Published OnlineFirst September 25, 2015; DOI: 10.1158/1078-0432.CCR-15-1054 Biology of Human Tumors Clinical Cancer Research Inactivation of the p53–KLF4–CEBPA Axis in Acute Myeloid Leukemia Katja Seipel1,2, Miguel Teixera Marques1, Marie-Ange Bozzini1, Christina Meinken1, Beatrice U. Mueller1, and Thomas Pabst2 Abstract Purpose: In acute myeloid leukemia (AML), the transcription of p53 function and concomitant reduction of KLF4 and CEBPA factors CEBPA and KLF4 as well as the universal tumor suppressor protein levels. Assessment of cellular p53 modulator proteins p53 are frequently deregulated. Here, we investigated the extent of indicated that p53 inactivation in leukemic cells correlated with dysregulation, the molecular interactions, and the mechanisms elevated levels of the nuclear export protein XPO1/CRM1 and involved. increase of the p53 inhibitors MDM2 and CUL9/PARC in the Experimental Design: One hundred ten AML patient samples cytoplasm. Finally, restoring p53 function following treatment were analyzed for protein levels of CEBPA, KLF4, p53, and p53 with cytotoxic chemotherapy compounds and p53 restoring non- modulators. Regulation of CEBPA gene expression by KLF4 and genotoxic agents induced CEBPA gene expression, myeloid dif- p53 or by chemical p53 activators was characterized in AML cell ferentiation, and cell-cycle arrest in AML cells. lines. Conclusions: The p53–KLF4–CEBPA axis is deregulated in Results: We found that CEBPA gene transcription can be AML but can be functionally restored by conventional chemo- directly activated by p53 and KLF4, suggesting a p53–KLF4– therapy and novel p53 activating treatments. Clin Cancer Res; 22(3); CEBPA axis. In AML patient cells, we observed a prominent loss 746–56. Ó2015 AACR. Introduction and/or translational expression, and altered protein degradation (3). Moreover, CEBPA is a DNA damage–inducible p53-regulated CCAAT/enhancer-binding protein alpha (CEBPA) is a lineage- mediator of the G checkpoint in keratinocytes, but not in specific transcription factor expressed in hematopoietic stem and 1 fibroblasts (4). myeloid progenitor cells (1). CEBPA functions as a differentiation Similarly, Kruppel-like€ factor 4 (KLF4/GKLF) is a lineage-spe- factor by directly targeting lineage-specific genes. CEBPA activates cific transcription factor expressed in a monocyte-restricted and granulocyte- and eosinophil-specific genes while inhibiting ery- stage-specific pattern during myelopoiesis, and it functions to throid- and lymphoid-specific factors. In addition, CEBPA acts as a promote monocyte differentiation (5, 6). Alike CEBPA, KLF4 can potent cell-cycle inhibitor. Cell-cycle arrest and terminal differ- arrest cell proliferation via induction of the CDKN1A gene, and it entiation are coupled processes, and the ability of CEBPA to represents a potent p53 mediator, thus acting as a tumor sup- mediate both may provide an explanation for its ability to act pressor (7). KLF4 expression is downregulated in NPM1-mutant as a switch between uncommitted proliferating cells and differ- AML by miR10a (8), and it can be repressed by CDX2 and HDAC1 entiated cell-cycle arrested cells. The mechanisms by which CEBPA (9–11). mediates cell-cycle arrest have been extensively investigated, and The tumor suppressor p53 has been dubbed the guardian of the they involve the ability of CEBPA to upregulate the CDKN1A gene genome (12) reflecting its crucial role in dealing with all kinds of encoding the cyclin-dependent kinase inhibitor p21/WAF1/Cip1, stressful conditions that can damage DNA. Physiologically, p53 its interaction with the cyclin-dependent kinases CDK2 and induces cell-cycle arrest to enable repair of damaged components CDK4, and the CEBPA-mediated repression of the E2F complex or, in the case of excessive damage, to induce cell death by (2). CEBPA function is commonly deregulated in patients apoptosis (13). p53 exerts its cell-cycle arrest and apoptotic func- with AML due to genomic mutations, suppressed transcriptional tions in the nucleus, but it is shuttled to the cytoplasm where it can be retained, reshuttled to the nucleus, or destroyed (14). The tumor suppressor p53 is inactivated in a wide variety of cancers, 1Department of Clinical Research, University and University Hospital of either by mutation or by dysregulation, in particular via induction 2 Berne, Berne, Switzerland. Department of Medical Oncology, Univer- of the p53 inhibitor MDM2 or by disturbing the nucleocytoplas- sity and University Hospital of Berne, Berne, Switzerland. mic shuttling of p53, either by dysregulation of the transporter Note: Supplementary data for this article are available at Clinical Cancer proteins importin and exportin or induction of the cytoplasmic Research Online (http://clincancerres.aacrjournals.org/). retention protein CUL9/PARC. In AML cells, the incidence of p53 Corresponding Author: Thomas Pabst, Department of Medical Oncology, mutations is low; however, dysregulation of p53 function in AML University Hospital, Berne 3010, Switzerland. Phone: 41-31-6328430; Fax: 41- appears to be a frequent event (15). 