Abstract A202: Evaluating the combination of anti-PD-1 and nitric oxide synthase inhibition therapy in 12 triple-negative breast cancer patient-derived xenografts using a human-derived immune system model
Authors: Davila-Gonzalez Daniel , Rosato Roberto R. , Dave Bhuvanesh , Choi Dong Soon , Qian Wie , Kozielski Anthony J. , Chen Wen , Ensor Joe E. , Chang Jenny C. 
Published: August 2018

Abstract
Introduction: Anti-PD-1 monotherapy has shown a limited therapeutic benefit in breast cancer (TNBC). In triple-negative breast cancer (TNBC), inducible nitric oxide synthase (iNOS) is associated with poor survival due to increased tumor aggressiveness. In cancer, iNOS product, nitric oxide (NO), is associated with the establishment of an immunosuppressive environment. Most patients do not, or only incompletely, respond to PD-1 inhibitors due to cancer-related immunosuppression. The purpose of the present study is to evaluate combination therapy anti-PD-1 plus NOS inhibition as a feasible combination for TNBC. Methods: BT-549, SUM-159, SUM-157, HCC-70, MDA-MB-231, and MDA-MD-468 TNBC cell lines and MDA-MB-436 HER2+ cell line were treated with NOS inhibition therapy (L-NMMA, L; 4mM) + amlodipine (A; 5 µM) daily for 48 h; Western blot was used to measure PDL-1 expression. BALBc mice growing orthotopically injected 4t1 cells were treated weekly with: 1) vehicle (saline, oral gavage/ Rat IgG2 i.p.); 2) NOS inhibitor [L-NMMA (200 mg/kg oral gavage)/mlodipine (10 mg/kg i.p.) on days 1-5]; 3) anti-PD-1 antibody (10 mg/kg i.p.; BioXcell Clone: RMP1-14, on days 1, 3, 5; or 4) NOS inhibitor + anti-PD-1. Humanized mice were developed by injecting hematopoietic stem cells (HSC, CD34+) into the tail vein of irradiated NOD-scid IL2Rγnull (NSG) immunodeficient mice. Six weeks later, human HSC engraftment was assessed by expression of human CD45, CD3, and CD20 in blood. Mice were sorted and TNBC patient-derived xenografts (PDXs) representing 12 different patients were implanted into the mammary fat pad (3 mice per PDX). Mice were sorted and treated weekly with: 1) vehicle; 2) pembrolizumab (anti-PD-1, 200 µg, day 1, IV); and 3) pembrolizumab/NOS inhibitor. Results: NOS inhibition increased PD-L1 levels in 6 out of 8 breast cancer cell lines. 4T1 syngeneic model was used to assess the potential benefit of NOS inhibition in combination with an anti-PD-1 therapy. No significant differences in tumor growth were observed between the single-agent therapies and the control group, but the combination anti-PD1/NOS inhibition resulted in a significant reduction in tumor growth. In the immune-humanized TNBC PDXs, 40% responded to anti-PD1 monotherapy, 60% had an improved response in combination with NOS inhibition, and 33% responded only to combination therapy. In total, 66% of the PDXs analyzed benefited from combination therapy. Conclusion: NOS inhibition upregulates PDL-1 expression in TNBC cell lines. Furthermore, coupling NOS inhibition with anti-PD1 therapy improved the antibody response. Additional analyses are currently in progress to identify biomarkers that will allow successful discrimination of responders and nonresponders. This combination treatment may benefit cancer patients who do not respond to anti-PD-1 monotherapy.
Citation Format: Daniel Davila-Gonzalez, Roberto R. Rosato, Bhuvanesh Dave, Dong Soon Choi, Wie Qian, Anthony J. Kozielski, Wen Chen, Joe E. Ensor, Jenny C. Chang. Evaluating the combination of anti-PD-1 and nitric oxide synthase inhibition therapy in 12 triple-negative breast cancer patient-derived xenografts using a human-derived immune system model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A202.