Abstract
BackgroundGTF2I-RARA is a newly identified RARA fusion gene in variant acute promyelocytic leukemia (APL) patients with t(7;17)(q11;q21). Clinical manifestation in the patient showed that it is a sort of ATRA-insensitive oncogene and is different from the classic PML-RARA in terms of therapeutic reaction.MethodsTo reveal the functional characteristics and regulating mechanism of the GTF2I-RARA fusion gene, we established a GTF2I-RARA-transfected HL60 cell model and examined its sensitivity to ATRA by western blot, MTT assay, flow cytometry, and Wright-Giemsa staining. Coimmunoprecipitation and confocal microscopy were used to examine the binding of GTF2I-RARA and transcriptional corepressors. We also performed ChIP-seq to search for potential target genes. Immunoprecipitation, ubiquitination assay, western blot, luciferase assay, and real-time PCR were used to analyze the effects of RNF8 on RARA. Flow cytometry and Wright-Giemsa staining were used to study the effect of MG132 and ATRA on the GTF2I-RARA-transfected HL60 cell model.ResultWe confirmed resistance of GTF2I-RARA to ATRA. Compared with PML-RARA, GTF2I-RARA has a higher affinity to HDAC3 under ATRA treatment. Using the ChIP-sequencing approach, we identified 221 GTF2I-RARA binding sites in model cells and found that the RING finger protein 8 (RNF8) is a target gene of GTF2I-RARA. RNF8 participates in disease progression and therapy resistance in APL with the GTF2I-RARA transcript. Elevated RNF8 expression promotes the interaction between RARA and RNF8 and induces RARA Lys-48 linkage ubiquitylation and degradation, resulting in attenuated transcriptional activation of RARA.ConclusionOur results suggest that RNF8 is a key GTF2I-RARA downstream event. Using the combination of MG132 and ATRA to treat GTF2I-RARA-HL60 cells, a synergistic effect leading to GTF2I-RARA-HL60 cell differentiation was confirmed. Taken together, the targeting of RNF8 may be an alternative choice for treatment in variant APL with GTF2I-RARA fusion.
Highlights
GTF2I-RARAis a newly identified RARAfusion gene in variant acute promyelocytic leukemia (APL) patients with t(7;17)(q11;q21)
Using the combination of MG132 and all-trans retinoic acid (ATRA) to treat GTF2I-RARA-HL60 cells, a synergistic effect leading to GTF2I-RARA-HL60 cell differentiation was confirmed
When ATRA is exposed to the GTF2I-RARApositive HL60 and NB4 for 48 h, the PML-RARAprotein expression in NB4 cells decreases in a dose-dependent manner, while there is no change regarding the effect in GTF2I-RARA-positive HL60 cells (Fig. 1b)
Summary
GTF2I-RARAis a newly identified RARAfusion gene in variant acute promyelocytic leukemia (APL) patients with t(7;17)(q11;q21). Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia that is mostly driven by the chimeric oncoprotein PML-RARA[1]. Accruing evidence indicates that PML-RARA-driven APL is sensitive to both all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) therapy [2, 3]. PMLRARAexhibits a high affinity for the corepressor proteins N-CoR and SMRT, and only the introduction of pharmacological doses of ATRA (1–2 μM) induces corepressor release and coactivator recruitment, as well as the degradation of PML-RARA[4, 5]. The extent to which the RARAfusion protein acquires altered DNA-binding capacities that may result in the aberrant expression of genes normally regulated by wild-type RARAis still not well understood. Subsequent genome-wide studies conducted by Martens et al and Wang et al identified nearly 3000 binding sites of PML-RARA, suggesting that PML-RARA-associated epigenetic alterations and regulatory mechanisms are very sophisticated [9, 10]
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