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A retinoid-resistant acute promyelocytic leukemia subclone expresses a dominant negative PML-RAR alpha mutation
W Shao1, L Benedetti, W W Lamph
1Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital and McGill University Department of Oncology, Montreal, Quebec, Canada.
Abstract:
The unique t(15;17) of acute promyelocytic leukemia (APL) fuses the PML gene with the retinoic acid receptor alpha (RAR alpha) gene. Although retinoic acid (RA) inhibits cell growth and induces differentiation in human APL cells, resistance to RA develops both in vitro and in patients. We have developed RA-resistant subclones of the human APL cell line, NB4, whose nuclear extracts display altered RA binding. In the RA-resistant subclone, R4, we find an absence of ligand binding of PML-RAR alpha associated with a point mutation changing a leucine to proline in the ligand-binding domain of the fusion PML-RAR alpha protein. In contrast to mutations in RAR alpha found in retinoid-resistant HL60 cells, in this NB4 subclone, the coexpressed RAR alpha remains wild-type. In vitro expression of a cloned PML-RAR alpha with the observed mutation in R4 confirms that this amino acid change causes the loss of ligand binding, but the mutant PML-RAR alpha protein retains the ability to heterodimerize with RXR alpha and thus to bind to retinoid response elements (RAREs). This leads to a dominant negative block of transcription from RAREs that is dose-dependent and not relieved by RA. An unrearranged RAR alpha engineered with this mutation also lost ligand binding and inhibited transcription in a dominant negative manner. We then found that the mutant PML-RAR alpha selectively alters regulation of gene expression in the R4 cell line. R4 cells have lost retinoid-regulation of RXR alpha and RAR beta and the RA-induced loss of PML-RAR alpha protein seen in NB4 cells, but retain retinoid-induction of CD18 and CD38. Thus, the R4 cell line provides data supporting the presence of an RAR alpha-mediated pathway that is independent from gene expression induced or repressed by PML-RAR alpha. The high level of retinoid resistance in vitro and in vivo of cells from some relapsed APL patients suggests similar molecular changes may occur clinically.
Insights
Acute promyelocytic leukemia (APL) cells can become resistant to retinoic acid (RA) due to mutations in the PML-RAR alpha fusion protein. This resistance involves a loss of RA binding and a dominant-negative transcriptional block, potentially explaining clinical retinoid resistance.
Area of Science:
- Hematology
- Molecular Biology
- Cancer Research
Background:
- Acute promyelocytic leukemia (APL) is characterized by the t(15;17) translocation, fusing PML and retinoic acid receptor alpha (RAR alpha) genes.
- Retinoic acid (RA) induces differentiation in APL cells, but resistance can develop, posing a clinical challenge.
- Understanding the molecular mechanisms of RA resistance is crucial for improving APL treatment.
Purpose of the Study:
- To investigate the molecular basis of RA resistance in human APL cell lines.
- To characterize the functional consequences of mutations in the PML-RAR alpha fusion protein.
- To identify potential pathways involved in retinoid resistance in APL.
Main Methods:
- Development of RA-resistant NB4 APL subclones (e.g., R4).
- Analysis of nuclear extracts for RA binding alterations.
- Identification of point mutations in the PML-RAR alpha fusion protein using molecular techniques.
- In vitro expression studies of wild-type and mutant PML-RAR alpha.
- Assessment of transcriptional regulation and gene expression changes.
Main Results:
- A specific point mutation (leucine to proline) in the ligand-binding domain of PML-RAR alpha was identified in RA-resistant R4 cells, causing loss of RA binding.
- The mutant PML-RAR alpha retained heterodimerization with RXR alpha and binding to RAREs, leading to a dominant-negative transcriptional block independent of RA.
- The R4 cell line exhibited altered retinoid regulation of specific genes (RXR alpha, RAR beta, CD18, CD38), suggesting distinct pathways.
- Unrearranged RAR alpha with the same mutation also lost ligand binding and exhibited dominant-negative effects.
Conclusions:
- A specific mutation in the PML-RAR alpha ligand-binding domain can confer dominant-negative transcriptional repression and RA resistance in APL.
- This mechanism, involving loss of ligand binding and dominant-negative effects, may contribute to clinical retinoid resistance observed in relapsed APL patients.
- The findings highlight the importance of the PML-RAR alpha fusion protein's ligand-binding function in mediating retinoid sensitivity and suggest potential therapeutic targets.