Abstract P3-07-09: Pan-NOS Inhibitor L-NMMA in combination with Docetaxel Enhances Antitumor Effect in Obesity-associated Triple Negative Breast Cancer
Authors: Sun Kai , anselme Ann C. , Qian Wei , Zhou Jianying , Cheng Robert , Rosato Roberto , Chang Jenny 
Published: March 2023

Abstract
Background: Obesity is associated with increased risk of triple negative breast cancer (TNBC). Obese patients with TNBC have worse outcomes compared to patients with normal weight. Both obesity and TNBC are associated with chronic inflammation including elevated nitric oxide (NO). In a recently completed Phase I/II clinical trial using a pan-NOS inhibitor L-NMMA in combination with chemotherapy docetaxel in chemorefractory and metastatic TNBC, a higher response rate was observed in obese patients. Furthermore, tumor microenvironment analysis from responders revealed a neutrophil phenotype shift from protumor N2 to antitumor N1. We thus hypothesize that combining pan-NOS inhibitor L-NMMA with docetaxel can modulate neutrophil mediated pathways, and further enhance antitumor effect in obese mice with TNBC. Methods: Three-week old syngeneic female C57/black mice were randomized to high fat diet (HFD, 18% protein, 21% carbohydrates, and 61% fat, n=50) and normal diet (ND, 20% protein, 70% carbohydrates, and 10% fat, n=50) for 10 weeks. Mouse weights were measured weekly. After 10 weeks on their respective diets, glucose tolerance test, serum cytokine and leptin analysis were performed. At week 13, TNBC E0771 tumor cells (1 × 105) were injected into the right mammary fat pads. Tumor progression was monitored twice weekly and tumor volume [0.5 × (long dimension) × (short dimension)2] was calculated. Once the tumor reached 80-100 mm3, mice in both HFD and ND groups were randomized to vehicle [saline, intraperitoneal (IP), n=10], docetaxel (20 mg/kg, IP, n=10), L-NMMA (400 mg/kg on day 1, 200 mg/kg on days 2-5, n=10), docetaxel and L-NMMA combination (same doses and schedule as in single agent groups, n=10). Tumor volume was measured throughout the experiment and tumor growth was calculated (tumor volume on day X/tumor volume at baseline). RNA sequencing of tumors from vehicle and combination groups in HFD and ND (n=6 in each arm) was performed (Azenta/Genewiz, NJ) and BioJupies was utilized for pathway analysis. GraphPad Software (La Jolla, CA, USA) was used to perform two-tailed Student’s t test and ANOVA statistical analysis. A p-value&lt; 0.05 was considered statistically significant. Results: Compared to ND group, HFD group mice had significantly higher weight gain (64.8% vs 83.2%, p&lt; 0.0001); they also demonstrated significant glucose intolerance, and higher serum leptin level consistent with metabolic changes observed in diet-induced obesity. After tumor injection, tumor growth rate was much higher in HFD mice compared to that in ND mice. At end-of-treatment, compared with vehicle, a significantly slower tumor growth in HFD mice (p=.015), and a trend of slower tumor growth was observed in ND mice (p=.92) in docetaxel and L-NMMA combination treatment. The reduction of tumor growth was significantly higher in HFD mice than that in ND mice (median of differences -2.0, p=.031). Differential gene expressions from RNA sequencing showed that HFD mice displayed higher expression of genes related to neutrophil degranulation and neutrophil mediated immunity compared to ND mice at baseline; while treatment with L-NMMA and docetaxel combination downregulated these genes. Conclusion: HFD mice had significantly faster tumor growth and higher expression of genes related to neutrophil mediated pathways, while treatment with pan-NOS inhibitor L-NMMA and docetaxel combination downregulated these genes. The combination treatment resulted in a more significant anti-tumor effect in HFD mice, likely through remodeling neutrophils. Future spatial analysis including CODEX and immunophenotyping are planned to further explore the role neutrophils play in the pathogenesis of obesity associated TNBC and its response to combination treatment.
Citation Format: Kai Sun, Ann C. anselme, Wei Qian, Jianying Zhou, Robert Cheng, Roberto Rosato, Jenny Chang. Pan-NOS Inhibitor L-NMMA in combination with Docetaxel Enhances Antitumor Effect in Obesity-associated Triple Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-09.