Discovery Of Novel Metformin Derivatives With Potent Antitumor Activity In Combination With Immunotherapy For Treatment Of Triple-Negative Breast Cancer

Abstract Triple negative breast cancer (TNBC) occurs in about 15% of breast cancer (BC) patients, yet it accounts for almost 50% of all BC deaths. TNBC is characterized by lack of expression of estrogen, progesterone and HER2 receptors, and it cannot be treated with current targeted therapies. TNBCs are heterogeneous and occur often in younger and African American women. Although initially responsive to some chemotherapies, TNBCs tend to relapse early and metastasize, leading to poor survival. Development of new therapeutic approaches for clinical translation is a high priority. Emerging evidence from epidemiologic and preclinical work suggests that metformin, the most widely used drug to treat type 2 diabetes mellitus, exerts anticancer activity in BC. Diabetic patients treated with metformin have a reduced incidence and better survival from BC. Moreover, TNBC cells are reported to be uniquely sensitive to metformin in vitro, but strong antitumor effects require relatively high metformin doses in vivo. To address this challenge, we used a structure-activity strategy to develop metformin analogues with more potent anticancer action and safety at lower doses in vivo. Using TNBC cell proliferation in vitro to screen drug candidates, selected new metformin analogues were identified that exerted dose-dependent inhibition of cell proliferation at significantly lower concentrations than that of parental metformin (P<0.01). As antitumor effects of metformin are attributed in part to activation of the LKB1-AMPK pathway, we find that metformin analogues also strongly induced phosphorylation of AMPK by Western blot assays and significantly reduced phosphorylation of mTOR downstream signaling pathway components (p70S6K, S6 ribosomal protein, 4E-BP1). Analogues also induced TNBC cell apoptosis at lower doses than metformin. In vivo, metformin analogues were more effective at lower doses given by oral gavage than metformin in suppressing TNBC human tumor xenograft progression in nude mouse models (P<0.001). It is notable that data from clinical trials with immune checkpoint inhibitors (ICI) indicate that TNBCs are susceptible to immunotherapy, but only a minority of patients to date have had clinical benefit. Importantly, our studies indicate that metformin analogues modulate the activity and trafficking of myeloid-derived suppressor cells (MDSC) and tumor infiltrating lymphocytes (TIL) that may significantly impact TNBC responses to ICIs. Thus, combination therapy with metformin analogues and anti-PD-L1 antibodies was more effective at blocking tumor growth in vivo than either treatment given alone using murine 4T1 TNBC implants in syngeneic, immune-competent BALB/C mouse models. In the TNBC microenvironment, analogue treatment with ICIs elicited significant infiltration of predominantly effector T-cells, increased populations of tissue-resident memory T-cells, and a reduction of exhausted T-cells as compared to controls (P<0.01). Moreover, MDSC were significantly decreased after combination therapy as compared to appropriate controls (P<0.05). Since there are currently no specific treatments for TNBC, identification of a new targeted therapeutic approach could ultimately be beneficial for managing patients afflicted with this deadly disease. Funding by California BC RP, NCI U54 CA143930. Citation Format: Diana C. Márquez-Garban, Gang Deng, Begonya Comin-Anduix, Vishaka Muhunthan, Gaoyuan Ma, Jennifer Murphy, Alejandro J. Garcia, Cristian D. Yanes, Lorena R. Burton, Nalo Hamilton, David Shackelford, Michael E. Jung, Richard J. Pietras. Discovery of novel metformin derivatives with potent antitumor activity in combination with immunotherapy for treatment of triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 692.