1. Sarcoma Research Laboratory University of Texas, MD Anderson Cancer Center Radiation-associated and sporadic UPS/MFH: Searching for novel therapeutic strategies Keila Enitt Torres

2. UPS/MFH Pleomorphic malignant fibrous histiocytoma (MFH)  unclassified pleomorphic sarcoma Pleomorphic malignant fibrous histiocytoma (MFH)  unclassified pleomorphic sarcoma Current technology  ? definable line of differentiation Current technology  ? definable line of differentiation

3. UPS Peak incidence in the 6th and 7th decades of life Peak incidence in the 6th and 7th decades of life Typically large, deep, aggressive tumors Typically large, deep, aggressive tumors 5% - metastases at presentation (lung) 5% - metastases at presentation (lung) <3% radiation-associated <3% radiation-associated

4. UPS Treatment Radical excision with negative margins Radical excision with negative margins Neo-Adjuvant RT is offered  widely negative margins is not possible. Neo-Adjuvant RT is offered  widely negative margins is not possible. Chemotherapy  Unresectable disease/metastatic disease Chemotherapy  Unresectable disease/metastatic disease

5. 5-year disease-specific survival 44- 66%* 5-year disease-specific survival 44- 66%*

6. Approach Low throughput: PI3K/AKT signaling pathway Low throughput: PI3K/AKT signaling pathway Determine if AKT/mTOR axis is a relevant target for UPS therapy High throughput: genomic/proteomic analysis High throughput: genomic/proteomic analysis Understand genetic and molecular de-regulations  UPS progression Understand genetic and molecular de-regulations  UPS progression

7. PI3K/AKT pathway

8. Nature Medicine 13, 748 - 753 (2007)

10. HGFp-c-Metp-MEKp-AKTc-Met

11. Goal #1 Confirm whether the AKT/mTOR axis is a potential target for UPS/MFH therapy Determine whether this pathway is differentially activated in radiation-associated versus sporadic UPS

12. Radiation-associated sarcoma Diagnosis of RAS based on the criteria published by Cahan, et al. in 1948.* Diagnosis of RAS based on the criteria published by Cahan, et al. in 1948.* Previous radiation tx for different tumor Previous radiation tx for different tumor Development of the sarcoma - radiation field Development of the sarcoma - radiation field Histologically different from the primary cancer Histologically different from the primary cancer Minimum latent period of 5 years between the radiation and development of the sarcoma Minimum latent period of 5 years between the radiation and development of the sarcoma Arlen et al. 1971- modified definition Arlen et al. 1971- modified definition Latency period of 3 years Latency period of 3 years *Cahan et al. Cancer 1948;1:3-29.

13. Bioresources in the laboratory Cell lines Cell lines Animal models Animal models Human Tissue Human Tissue Frozen Frozen FFPE FFPE

14. Cell strain/cell line bank > 60 human tumors 13 Sporadic UPS cell strains 6 Radiation- associated UPS cell strains RIS-819.1 RIS-023 UPS-060 UPS-485 UPS-186

15. UPS-060

16. AKT/mTOR pathway is activated in UPS UPS-186RIS-DL620RIS-DL618hMSC p4EBP1 pAKT 4EBP1 AKT S6K  -actin UPS-060UPS-356 UPS-DL511 RIS-023 RIS-819 Radiation-associated Sporadic pS6K

17. Rapamycin 0 10 100 0 10 100 0 10 100 RIS -819RIS -023UPS-485UPS-060 Radiation-associated Sporadic 0 10 100 p4EBP1 pAKT 4EBP1 AKT S6K  -actin pS6K

18. Rapamycin Concentration (  M) 0 0.05 0.1 0.5 1 5 Radiation-associated Concentration (  M) Sporadic Concentration (  M)

19. pAKT AKT pS6K S6K  actin p4EBP1 4EBP1  M/4hr 0 0.05 0.1 0 0.05 0.1 UPS-060RIS-819 PI103

20. PI103 Concentration (  M) Radiation-associated Concentration (  M) Sporadic Concentration (  M)

21. RIS-819 ControlPI103 UPS-060 ControlPI103 Cell Cycle

22. Migration ControlPI103 RIS-819 UPS-060

23. Invasion ControlPI103 UPS-060 RIS-819

24. In vivo studies In vivo studies Orthotopic Xenograft Model 1-2mm pieces of tumor

25. * * RIS-819 tumor growth

26. pAKTp4EBP1pS6RPKi67H&E PI103 Control RIS-819 Immunohistochemistry

27. UPS-060.1 tumor growth PI103 Control *

28. pAKTp4EBP1pS6RPKi67H&E PI103 Control UPS-060.1 Immunohistochemistry

29. Summary PI3K/AKT pathway is activated in radiation- associated and sporadic UPS PI3K/AKT pathway is activated in radiation- associated and sporadic UPS RA-UPS and sporadic UPS are sensitive to PI103 RA-UPS and sporadic UPS are sensitive to PI103 Effects in vivo model => Cell death Effects in vivo model => Cell death

31. Current studies Proteomics miRNA/ non-coding RNAs mRNA Methylation DNA copy number variation DNA Sequencing Sequenome Exon Sequencing Array- comparative genomic hybridization miRNA analysis RPPA TMA

32. Reverse-phase protein lysate array for cell signaling analysis

33. EIF4E 29 molecules differentially expressed HER-2 Signaling ERK/MAPK PI3K/AKT/mTOR ErbB AMPK HGF signaling IL-15 Signaling -3.5058 5.1031 (Log 2) Mesenchymal cells UPS >50% P value <0.01 Unsupervised hierarchical clustering

34. Unsupervised hierarchical clustering RA-UPS versus sporadic UPS -3.5058 5.1031 (Log 2) RA-UPS Sporadic-UPS

35. Sarcoma Research Laboratory University of Texas, MD Anderson Cancer Center Dina Lev Dina Lev Raphael E. Pollock Raphael E. Pollock Billy Wang Billy Wang Alexander J. Lazar Alexander J. Lazar Vinod Ravi Vinod Ravi SARC (Sarcoma Alliance for Research through Collaboration) SARC (Sarcoma Alliance for Research through Collaboration) Acknowledgments