Sensitizing tumors to anti-PD-1 therapy by promoting NK and CD8+ T cells via pharmacological activation of FOXO3

Stimulating antitumor immunity by blocking programmed death-1 (PD-1) or its ligand (programmed death-ligand 1 (PD-L1) is a promising antitumor therapy. However, numerous patients respond poorly to PD-1/PD-L1 blockade. Unresponsiveness to immune-checkpoint blockade (ICB) can cast significant challenges to the therapeutic options for patients with hard-to-treat tumors. There is an unmet clinical need to establish new therapeutic approaches for mitigating ICB unresponsiveness in patients. In this study, we investigated the efficacy and role of low-dose antineoplastic agent SN-38 or metformin in sensitizing unresponsive tumors to respond to ICB therapy. We assessed the significant pathological relationships between PD-L1 and FOXO3 expression and between PD-L1 and c-Myc or STAT3 expression in patients with various tumors. We determined the efficacy of low-dose SN-38 or metformin in sensitizing unresponsive tumors to respond to anti-PD-1 therapy in a syngeneic tumor system. We deciphered novel therapeutic mechanisms underlying the SN-38 and anti-PD-1 therapy-mediated engagement of natural killer (NK) or CD8+ T cells to infiltrate tumors and boost antitumor immunity. We showed that PD-L1 protein level was inversely associated with FOXO3 protein level in patients with ovarian, breast, and hepatocellular tumors. Low-dose SN-38 or metformin abrogated PD-L1 protein expression, promoted FOXO3 protein level, and significantly increased the animal survival rate in syngeneic mouse tumor models. SN-38 or metformin sensitized unresponsive tumors responding to anti-PD-1 therapy by engaging NK or CD8+ T cells to infiltrate the tumor microenvironment (TME) and secret interferon-γ and granzyme B to kill tumors. SN-38 suppressed the levels of c-Myc and STAT3 proteins, which controlled PD-L1 expression. FOXO3 was essential for SN38-mediated PD-L1 suppression. The expression of PD-L1 was compellingly linked to that of c-Myc or STAT3 in patients with the indicated tumors. We show that SN-38 or metformin can boost antitumor immunity in the TME by inhibiting c-Myc and STAT3 through FOXO3 activation. These results may provide novel insight into ameliorating patient response to overarching immunotherapy for tumors.