31-6323410; E-mail: [email protected] In AML as well as in other hematologic malignancies, CEBPA, doi: 10.1158/1078-0432.CCR-15-1054 KLF4, and p53 can be deregulated. The extent of dysregulation, Ó2015 American Association for Cancer Research. the molecular interactions, and mechanisms involved in this 746 Clin Cancer Res; 22(3) February 1, 2016 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst September 25, 2015; DOI: 10.1158/1078-0432.CCR-15-1054 p53–KLF4–CEBPA Axis in AML CAC-30)andp53_mut_R (50-GTGGATAGATGCGGGAATTCA- Translational Relevance 0 GAGG-3 ) and to create pGL4.23-36-kbM1 using primer pairs The transcription factors CEBPA and KLF4 act as key reg- p53-mut F2 (50-CCTCTGCATGCCCGCATCTATCCAC-30)and ulators of normal differentiation and suppress malignant p53_mut_R2 (50- GTGGATAGATGCGGGCATGCAGAGG-30). transformation, whereas in acute myeloid leukemia (AML), Oligonucleotide primers were supplied by Microsynth. the functions of CEBPA, KLF4, and of the tumor suppressor To test the KLF4-binding sites in the 9-kb 30 HCNE, a partial p53 are frequently suppressed. Our study indicates that CEBPA construct pGL4.23-9kb3C (190 bp, 9-kb 30) was created by gene transcription can be directly activated by p53 and KLF4, internal SacI deletion in pGL4.23-9kb30 (730 bp, 9-kb 30). The suggesting a p53–KLF4–CEBPA axis. We observed in primary other 540-bp sequence was PCR-amplified using primers with AML samples a characteristic loss of p53 function and a restriction sites XhoI(50) and HindIII (30) and subcloned into concomitant reduction in KLF4 and CEBPA protein levels. pGL4.23 to create pGL4.23-9kb3N (540 bp, 9-kb 30). The 730-bp Importantly, restoring p53 function following treatment with 9-kb 30 region encoded four KLF4 sites, whereas the 190-bp 9-kb 30 cytotoxic chemotherapy together with p53 activating non- and the 540-bp 9-kb 30 constructs encoded two KLF4 sites. genotoxic agents induced CEBPA gene expression and initiated myeloid differentiation and cell-cycle arrest in AML cells. Our Luciferase reporter assays data pave the way toward therapeutic concepts combining CEBPA-firefly luciferase reporter and Renilla luciferase reference conventional chemotherapy and novel p53 activating treat- plasmids together with pcDNA3.3_KLF4 (16) and pcDNA_TP53 ment based on the observation that conventional chemother- expression plasmids were transfected into H1299 cells using apy together with novel p53 activating treatment can restore Lipofectamine2000 (Invitrogen). After 24 hours, cells were lysed CEBPA function. and luciferase activity was assessed using the Dual-Luciferase- Reporter Assay System (Promega) on a microplate reader Infinite 200 (Tecan). Assays were performed in at least three independent experiments. Statistical analysis was done with GraphPad Prism dysregulation are unclear. We investigated whether KLF4 and p53 software using two-tailed t tests. Data are depicted in column bar can activate CEBPA gene expression, and we assessed the extent of graphs plotting mean with SD values. The TP53 expression dysregulation in the protein levels of the three transcription plasmid was provided by Carol Prives and the KLF4 expression factors and of cellular p53 modulators in primary AML patient plasmid by Derrick Rossi (Addgene #26815). samples. CEBPA-luc-BAC stable cell lines Materials and Methods The CEBPA-BAC transgene P391 was created by insertion of a CEBPA reporter plasmids luciferase-puromycin cassette into RP11-270I13 (Gene Bridges). Highly conserved noncoding elements of the CEBPA gene P391 BAC DNA was prepared from the E. coli HS996 bacterial (HCNE) were defined using the genome browser genome.ucsc.edu. culture grown in LB medium with Cm (15 mg/mL), linearized by HCNEs were PCR-amplified from human gDNA using primers SgfI digestion, and subsequently used to generate stable cell lines. with restriction sites XhoI(50) and HindIII (30) for all HCNEs, with Leukemic HL60 cells were transfected with linearized P391 BAC 6 the exception of SacI(50) and BamHI (30) for the 33-kb 30 and 3- DNA (4 mg/10 cells) using the Nucleofector Kit V Program X-001 1kb 30 HCNEs, and BamHI (50 and 30) for the 6-kb 30 HCNE, and (Lonza). Cells were cultured for 24 hours before addition of they were cloned into pCR4-Topo (Invitrogen) before being puromycin (0.5 mg/mL). Colonies emerged after 10 to 14 days. transferred to the pGL4.23 firefly luciferase