Human acute myeloid cell lines can be induced to differentiate into macrophage-like cells by phorbol esters. Differentiation is accompanied by cessation of cell proliferation and initiation of apoptosis. Many of these cell lines, like HL-60, are p53 deficient and therefore execute a differentiation-induced, p53-independent program of cell death. Evaluating changes in gene expression during a time course of PMA-induced HL-60 cell differentiation by DNA microarray analyses and RT-qPCR reveals up-regulation of several pro-apoptotic genes (ex. TNF, MCL1, EGR2, BCL3) and down-regulation of several anti-apoptotic genes (ex. BCL2, ANXA3, TSC22D3). Interestingly, RNA levels of p73 are unchanged, suggesting p73 does not substitute for the missing p53 function via activation of p73 gene transcription, however, we report here that p73 protein levels increase dramatically during the differentiation time course. By reintroducing p53 into HL-60 cells, we compare the changing gene expression profile during p53-mediated apoptosis versus that which occurs during phorbol ester-induced p73 accumulation through protein stabilization. Common and unique gene expression changes define the putative p73-dependent apoptosis program compared to p53-dependent apoptosis. Citation Format: Michael Roberts, Rizwan Saffie, Harold Salmons, Mansoor Ghoto, Juliana Schneider, Jeffrey Forrester. Differentiation induced apoptosis in AML cells: The role of p73 in p53-independent versus p53-mediated apoptosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 346. doi:10.1158/1538-7445.AM2014-346

The human acute myeloid leukemia cell line, HL-60, in response to phorbol esters, undergoes genetic re-programming leading to cell cycle arrest, macrophage differentiation, and apoptosis. The induced program overrides the oncogenic mutations driving the leukemia program, illustrating that changes in gene expression can reverse the cancer phenotype. Whole genome exon DNA microarray analyses identifies 1260 genes whose RNA expression levels change significantly during the reprogramming. We define a subset of 186 genes functionally assigned to the biological processes of regulation of cell proliferation, differentiation, and/or programmed cell death showing specific temporal expression patterns, and suggest that a subset of these genes, which are down-regulated during the reversal of the leukemia phenotype while showing overexpression in AML patient samples, may represent new therapeutic targets in AML. Among the genes showing immediate early up-regulation (2-fold or greater by one hour post-PMA treatment), promoter analysis reveals highly significant overrepresentation of binding sites for the transcription factors STAT5A, HIF1A and NF-kB. Delayed early up-regulated genes (2-fold or greater by three hours post-PMA treatment), have promoters highly enriched in binding sites for EGR1, SP1:SP3, ZFP206 and E2F4. A phorbol ester response “signature” related to reversal of the leukemic phenotype and initiation of apoptosis has been defined and examined in two additional AML cell lines (THP-1 and Kasumi-1). Among the significantly up-regulated genes are MCL1, CDKN1A, NR4A1, and BCL6. Down-regulated genes include MYC, KIT, LGALS12, and ZBTB16. HL-60 cell lines with CRISPR-mediated knockouts of several of these genes are currently being evaluated for their effects on the leukemic phenotype and phorbol ester-induced apoptosis. Citation Format: Michael Roberts, Cassandra Holbert, Mansoor Ghoto, Juliana Schneider, Abigail Marriott, Jeffrey Forrester. Genetic re-programming of the acute myeloid leukemia cell line HL-60: Regulation of cell proliferation, differentiation, and programmed cell death. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 22. doi:10.1158/1538-7445.AM2015-22