RNA-MEDIATED CORRECTION OF ABERRANT DNA METHYLATION AT THE P15 LOCUS
(Abstract release date: 05/18/17)
EHA Library. Li M. 05/18/17; 182378; PB1664
Mailin Li
Contributions
Contributions
Abstract
Abstract: PB1664
Type: Publication Only
Background
P15 (a.k.a cell cycle dependent kinase inhibitor 2B; CDKN2B; INK4B) is a methylation sensitive gene located on chromosome 9p21 and commonly found silenced during Myelodysplastic Syndrome (MDS) progression to Acute Myeloid Leukemia (AML). P15 encodes for a cyclin-dependent kinase inhibitor increasingly expressed during granulomonocytic maturation (Teofili et al., Exp Hematol 2000). P15 deletion or promoter methylation has been shown to independently correlate with disease progression and poor patient prognosis (Tien et al., Br J Hematol 2001). Additionally, P15 expression was also sensitive to regulation by myeloid-specific transcription factor PU.1 (Schmidt Blood 2004). As MDS evolution to AML includes both myeloid proliferation and blocked differentiation stages, restoration of the natural P15 transcript will provide not only valuable information regarding disease progression but may also alleviate some of their characteristic symptoms.
Aims
Currently available demethylating agents approved for therapeutic applications, e.g. 5-azacytidine and decitabine, have major side effects of high toxicity and non-specific DNA methylation that limit their clinical application. Therefore, the aim of this study is to achieve RNA-mediated correction of the aberrantly methylated P15 locus using small activating RNAs (saRNAs; Li et al PNAS 2006).
Methods
Myeloid Leukemia cell lines HL-60, KG1a, and K562 were screened for basal p15 expression by western blotting and qRT-PCR. As the P15 locus is also often deleted, deletion of the locus was assayed for by PCR and by Fluorescent In Situ Hybridization. Because the methylation status of P15 was shown to be inversely correlated with ANRIL (Antisense Non-coding RNA in the INK4 Locus) expression (Kotake et al Oncogene 2010), p15 and ANRIL gene expression were measured in parallel. HEK293 cells serve as positive control in all studies.
SaRNAs were designed against the proximal promoter, first exon, and intron regions of the P15 gene body. SaRNAs were introduced to cell lines through electroporation, and re-activation of the locus was measured at the transcript level by qRT-PCR and protein level by western blotting. Changes in P15 promoter level methylation were determined by Methylation Specific PCR.
Results
Transfection of saRNAs into the HL60 cell line showed upregulated p15 expression 24 and 48 hrs post-transfection. Analysis of ANRIL after saRNA-transfection showed no concomitant changes, suggesting locus-specific activity of the saRNAs. Future experiments will elucidate the mechanisms of saRNA activation of P15 gene expression and genome-scale specificity of saRNA-mediated methylation changes.
Conclusion
There is much interest in using RNA molecules as a therapeutic tool (Kole et al., Nat Rev Drug Discovery 2012; Reebye et al., Hepatology 2014). Introduction of such an approach offers greater advantages over existing hypomethylating-based protocols: a) high gene specificity b) lower cytotoxicity and c) absence of drug based off-target side-effects. In the short term, this research can lead to the identification of novel key regulators of leukemogenesis and new targets for therapeutic treatments; in the long term pave the way for development of RNA-based gene demethylating agents for cancer treatment.
Session topic: 3. Acute myeloid leukemia - Biology
Keyword(s): MDS/AML, Decitabine, Cell cycle, Methylation
Abstract: PB1664
Type: Publication Only
Background
P15 (a.k.a cell cycle dependent kinase inhibitor 2B; CDKN2B; INK4B) is a methylation sensitive gene located on chromosome 9p21 and commonly found silenced during Myelodysplastic Syndrome (MDS) progression to Acute Myeloid Leukemia (AML). P15 encodes for a cyclin-dependent kinase inhibitor increasingly expressed during granulomonocytic maturation (Teofili et al., Exp Hematol 2000). P15 deletion or promoter methylation has been shown to independently correlate with disease progression and poor patient prognosis (Tien et al., Br J Hematol 2001). Additionally, P15 expression was also sensitive to regulation by myeloid-specific transcription factor PU.1 (Schmidt Blood 2004). As MDS evolution to AML includes both myeloid proliferation and blocked differentiation stages, restoration of the natural P15 transcript will provide not only valuable information regarding disease progression but may also alleviate some of their characteristic symptoms.
Aims
Currently available demethylating agents approved for therapeutic applications, e.g. 5-azacytidine and decitabine, have major side effects of high toxicity and non-specific DNA methylation that limit their clinical application. Therefore, the aim of this study is to achieve RNA-mediated correction of the aberrantly methylated P15 locus using small activating RNAs (saRNAs; Li et al PNAS 2006).
Methods
Myeloid Leukemia cell lines HL-60, KG1a, and K562 were screened for basal p15 expression by western blotting and qRT-PCR. As the P15 locus is also often deleted, deletion of the locus was assayed for by PCR and by Fluorescent In Situ Hybridization. Because the methylation status of P15 was shown to be inversely correlated with ANRIL (Antisense Non-coding RNA in the INK4 Locus) expression (Kotake et al Oncogene 2010), p15 and ANRIL gene expression were measured in parallel. HEK293 cells serve as positive control in all studies.
SaRNAs were designed against the proximal promoter, first exon, and intron regions of the P15 gene body. SaRNAs were introduced to cell lines through electroporation, and re-activation of the locus was measured at the transcript level by qRT-PCR and protein level by western blotting. Changes in P15 promoter level methylation were determined by Methylation Specific PCR.
Results
Transfection of saRNAs into the HL60 cell line showed upregulated p15 expression 24 and 48 hrs post-transfection. Analysis of ANRIL after saRNA-transfection showed no concomitant changes, suggesting locus-specific activity of the saRNAs. Future experiments will elucidate the mechanisms of saRNA activation of P15 gene expression and genome-scale specificity of saRNA-mediated methylation changes.
Conclusion
There is much interest in using RNA molecules as a therapeutic tool (Kole et al., Nat Rev Drug Discovery 2012; Reebye et al., Hepatology 2014). Introduction of such an approach offers greater advantages over existing hypomethylating-based protocols: a) high gene specificity b) lower cytotoxicity and c) absence of drug based off-target side-effects. In the short term, this research can lead to the identification of novel key regulators of leukemogenesis and new targets for therapeutic treatments; in the long term pave the way for development of RNA-based gene demethylating agents for cancer treatment.
Session topic: 3. Acute myeloid leukemia - Biology
Keyword(s): MDS/AML, Decitabine, Cell cycle, Methylation
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