IRF1 Is Crucial for ATRA-Induced Differentiation By Regulating Its Multiple Functional Targets

Correspondence: Kankan Wang, State Key Laboratory of Medical Genomics and Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine; E-mail: kankanwang@shsmu.edu.cn.

Abstract

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML), characterized by the accumulation of the blasts arrest at the promyelocyte stage and cytogenetically defined by the PML-RARa oncofusion gene generated by the t(15;17) translocation. All-trans retinoic acid (ATRA), as a front-line agent in the treatment of APL, can reactivate PML-RARα targets by transcriptional switch and degradation of PML-RARα fusion protein, finally induce promyelocytic blasts to terminal differentiation and elicit complete remission of APL. However, a significant amount of evidence has demonstrated that the effect of ATRA treatment is not simply a direct consequence of the reversion of pathological processes caused by PML-RARα. Furthermore, the removal of PML-RARa only has been illustrated to stimulate cell apoptosis and demonstrated to be insufficient for ATRA-mediated differentiation. There must be other important signaling pathways synergizing with ATRA to induce differentiation. And several lines of studies have indicated early ATRA-responsive genes are more important in the ATRA-induced transcriptional regulatory cascades by mediating crosstalk among various ATRA downstream pathways. Our previous studies, together with others, indicate that interferon regulator factor 1 (IRF1) is upregulated rapidly at a very early stage after ATRA treatment. It’s not only a direct target of ATRA, but also a central transducer of IFN signaling. IFN and ATRA can potentiate each other to induce gene expression and various biological responses. Gene set enrichment analysis (GSEA) showed that IRF1 was the major transcriptional factor participating in the regulation of ATRA-upregulated genes, especially at the early stage after ATRA treatment. Thus, IRF1 may play an important role in ATRA-induced transcriptional regulatory cascades by regulating its targets. To understand molecular mechanisms of IRF1 after ATRA induction, we performed ChIP-seq to identify the genome-wide binding sites of IRF1 at 4 hours after ATRA treatment. ChIP-QPCR and luciferase assays were conducted to validate the ChIP-seq enriched target genes of IRF1 and further demonstrated that IRF1 can directly transactivated its targets in 293T cells. Furthermore, the binding sites of IRF1 mainly located near the transcription start sites (TSS), especially at the proximal promoter region, and conserved. Motif analysis showed that there were two binding motifs, classical IRF1 motif (short motif) and long motif. And these differential motifs appeared at different stages during ATRA-induced differentiation and involved in different sets of biological processes. After gene ontology analysis, IRF1 targets were identified to be involved in a variety of important biological processes, such as hematopoiesis, cell cycle, apoptosis, JAK-STAT cascade, immune response, etc. Furthermore, knockdown of IRF1 with siRNA led to a significantly repression of ATRA-induced differentiation, degradation of PML-RARα, cell cycle arrest and proliferation inhibition in APL cells. These results collectively demonstrate that IRF1 plays a crucial role in ATRA-induced differentiation and mediates multiple signaling pathways by regulating its various functional targets. Thus, our results will provide a better understanding of treatment mechanisms in APL and extend the application of ATRA to the treatment of other cancers.