Jornal de Hematologia e Doenças Tromboembólicas
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Abstrato

Restoration of PRG2 Expression by 5-Azacytidine Involves in Sensitivity of PKC-412 (Midostaurin) Resistant FLT3-ITD Positive Acute Myeloid Leukaemia Cells

Hamid Ali Nagi Al-jamal, Siti Asmaa Mat Jusoh, Mohamad Ros Sidek, Rosline Hassan and Muhammad Farid Johan

Background: Tumor-suppressor genes Tumour suppressor genes (TSG) are inactivated by methylation in several cancers including acute myeloid leukemia (AML). Transcriptional silencing of PRG2 gene could be involved in development and progression of cancers. 5-Azacytidine (5-Aza) is a DNA methyltransferase inhibitor that causes DNA de-methylation resulting in re-expression of silenced TSG. Midostaurin (PKC412) is a multitargeted tyrosine kinase inhibitor that potently inhibits FLT3 tyrosine kinase and induces hematological remission in patients with AML. However, majority of AML patients in clinical trials developed resistance to PKC412.

Methods: Resistant cells were developed by overexposure of MV4-11 cells to PKC-412 and treated with 5-Aza. Apoptosis and cytotoxicity of PKC-412 were determined using Annexin V and MTS assays, respectively. Gene expression profiling was performed using microarray followed by quantitative real-time PCR. STATs activity was examined using Western bloting and methylation of PRG2 was studied using pyrosequencing analysis.

Results: The cytotoxic dose of PKC-412 on resistant cells was significantly higher than parental and MV4-11Rpkc+5-Aza cells (p=0.003). The resistant cells showed significant higher viability and lower apoptotic cells compared to other cells (p<0.001). Expression of PRG2 was more than 300 folds higher in MV4-11R-pkc+5-Aza cells compared to parental and resistant cells (p=0.001). STAT3 was activated in resistant cells. Methylation of PRG2 gene was significantly decreased in MV4-11R-pkc+5-Aza cells.

Conclusion: The restoration of PRG2 expression induces sensitivity towards PKC-412 and an increased in cell death. Our findings suggest that PRG2 gene could play a role in the sensitivity to PKC-412 and serve as a target for treatment of AML patients resistant to PKC-412 tyrosine kinase inhibitor.