RNF8 is responsible for ATRA resistance in variant acute promyelocytic leukemia with GTF2I/RARA fusion, and inhibition of the ubiquitin-proteasome pathway contributes to the reversion of ATRA resistance

Abstract

Background: GTF2I-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. Methods: To 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. Result: We 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. Conclusion: Our 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.