Volume 140, Issue 2, Pages 709-720.e9 (February 2011) Acycloguanosyl 5′-thymidyltriphosphate, a Thymidine Analogue Prodrug Activated by Telomerase, Reduces Pancreatic Tumor Growth in Mice  Simone Polvani, Massimo Calamante, Valeria Foresta, Elisabetta Ceni, Alessandro Mordini, Alessandro Quattrone, Massimo D'Amico, Claudio Luchinat, Ivano Bertini, Andrea Galli  Gastroenterology  Volume 140, Issue 2, Pages 709-720.e9 (February 2011) DOI: 10.1053/j.gastro.2010.10.050 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Structure and schematic mode of action of ACV-TP-T in comparison with ACV. For activation, ACV requires to be phosphorylated to ACV monophosphate by viral TK carried either by wild-type herpes virus or, in the suicide gene therapy, engineered adenovirus (lower part of the Figure). ACV monophosphate is then further phosphorylated by cellular kinases to the triphosphated active form. Conversely, ACV-TP-T is a substrate of telomerase that incorporates the thymidine in the replicating telomeres and directly releases ACV diphosphate skipping the viral TK phosphorylation step (upper part of the Figure). Gastroenterology 2011 140, 709-720.e9DOI: (10.1053/j.gastro.2010.10.050) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Cellular uptake and detection of ACV-TP-T and ACV diphosphate. Mass spectrometry analysis of PANC-1 treated for 2 hours with 1 mmol/L ACV-TP-T (A) shows the presence of signaling peaks (retention time between 7.0 and 9.0 minutes) corresponding to the prodrug and ACV diphosphate. No such peaks were detectable in the culture medium (B). Gastroenterology 2011 140, 709-720.e9DOI: (10.1053/j.gastro.2010.10.050) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 In vitro effects of ACV-TP-T. (A) Growth-inhibitory effects. Pancreatic cancer cell lines were treated with increasing doses of the prodrug for 24 hours, and proliferation was tested by BrdU incorporation. ACV-TP-T inhibited the growth of all tested cell lines in a dose-dependent manner. RTA, relative telomerase activity. (B) Colony formation assay of PANC-1-treated cells. PANC-1 cells were treated with 10 μmol/L ACV-TP-T then plated in 60-mm dishes and allowed to grow. Colonies were counted 14 days after seeding. The graph summarizes the results from 2 independent experiments. *P = .01. Gastroenterology 2011 140, 709-720.e9DOI: (10.1053/j.gastro.2010.10.050) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Effect of prolonged ACV-TP-T treatment. (A) Growth-inhibitory effects. PANC-1 and MiaPaca2 cells were treated with 10 and 100 μmol/L ACV-TP-T from 24 to 96 hours. (B) Cell cycle analysis. Synchronized PANC-1 cells, treated from 24 to 96 hours with 10 μmol/L ACV-TP-T, show a S-phase arrest of the cell cycle. Grey area represents G1 phase; black area represents G2 phase; hatched area represents S phase. (C) ACV-TP-T induces apoptosis. PANC-1 and MiaPaca2 cells were treated for various times (from 24 to 96 hours) with 10 and 100 μmol/L ACV-TP-T and then tested for caspase 3/7 activity. *P < .05; **P < .01; ***P < .001. Gastroenterology 2011 140, 709-720.e9DOI: (10.1053/j.gastro.2010.10.050) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 ACV-TP-T is activated by telomerase. (A) Su.86.86 cell lines were transfected with DN-hTERT and hTERT expression plasmids; control cells were transfected with the empty vector. Twenty-four hours after transfection, the cells were treated for 24 hours with ACV-TP-T. Proliferation was tested by BrdU incorporation, and telomerase activity was evaluated. hTERT-transfected cells show a higher telomerase activity and a lower IC50, compared with control and DN-hTERT transfected cells. RTA, relative telomerase activity. (B) hTERT transfected cells treated with 200 μmol/L ACV-TP-T show a higher susceptibility to the prodrug compared with control and DN-hTERT transfected cells. (C) hTERT silencing reduces MiaPaca2 cells susceptibility to the prodrug, compared with cells transfected with a negative control siRNA. (D) ACV-TP-dC does not inhibit cell proliferation. PANC-1 cells were treated for 24 hours with 3 ACV derivatives where the thymidine base was replaced by either cytosine, adenosine, or guanosine (ACV-TP-dC, ACV-TP-dA, ACV-TP-dG). Proliferation was tested by BrdU incorporation. *P < .05; **P < .01. Gastroenterology 2011 140, 709-720.e9DOI: (10.1053/j.gastro.2010.10.050) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 ACV-TP-T antitumor effect in PANC-1 xenografts. Tumor-bearing nude-Foxn1nu/nu mice were treated 3 times per week for 3 weeks with intraperitoneal injection of ACV-TP-T (200, 12.5, 1.25, and 0.125 mg/kg). Control mice were treated with saline solution. (A) Growth curves of PANC-1 xenografts in treated and control groups are shown. (B) Representative tumor masses