Coordinated Repression of Cyclin-Dependent Kinase Inhibitors By PML/RARa in Acute Promyelocytic Leukemia

Inappropriate cell proliferation during oncogenesis is often attributed to dysregulation of components of the cell cycle machinery, which is strictly controlled by the activity of cyclins, cyclin-dependent kinases (CDKs) and cyclin-dependent kinases inhibitors (CKI). In our previous study, we have reported that the oncogenic fusion protein PML/RARa directly targets and represses the expression of CDKN2D, one of the CKI member, in acute promyelocytic leukemia (APL). Since different members of CKIs can mediate separate pathways to negatively regulate CDKs so as to control the cell cycle progression, thus implicating that multiple components of CKIs might be affected during the malignant transformation. However, the transcription regulation of the overall machinery of CKIs in APL remains unclear. Here, we report that PML/RARa coordinately represses multiple members of CKIs by directly binding to their regulatory regions. To identify potential CKIs that were dys-regulated in APL cells, we first studied the chromatin status of all the CKIs in APL by chromatin immunoprecipitation combined with next generation sequencing (ChIP-seq) using antibodies against histone modification markers (H3K4me3, H3K4me1 and H3K27me3). We found that the regulatory regions of CDKN2C, CDKN2D and CDKN1A were accessible in APL, suggesting that these CKIs were dynamically regulated in APL. We then performed ChIP-seq using antibody against PML/RARa and found that these three CKIs were occupied with PML/RARa binding, indicating that PML/RARa coordinately regulated CKIs in APL. To further study the role of PML/RARa in transcription regulation of CKIs, we used luciferase reporter assays to detect the effect of PML/RARα on CKIs transcription regulation and found that PML/RARα directly inhibited the promoter activity of CDKN2C and CDKN2D. Furthermore, we demonstrated that ATRA treatment increased the expression of CDKN2C, CDKN2D and CDKN1A through releasing the PML/RARα binding on chromatin in NB4 cells. Ectopic expression of these three CKIs could induce both cell cycle arrest and differentiation. Finally, the finding on the transcriptional regulation of CDKN1A, CDKN2C and CDKN2D in NB4 cells was validated in primary APL patient samples. Collectively, this study emphasizes that PML/RARa represses the CKI machinery and leads to cell cycle progression and differentiation block of APL. The coordinated regulation of multi-factors involved in the same pathway may shed new lights on the study of pathogenesis and target therapy of APL.