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2',5'-Oligoadenylate-antisense chimeras cause RNase L to selectively degrade bcr/abl mRNA in chronic myelogenous
A Maran1, C F Waller, J M Paranjape
1Department of Cancer Biology, The Lerner Research Institute, and Department of Hematology and Oncology, Cleveland Clinic Foundation, Cleveland, OH,USA.
Abstract:
We report an RNA targeting strategy, which selectively degrades bcr/abl mRNA in chronic myelogenous leukemia (CML) cells. A 2', 5'-tetraadenylate activator (2-5A) of RNase L was chemically linked to oligonucleotide antisense directed against either the fusion site or against the translation start sequence in bcr/abl mRNA. Selective degradation of the targeted RNA sequences was demonstrated in assays with purified RNase L and decreases of p210(bcr/abl) kinase activity levels were obtained in the CML cell line, K562. Furthermore, the 2-5A-antisense chimeras suppressed growth of K562, while having substantially reduced effects on the promyelocytic leukemia cell line, HL60. Findings were extended to primary CML cells isolated from bone marrow of patients. The 2-5A-antisense treatments both suppressed proliferation of the leukemia cells and selectively depleted levels of bcr/abl mRNA without affecting levels of beta-actin mRNA, determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The specificity of this approach was further shown with control oligonucleotides, such as chimeras containing an inactive dimeric form of 2-5A, antisense lacking 2-5A, or chimeras with altered sequences including several mismatched nucleotides. The control oligonucleotides had either reduced or no effect on CML cell growth and bcr/abl mRNA levels. These findings show that CML cell growth can be selectively suppressed by targeting bcr/abl mRNA with 2-5A-antisense for decay by RNase L and suggest that these compounds should be further explored for their potential as ex vivo purging agents of autologous hematopoietic stem cell transplants from CML patients.
Insights
This study introduces a novel RNA targeting method that selectively degrades bcr/abl mRNA in chronic myelogenous leukemia (CML) cells. This approach shows promise for CML treatment and stem cell purging.
Area of Science:
- Molecular Biology
- Oncology
- RNA Therapeutics
Background:
- Chronic myelogenous leukemia (CML) is characterized by the BCR-ABL oncoprotein.
- Targeting BCR-ABL mRNA offers a potential therapeutic strategy.
- Existing treatments may have limitations, necessitating novel approaches.
Purpose of the Study:
- To develop and evaluate an RNA targeting strategy for selective BCR-ABL mRNA degradation.
- To assess the efficacy of 2', 5'-tetraadenylate (2-5A)-antisense chimeras in CML cells.
- To investigate the potential of this approach for ex vivo purging of CML stem cells.
Main Methods:
- Chemically linking a 2', 5'-tetraadenylate (2-5A) activator to antisense oligonucleotides targeting BCR-ABL mRNA.
- Assessing selective RNA degradation using purified RNase L and CML cell lines (K562).
- Evaluating the impact on cell proliferation and mRNA levels (BCR-ABL, beta-actin) in patient-derived CML cells using RT-PCR.
Main Results:
- 2-5A-antisense chimeras selectively degraded BCR-ABL mRNA and reduced p210(BCR-ABL) kinase activity in K562 cells.
- Significant suppression of K562 cell growth was observed, with minimal effect on HL60 cells.
- Treatment of primary CML cells reduced proliferation and selectively depleted BCR-ABL mRNA without affecting beta-actin mRNA levels.
- Control oligonucleotides showed reduced or no effect, confirming the specificity of the approach.
Conclusions:
- The 2-5A-antisense strategy effectively targets and degrades BCR-ABL mRNA in CML cells.
- This method selectively suppresses CML cell growth and proliferation.
- The findings support further exploration of these compounds for ex vivo purging of CML stem cells for transplantation.