Inhibition of the Methyltransferase G9a with Small Molecules As a New Therapeutic Strategy for Treatment of Hematological Malignancies

The indisputable role of epigenetics in hematological malignancies, along with the fact that unfavorable epigenetic alterations might be reversible, has favored the development of novel epigenetic drugs. Although according to the Structural Genomics Consortium there are 377 known epigenetic proteins, currently most epigenetic drugs are inhibitors of DNA methyltransferases (DNMTS) and histone deacetylases (HDACS). Therefore, many epigenetic targets remain to be discovered and exploited within the vast field of epigenetics. With the aim of identify novel epigenetic targets we carried out a screening using small interfering RNAs (siRNAS): we interfered the expression of 134 genes belonging to the main protein families implicated in the epigenetic mechanism regulation (methyltransferases, demethylases, bromodomain proteins and MBT proteins) in several cell lines of Acute Lymphoblastic Leukemia (ALL), Multiple Myeloma (MM) and Lymphoma and determined the effect of the interference in cell proliferation. Those candidates with a decrease in cell proliferation greater than 40% in all cell lines analyzed were selected. In this way, we identified the histone H3 Lysine 9 (K9H3) methyltransferase G9a as a potential target in hematological malignancies. In parallel, ChIP on chip assays was performed, and a considerable number of genes showed an increase in K9H3me2 and K9H3me3 marks in ALL cell lines in comparison with healthy donor samples, suggesting an important role of G9a in this disease. In order to corroborate the results obtained, we nucleofected a greater number of cell lines with two different siRNAs against G9a. As expected, inhibition of G9a expression induced a significant decrease in cell proliferation. We verified that G9a was overexpressed at mRNA and protein level in ALL, AML and MM cell lines and ALL primary samples by Q-RT-PCR and western blot, respectively. Besides, treatment of ALL, AML, MM and Lymphoma cell lines with the G9a inhibitors BIX01294 and UNC0638 induced a decreased in cell proliferation and an increased in apoptosis. All these data suggest that G9a is an ideal epigenetic target for the treatment of hematological malignancies, so we designed and synthesized small molecules against G9a. The design was based on the mechanisms of action of G9a, taking into account the available structural and biochemical information. We selected those compounds that presented an optimal affinity with G9a (biochemical assay) and a good in vitro G9a activity inhibition, decreasing H3K9me2 mark by western blot and flow cytometry. These compounds showed an important in vitro antitumoral effect with GI₅₀ values lying in the nanomolar concentration range. In fact, our compounds are much better than reference compounds BIX01294 and UNC0638, both in cellular and H3K9me2 inhibition level. Moreover, the small molecules against G9a induced apoptosis in addition to decrease cell proliferation. Once the lead compounds were selected, we performed ADME, toxicity and pharmacokinetic studies in order to carry out the in vivo analysis in human CEMO-1 ALL mouse model. Treatment of mice engrafted with CEMO-1 cells with G9a small inhibitory molecules induced an in vivo decrease of the H3K9me2 mark, which was accompanied with a significant prolonged survival of G9a inhibitors-treated mice (57.3 days) in comparison with control group (90.8 days). This is the first evidence of in vivo efficacy of G9a inhibitors. Further studies using RNA-sequencing will be performed in the presence of G9a inhibitors to determine mechanism of action. In conclusion, we have identified and validated G9a as an epigenetic target for the treatment of hematological malignancies and we have developed small inhibitory molecules against this target. Finally, our results show the therapeutic activity of G9a small inhibitory molecules in models of ALL and suggest that these compounds may be of clinical value in the treatment of patients with ALL and other hematological malignancies.