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Supplementary Figure S1. PHGDH is highly expressed in Ewing sarcoma cell lines; Supplementary Figure S2. Measurement of PHGDH protein expression. Immunoblot analysis of PHGDH in the specified cell lines expressing lentiviral control shRNA (shControl) or no shRNA (Parental); Supplementary Figure S3. PHGDH knockdown and ES proliferation in vitro; Supplementary Figure S4. Effect of GNE-618 on ES cellular proliferation; Supplementary Figure S5. Dose-effect Curves for GNE-618 and NCT-503; Supplementary Figure S6. Mouse body weight is unaffected by inhibitor treatments; Supplementary Figure S7. EWS-FLI1 is important for sarcoma cellular proliferation in the absence of serine; Supplementary Figure S8. Mouse body weight is unaffected by serine/glycine deficient diet

Supplementary Table S1. Evaluation of drug combinations in TC32 and TC71 Ewing sarcoma cells; Supplementary Table S2. Evaluation of drug combinations in EW8 Ewing sarcoma cells.

Purpose:Ewing sarcoma is an aggressive solid tumor malignancy of childhood. Although current treatment regimens cure approximately 70% of patients with localized disease, they are ineffective for most patients with metastases or relapse. New treatment combinations are necessary for these patients.Experimental Design:Ewing sarcoma cells are dependent on focal adhesion kinase (FAK) for growth. To identify candidate treatment combinations for Ewing sarcoma, we performed a small-molecule library screen to identify compounds synergistic with FAK inhibitors in impairing Ewing cell growth. The activity of a top-scoring class of compounds was then validated across multiple Ewing cell lines in vitro and in multiple xenograft models of Ewing sarcoma.Results:Numerous Aurora kinase inhibitors scored as synergistic with FAK inhibition in this screen. We found that Aurora kinase B inhibitors were synergistic across a larger range of concentrations than Aurora kinase A inhibitors when combined with FAK inhibitors in multiple Ewing cell lines. The combination of AZD-1152, an Aurora kinase B–selective inhibitor, and PF-562271 or VS-4718, FAK-selective inhibitors, induced apoptosis in Ewing sarcoma cells at concentrations that had minimal effects on survival when cells were treated with either drug alone. We also found that the combination significantly impaired tumor progression in multiple xenograft models of Ewing sarcoma.Conclusions:FAK and Aurora kinase B inhibitors synergistically impair Ewing sarcoma cell viability and significantly inhibit tumor progression. This study provides preclinical support for the consideration of a clinical trial testing the safety and efficacy of this combination for patients with Ewing sarcoma.

Supplemental Table S7. Reverse Phase Protein Array (RPPA) data. Total protein and phosphorylation levels in untreated and treated A673 and TC32 cells are normalized to total protein levels in each sample.

Supplemental Table S3. Target sequences for shRNAs targeting AURKB expression in the in vivo dependency screen

Supplemental Table S2. Target sequences for shRNAs targeting AURKB expression in the Achilles Project

Supplemental Methods and Figures Supplemental Figure S1. Aurora kinase expression in Ewing sarcoma Supplemental Figure S2. Effects of cell growth on response to AZD-1152 as a function of duration of treatment. Supplemental Figure S3. Aurora kinase and FAK inhibitor combinations are synergistic in Ewing sarcoma cell lines Supplemental Figure S4. Response of Ewing cell lines treated with combinations of Aurora kinase and FAK inhibitors Supplemental Figure S5. Cell cycle and apoptotic effects of Aurora kinase B knock out in Ewing sarcoma cell lines Supplemental Figure S6. PYK2 is poorly expressed in Ewing sarcoma cells Supplemental Figure S7. Zebrafish and Murine studies

Supplemental Table S1. List of compounds in MIPE 4.0 compound library

Supplemental Table S6. Muliple metrics were used to calculate synergy of all compounds tested from MIPE 4.0 library in combination with PF-562271. Highlighted in green are Pan-Aurora Inhibitors, red are Aurora B inhibitors, and blue are Aurora A inhibitors.

Supplemental Table S4. Target sequences for sgRNA guides targeting AURKB, AURKA, and FAK genes