Periodic Reporting for period 1 - TARGEPILIVER (Characterization of Key Epigenetic Targets in Hepatic Fibrosis and Hepatocellular Carcinoma Development. Generation of New Antifibrotic and Antitumoral Drugs.) Reporting period: 2015-12-01 to 2017-11-30 Summary of the context and overall objectives of the project "Hepatocellular carcinoma is a malignant tumors that, globally, affect one million new patients each year. For patients with advanced liver cancer the alternative treatment options are very limited because these tumors are poorly sensitive to antitumor drugs. In addition, the dose that can be administered is restricted by the fact that very often they are patients with precarious health by the existence of multiple pathologies and in whom the tumor has developed on a cirrhotic liver, so that there is very low tolerability to the antitumor drugs that the oncologist uses in the attempt to selectively ""poison"" the tumor, being as innocuous as possible for the patient. The expectation for survival of these patients is less than 30% at one year and about half to that at two years. For this reason emerged this project, whose basic idea is that ""not everything that determines tumor development and subsequent behavior, as their response to antitumor drugs, is written in the genes"", but there is a complex cellular machinery, the so-called epigenetic mechanisms, which affects the proper functionality of their expression and that certain elements of that gear can be altered in situations where the initiation of liver cancer is favored, such as chronic liver disease situations. This project aim to identifying such epigenetic changes and their potential enzymes implicated to be used as predictive markers of the risk of developing liver cancer and as molecular targets for novel pharmacological strategies to treat these tumors.The exploration of the epigenetic mechanisms involved in hepatocarcinogenesis processes can open the door to the development of promising new therapies. This has been a pioneering project in the field of hepatic tumor disease, in which, in addition to identifying and characterizing new epigenetic targets, new pharmacological agents potentially capable of preventing and / or treating hepatic fibrosis and HCC have been developed. The weight of chronic liver diseases in Europe has increased greatly in recent decades, assuming a clinical and economic problem, and the prognosis is that they continue to increase in the coming years. Liver cirrhosis along with HCC cause 220,000 deaths a year in the European Union (http://www.easl.eu/_eu-policy/publications) highlighting the need to develop effective therapies. The objectives of this project have been to address this problem with an innovative technology and a multidisciplinary approach and can therefore be susceptible to further clinical development. Our proposal presented two general objectives: i) a comprehensive analysis of the expression and pathological significance of two epigenetic modifiers, the DNA and histone methyltransferases DNMT1 and G9a, in experimental fibrosis, HCC and ii) the development of novel and efficacious DNMT1 and G9a specific inhibitors with a good safety profile, which is critical when treating patients with compromised liver function. These new epigenetic pharmacological tools may be further developed into drugs to prevent chronic liver diseases progression, and to treat HCC alone or in combination with existing drugs." Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Epigenetic modifications like DNA and histone methylation functionally cooperate fostering tumor growth, including that of hepatocellular carcinoma (HCC). Pharmacological targeting of these mechanisms may open new therapeutic avenues. In this proposal we finally focused in determining the therapeutic efficacy and potential mechanism of action of our new dual G9a histone-methyltransferase and DNA-methytransferase 1 DNMT1 inhibitor in human HCC cells and their crosstalk with fibrogenic cells. The expression of these two epigenetic modifiers, along with that of their molecular adaptor ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was measured in human HCCs, peritumoral tissues and HCC cell lines. We evaluated the effect of individual and combined inhibition of G9a and DNMT1 on HCC cells growth by pharmacological and genetic approaches. The activity of our lead compound, CM-272, was examined in HCC cells under normoxia and hypoxia, human hepatic stellate cells and LX2 cells, and xenograft tumors formed by HCC or combined HCC+LX2 cells. We found a significant and correlative overexpression of G9a, DNMT1 and UHRF1 in HCCs in association with poor prognosis. Independent G9a and DNMT1 pharmacological targeting synergistically inhibited HCC cell growth. CM-272 potently reduced HCC and LX2 cells proliferation and quelled tumor growth, particularly in HCC+LX2 xenografts. Mechanistically, CM-272 inhibited the metabolic adaptation of HCC cells to hypoxia, and induced a differentiated phenotype in HCC and fibrogenic cells. The expression of the metabolic tumor suppressor gene fructose-1,6- bisphosphatase (FBP1), epigenetically repressed in HCC, was restored by CM-272.Conclusion: Combined targeting of G9a/DNMT1 with compounds like CM-272 is a promising strategy for HCC treatment. Our findings also underscore the potential of differentiation therapy in HCC. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Epigenetic mechanisms are emerging as attractive therapeutic targets in solid tumors and epigenetic drugs are already being tested in HCC clinical trials. However, the extensive functional crosstalk between chromatin marks such as DNA and H3K9 methylation, suggests that simultaneous targeting of different epigenetic modifiers may improve therapeutic efficacy. We recently demonstrated this contention by showing the remarkable antitumoral activity of CM-272, a dual G9a/DNMT1 inhibitor, in different hematological neoplasms. Now we provide evidence supporting the potential of combined G9a/DNMT1 antagonism for HCC treatment.First, we demonstrated the coordinated overexpression of G9a and DNMT1, along with their key adaptor UHRF1, in human HCC tissues. The pathophysiological relevance of this matched overexpression is suggested by its significant association with molecular, histological and clinical characteristics indicative of aggressive disease and poor prognosis. The rationale for the simultaneous inhibition of G9a/DNMT1 in HCC was further established by the synergistic anti-proliferative action of combined G9a and DNMT1 specific antagonists. Thus, CM-272 exerted a very potent antiproliferative effect in a wide panel of human HCC cell lines, which also showed aclose correlation in the expression of G9a, DNMT1 and the UHRF1 adaptor. CM-272 inhibited HCC cells growth in vivo in the absence of apparent toxicity. The high specificity of CM-272 for G9a/DNMT1, and a mechanism of action based on the reversible inhibition of G9a and DNMT1 binding to their substrates, but not to Sadenosylmethionine, may contribute to explain its lack of off-target effects and systemic toxicity.A patent application for the small molecules mentioned in this project was initially filed (June 2014), covering chemical series for these compounds and finally accepted (Agirre, X. et al. Novel compounds as dual inhibitors of histone methyltransferases and DNA methyltransferases. WO2015192981A1, 30 March (2015). Any potential new application derived of these findings will be also susceptible to be protected from IP perspective. If the output of this research is amenable for industrial development it will be readily transferred to interested companies, with positive repercussions for the advancement of this research line, the university's wealth and the scientific and patient’s community at large. Dual targeting of G9a and DNMT1 in HCC treatment.
