Young K Hong1, Yan Li1, Harshul Pandit2, SuPing Li1, Zachary Pulliam1, Qianqian Zheng3, Youxi Yu4, Robert C G Martin5. 1. Division of Surgical Oncology, Hiram C. Polk Jr. M.D. Department of Surgery, University of Louisville School of Medicine, Louisville, KY 40202, USA. 2. Division of Surgical Oncology, Hiram C. Polk Jr. M.D. Department of Surgery, University of Louisville School of Medicine, Louisville, KY 40202, USA; Department of Pharmacology & Toxicology, University of Louisville School of Medicine, Louisville, KY 40202, USA. 3. Department of Pathophysiology, Basic Medicine College, China Medical University, Shenyang 110122, China. 4. Department of Hepatobiliary and Pancreatic Surgery, The First Hospital of Jilin University, Changchun 130021, China. 5. Division of Surgical Oncology, Hiram C. Polk Jr. M.D. Department of Surgery, University of Louisville School of Medicine, Louisville, KY 40202, USA; Department of Pharmacology & Toxicology, University of Louisville School of Medicine, Louisville, KY 40202, USA. Electronic address: Robert.Martin@louisville.edu.
Abstract
BACKGROUND: Anti-PDL-1 immunotherapy for Hepatocellular Carcinoma (HCC) demonstrated a mixed response. Polycomb Repressor Complex 2(PRC2) contributes to the initiation and progression of HCC by suppressing tumor antigens and inhibiting an immune response. Two components of epigenetic modulation are Enhancer of Zeste Homolog 2 (EZH2, the catalytic component of PRC2) and DNA Methyltransferase 1 (DNMT1). We aim to investigate the potential role of epigenetic therapy targeting EZH2 and DNMT1 as a novel strategy to modulate immunotherapy response in HCC. METHODS: HepG2, Hep3B, and Hepa1-6 HCC cell lines were treated with EZH2 inhibitor (DZNep) and DNMT1 inhibitor (5-Azacytidine) with and without anti-PDL-1. Quantitative RT-PCR and immunohistochemistry were performed to evaluate the expression of tumor suppressors, tumor antigens, and Th1 chemokines. In-vivo C57/LJ immunocompetent mice model with subcutaneous tumor inoculation was performed with intraperitoneal drug injections. RESULTS: There was a significant upregulation of Th1 chemokines in HepG2 (CXCL9 5.5 ± 0.2 relative fold change; CXCL10 1.44 × 103 ± 37 relative fold change) and Hep3B (CXCL 9 6.85 × 103 ± 1.3 × 103 relative fold change; CXCL 10 2.15 × 103 ± 3.1 × 102 relative fold change). Additionally, there was a significant induction of cancer testis antigens NY-ESO-1 (3.6-3.7 ± 0.3 relative fold change) and LAGE (8.3-11.7 ± 1.9 relative fold change). In vivo model demonstrated statistically significant tumor regression in the combination treatment group (0.02 g ± 0.02) compared to epigenetic therapy (0.63 g ± 0.61) or immunotherapy alone (0.15 g ± 0.21) with untreated control (2.4 g ± 0.71). There was significantly increased trafficking of cytotoxic T- lymphocytes and associated apoptosis for the combination treatment group compared to epigenetic or immunotherapy alone. CONCLUSIONS: This study demonstrates that epigenetic modulation could be a novel potential strategy to augment immunotherapy for HCC by stimulating T cell trafficking into tumor microenvironment via activation of transcriptionally repressed chemokine genes responsible for T-cell trafficking, inducing previously silent neoantigens for immune targets, and allowing tumor regression as a result. A clinical trial of this feasible combination therapy of these clinically available agents is warranted.
BACKGROUND: Anti-PDL-1 immunotherapy for Hepatocellular Carcinoma (HCC) demonstrated a mixed response. Polycomb Repressor Complex 2(PRC2) contributes to the initiation and progression of HCC by suppressing tumor antigens and inhibiting an immune response. Two components of epigenetic modulation are Enhancer of Zeste Homolog 2 (EZH2, the catalytic component of PRC2) and DNA Methyltransferase 1 (DNMT1). We aim to investigate the potential role of epigenetic therapy targeting EZH2 and DNMT1 as a novel strategy to modulate immunotherapy response in HCC. METHODS: HepG2, Hep3B, and Hepa1-6 HCC cell lines were treated with EZH2 inhibitor (DZNep) and DNMT1 inhibitor (5-Azacytidine) with and without anti-PDL-1. Quantitative RT-PCR and immunohistochemistry were performed to evaluate the expression of tumor suppressors, tumor antigens, and Th1 chemokines. In-vivo C57/LJ immunocompetent mice model with subcutaneous tumor inoculation was performed with intraperitoneal drug injections. RESULTS: There was a significant upregulation of Th1 chemokines in HepG2 (CXCL9 5.5 ± 0.2 relative fold change; CXCL10 1.44 × 103 ± 37 relative fold change) and Hep3B (CXCL 9 6.85 × 103 ± 1.3 × 103 relative fold change; CXCL 10 2.15 × 103 ± 3.1 × 102 relative fold change). Additionally, there was a significant induction of cancer testis antigens NY-ESO-1 (3.6-3.7 ± 0.3 relative fold change) and LAGE (8.3-11.7 ± 1.9 relative fold change). In vivo model demonstrated statistically significant tumor regression in the combination treatment group (0.02 g ± 0.02) compared to epigenetic therapy (0.63 g ± 0.61) or immunotherapy alone (0.15 g ± 0.21) with untreated control (2.4 g ± 0.71). There was significantly increased trafficking of cytotoxic T- lymphocytes and associated apoptosis for the combination treatment group compared to epigenetic or immunotherapy alone. CONCLUSIONS: This study demonstrates that epigenetic modulation could be a novel potential strategy to augment immunotherapy for HCC by stimulating T cell trafficking into tumor microenvironment via activation of transcriptionally repressed chemokine genes responsible for T-cell trafficking, inducing previously silent neoantigens for immune targets, and allowing tumor regression as a result. A clinical trial of this feasible combination therapy of these clinically available agents is warranted.