Epigenetic modulation enhances immunotherapy for pancreatic ductal adenocarcinoma.

Objectives: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a poor prognosis. PDAC has poor response to immunotherapy because of its unique tumour microenvironment (TME). In an attempt to stimulate immunologically silent pancreatic cancer, we investigated the role of epigenetic therapy in modulating the TME to improve immunogenicity. Methods: In vitro human PDAC cell lines MiaPaca2 and S2-013 were treated with 5µ m 3-Deazaneplanocin A (DZNep, an EZH2 inhibitor) and 5 µ m 5-Azacytidine (5-AZA, a DNMT1 inhibitor). In vivo orthotopic murine tumour models using both murine PAN02 cells and KPC cells inoculated in immunocompetent C56/BL7 mice were treated with anti-PD-L1 combined with DZNep and 5-AZA. Short hairpin knockdown (KD) of EZH2 and DNMT1 in PAN02 cells for the orthotopic murine tumour model was established to validate the drug treatment (DZNep and 5-AZA). qRT-PCR and microarray assays were performed for the evaluation of Th1-attracting chemokines and cancer-associated antigen induction. Results: Drug treatments induced significant upregulation of gene expressions of Th1-attracting chemokines, CXCL9 and CXCL10, and the cancer-testis antigens, NY-ESO-1, LAGE and SSX-4 (P < 0.05). In orthotopic tumour models, inoculation of PAN02 cells or KPC cells demonstrated significant tumour regression with corresponding increased apoptosis and infiltration of cytotoxic T lymphocytes in the combination treatment group. In the orthotopic Pan02-KD model, the anti-PD-L1 treatment also caused significant tumour regression. Conclusion: We demonstrate that immunotherapy for PDAC can be potentiated with epigenetic therapy by increasing cancer-associated antigen expression and increased T-cell trafficking across the immunosuppressive tumour microenvironment via upregulation of the repressed chemokines and increased apoptosis with subsequent tumour regression.

Electrochemotherapy with Irreversible Electroporation and FOLFIRINOX Improves Survival in Murine Models of Pancreatic Adenocarcinoma.

BACKGROUND: Previously published work has demonstrated that combining gemcitabine with irreversible electroporation (IRE) results in increased drug delivery to pancreatic adenocarcinoma cells in vivo. This study assessed the efficacy of IRE + gemcitabine and IRE + FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, and oxaliplatin), the impact of the superior regimen on survival, and the safety of electrochemotherapy in human subjects. METHODS: Histologic analysis was performed after in vitro and in vivo treatment of S2013 and Panc-1 pancreatic cancer cells and S2013 orthotopic tumors, respectively, and levels of apoptotic machinery and cell cycle proteins were evaluated using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot. RESULTS: Electrochemotherapy (ECT) with IRE and FOLFIRINOX resulted in increased tumor cells apoptosis compared with gemcitabine, gemcitabine + IRE, and FOLFIRINOX alone, and significantly improved overall survival when compared with mice treated with IRE or FOLFIRINOX. Increased tumor cell apoptosis, caspase-3 mRNA, active caspase-3 protein, and decreased cell proliferation were noted at the time of death or euthanasia in the ECT group compared with folinic acid alone. In five patients, ECT with either FOLFIRINOX or gemcitabine was well-tolerated and resulted in no dose-limiting toxicities. CONCLUSIONS: ECT thus results in synergistic antitumor activity compared with either treatment modality used alone, resulting in increased tumor cell apoptosis as well as decreased tumor cell proliferation and improved overall survival. Pilot data suggest that ECT represents a promising modality for the treatment of patients with locally advanced pancreatic cancer. TRIAL REGISTRATION: The human subject portion of this work was conducted as part of an investigator-initiated clinical trial at the University of Louisville (NCT03484299).

Epigenetic modulation enhances immunotherapy for hepatocellular carcinoma.

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 ±â€¯0.2 relative fold change; CXCL10 1.44 × 103 ±â€¯37 relative fold change) and Hep3B (CXCL 9 6.85 × 103 ±â€¯1.3 × 103 relative fold change; CXCL 10 2.15 × 103 ±â€¯3.1 × 102 relative fold change). Additionally, there was a significant induction of cancer testis antigens NY-ESO-1 (3.6-3.7 ±â€¯0.3 relative fold change) and LAGE (8.3-11.7 ±â€¯1.9 relative fold change). In vivo model demonstrated statistically significant tumor regression in the combination treatment group (0.02 g ±â€¯0.02) compared to epigenetic therapy (0.63 g ±â€¯0.61) or immunotherapy alone (0.15 g ±â€¯0.21) with untreated control (2.4 g ±â€¯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.

Evaluating the Regulatory Immunomodulation Effect of Irreversible Electroporation (IRE) in Pancreatic Adenocarcinoma.

BACKGROUND: Irreversible electroporation (IRE) has been demonstrated as an effective local method for locally advanced (stage 3) pancreatic adenocarcinoma. Immune regulatory T cells (Tregs) induce immunosuppression of tumors by inhibiting patients' anti-tumor adaptive immune response. This study aimed to evaluate the immunomodulation effect of IRE to identify an ideal time point for potential adjuvant immunotherapy. METHODS: This study prospectively evaluated an institutional review board-approved study of patients undergoing either in situ IRE or pancreatectomy. Patient blood samples were collected at different time points (before surgery [preOP] and on postoperative day [POD] 1, POD3, and POD5). Peripheral blood mononuclear cells (PBMCs) were isolated and evaluated for three different CD4 + Treg subsets (CD25 + CD4 +, CD4 + CD25 + FoxP3 +, CD4 + CD25 + FoxP3 -) by flow cytometry and analyzed for median fold change (MFC) between each two consecutive time points (MFC = log2(T2/T1)). RESULTS: The study analyzed 15 patients with in situ IRE (n = 11) or pancreatectomy (PAN) (n = 4). In both groups, CD25 + CD4 + Tregs decreased on POD1 followed by a steady increase in pancreatectomy, whereas the trend in the IRE group reversed between D3 and D5 (MFC: IRE [- 0.01], PAN [+ 0.39]). For each period, CD4 + CD25 + FoxP3 + Tregs showed the most dramatic inverse effect, with D3 to D5 showing the most change (MFC: IRE [- 0.18], PAN [+ 0.39]). Also, CD4 + CD25 + FoxP3 - Tregs showed an inverse effect between D3 and D5 (MFC: IRE [- 0.25], PAN [+ 0.49]). Altogether, the Treg trend was inversely affected by the in situ IRE procedure, with the greatest cumulative significant change for all three Treg subsets between D3 and D5 (MFC ± SEM: IRE [- 0.24 ± 0.05], PAN [+ 0.37 ± 0.02]; p = 0.016). CONCLUSIONS: The study data suggest that in situ IRE procedure-mediated Treg attenuation between POD3 and POD5 can provide a clinical window of opportunity for potentiating clinical efficacy in combination with immunotherapy.