1. Combination of Vaccine-derived Measles and Mumps Virus Enhanced anti-tumor Effects: Special Focus on Acute Leukemia and Prostate Cancer Sen. Colonel A/Pr. Dr. Nguyen Linh Toan 1$, Ho Anh Son 1#, LiFeng Zhang 3#, Bui Khac Cuong 1, Hoang Van Luong 2, Naoki Yamamoto 3$, et al. 1 Vietnam Military Medical University, Vietnam. 2 Department of Microbiology, NUS, Singapore. Nguyen Linh Toan - VMMU - Vietnam 2015 Asia Pacific Military Health Exchange Da Nang - 2015

2. BACKGROUND (Torre LA et al. CA Cancer J Clin. 2015). 14.1 million new cancer cases & 8.2 million cancer deaths, 2012 Cancer is major leading cause of death in worldwide.

3. (Torre LA et al. CA Cancer J Clin. 2015). Lung and breast cancer are the most frequently and the leading causes of cancer death in men and women worldwide. However, prostate cancer is the second most frequently diagnosed cancer in men worldwide, with 1.1mil. new cases to occurred in 2012

4. Despite the considerable progress accomplished in recently years, most advanced cancers remain incurable prompting the need for novel, less toxic and more targeted therapy.

5. Freidman et al., Pediatric Research (2012) CSC: cancer stem cells OLV is a novel treatment approach that exploits the ability of viruses to selectively infected and destroy cancer cells. OLV replication, cell killing, virus release and spread within cancer cells but not normal cells Kirn D et al. (2001). Nat Med Liu T-C et al. (2007) Nat Clin Pract Oncol 5 Oncolytic virotherapy (OLV) CD46 Nectin 4 CD46 Nectin 4 CD150: B,T, M, DC

7. Summay: phase I-III clinical trials of OLV (Buijs PR et al. 2015; Lapp et al. 2014; Msaouel et al. 2013)

8. It is highly anticipated that the oncolytic activity of combined OLVs should be improved quantitatively as well as qualitatively. This situation is rather reminiscent of drug therapy in the clinical settings. It is highly anticipated that the oncolytic activity of combined OLVs should be improved quantitatively as well as qualitatively. This situation is rather reminiscent of drug therapy in the clinical settings. 1) Improved targeting 2) Synergy and complementation of multiple viruses 3) Produces enhanced anti-tumor effects Our strategy is to exploit Viral Synergy Nguyen Linh Toan - VMMU - Vietnam8

9. HK1 (Nasopharyngeal) HCT116 (Colorectal) H358 (Lung) EC109 (Esohageal) PC-3 (prostate) MCF-7 (Breast) HepG2 (Liver) Mock MMR-Infected Mock MMR-Infected Cytopathic effects (CPE) were observed in a wide range of human solid malignancy cell lines infected with the MMR viruses MMR: Measles Mumps Rubella 9

10. Cytopathic effects (CPE) was observed in a wide range of hematological malignancy cell lines infected with the MMR viruses KI-4 Raji Mock MMR-Infected U937 HuT 102 Lymphoma cell lines Mock MMR-Infected Leukemia cell lines HL-60 Jurkat 10 (Zhang….. Toan...et al, Cancer Letter, 2014)

11. Post infection (P.I.) MeVMuVMM The MM targeted a wider range of solid cancer cell lines and displayed greater oncolytic effect on several cell lines compared with single virus PC3:Prostate C., MCF-7: Breast C., KATO III: Gastric C., HepG2, HuH4: Liver C., H358, H1299: Lung C., Hela and SiHa: cervix C. HK-1:NPC, HONE-2 EC109: Esohageal, KYSE70, HT29 and HCT166: Colorectal C., 11

12. The MM targeted a wider range of hematological cancer cell lines and displayed greater oncolytic effect on several cell lines compared with single virus MeV MuV MM (Zhang….. Toan...et al, Cancer Letter, 2014)

13. LungColorectal HCT-116H358 MCF-7 (Breast) HT-29H1299 More potential oncolytic effect of MM combination on human solid tumor cell lines Kato III (Gastric) PC-3 (Prostate) Human solid cancer cell lines infected with MeV, MuV or MM followed by cell viability testing on day 6 P.I. The oncolytic effect of the MM compared with single virus was significantly enhanced in PC-3, MCF-7, Kato III, HT29, H358 and H1299 cell lines 13

14. Oncolytic effects between MeV, MuV and MM on human hematological malignant cell lines. HuT 102 KI-4 U937HL-60 MeVMuVMM Jurkat THP-1 MeVMuVMMMeVMuVMM MeVMuVMMMeVMuVMMMeVMuVMM The oncolytic effect of the MM compared with single virus was significantly enhanced in U937, THP-1, HL-60, Jurkat, KI-4 and HuT102 cell lines. 14 (Zhang….. Toan...et al, Cancer Letter, 2014)

15. Nude Mice bearing human solid tumors Colon cancer (HT 29) Larynx cancer (Hep2) Liver cancer (Hep 3B) Lung cancer (H211) Muscles PC tissue with prostate tubes PC PC invaded venous system Prostate Cancer (PC-3) 15

16. GroupsVirus concentration FrequencyInoculation site Negative control M199 medium1 time or 2 time/wk x 3wks Intra-tumor Me10 6 pfu/100ul1 time or 2 time/wk x 3wks Intra-tumor Mu10 6 pfu/100ul1 time or 2 time/wk x 3wks Intra-tumor Me+Mu10 6 pfu/100ul + 10 6 pfu/100ul 1 time or 2 time/wk x 3wks Intra-tumor Dose of intra-tumoral injections OLV were inoculated intra-tumor when PC-3 tumors reached 0.5 mm 3 volume 16

17. Multi-dose MM enhances killing human PC-3 tumor xenograft Time (day) Tumor size (mm 3 ) * * * * P<0.05 Single dose Tumor size (mm 3 ) Time (day) * * P<0.05 17

18. MM combination prolonging survival time Survival time (day) Percent Survival Multi-doseSingle dose 18

19. Enhanced killing of U937 cells and prolonged mice survival by MM treatment in nude mice bearing U937 tumors a. Tumor volume b. Survival Days post treatment 19 (Zhang….. Toan...et al, Cancer Letter, 2014)

20. MM Kp7-6 (1mg/ml) Mock Annexin V PI Kp7-6 (1mg/ml)MM 6.9% 21.7% 8.6% 27.9% 1.6% 2.3% 1.2% 2.9% THP-1 U937 Mechanistic studies: Involvement in apoptosis of U937 and THP-1 cells induced by MM infection. 7.9% 31.1% 6.9% 41.1% 1.0% 2.7% 1.4% 2.2% PI (Zhang….. Toan...et al, Cancer Letter, 2014)

21. SUMMARY In vitro MM displayed a more potent oncolytic effect than single virus on human solid and hematological cancer cell lines In vivo, MM displayed significant tumor suppression and prolonged survival, compared to single virus, in a PC-3 and U937 xenograft tumor models. Our results demonstrate that MM is a promising candidate for the treatment human cancers.

22. ACKNOWLEDGMENTS Centre Biomedical & Pharmaceutical Lab., VMMU, Vietnam Department of Microbiology, NUS, Singapore NUS-VMMU collaboration project; Vietnam NAFOSTED project, Viet Nam

24. VIETNAM MILITARY MEDICAL UNIVERSITY