Abstract 2945: Nilotinib abrogates DNMT1-depenent DNA methylation: A novel mechanism for induction of AML leukemia regression
Authors: Shen Na , Yan Fei , Pang Jiuxia , Gangat Naseema , Litzow Mark R. , Al-Kali Aref , Liu Shujun 
Published: August 2015

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