from control and ACV-TP-T-treated mice. (C) Representative IHC for proliferating cell nuclear antigen (PCNA), activated caspase 3, and CD34 from control and ACV-TP-T-treated mice. Original magnification, 200×. (D) Quantitative analysis of the histochemical results. *P < .05, **P < .01. Gastroenterology 2011 140, 709-720.e9DOI: (10.1053/j.gastro.2010.10.050) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 ACV-TP-T antitumor effect in MiaPaca2 xenografts. Tumor-bearing nude-Foxn1nu/nu mice were treated 3 times per week for 3 weeks with intraperitoneal injection of ACV-TP-T (12.5 mg/kg). Control mice were treated with saline. (A) Growth curves of MiaPACA2 xenografts in treated and control groups. (B) Representative tumor masses from control and ACV-TP-T-treated mice. (C) Representative IHC for proliferating cell nuclear antigen (PCNA), activated caspase 3, and CD34 from control and ACV-TP-T-treated mice. Original magnification, 200×. (D) Quantitative analysis of the histochemical results. *P < .05. Gastroenterology 2011 140, 709-720.e9DOI: (10.1053/j.gastro.2010.10.050) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 (A) Structures of analytes. (B) Scheme of ACV-TP-T synthesis. Gastroenterology 2011 140, 709-720.e9DOI: (10.1053/j.gastro.2010.10.050) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 Cell cycle analysis of MiaPaca2 cells. Synchronized MiaPaca2 cells were treated from 24 to 96 hours with ACV-TP-T (100 μmol/L final concentration). ACV-TP-T induces an S-phase arrest in treated cells. Grey area represents G1 phase; black area represents G2 phase; hatched area represents S phase. Gastroenterology 2011 140, 709-720.e9DOI: (10.1053/j.gastro.2010.10.050) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 hTERT specific siRNA identification. hTERT messenger RNA (mRNA) levels were determined by RQ-polymerase chain reaction on MiaPaca2 cells 48 hours after transfection with hTERT specific small interfering RNA (siRNA) or with a control siRNA (All Star Negative siRNA). Expression levels were normalized for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gastroenterology 2011 140, 709-720.e9DOI: (10.1053/j.gastro.2010.10.050) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4 Histologic analysis of the liver of PANC-1 xenograft bearing mice. Tumor-bearing nude-Foxn1nu/nu mice were treated 3 times per week for 3 weeks with intraperitoneal injection of ACV-TP-T. Control mice were treated with saline solution. Representative liver images from control (A), 200 mg/kg (B), and 12.5 mg/kg (C) ACV-TP-T treated mice. No evident alterations were observed. Original magnification, 200×. Gastroenterology 2011 140, 709-720.e9DOI: (10.1053/j.gastro.2010.10.050) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 5 Immunohistochemistry on gastrointestinal tract of PANC-1 xenograft bearing mice. Tumor-bearing nude-Foxn1nu/nu mice were treated 3 times per week for 3 weeks with intraperitoneal injection of ACV-TP-T. Control mice were treated with saline solution. Representative proliferating cell nuclear antigen (PCNA) and Caspase 3 immunohistochemistry gastrointestinal images from control (A), 200 mg/kg (B) and 12.5 mg/kg (C) ACV-TP-T treated mice. No alterations in the proliferation pattern and caspase 3 immunoreactivity were observed. Original magnification, 400×. Gastroenterology 2011 140, 709-720.e9DOI: (10.1053/j.gastro.2010.10.050) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 6 ACV-TP-T antitumor effect in MiaPaca2 xenografts. Tumor-bearing nude-Foxn1nu/nu mice were treated 3 times per week for 6 weeks with intraperitoneal injection of ACV-TP-T (12.5 mg/kg). Control mice were treated with saline. (A) Growth curve of PANC-1 xenografts in treated and control groups. (B) Representative tumors masses from control and ACV-TP-T-treated mice. (C) Quantitative analysis of the immunohistochemistry for proliferating cell nuclear antigen (PCNA), activated caspase 3, and CD34 from control and ACV-TP-T-treated mice. *P < .05. Gastroenterology 2011 140, 709-720.e9DOI: (10.1053/j.gastro.2010.10.050) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 7 ACV-TP-T antitumor effect against PANC-1 xenografts. Tumor-bearing nude-Foxn1nu/nu mice were treated 3 times per week for 6 weeks with intraperitoneal injection of ACV-TP-T (12.5 mg/kg). Control mice were treated with saline. (A) Growth curve of PANC-1 xenografts in treated and control groups. (B) Representative tumors masses from control and ACV-TP-T-treated mice. (C) Quantitative analysis of the IHC for proliferating cell nuclear antigen (PCNA), activated caspase 3, and CD34 from control and ACV-TP-T-treated mice. *P < .05. Gastroenterology 2011 140, 709-720.e9DOI: (10.1053/j.gastro.2010.10.050) Copyright © 2011 AGA Institute Terms and Conditions