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

Figures

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  Figure 1. MLL1 is expressed by Ewing sarcoma (A) CCLE data depicting MLL1 transcript expression. (B) RT-PCR and (C) western blot depicting MLL1 transcript and protein expression in Ewing sarcoma cells, MRC5 fibroblasts and adult bone marrow-derived mesenchymal stem cells (hMSC). (D) Expression array data for MLL1 in 32 Ewing sarcoma tumors compared to 33 samples from 11 normal adult tissues. ****p < .0001. (E) Representative immunohistochemical staining of Ewing sarcoma tumor, showing CD99 (top) and MLL1 (middle). Thyroid (bottom) was negative for MLL1 expression. (F) Scoring of MLL staining in 42 Ewing sarcoma tumors and 22 normal tissue samples (liver, bladder, heart, fat, lung, spleen, pancreas, thymus, stomach, salivary gland, thyroid, uterus, kidney, prostate, testes, tonsil, breast, skin). Normal tissues with high MLL1 expression (Allred score of 6 or greater) included heart, spleen, stomach, pancreas, and salivary gland. ****p < .0001.

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  Figure 2. Knockdown of MLL1 in Ewing sarcoma cells reduces proliferation and tumorigenicity qRT-PCR (A) and western blot (B) showing MLL1 knockdown. (C) Brightfield images (left) and histograms (right) show reduced cell expansion after 6 days. (D) Histograms (top) and representative images (bottom) showing reduced tumorigenicity with MLL1 knockdown. N = 3 experiments. *p < .05, **p = .002, ****p < 0.0001. (E) Knockdown of MLL1 significantly reduced tumor growth rate in vivo. ****p = .0001. N = 5 mice. (F) qRT-PCR for MLL1 shows restoration of MLL1 expression in tumors derived from shMLL1 cells.

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  Figure 3. HOXD13 is a target of MLL1 (A) qRT-PCR depicting MLL1 transcript levels. (B) qRT-PCR of posterior HOXD gene expression in cells with MLL1 knockdown. Error bars depict mean ± SEM. *p < .05, **p < .01, ***p < .001, ****p < 0.0001. (C) Left: Diagram of the HOXD13 promoter showing the location of primers used for qPCR. Right: MLL1 ChIP at the HOXD13 promoter. N = 2 experiments.

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  Figure 4. Menin has an oncogenic function in Ewing sarcoma qRT-PCR (A) and western blot (B) showing menin knockdown. (C) Brightfield images (top) and histograms (bottom) show reduced cell growth with menin knockdown. (D) Histograms (left) and representative images (right) of soft agar assays. (E) Menin ChIP at the HOXD13 promoter. N = 2 experiments. (F–G) qRT-PCR depicting expression of menin (F) and posterior HOXD genes (G). *p < .05, **p < .01, ***p < .001, ****p < 0.0001. N = 3 experiments.

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  Figure 5. MI-503 inhibits Ewing sarcoma cell expansion and tumorigenicity (A) IC50 curves and (B) trypan blue assay in MI-503 treated cells. (C) Histograms (top) and representative images (bottom) of soft agar assays in cells pre-treated with MI-503 or vehicle. N = 3 experiments. **p = .002, ****p < 0.0001. (D) Left: Spider plot depicting growth rates of individual tumors pre-treated with vehicle (Black Circle) or 6 μM MI-503 (Grey Square). N = 8 mice. Right: Image of tumors extracted on day 28.

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  Figure 6. MI-503 treatment of Ewing sarcoma leads to loss of posterior HOXD gene and MLL and menin protein expression (A) qRT-PCR data depicting reduced HOXD10, HOXD11, and HOXD13 expression with MI-503 treatment. *p < .05, **p = .003. (B) Increased histone H3 at the HOXD13 promoter after MI-503 treatment. (C) Reduced H3K4me3 at the HOXD13 promoter with MI-503 treatment. Control is a gene desert region telomeric to the posterior HOXD cluster. Error bars in all panels depict mean ± SEM. (D). Western blot depicting MLL1 and menin protein expression after 6 day treatment with MI-503 or MI-NC. (E) Western blot showing dose-dependent down-regulation of MLL1 and menin expression with MI-503 treatment. (F) Model of epigenetic activation of posterior HOXD gene cluster in Ewing sarcoma that is disrupted by exposure of cells to MLL-menin interaction inhibitor.