Abstract 2199: Death Associated Protein Kinase 2 (DAPK2) is a novel transcriptional target of PML-RARA and PU.1 (SPI1) during neutrophil differentiation of acute promyelocytic leukemia (APL) cells

Abstract

Abstract APL is characterized by the fusion protein promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) which blocks myeloid differentiation. Pharmacological doses of all-trans retinoic acid (ATRA) relieve transcriptional repression inducing terminal differentiation of the leukemic blasts. DAPK2 mRNA levels are much lower in acute myeloid leukemia cells than in normal granulocytes, particularly so in APL cells, and are up-regulated during ATRA-induced neutrophil differentiation of APL cells. To extend our data from primary APL patients, we differentiated two APL cell lines, namely NB4 and HT93 as well as an ATRA-resistant NB4 subline, NB4-R2, towards neutrophils using ATRA. DAPK2 mRNA levels were increased 210- and 280-fold upon ATRA-treatment in NB4 and HT93 cells, respectively, and were paralleled by marked induction of protein expression. No significant increase was measured in NB4-R2 suggesting that upregulation of DAPK2 is clearly associated with neutrophil differentiation. Next, MatInspector software analysis allowed the identification of two putative retinoic acid responsive elements (RAREs) in the DAPK2 promoter region. To verify PML-RARA binding to these sites we performed PML and RARA chromatin immunoprecipitations (ChIP) in NB4 cells. We showed in vivo binding of PML-RARA to the RARE at position −739 of the DAPK2 promoter. A repressive function of PML-RARA on DAPK2 expression was further shown by overexpression of PML-RARA in U937 AML cells. Exogenous expression of PML-RARA reduced DAPK2 expression levels by 20%. RAREs often coexist with PU.1 (SPI1) binding motifs. Indeed, we also identified three putative PU.1 binding sites in the DAPK2 promoter. The PU.1 transcription factor is a master regulator of myeloid differentiation and PU.1-mediated transactivation of myeloid genes is repressed by PML-RARA. We confirmed in vivo binding of PU.1 to two binding sites in the DAPK2 promoter using ChIP analysis. In addition, a DAPK2 promoter reporter plasmid was significantly (p<0.05) induced 1.6-fold in the presence of PU.1. Moreover, knocking down PU.1 in NB4 cells using two different shRNA constructs resulted in a 4-fold reduction of DAPK2 mRNA (p<0.05) and protein expression during ATRA-induced neutrophil differentiation as compared to control transduced cells. To show that DAPK2 is a relevant PU.1 target during myeloid differentiation, we used lentiviral vectors to re-express DAPK2 in NB4 PU.1 knockdown cells (DAPK2 rescue). CD11b and CEBPE, two neutrophil differentiation markers, were significantly increased in DAPK2 restored PU.1 knockdown NB4 cells upon neutrophil differentiation. In summary, we found that transcription of DAPK2 in APL cells is repressed by PML-RARA. We further identified DAPK2 as novel PU.1 transcriptional target during neutrophil differentiation of APL cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2199. doi:1538-7445.AM2012-2199