Abstract P1-04-12: Pathway analysis of immune checkpoint gene regulation as altered in Type 2 diabetes: Implications for breast cancer patients treated with checkpoint inhibitors
Authors: Ennis Christina S. , LLevenes Pablo , Kolla Manohar , Jafari Naser , Belkina Anna C , Denis Gerald V. 
Published: March 2022

Abstract
Background: Mediators of immune exhaustion are an active area of breast cancer research. Type II diabetes mellitus (T2D), the most common metabolic disorder, both increases breast cancer incidence and decreases survival. Despite this clear link, impacts of the T2D immune phenotype on cancer remain poorly understood. We have previously demonstrated that the chronic inflammatory state of T2D leads to immune exhaustion, but how the T2D breast microenvironment interacts with T cells is unclear. Exosomes are crucial components of intercellular communication and are associated with increased breast cancer aggressiveness. Given this function, we hypothesized that T2D adipocyte-derived exosomes drive T cell exhaustion. Here, we characterize expression patterns and regulatory networks driving T2D immune phenotypes. Methods: Exosomes were first isolated from culture media of mature human primary breast adipocytes from, either insulin-sensitive (IS), or rendered insulin-resistant (IR) by ex vivo TNFα-treatment. Human primary peripheral blood mononuclear cells (PBMCs) from nondiabetic (ND) and T2D donors were stimulated ex vivo with plate bound anti-CD3 (5 ug/ml) and soluble CD28 (2 ug/ml) for 48 hours and treated with exosomes. Different small molecule inhibitors of the bromo and extraterminal (BET) protein family, including the pan-BET inhibitor JQ1 and the BRD4-selective PROTAC degrader MZ-1, were used to identify BET protein-regulated targets. We also used the AMP-activating protein kinase (AMPK) inhibitor Compound-C to identify the role of this pathway in activating this major epigenetic player. Multicolor flow cytometry was subsequently performed with an LSRII cytometer to assess expression of inhibitory receptors PD-1, CTLA-4, TIM-3 and TIGIT on immune subsets. Events for live cells were analyzed in FlowJo. Cytokines were collected from conditioned media and analyzed via Th17 cytokine staining panel. Results: We observed that exosomes derived from IR breast adipocytes increase expression of immune exhaustion markers in CD4+ and CD8+ T cells, compared to IS or ND matched controls. Additionally, we define signal transduction among BET proteins, AMPK signaling, and immune checkpoint expression. Lastly, we identify changes in cytokine profile between IS and IR treated groups. Taken together, our findings suggest a network of immune regulation imparted by exosomes. Impacts: Metabolic health does not inform the current standard of care in breast medical oncology, which contributes to a large, underserved population in which treatment plans are not well established or optimized for their comorbidities. Our findings offer a deeper understanding of immune checkpoint regulation in T2D and suggest new insights into treatment of diabetic breast cancer patients.
Citation Format: Christina S. Ennis, Pablo LLevenes, Manohar Kolla, Naser Jafari, Anna C Belkina, Gerald V. Denis. Pathway analysis of immune checkpoint gene regulation as altered in Type 2 diabetes: Implications for breast cancer patients treated with checkpoint inhibitors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-12.