Overexpression of MN1 Confers Resistance to Chemotherapy, Accelerates Leukemia Onset, and Suppresses p53 and Bim Induction

and Suppresses p53 and Bim Induction. PLoS ONE 7(8): e43185. doi:10.1371/journal.pone.0043185 Overexpression of MN1 Confers Resistance to Chemotherapy, Accelerates Leukemia Onset, and Suppresses p53 and Bim Induction Timothy S. Pardee 0 0 1 Wake Forest University Health Sciences, Department of Internal Medicine, Section on Hematology and Oncology, Winston-Salem, North Carolina, United States of America, 2 Wake Forest School of Medicine, Department of Cancer Biology, Comprehensive Cancer Center , Winston-Salem, North Carolina , United States of America Background: The transcriptional co-activator MN1 confers a worse prognosis for patients with acute myeloid leukemia (AML) when highly expressed; however, the mechanisms involved are unknown. We sought to model the effects of high MN1 expression in AML models to explore the underlying mechanisms. Methodology/Principal Findings: We used cell lines and a genetically defined mouse model of AML to examine the effects of MN1 overexpression on prognosis and response to cytarabine and doxorubicin in vitro and in vivo. Murine AML that was engineered to overexpress MN1 became more aggressive in vivo, leading to shortened survival in both treated and control groups. In vitro murine AML cells that overexpressed MN1 became resistant to treatment with cytarabine and highly resistant to doxorubicin. This resistant phenotype was also seen in vivo, where treatment with the combination of cytarabine and doxorubicin selected for cells expressing MN1. When therapy-induced DNA damage levels were assessed by cH2AX foci, no reduction was seen in MN1 expressing cells arguing against a drug efflux mechanism. Despite no reduction in DNA damage, MN1-expressing cells showed less apoptosis as assessed by annexin V and propidium iodide staining. Following treatment, p53 and BIM induction were markedly reduced in cells expressing MN1. Pharmacologic inhibition of the p53 E3 ligase MDM2 resulted in increased p53 levels and improved response to doxorubicin in vitro. Conclusions/Significance: MN1 overexpression accelerates an already aggressive leukemia, confers resistance to chemotherapy, and suppresses p53 and BIM induction, resulting in decreased apoptosis. This provides a mechanistic explanation of the poor prognosis observed with high MN1 expression and suggests that therapies directed at increasing p53 function may be useful for these patients. - Funding: Funding for the studies contained in the manuscript was obtained from: The Doug Coley Leukemia Research Foundation (http://dougcoleyfoundation. org/), The MacKay Foundation for Cancer Research (http://www.mackayfoundation.org/), The Leight Endowed Research Fund and the Frances P. Tutwiler Fund. Additional support provided by P30CA012197 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The author has declared that no competing interests exist. Acute myeloid leukemia (AML) is an aggressive malignancy of the myeloid lineage that leads to progressive marrow failure and death. It affects approximately 13,000 Americans every year, leading to over 9,000 deaths [1]. Despite decades of research, therapies for most patients have remained unchanged and outcomes are poor [2]. AML is a very genetically heterogenous disease; this heterogeneity is thought to underlie the diverse responses to therapy. Cytogenetics are the single most important prognostic marker in AML. Patients can be placed into poor, intermediate, and good risk categories depending on the presence of different chromosomal rearrangements [3,4,5,6]. Most patients with AML have a normal karyotype, placing them in the intermediate category; however, within this category, patients have very diverse outcomes. In these patients prognosis is affected by the presence of mutations or overexpression of certain genes [7,8]. The meningioma 1 (MN1) gene adversely affects prognosis when highly expressed in AML patients [9], as reported in multiple independent cohorts and in both younger and older patients [10,11]. MN1 is thought to be a transcriptional co-activator and, through its interactions with p300 and RAC3, to be involved in regulating transcriptional targets of the retinoic acid and vitamin D receptors [12,13,14,15]. Overexpression of MN1 generates AML in murine bone marrow experiments and predicts resistance to all-trans retinoic acid (ATRA) therapy in elderly AML patients [16]. MN1 overexpression also cooperates with MLL-ENL and CBFb-SMMHC in the generation of murine AML [17,18]. Despite these studies, the mechanisms by which MN1 overexpression confers an adverse prognosis remain unclear. To address this question, we have altered a genetically defined murine AML model and human cell lines to express high levels of MN1, and determined the effects on prognosis and response to standard therapy. Materials and Methods Ethics Statement All animal studies were approved by the Wake Forest University Health Sciences IACUC committee. Retroviral Constructs All retroviruses were constructed in the MSCV backbone (Clontech, Mountain View, CA). MN1-overexpressing vector was generated using the gateway cloning system (Invitrogen, Grand Island, NY). The destination vector (pMGWIG, a kind gift of Dr Uli Bialucha) was created with attR1 and R2 sites placed upstream of an IRES GFP in MSCV. MN1 full-length ORF in pENTR223.1 was purchased from Open Biosystems. The resulting MN1 IRES GFP vector was confirmed by sequencing. The GFP-only vector contained the full-length GFP ORF only. Cloning strategies are available on request. Cell Culture and Viability Assays OCI-AML3 cells [19] were a kind gift of Dr Mark Minden at University of Toronto. Molm-13 cells [20] were purchased from the DSMZ. All human cell lines were maintained in RPMI media (GIBCO, Carlsbad, CA) supplemented with 10% FBS, penicillin, and streptomycin. Cells were grown at 37uC with 5% CO2. Viability assays were done using the Cell Titer-Glo assay (Promega, Madison, WI) according to the manufacturers protocol, or by Trypan blue exclusion assay using the Countess cell counting system (Invitrogen, Carlsbad, CA). All murine cells were derived from fetal liver cells infected with MLL-ENL alone or with Flt3-ITD-expressing vectors (M1p5, MF3 and MFL2) [21]. Murine cells were maintained in stem cell media (40% DMEM, 40% IMDM, 20% FBS, supplemented with murine SCF to 10 ng/ml, murine IL6 to 2 ng/ml, and murine IL3 to 0.4 ng/ml). Competition Assays Two days after infection or thereafter, leukemia cells were split into replicate wells of ,50,000 cells in 24-well plates. After 72hour treatments, the GFP-positive percentage was quantified in the live cell population by using a FACSCalibur flow (...truncated)