Attenuated Expression of Apoptosis Stimulating Protein of p53-2 (ASPP2) in Human Acute Leukemia Is Associated with Therapy Failure

et al. (2013) Attenuated Expression of Apoptosis Stimulating Protein of p53-2 (ASPP2) in Human Acute Leukemia Is Associated with Therapy Failure. PLoS ONE 8(11): e80193. doi:10.1371/journal.pone.0080193 Attenuated Expression of Apoptosis Stimulating Protein of p53-2 (ASPP2) in Human Acute Leukemia Is Associated with Therapy Failure Marcus M. Schittenhelm 0 Barbara Illing 0 Figen Ahmut 0 Katharina Henriette Rasp 0 Gunnar Blumenstock 0 Konstanze Do hner 0 Charles D. Lopez 0 Kerstin M. Kampa-Schittenhelm 0 Klaus Roemer, University of Saarland Medical School, Germany 0 1 University Hospital Tu bingen, Department of Hematology , Oncology, Rheumatology, Immunology and Pulmology, T u bingen, Germany , 2 Department of Clinical Epidemiology and Applied Biometry, Eberhard Karls University Tu bingen, Germany, 3 University Hospital Ulm, Department of Internal Medicine III , Ulm, Germany , 4 Department of Medicine, Division of Hematology and Medical Oncology, Oregon Health & Science University and The OHSU Knight Cancer Institute , Portland, Oregon , United States of America Inactivation of the p53 pathway is a universal event in human cancers and promotes tumorigenesis and resistance to chemotherapy. Inactivating p53 mutations are uncommon in non-complex karyotype leukemias, thus the p53-pathway must be inactivated by other mechanisms. The Apoptosis Stimulating Protein of p53-2 (ASPP2) is a damage-inducible p53binding protein that enhances apoptosis at least in part through a p53-mediated pathway. We have previously shown, that ASPP2 is an independent haploinsufficient tumor suppressor in vivo. Now, we reveal that ASPP2 expression is significantly attenuated in acute myeloid and lymphoid leukemia - especially in patients with an unfavorable prognostic risk profile and patients who fail induction chemotherapy. In line, knock down of ASPP2 in expressing leukemia cell lines and native leukemic blasts attenuates damage-induced apoptosis. Furthermore, cultured blasts derived from high-risk leukemias fail to induce ASPP2 expression upon anthracycline treatment. The mechanisms of ASPP2 dysregulation are unknown. We provide evidence that attenuation of ASPP2 is caused by hypermethylation of the promoter and 59UTR regions in native leukemia blasts. Together, our results suggest that ASPP2 contributes to the biology of leukemia and expression should be further explored as a potential prognostic and/or predictive biomarker to monitor therapy responses in acute leukemia. - Funding: Grant support for this study in part by the Deutsche Krebshilfe Foundation (MMS, KKS), the IZKF Program of the Medical Faculty Tu bingen (MMS) and the Jose Carreras Scholarship Program (KKS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The p53 pathway is well known as a central player in the cellular stress response and tumor suppression. Depending on the nature of the stress, the outcome of p53 activation can range from cell cycle arrest and DNA repair to senescence, autophagy and apoptosis. Not surprisingly, inactivation of the p53 pathway is a universal event in human cancers and p53 is one of the most highly mutated genes with greater than 60% of human malignancies harboring inactivating p53 mutations (reviewed by Vousden and Prives[1]; UMD TP53 mutation database at http://p53.fr/ [2]). In some hematopoietic malignancies, inactivating mutations of p53 are involved with chromosomal instability and progression towards acute leukemia, such as complex karyotype myelodysplastic syndromes[3,4,5,6] and chronic phase Philadelphia-chromosome positive chronic myeloid leukemia (CML)[7]. Moreover, chromosomal aberrations of the long arm of chromosome 17 (locus of p53) or inactivating p53 mutations impede cancer therapies, such as fludarabine-based chemotherapy in CLL[8], BCR/ABL-targeted therapies in CML[9] as well as induction chemotherapy in AML[10]. However, in most cases of de novo acute leukemia, p53 mutations or chromosomal aberrations of chromosome 17 are uncommon, but primarily associate with therapy-related acute myeloid leukemias[11] and MDS-related complex karyotype leukemia[10,12,13]. We therefore speculated that in acute leukemia the p53 pathway may be altered by other means besides mutation. However the molecular mechanisms that inactivate the p53 pathway in acute leukemia remain unclear. Apoptosis Stimulating Protein of p53-2 (ASPP2), also referred to as 53BP2L, encoded by TP53BP2 enhances damage-induced apoptosis at least in part through a p53-mediated pathway[14]. Mouse models targeting ASPP2 using homologous recombination demonstrate that ASPP2 is a haploinsufficient tumor suppressor[15,16]. Indeed, ASPP2 expression is frequently suppressed in a variety of human cancers, such as breast cancer[14] and lymphoma subtypes, where low ASPP2 mRNA expression levels are associated with biologically more aggressive lymphoma subtypes and with poor clinical outcome[17]. ASPP2 is also a damage-inducible protein. Depending on cell context and type of stress, ASPP2 levels typically increase via transcriptional and/or posttranslational mechanisms after cellular damage[18,19]. Thus, the complex regulation of ASPP2 expression may provide important prognostic or predictive clinical information. However, to date there have been no studies examining ASPP2 expression in acute leukemia. In this report, we now demonstrate that lower ASPP2 mRNA and protein expression levels are statistically significantly associated with clinical unfavorable disease and early chemotherapyinduction failure in de novo as well as secondary acute myeloid and lymphoid leukemia. Moreover, ASPP2 siRNA knockdown in leukemia cell lines and ex vivo cultured patient derived primary leukemic blasts results in resistance to anthracycline-induced apoptosis. Our findings provide evidence that ASPP2 plays a role in the biology of acute leukemia and might serve as a biomarker to risk stratify patients and monitor therapy responses. Design and Methods Cell lines The promyelocytic AML cell line HL60 was purchased from the Leibniz Institute-German Collection of Microorganisms and Cell Cultures (DSMZ), Germany. The acute T-cell lymphoblastic leukemia cell line Jurkat was a gift from Dr. Salih, University of Tu bingen. The CML blast crisis cell line K562 was a generous gift of Dr. Lopez, Oregon Health and Science University, Portland, OR. The core binding factor leukemia cell line Kasumi-1 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). Cells were cultured in RPMI 1640, supplemented with 10% fetal bovine serum (GIBCO/Invitrogen, Darmstadt, Germany), 1% penicillin G (10,000 units/mL) and streptomycin (10,000 mg/ mg) and 2 mmol/L l-glutamine (GIBCO/Invitrogen, Darmstadt, Germany or Biochrom AG, Berlin, Germany). Antibodies and reagents An anti-ASPP2 iso (...truncated)