Pang, Jiuxia

Introduction: It is known that overexpression of DNA methyltransferases (e.g., DNMT1) is frequent and changes of DNA cytosine methylation (5mC) are a constant feature of cancers. DNA methylation inhibitors, such as 5-aza-2'-deoxycytidine (Dec), have been in clinics for patients with leukemia. It is classically believed that promoter hypomethylation coupled by reexpression of epigenetically-suppressed tumor suppressors is a core mechanism behind Dec-impaired leukemia cell growth. However, the fact that global DNA methylation profiling barely predicts Dec-response suggests a demethylation-independent mechanism of Dec-induced cell death. N6-methyladenine (m6A) has been identified recently as an abundant DNA modification in eukaryotes (Wu, Nature 2016;532:329). Importantly, m6A is extensively present in the human genome, and m6A abundance is associated with tumorigenesis (Xie, Cell 2018;71:306). Furthermore, the DNA m6A is dynamically modulated by the methyltransferases (i.e., METTL3, N6AMT1) and demethylases (i.e., ALKBH1), and changes in m6A predict gene expression (Wu, Nature 2016;532:329). Given a potential crosstalk between m6A and distinct epigenetic mechanisms (Yao, Nat. Commun 2017;8:1122), we hypothesized that the anticancer actions of Dec may partially result from changes in DNA m6A in leukemia cells. Methods: Protein expression of target genes was assessed by Western blotting. The levels of DNA cytosine methylation (5mC) and N6-methyladenine (m6A) were measured by dotblotting or liquid chromatography-mass spectrometry (LC-MS/MS). The cell viability and apoptosis were determined by the Cell Counting Kit 8 (CCK8) assays as well as the Annexin V/Propidium Iodide staining and flow cytometry. The peripheral blood mononuclear cells (PBMCs) of leukemia patients from Mayo Clinic were prepared by Ficoll-Hypaque gradient centrifugation. Results: To test our hypothesis, leukemia cells, Kasumi-1, MV4-11, K562 and KU812, were exposed to 2 µM Dec, a clinical achievable concentration, for 72 hours. As expected, Dec treatment led to a downregulation of DNMT1 and DNMT3a, a reduction of 5mC levels by dotblotting using anti-5mC antibody, a blockage of cell proliferation and a promotion of cell apoptosis. When genomic DNA was subjected to dotblotting using anti-m6A antibody, the results revealed a marked decrease of DNA m6A levels in all Dec-treated cells. Then genomic DNA from K562 and MV4-11 cells was enzymatically digested to 2'-deoxynucleosides. dA was quantified by HPLC-UV, while the amount of m6A was measured by isotope dilution HPLC-ESI-MS/MS using 15N labeled internal standard. The standard curves were generated using pure standards, from which the m6A/A ratio was calculated. In agreement with dotblotting results, Dec treatment significantly decreased DNA m6A abundance in both cell lines. Mechanistically, exposure to Dec led to a consistent increase of demethylase fat mass and obesity-associated protein (FTO), but not METTL3 nor ALKBH1 and ALKBH5. Further, knockdown of FTO increased DNA m6A, which was further confirmed by treatment with FTO inhibitors rhein and meclofenamic acid (MA). These data indicate that FTO may be responsible for Dec-induced m6A changes in leukemia cells. To investigate the clinical implications of DNA m6A, we obtained PBMCs from AML patients (n = 10), who received Dec therapy (20 mg/m2 daily for 5 days every 4 weeks) in Mayo Clinic. These PBMCs were further cultured for 48 hours, frozen and stored in 100% ethanol before DNA extraction. The results from dotblotting using anti-5mC or anti-m6A showed that a trend of decrease in both m6A and 5mC abundance is observed, and the pattern of changes in m6A and 5mC displays a positive correlation. Finally, exposure of leukemia cells to the combination of Dec (2 µM) with FTO inhibitor MA (50 µM) induced more cell apoptosis and greater inhibition on cell proliferation as compared to single agent in vitro, supporting FTO inhibitors as new therapeutic agents in leukemia. Conclusion: Our studies suggest that the FTO-DNA m6A axis may partially mediate the therapeutic outcomes of Dec in leukemia. Our findings provide a new mechanistic paradigm for the anticancer activities of Dec, and define the m6A methylation status in leukemia cells as a new pharmacodynamic marker for their response to Dec-based therapy, pointing to a novel treatment strategy for incorporating m6A modulators to enhance the therapeutic index of Dec. Disclosures Al-Kali: Astex Pharmaceuticals, Inc.: Research Funding.

