Hypermethylation of TAp73 Suppresses ABL1-Involved DNA Damage Response in Multiple Myeloma

Background: More recently, multiple myeloma (MM) cells evade apoptosis despite pervasive DNA damage was demonstrated. However, the relevance of ongoing DNA damage and the mechanisms by which apoptosis is suppressed remain to be fully elucidated. p53 deletion and mutations do not appear to be a pivotal event in the evolution from pre-malignancy toward malignancy in MM. The protooncogene ABL1 was an alternative pathway to p53 down stream of ATM/ATR, which is commonly translocated in Chronic Myelogenous Leukemia (CML). ABL1 forms a complex with the tumor suppressor gene TP73, which belongs to the p53 family. P73 is expressed as multiple isoforms due to the usage of two different promoters, P1 promoter of TAp73 inducing apoptosis and P2 promoter of Δ Np73 promoting survival. In this study, we explored the role of ABL1/p73 axis in MM cells evading ongoing DNA damage induced apoptosis.

Materials and methods: Real-time-PCR was used to detect the ABL1 and miR-203a expression in MM primary samples and MM cell lines. Immunofluency staining was performed to detect the ɣ-H2A.X level in MM cells. Flow cytometry was performed to detect the apoptosis in MM cells. Bisulfite Pyrosequencing and Methylation Specific-PCR were used to detect the p73 promoter methylation.

Results: Our results revealed that ABL1 level was up-regulated both in primary MM samples and MM cell lines (-1.25±0.28 vs. 0.06±0.24, p=0.02). MiR-203 which suppresses ABL1 expression was down-regulated (0.01±0.01 vs. 0.97±0.08, p=0.01). MM cell lines and primary cells showed high ɣ-H2A.X staining. Immunofluency staining showed that ABL1 relocalized in the nucleus of MM cells after treated with doxorubicin. The apoptosis of MM cells was significantly up-regulated (7.8±2.1)% vs. (25.4±4.5)%, p<0.05. Doxorubicin treatment combined with ABL1 inhibitor (STI571) suppressed the apoptosis significantly, (25.4±4.5)% vs. (12.2±3.4)%, p=0.03. Bisulfite Pyrosequencing and MS-PCR of 42 newly diagnosed MM patient sample revealed that the P1 promoter of p73 was hypermethylated compared with normal plasma cells (86% ±7% vs. 58%±4%, p=0.032). RT-qPCR and western blotting showed that Δ Np73 levels were significantly higher than TAp73 (148.6±19.3 vs. 6.8±2.4, p=0.021) in plasma cells of those patients and MM cell lines.

Conclusion: Hypermethylation of p73 promoter suppresses TAp73 expression. The deregulated of ABL1-p73 pathway in MM cells resulted in marked reduction of apoptosis induced by ongoing DNA damage.