A novel PAD4/SOX4/PU.1 signaling pathway is involved in the committed differentiation of acute promyelocytic leukemia cells into granulocytic cells

// Guanhua Song 1 , Lulu Shi 1 , Yuqi Guo 1 , Linchang Yu 1 , Lin Wang 2 , Xiaoyu Zhang 1 , Lianlian Li 1 , Yang Han 1 , Xia Ren 1 , Qiang Guo 1 , Kehong Bi 3 , Guosheng Jiang 1 1 Department of Hemato-Oncology, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Key Medical Laboratory for Tumor Immunology and Traditional Chinese Medicine Immunology of Shandong, Jinan, Shandong, China 2 Research Center for Medicinal Biotechnology, Shandong Academy of Medicinal Sciences, Jinan, Shandong, China 3 Qianfoshan Hospital of Shandong, Jinan, Shandong, China Correspondence to: Kehong Bi, e-mail: kehongbi@126.com Guosheng Jiang, e-mail: jiangguosh@163.com Keywords: PADI4, methylation, differentiation, leukemia Received: April 24, 2015      Accepted: November 20, 2015      Published: December 10, 2015 ABSTRACT All-trans retinoic acid (ATRA) treatment yields cure rates > 80% through proteasomal degradation of the PML-RARα fusion protein that typically promotes acute promyelocytic leukemia (APL). However, recent evidence indicates that ATRA can also promote differentiation of leukemia cells that are PML-RARα negative, such as HL-60 cells. Here, gene expression profiling of HL-60 cells was used to investigate the alternative mechanism of impaired differentiation in APL. The expression of peptidylarginine deiminase 4 (PADI4), encoding PAD4, a protein that post-translationally converts arginine into citrulline, was restored during ATRA-induced differentiation. We further identified that hypermethylation in the PADI4 promoter was associated with its transcriptional repression in HL-60 and NB4 (PML-RARα positive) cells. Functionally, PAD4 translocated into the nucleus upon ATRA exposure and promoted ATRA-mediated differentiation. Mechanistic studies using RNAi knockdown or electroporation-mediated delivery of PADI4, along with chromatin immunoprecipitation, helped identify PU.1 as an indirect target and SOX4 as a direct target of PAD4 regulation. Indeed, PAD4 regulates SOX4-mediated PU.1 expression, and thereby the differentiation process, in a SOX4-dependent manner. Taken together, our results highlight an association between PAD4 and DNA hypermethylation in APL and demonstrate that targeting PAD4 or regulating its downstream effectors may be a promising strategy to control differentiation in the clinic.