Role of cytoplasmic forms of PML and PML-RAR alpha in the response to therapy

The PML tumour suppressor of acute promyelocytic leukaemia (APL) regulates major apoptotic and growth suppressive pathways. In APL, one PML allele is involved in a chromosomal translocation generating the PML-RARα fusion protein. Two missense mutations in the remaining PML allele have been identified, which give rise to a truncated cytoplasmic PML protein (Mut PML). APL patients carrying these mutations display resistance to retinoic acid (RA) and very poor prognosis.The aim of this study was to analyze the subcellular distribution of Mut PML and determine its role in the regulation therapy-induced differentiation. Finally, we set to determine the relationship between Mut PML and PML-RARα.Both non-haematopoietic and haematopoietic cell lines were used in this study. Retroviral transduction was achieved using retroviral vectors encoding Mut PML, wild type and mutant PML-RARα. Both bcr1 and bcr3 PML-RARα variants were used in this study. PML-RARα nuclear localization signals were mutagenized in order to induce its exclusive cytoplasmic localization. Differentiation was determined both by surface markers expression and morphological analysis. Transcriptional assays were performed using multimerized retinoic-acid (RA) responsive elements cloned upstream the luciferase cDNA, and transcriptional activities were measured in transient transfection experiments.We found that Mut PML associates with cytoplasmic regions we refer to as PML-cytoplasmic bodies (PML-CBs). Mut PML interacted with PML-RARα in PML-CB and potentiated PML-RARα-mediated inhibition of RA-dependent transcription. Remarkably, Mut PML stabilized PML-RARα and inhibited differentiation induced by pharmacological doses of RA. A mutant form of PML-RARα that accumulates in the cytoplasm inhibits RA-dependent transcription and differentiation, thus suggesting that cytoplasmic localization of PML-RARα may contribute to transformation. Finally, we show that the bcr3 PML-RARα form is predominantly cytoplasmic and accumulates in PML-CBs.Taken together, these findings reveal novel insights into the molecular mechanisms contributing to APL and the response to therapy.