Abstract Purpose: Receptor tyrosine kinases (RTKs) are frequently deregulated in leukemia, yet the biological consequences of this deregulation remain elusive. The mechanisms underlying aberrant methylation, a hallmark of leukemia, are not fully understood. Here we investigated the role of RTKs in methylation abnormalities and characterized the hypomethylating activities of RTK inhibitors. Experimental Design: Whether and how RTKs regulate expression of DNA methyltransferases (DNMTs), tumor suppressor genes (TSGs) as well as global and gene-specific DNA methylation were examined. The pharmacologic activities and mechanisms of actions of RTK inhibitors in vitro, ex vivo, in mice, and in nilotinib-treated leukemia patients were determined. Results: Upregulation of RTKs paralleled DNMT overexpression in leukemia cell lines and patient blasts. Knockdown of RTKs disrupted, whereas enforced expression increased DNMT expression and DNA methylation. Treatment with the RTK inhibitor, nilotinib, resulted in a reduction of Sp1-dependent DNMT1 expression, the diminution of global DNA methylation, and the upregulation of the p15INK4B gene through promoter hypomethylation in AML cell lines and patient blasts. This led to disruption of AML cell clonogenicity and promotion of cellular apoptosis without obvious changes in cell cycle. Importantly, nilotinib administration in mice and human patients with AML impaired expression of DNMTs followed by DNA hypomethylation, TSG re-expression, and leukemia regression. Conclusions: Our findings demonstrate RTKs as novel regulators of DNMT-dependent DNA methylation and define DNA methylation status in AML cells as a pharmacodynamic marker for their response to RTK-based therapy, providing new therapeutic avenues for RTK inhibitors in overcoming epigenetic abnormalities in leukemia. Clin Cancer Res; 23(20); 6254–66. ©2017 AACR.

Abstract Nilotinib has been approved to treat newly diagnosed, imatinib-resistant or -intolerant leukemia, but the mechanisms underlying its antileukemia actions remain elusive. Here, we report that nilotinib blocks AML growth through its DNA hypomethylating activity. We showed that, in addition to inducing a reduction of KIT and FLT3 protein expression and phosphorylation, nilotinib downregulated DNMT1, 3a and 3b in Kasumi-1, MV4-11 and C1498 cells. Since DNMT1 is maintenance and de novo DNMT, we focused on DNMT1 and proved that nilotinib impaired DNMT1 expression via Sp1 dysfunction, since the luciferase activity driven by DNMT1 promoter containing Sp1 binding sites was decreased by 2.6 fold by nilotinib, while increased by 10.6 fold after Sp1 overexpression. Electrophoretic mobility shift assay disclosed that nilotinib abolished Sp1 binding on DNMT1 promoter. Dotblot using 5mC antibody revealed global DNA hypomethylation and qPCR showed p15INK4B upregulation in Kasumi-1 (3.4 fold) or MV4-11 (5.2 fold). Bisulfite sequencing found a decrease of DNA methylation on p15INK4B promoter from 100% to 89% in Kasumi-1 or 13% to 0% in MV4-11. The functional assays showed a decrease of colony formation in Kasumi-1 (60%) or MV4-11 (58%), and an increase of cell apoptosis in Kasumi-1 (6.9 fold) or MV4-11 (3.2 fold). We verified these findings in AML patient blasts (n = 3) by showing that nilotinib treatment led to the dephosphorylation of KIT, the downregulation of DNMT1, 3a, 3b and Sp1, the reduction of global DNA methylation and the re-expression of p15INK4B (4.4 fold) via promoter demethylation (from 14% to 6%), which was followed by suppressed colony formation (77%), promoted cell apoptosis (1.84 fold) and increased caspase-3 and caspase-8. To test if nilotinib induces AML regression via DNA hypomethylation in vivo, 0.1×106 C1498 cells were injected into C57BL/6 mice (n = 3 mice/group) via the tail vein. The leukemic mice were treated with 5, 10 or 15 mg/kg of nilotinib in PEG400 plus saline via intraperitoneal injection twice a week for a total of 6 doses. No toxicity was observed for the tested drug dosage and schedule, because of no evident change of mouse body weight. Compared to the control group, nilotinib-treated mice had a decrease in spleen size. H&E staining showed that the control mice exhibited increased infiltration of AML cells into the spleen, lung and liver of recipients. The bone marrow (BM) histopathology identified a higher degree of mature post-mitotic cells containing metamyelocytes, bands and segmented neutrophils in nilotinib-treated mice than control. DNMT1 was downregulated (39%), p15INK4B was upregulated and global DNA methylation was reduced (60%) in BM cells from nilotinib-treated mice. Thus, our findings unveil DNA methylation machinery as an innovative target behind nilotinib's anticancer activities, and provide a new therapeutic avenue for nilotinib in overcoming aberrant epigenetics in AML. Note: This abstract was not presented at the meeting. Citation Format: Na Shen, Fei Yan, Jiuxia Pang, Naseema Gangat, Mark R. Litzow, Aref Al-Kali, Shujun Liu. Nilotinib abrogates DNMT1-depenent DNA methylation: A novel mechanism for induction of AML leukemia regression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2945. doi:10.1158/1538-7445.AM2015-2945