Glutaminase 1 and the Remodeling of the Immune Microenvironment in Triple-Negative Breast Cancer

• Grant number: 25/08548-2
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: July 01, 2025
• End date: June 30, 2028
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Sandra Martha Gomes Dias
• Grantee: Mariana Camargo Silva Mancini
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Associated research grant: 21/05726-6 - Metabolism in the microenvironment and the role of metabolic exchanges in tumor progression, AP.TEM

Abstract

The relationship between tumor cells and their microenvironment governs essential aspects of tumor biology, including therapeutic response and clinical prognosis. Immune cells-whether myeloid or lymphoid-interact with tumor cells, but their actions can be suppressed by potent immunosuppressive mechanisms. Immune function is also impacted by tumor metabolism, as cancer cells rapidly deplete carbon sources in the tumor microenvironment, leaving them scarce for the immune machinery. In addition to being avid consumers of carbon, cancer cells secrete immunosuppressive metabolic by-products. The enzyme glutaminase (GLS) is frequently overexpressed in tumors, and its inhibition is being tested in clinical studies, including for triple-negative breast cancer (TN; lacking estrogen and progesterone receptors and HER2 amplification). TN is highly aggressive and has poor prognosis, as the most effective targeted therapies used in other breast cancer subtypes are not applicable here.Immune checkpoint blockade therapy (ICB), which aims to reactivate the immune system to attack tumor cells, has shown promise in TN treatment but benefits only a fraction of patients. Thus, combination treatments that may synergize with ICB are under investigation. In silico data from our group show that breast tumors with high GLS expression are enriched for an immune infiltrate profile marked by abundant CD8¿ cytotoxic T lymphocytes, strong IFN-¿ activity signatures, and high T cell receptor (TCR) diversity. Additionally, compared to GLS-low tumors, these GLS-high tumors express higher levels of PD-1 and PD-L1, which together form a co-repressor complex that inhibits T cell activation (as shown in our Thematic Project). We performed orthotopic syngeneic implantation of the murine TN cell line 4T1 into immunocompetent mice and treated them with CB-839 (a glutaminase inhibitor), anti-PD-1 antibody, or their combination. CB-839 increased intratumoral glutamine levels, while anti-PD-1 activated CD8¿ T cells both locally and systemically, as evidenced by increased frequencies of GZMA¿, GZMB¿, and perforin¿ CD8¿ T cells. We also observed higher Th1/Th2 ratios and increased CD8¿ T cell/Treg ratios in tumors from mice treated with CB-839 or the combination therapy.Despite this apparent CD8¿ T cell activation and a favorable Th1/Th2 profile from the combined CB-839 and anti-PD-1 treatment, we did not observe tumor growth inhibition; on the contrary, tumors became heavier and more invasive to the lungs. qPCR analysis of lymphocyte-related genes revealed five genes significantly downregulated following combination therapy (data shown in Thematic Report_II). Altogether, these changes suggest that CB-839 and anti-PD-1 reshape the tumor microenvironment (TME), making it less rigid and more permissive to immune infiltration, yet somehow more favorable to tumor invasion. This incongruity between the immune cell infiltration profile and gene expression versus in vivo growth/metastasis-indicating increased aggressiveness-raises concerns about ICB use in TN and highlights the need to understand the underlying molecular mechanisms and markers of such responses, as proposed in this project. (AU)