Small molecules targeting the NADH-binding pocket of VDAC enhance T cell cytotoxicity and selectively inhibit cancer cell proliferation

Adoptive cell therapy involves administration of cytotoxic immune cells. Upon activation of naïve T cells, CD8+ (cytotoxic) T cells increase both mitochondrial metabolism and glycolysis. Upregulated mitochondrial metabolism is essential for the activation of effector T cells. Voltage dependent anion channels (VDAC 1/2/3) regulate the flux of most metabolites entering and exiting mitochondria. VDAC opening increases mitochondrial metabolism. NADH binding to a VDAC pocket, conserved in all isoforms, closes the channel. The small molecules SC01 and SC18 bind to the VDAC-NADH binding pocket. We hypothesize that SC01 and SC18 stabilize VDAC in an open state enhancing T cell oxidative metabolism, survival and cytotoxicity. We used melanoma epitope gp100 reactive mouse splenic CD8+ T cells, HepG2 and SNU-449 human hepatocarcinoma cancer cells, and B16 mouse melanoma cells. We determined T cell activation (CD25 expression), proliferation (Ki67), cytokine secretion (IFNγ, TNFα), cytolytic activity (granzyme-B), and expression of memory markers CD44 and CD62L, by FACS. We assessed mitochondrial membrane potential (ΔΨm) and NADH by confocal microscopy. Cell proliferation and cell death were determined by cell count and propidium iodide uptake, respectively. VDAC isoform distribution in T cells was ∼54%, 24% and 22% for VDAC 1, 2 and 3, respectively. SC18 moderately decreased T cell ΔΨm and NADH. SC01 and SC18 did not affect proliferation or activation of splenic CD8+ T cells exposed to cognate antigen (gp100). By contrast, SC18 inhibited cell proliferation in SNU-449, HepG2, and B16 cells. SC18 also increased the secretion of IFNγ, TNF-α, and granzyme-B. Memory markers determined by CD44 and CD62L co-expression were unchanged. These data suggest a two-sided antitumor property of SC18, that enhances T cell cytotoxicity and selectively inhibits cancer cell proliferation.