Efficacy and epigenetic interactions of novel DNA hypomethylating agent guadecitabine (SGI-110) in preclinical models of hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a deadly malignancy characterized at the epigenetic level by global DNA hypomethylation and focal hypermethylation on the promoter of tumor suppressor genes. In most cases it develops on a background of liver steatohepatitis, fibrosis, and cirrhosis. Guadecitabine (SGI-110) is a second-generation hypomethylating agent, which inhibits DNA methyltransferases. Guadecitabine is formulated as a dinucleotide of decitabine and deoxyguanosine that is resistant to cytidine deaminase (CDA) degradation and results in prolonged in vivo exposure to decitabine following small volume subcutaneous administration of guadecitabine. Here we found that guadecitabine is an effective demethylating agent and is able to prevent HCC progression in pre-clinical models. In a xenograft HCC HepG2 model, guadecitabine impeded tumor growth and inhibited angiogenesis, while it could not prevent liver fibrosis and inflammation in a mouse model of steatohepatitis. Demethylating efficacy of guadecitabine on LINE-1 elements was found to be the highest 8 d post-infusion in blood samples of mice. Analysis of a panel of human HCC vs. normal tissue revealed a signature of hypermethylated tumor suppressor genes (CDKN1A, CDKN2A, DLEC1, E2F1, GSTP1, OPCML, E2F1, RASSF1, RUNX3, and SOCS1) as detected by methylation-specific PCR. A pronounced demethylating effect of guadecitabine was obtained also in the promoters of a subset of tumor suppressors genes (CDKN2A, DLEC1, and RUNX3) in HepG2 and Huh-7 HCC cells. Finally, we analyzed the role of macroH2A1, a variant of histone H2A, an oncogene upregulated in human cirrhosis/HCC that synergizes with DNA methylation in suppressing tumor suppressor genes, and it prevents the inhibition of cell growth triggered by decitabine in HCC cells. Guadecitabine, in contrast to decitabine, blocked growth in HCC cells overexpressing macroH2A1 histones and with high CDA levels, despite being unable to fully demethylate CDKN2A, RUNX3, and DLEC1 promoters altered by macroH2A1. Collectively, our findings in human and mice models reveal novel epigenetic anti-HCC effects of guadecitabine, which might be effective specifically in advanced states of the disease.

Induction of cancer cell stemness by depletion of macrohistone H2A1 in hepatocellular carcinoma

“…In well‐differentiated tumors, cells resembled organized hepatocytes, and almost all nuclei were positive for macroH2A1 (Fig. B, left panel), as reported . We then confirmed that the observed decreases in macroH2A1 expression were not restricted to a specific isoform: both alternatively spliced macroH2A1 variants, macroH2A1.1 and macroH2A1.2, were significantly lower at the mRNA and protein levels in poorly differentiated versus well‐differentiated HCC, as assessed by quantitative PCR and immunoblotting (Fig.…”

“…Increased levels of macroH2A1 expression mark differentiated human HCC . Here, we examined whether macroH2A1 expression varies between human HCCs with different degrees of stem‐like characteristics (differentiation, aggressiveness, refractoriness to chemotherapy, and high recurrence).…”

“…MacroH2A proteins are composed of a domain that is 66% homologous to histone H2A and a C‐terminal linker that connects the histone fold domain to a macro domain . Transcriptional regulation by macroH2A and, in particular, of macroH2A1 has also been linked to the pathogenesis of multiple cancers including skin, breast, lung, and colon . Importantly, macroH2A1 levels negatively correlate with the self‐renewal capacity of the pluripotent stem cells and regulate the delicate balance between self‐renewal and differentiation of embryonic and adult stem cells .…”

“…Importantly, macroH2A1 levels negatively correlate with the self‐renewal capacity of the pluripotent stem cells and regulate the delicate balance between self‐renewal and differentiation of embryonic and adult stem cells . In the context of HCC, we recently demonstrated that macroH2A1 is a marker of differentiated liver tumor cells and protects them from decitabine‐induced senescence …”

Hepatoepigenetic Alterations in Viral and Nonviral-Induced Hepatocellular Carcinoma

“…This synergistic effect results in the suppression of a subset of tumour suppressor genes (CDKN2A, DLEC1, and RUNX2) and enhanced HCC cell growth in Hep G2 and Huh-7. Treatment with DNA hypomethylating agent guadecitabine reversed this effect leading to inhibition of HCC cell growth [62]. EZH2 is an important component of PRC2 that interacts with EED and SUZ12 to establish trimethylation of H3K27 (H3K27me3) leading to tumour initiation and progression [63, 64].…”