Abstract LB-196: Evaluation of anti PD-1 plus nitric oxide synthase inhibition combination therapy in 12 triple-negative breast cancer patient-derived xenografts using a human-derived immune system model
Authors: Davila-Gonzalez Daniel , Rosato Roberto R. , Qian Wei , Kozielski Antony J. , Chen Wen , Choi Dong S. , Dave Bhuvanesh , Kranjac Dinko , Ensor Joe E. , Chang Jenny C. 
Published: July 2017

Abstract
Introduction: Breast cancer exhibits a relatively low somatic mutation burden frequency, and anti-PD-1 monotherapy therefore has limited benefit. 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), establishes an immunosuppressive environment. Most patients do not, or do only incompletely, respond to PD-1 inhibitors due to cancer related immunosuppression. The goal 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) /Amlodipine (10mg/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; and 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 week later, human HSCs 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 up-regulated PD-L1 levels in 6 out of 8 breast cancer cell lines. 4T1 syngeneic model was used to test 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 therapy and the control group, but the combination anti-PD1/NOS inhibitor 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: Coupling the NOS inhibitor with anti-PD1 therapy modulates the tumor immunosuppressive microenvironment that may benefit cancer patients who do not respond to anti-PD-1 monotherapy. Additional analyses are currently in progress to identify biomarkers that will allow successful discrimination of responders and non-responders.
Citation Format: Daniel Davila-Gonzalez, Roberto R. Rosato, Wei Qian, Antony J. Kozielski, Wen Chen, Dong S. Choi, Bhuvanesh Dave, Dinko Kranjac, Joe E. Ensor, Jenny C. Chang. Evaluation of anti PD-1 plus nitric oxide synthase inhibition combination therapy in 12 triple-negative breast cancer patient-derived xenografts using a human-derived immune system model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-196. doi:10.1158/1538-7445.AM2017-LB-196