Abstract B011: Rac and Cdc42 inhibition in the tumor microenvironment as a strategy to prevent metastasis
Authors: Dorta-Estremera Stephanie , Torres Anamaris , Rivera-Robles Michael , Cruz-Collazo Ailed , Borrero-Garcia Luis , Dharmawardhane Suranganie 
Published: January 2023

Abstract
Metastatic disease lacks effective treatments and remains the primary cause of mortality from epithelial cancers, especially breast cancer. The metastatic cascade involves cancer cell migration and invasion, modulation of the tumor microenvironment (TME), plasticity, and ability to colonize secondary tissues. Therefore, a viable anti-metastasis strategy needs to simultaneously target the migration of cancer cells and the tumor-infiltrating immunosuppressive inflammatory cells such as activated macrophages, neutrophils, and myeloid-derived suppressor cells (MDSC). The Rho GTPases Rac and Cdc42 are ideal molecular targets that regulate both cancer cell and immune cell migration, as well as their crosstalk signaling at the TME. Therefore, the purpose of this study was to test the hypothesis that Rac and Cdc42 inhibitors target both migratory cancer cells and immunosuppressive cells in the TME. Our published data demonstrate that the Vav/Rac inhibitor EHop-016 and the Rac/Cdc42 guanine nucleotide association inhibitor MBQ-167 reduce mammary tumor growth and prevent breast cancer metastasis from pre-clinical mouse models without toxic effects. Since the targets of these inhibitors may also affect immune cell behavior, we first tested their potential to target Rac/Cdc42 function in human and rat macrophage cell lines and show that EHop-016 and MBQ-167 inhibit Rac activation (at 5X higher concentration than the IC50 in breast cancer cells), actin cytoskeletal extensions, and migration, without affecting cell viability. EHop-016 and MBQ-167 significantly decrease the proinflammatory cytokine Interleukin-6 (Il-6) from plasma and the TME. Next, we tested the in vivo effect of MBQ-167 on immune cells from breast cancer mouse models. Tumors from immunodeficient mice bearing mammary tumors that were treated with vehicle or 50mg/kg MBQ-167 5x a week for 5 weeks, were analyzed by flow cytometry to quantify different immune cell types. MBQ-167 treated mice demonstrated elevated CD11+F4/80+CD86+ M1 macrophages and reduced MDSCs in mammary tumors. In the immunocompetent 4T-1 breast cancer model, spleens contained a lower frequency of macrophages and neutrophils, accompanied by an increase in activated cytotoxic CD69+, CD8+T cells. Moreover, when metastasis to the lungs was reduced by ~90% following MBQ-167 treatment, granzyme B+ CD8+ T cells were elevated in the lung extracts, without affecting the immunosuppressive CD4+ regulatory T cell counts. In vitro, we also demonstrated that increasing doses of MBQ-167 induced T cell activation and interferon-gamma secretion, a cytokine known to have antitumor properties. Taken together, Rac/Cdc42 inhibitors induce anti-metastatic cancer effects via inhibition of both metastatic cancer cells and immunosuppressive myeloid cells, while promoting the activation of cytotoxic T cells, in the tumor and the metastasis microenvironment. Therefore, MBQ-167 promises to be an effective antimetastatic cancer therapeutic that is poised to enter Phase I clinical trials.
Citation Format: Stephanie Dorta-Estremera, Anamaris Torres, Michael Rivera-Robles, Ailed Cruz-Collazo, Luis Borrero-Garcia, Suranganie Dharmawardhane. Rac and Cdc42 inhibition in the tumor microenvironment as a strategy to prevent metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B011.