title: Enhancing SHP-1 expression with 5-azacytidine may inhibit STAT3 activation and confer sensitivity in lestaurtinib (CEP-701)-resistant FLT3-ITD positive acute myeloid leukemia.
abstract: BACKGROUND
  Tumor-suppressor genes are inactivated by methylation in several cancers including acute myeloid leukemia (AML). Src homology-2 (SH2)-containing protein-tyrosine phosphatase 1 (SHP-1) is a negative regulator of the JAK/STAT pathway. Transcriptional silencing of SHP-1 plays a critical role in the development and progression of cancers through STAT3 activation. 5-Azacytidine (5-Aza) is a DNA methyltransferase inhibitor that causes DNA demethylation resulting in re-expression of silenced SHP-1. Lestaurtinib (CEP-701) is a multi-targeted tyrosine kinase inhibitor that potently inhibits FLT3 tyrosine kinase and induces hematological remission in AML patients harboring the internal tandem duplication of the FLT3 gene (FLT3-ITD). However, the majority of patients in clinical trials developed resistance to CEP-701. Therefore, the aim of this study, was to assess the effect of re-expression of SHP-1 on sensitivity to CEP-701 in resistant AML cells.
  
  
  METHODS
  Resistant cells harboring the FLT3-ITD were developed by overexposure of MV4-11 to CEP-701, and the effects of 5-Aza treatment were investigated. Apoptosis and cytotoxicity of CEP-701 were determined using Annexin V and MTS assays, respectively. Gene expression was performed by quantitative real-time PCR. STATs activity was examined by western blotting and the methylation profile of SHP-1 was studied using MS-PCR and pyrosequencing analysis. Repeated-measures ANOVA and Kruskal-Wallis tests were used for statistical analysis.
  
  
  RESULTS
  The cytotoxic dose of CEP-701 on resistant cells was significantly higher in comparison with parental and MV4-11R-cep + 5-Aza cells (p = 0.004). The resistant cells showed a significant higher viability and lower apoptosis compared with other cells (p < 0.001). Expression of SHP-1 was 7-fold higher in MV4-11R-cep + 5-Aza cells compared to parental and resistant cells (p = 0.011). STAT3 was activated in resistant cells. Methylation of SHP-1 was significantly decreased in MV4-11R-cep + 5-Aza cells (p = 0.002).
  
  
  CONCLUSIONS
  The restoration of SHP-1 expression induces sensitivity towards CEP-701 and could serve as a target in the treatment of AML. Our findings support the hypothesis that, the tumor-suppressor effect of SHP-1 is lost due to epigenetic silencing and its re-expression might play an important role in re-inducing sensitivity to TKIs. Thus, SHP-1 is a plausible candidate for a role in the development of CEP-701 resistance in FLT3-ITD+ AML patients.
authors.affiliation: Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia. aljamalhamid08@gmail.com.
authors.forename: Hamid Ali Nagi
authors.initials: HA
authors.lastname: Al-Jamal
authors.affiliation: Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia. asma_sof88@yahoo.com.
authors.forename: Siti Asmaa
authors.initials: SA
authors.lastname: Mat Jusoh
authors.affiliation: Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia. roslin@usm.my.
authors.forename: Rosline
authors.initials: R
authors.lastname: Hassan
authors.affiliation: Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia. faridjohan@usm.my.
authors.forename: Muhammad Farid
authors.initials: MF
authors.lastname: Johan
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001374:Azacitidine / Q000494:pharmacology*; D002227:Carbazoles / Q000494:pharmacology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D019175:DNA Methylation; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005663:Furans; D020440:Gene Duplication; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism*; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000235:genetics*; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics*
chemical_list: D002227:Carbazoles; D005663:Furans; D050796:STAT3 Transcription Factor; C119379:lestaurtinib; D051941:fms-Like Tyrosine Kinase 3; C509249:PTPN6 protein, human; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D001374:Azacitidine