1. Asparagine starvation inhibits rhabdomyosarcoma growth Connective Tissue Oncology Society 2014 Annual Meeting in Berlin Simone Hettmer, Anna C Schinzel, Daria Tchessalova, Nigel GJ Richards, William C Hahn, Amy J Wagers

2. Ras pathway activation in fusion-negative rhabdomyosarcoma Skeletal muscle Ras pathway activation

3. Sarcoma induction in skeletal muscle Transcriptional profiling identified 141 discrete genes that are upregulated in Kras; p16p19 null mouse sarcomas and in human rhabdomyosarcomas compared to skeletal muscle.

4. Negative control shRNA Sarcoma genes Positive control shKras shASNS Functional screening of sarcoma-relevant candidate genes q<0.001 Customized shRNA proliferation screen (in vitro)

5. Asparagine Synthetase (ASNS)

6. The growth-inhibitory effects of ASNS silencing are due to Asparagine starvation Kras;p16p19 null mouse RMS cell line

7. Asparagine starvation reduces the synthesis of nascent polypeptide chains in rhabdoyosarcoma cells

8. Asparagine starvation reduces human rhabdomyosarcoma growth and peptide synthesis Human RMS cell line RH30 (PAX3:FOXO1a +)

9. Asparagine starvation reduces mouse rhabdomyosarcoma growth in vivo shASNS shLuc 100 cells per injection

10. Asparagine starvation reduces mouse rhabdomyosarcoma growth in vivo shASNS shLuc Aspara- ginase 100 cells per injection

11. Asparagine homeostasis is an actionable cellular process Amino Sulfoximines

12. Asparaginase and Amino Sulfoximine 5 inhibit rhabdomyosarcoma growth Amino Sulfoximine 5Asparaginase Kras;p16p19 null mouse RMS cell line

13. Asparaginase effects on rhabdomyosarcoma growth in vivo Aspara- ginase Mouse RMSRh30 (Hu RMS)Rh41 (Hu RMS) Aspara- ginase Control Aspara- ginase Control Aspara- ginase Control p=0.02p=0.09 p=0.0004

14. Asparagine homeostasis is an actionable metabolic vulnerability in sarcoma  Sarcoma induction by Ras pathway activation in skeletal muscle identified a cluster of sarcoma-relevant genes.  Functional screening identified Asparagine homeostasis as a metabolic vulnerability in sarcoma.  Asparagine starvation inhibits growth and nascent peptide synthesis in mouse and human sarcomas.  Chemical targeting of Asparagine homeostasis reduces mouse and human rhabdomyosarcoma growth.

15. Acknowledgements Amy Wagers Wagers lab: Daria Tchessalova Tata Nageswara Rao DFCI shRNA screening core: Anna Schinzel William Hahn David Langenau Nigel Richards Peter Houghton Richard Lock Joslin Flow Cytometry Core Funding: PALS/St Baldrick’s Foundation Alex’s Lemonade Stand Bear Necessities SARC