Abstract 3525: N1-hexyl-N5-benzyl-biguanide promotes proliferation of CD4+ and CD8+ T lymphocytes
Authors: Zhijun Guo, David Owen, Pamela Rosato, David Masopust, Michael A. Farrar, David A. Potter
Journal: Cancer Research (2018)

Abstract Introduction: Solid tumors are often resistant to immunotherapy due to a hostile microenvironment for immune cells, characterized by hypoxia and low nutrients. Metformin has been shown to potentiate PD-1 blockade and improve intratumoral T-cell function and tumor response through reduction of hypoxia (1). A novel biguanide (neo-biguanide) that potentiates immunotherapy similar to metformin, but is more potent, would be more ideal for combination with immunotherapy. Recently, we have discovered that metformin binds to the mitochondria associated enzyme CYP3A4 and thereby suppresses the electron transport chain (ETC) and oxygen consumption rates (OCR) (2). Structural biology approaches led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which binds to the CYP3A4 heme with higher affinity than metformin and much more potently inhibits the ETC and OCR (2). HBB potently and specifically inhibits CYP3A4 arachidonic acid (AA) epoxygenase activity, causing OCR and growth inhibition of breast cancer cell lines and established mouse mammary tumors (IC50=3-30 uM). Because lymphocytes express relatively lower levels of the CYP3A4 target of HBB (3), we hypothesized that HBB may permit T lymphocyte growth at concentrations that inhibit tumor epithelia. Therefore, effects of HBB on T cell proliferation and activation were tested with mouse splenocytes. Methods: HBB effects on T cell growth were tested across a range of concentrations (0 to 50 uM). C57BL/6J mouse splenocytes were incubated with or without biguanides in the presence of anti-mouse CD3 and anti-mouse CD28 antibodies. After 24 hours, cells were labeled with fluorescently tagged antibodies to mouse Thy-1, CD4, CD8, CD25, CD44, CD62L and CD73 surface markers and T cell subsets were analyzed by flow cytometry. Results: At low concentrations (0.09 to 1.5 uM) HBB did not inhibit T cell growth, but at higher concentrations (3 to 25 uM) HBB caused proliferation of CD4+ and CD8+ T lymphocytes with optimum concentrations of 6 to 12 uM (1.9-fold for CD4+ and 1.6-fold for CD8+; P&amp;lt;0.05 for both), conditions that inhibited breast cancer cells. Notably, CD4+FoxP3+ regulatory T cells were not increased by HBB. Metformin treatment caused an increase of CD4+ and CD8+ T lymphocytes only at concentrations &amp;gt;5 mM. Conclusions: HBB potently increases the proliferation of CD4+ and CD8+ mouse T lymphocytes stimulated with anti-CD3 and anti-CD28 antibodies at concentrations that suppress human and mouse mammary carcinoma cells. In contrast, metformin failed to show proliferation-inducing activity for T cells at concentrations below 5 mM. HBB therefore is a potent T lymphocyte activator, while inhibiting breast cancer epithelial cells, supporting further study of HBB to facilitate immune therapy in animal models. 1. Cancer Immunol Res. 2017 Jan 1; 5 (1) 9-16. 2. Cell Chemical Biology. 2017 Oct 19; 24(10) 1259 - 1275. 3. Drug Metab Dispos. 2008 Jan;36(1):182-9. Citation Format: Zhijun Guo, David Owen, Pamela Rosato, David Masopust, Michael A. Farrar, David A. Potter. N1-hexyl-N5-benzyl-biguanide promotes proliferation of CD4+ and CD8+ T lymphocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3525.