Abstract
Introduction
The majority of acute promyelocytic leukemia (APL) cases are characterized by PML-RARα fusion gene. Although PML-RARα fusion gene can be detected in more than 98% of APL cases, RARα is also found to be fused with other partner genes, which are also related to ATRA-dependent transcriptional activity and cell differentiation. In this study, we identified a novel RARα fusion gene, TBLR1-RARα, in a rare case of APL with a t(3;17)(q26;q21),t(7;17)(q12;q21) complex chromosomal rearrangement. The structure, pathogenesis and response to drug therapy of the novel fusion gene were investigated to illustrate the characteristics, pathogenesis and the therapeutic effect in this variant APL.
Methods
To identify and amplify the novel chimeric fusion transcript, 5’ RACE and RT-PCR was performed. The TBLR1-RARα expression vector was constructed and transfected into 293T cell line by Lipofectamine2000 reagent. When the transfected 293T cell line was treated with or without ATRA, the expression level and the subcellular localization of TBLR1-RARα were investigated by Western blot and immunofluorescence analysis, and then coimmunoprecipitation and immunofluorescence analysis were performed to investigate the formation of homodimer and the recruitment of the corepressors by TBLR1-RARα. Dual-luciferase assay was used to clarify the transcriptional activity of TBLR1-RARα. Then, a lentiviral vector of TBLR1-RARα was constructed and infected the HL-60 cell line. The HL-60 cells which highly expressed TBLR1-RARα were sorted by flow cytometry. Colony formation assay and flow cytometry analysis were performed to detect the differentiation status in the TBLR1-RARα highly expressed HL-60 cells.
Results
In our study, the novel TBLR1-RARα fusion gene was cloned from an APL patient who demonstrated the typical clinical features of APL, such as bleeding tendency, leukocytosis, hypergranular promyelocytes accumulated in the bone marrow, coagulopathy. However, RT-PCR analysis and FISH studies failed to detect PML-RARα fusion gene in this case, while karyotype analysis revealed a rare complex translocation, t(3;17)(q26;q21),t(7;17)(q12;q21). When treated with ATRA, As2O3 and chemotherapeutic drugs, this patient achieved complete remission. After three courses of consolidation therapies, the patient relapsed with leukocytosis and the bone marrow karyotype analysis displayed a recurring chromosomal rearrangement, 46,xy,t(3;17)(q26;q21),t(7;17)(q12;q21),5q+,6q-,10q+,11p-, which was more complex in comparison with the karyotype at diagnosis.
There have been nine RARα fusion genes reported so far. Like other RARα fusion genes, TBLR1-RARα contains the exon 3 and the 3’ sequence of RARα. TBLR1-RARα oncoprotein contains the LisH domain from TBLR1 and the B-F domains from RARα. TBLR1-RARα diffusely locates in nucleus and cytoplasm. Like other RARα fusion protein, TBLR1-RARα can form homodimer and recruit corepressors to inhibit the transcription of the RARα target gene. TBLR1-RARα inhibits the RARα transcriptional activation in a dominant-negative manner and the transcriptional inhibition can be rescued by overexpression of wild-type RARα. In the presence of pharmacological doses of ATRA, TBLR1-RARα could be degraded and its homodimerization was abrogated. Moreover, when treated with ATRA, TBLR1-RARα could mediate the dissociation and degradation of transcriptional corepressors and consequently transactivated transcription of RARα target genes and induced cell differentiation in a dose- and time- dependent manner. Finally, TBLR1-RARα was also detected in another two cases of APL with t(3;17) chromosomal translocation.
Conclusions
In this study, we discovered a novel RARα fusion gene TBLR1-RARα from a APL patient with t(3;17) chromosomal translocation, and investigated its function, pathogenesis and response to drug treatment. Unlike wild-type RARα, TBLR1-RARα can form homodimer, recruit more corepressors to inhibit the transcription of RARα target gene and play a role in APL pathogenesis. The transcriptional inhibition of TBLR1-RARα can be rescued by overexpression of RARα and ATRA treatment and finally leads to cell differentiation.
Disclosures:
Wang: Bristol Myers Squibb: Consultancy; Novartis: Consultancy.
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