Targeting KLF2 in macrophages to improve immune checkpoint therapy for hepatocellular cancer - ABSTRACT Anti-programmed death-1 antibody (αPD-1 Ab) as a single agent for treating human hepatocellular cancer (HCC) was withdrawn from the US market on July 26, 2021, because a multi-center phase III study did not demonstrate its efficacy in improving patient survival over controls. Thus, there is an urgent need to identify and target critical cellular and molecular regulators to design new immune checkpoint therapy (ICT) strategies against HCC. A unique, orthotopic, and clinically relevant murine HCC model was established that mimics HCC initiation and progression in humans and reflects the tumor biology, immunology, and histology typical of human disease. In this model, SV40 T antigen (TAg) is expressed solely in tumors as a trackable tumor-specific antigen (TSA), enabling TSA immunity study during tumor initiation, progression, and response to treatments. Using this model, several immune-based therapeutic strategies for HCC were developed and a novel microbe-based strategy was recently established that significantly improves the therapeutic efficacy of αPD-1 Ab for HCC. Specifically, Bacteroides thetaiotaomicron (B.th), one member of genus Bacteroides, with CpG-rich nucleic acid which functions as TLR9 agonist, was identified as a microbial regulator to significantly boost αPD-1 Ab therapeutic efficacy for HCC. This exciting finding led to studies of the underlying cellular and molecular mechanisms. Single- cell RNA sequencing (scRNA-seq) revealed that HCC growth upregulated Kruppel like factor-2 (KLF2) in tumor- associated macrophages (TAMs), and B.th addition activated effector CD8+ T cells and improved αPD-1 therapeutic effect against HCC, which was associated with reduced KLF2 expression in TAMs. Moreover, adoptive cell transfer (ACT) of macrophages (MΦs) with KLF2 knockdown (KD) enabled TSA immunization to significantly suppress HCC growth. Conversely, KLF2 overexpression (OE) in MΦs compromised B.th/αPD-1- induced therapeutic suppression of HCC. These compelling results highlight KLF2 as a key regulator mediating microbes’ impact on hepatocarcinogenesis and B.th/αPD-1 immunotherapy by modulating MΦs. Further studies indicate that KLF2 controls MΦ expression of TLR9 and signal-regulatory protein α (SIRPα) to regulate MΦ tumor phagocytosis and immune regulatory function. These findings generate the following hypothesis: B.th, with CpG-rich nucleic acid, reinvigorates αPD-1 Ab ICT in HCC by phenotypically and functionally programming MΦ via KLF2-controlled expression of TLR9 and SIRPα. In Aim 1, KLF2-directed MΦs as the cellular basis mediating HCC pathogenesis and immune tolerance will be studied toward the development of a new therapeutic approach by integrating KLF2-KO MΦs with αPD-1 Ab. In Aim 2, the molecular mechanism and regulators by which B.th/αPD-1 suppress KLF2 to reprogram MΦ by controlling TLR9 and SIRPα expression will be studied. The knowledge generated from this study will not only identify unrecognized endogenous regulators with a role in programming MΦ in HCC, but also make a case that these factors are effective targets to trigger MΦs and lead to improved ICT for HCC.
