Int J Biol Sci 2021; 17(9):2240-2251. doi:10.7150/ijbs.60473

Research Paper

Inhibition of de novo pyrimidine synthesis augments Gemcitabine induced growth inhibition in an immunocompetent model of pancreatic cancer

Thuy Phan, PhD1, Vu H. Nguyen, PhD2, Ralf Buettner, PhD2, Corey Morales, BA2, Lifeng Yang, PhD3, Paul Wong, BS1, Weiman Tsai, BA2, Marcela d'Alincourt Salazar2, Ziv Gil, MD4, Don J Diamond, PhD2, Joshua D. Rabinowitz, MD3, Steven Rosen, MD2, Laleh G. Melstrom, MD MSCI1✉

1. Department of Surgery, City of Hope National Medical Center, Duarte, CA 91010.
2. Department of Hematology, City of Hope National Medical Center, Duarte, CA 91010.
3. Lewis Sigler Institute for Integrative Genomics and Department of Chemistry, Princeton University, Princeton, NJ 08544.
4. Rambam Medical Center, Israel.

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Citation:
Phan T, Nguyen VH, Buettner R, Morales C, Yang L, Wong P, Tsai W, Salazar Md, Gil Z, Diamond DJ, Rabinowitz JD, Rosen S, Melstrom LG. Inhibition of de novo pyrimidine synthesis augments Gemcitabine induced growth inhibition in an immunocompetent model of pancreatic cancer. Int J Biol Sci 2021; 17(9):2240-2251. doi:10.7150/ijbs.60473. Available from https://www.ijbs.com/v17p2240.htm

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Abstract

Leflunomide (Lef) is an agent used in autoimmune disorders that interferes with DNA synthesis. De Novo pyrimidine synthesis is a mechanism of Gemcitabine (Gem) resistance in pancreatic cancer. This study aims to assess the efficacy and changes in the tumor microenvironment of Lef monotherapy and in combination with Gem, in a syngeneic mouse model of pancreatic cancer.

Methods: MTS proliferation assays were conducted to assess growth inhibition by Gem (0-20 nM), Lef (0-40 uM) and Gem+Lef in KPC (KrasLSL.G12D/+;p53R172H/+; PdxCretg/+) cells in vitro. An in vivo heterotopic KPC model was used and cohorts were treated with: PBS (control), Gem (75 mg/kg/q3d), Lef (40 mg/kg/d), or Gem+Lef. At d28 post-treatment, tumor burden, proliferation index (Ki67), and vascularity (CD31) were measured. Changes in the frequency of peripheral and intratumoral immune cell subsets were evaluated via FACS. Liquid chromatography-mass spectrometry was used for metabolomics profiling.

Results: Lef inhibits KPC cell growth and synergizes with Gem in vitro (P<0.05; Combination Index 0.44 (<1 indicates synergy). In vivo, Lef alone and in combination with Gem delays KPC tumor progression (P<0.001). CTLA-4+T cells are also significantly decreased in tumors treated with Lef, Gem or in combination (Gem+Lef) compared to controls (P<0.05). Combination therapy also decreased the Ki67 and vascularity (P<0.01). Leflunomide inhibits de novo pyrimidine synthesis both in vitro (p<0.0001) and in vivo (p<0.05).

Conclusions: In this study, we demonstrated that Gem+Lef inhibits pancreatic cancer growth, decrease T cell exhaustion, vascularity and as proof of principle inhibits de novo pyrimidine synthesis. Further characterization of changes in adaptive immunity are necessary to characterize the mechanism of tumor growth inhibition and facilitate translation to a clinical trial.

Keywords: pancreatic cancer, leflunomide, de novo pyrimidine synthesis