Histogram of publicly available RNA-seq in RS4;11, and D, MOLM13 cell lines after 7 days of treatment with 330 nM VTP50469 (N=3) [36]. E, Volcano plot of publicly available RNA-seq data from MOLM13 cells following 6 days of MEN1 knockout [36]. Upregulated genes with adjusted p > 30 are excluded. Genes marked in red show <−2-fold change in gene expression and p <0.05. F, Working model in which the EWS-FLI1 oncogene in Ewing sarcoma and high menin expression converge on regulation of ATF4. Error bars represent SEM from independent biological replicates. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; Two-tailed t-test.