Imaging, Diagnosis, Prognosis Activation of the Unfolded Protein Response Is Associated with Favorable Prognosis in Acute Myeloid Leukemia

Purpose: The unfolded protein response is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum. Previous studies suggest that the unfolded protein response is activated in some cancer cell lines and involved in tumor development. The role of the unfolded protein response during leukemogenesis is unknown thus far. Experimental Design: Here, we assessed the induction of key effectors of the unfolded protein response in leukemic cells at diagnosis of 105 acute myeloid leukemia (AML) patients comprising all subtypes. We determined the formation of the spliced variant of the X-box–binding protein 1 (XBP1) mRNA, as well as expression levels of calreticulin, GRP78, and CHOP mRNA. Results: The formation of the spliced variant of XBP1s was detectable in 16.2% (17 of 105) of AML patients. Consistent with activated unfolded protein response, this group also had significantly increased expression of calreticulin, GRP78, and CHOP. AML patients with activated unfolded protein response had lower WBC counts, lactate dehydrogenase levels, and more frequently, secondary AML. The incidence of fms-related tyrosine kinase 3 (FLT3) mutations was significantly lower in patients with activated unfolded protein response. In addition, an association was observed between activated unfolded protein response and deletion of chromosome 7. Finally, the clinical course of AML patients with activated unfolded protein response was more favorable with lower relapse rate (P = 0.0182) and better overall (P = 0.041) and disease-free survival (P = 0.022). Conclusions: These results suggest that the unfolded protein response is activated in a considerable subset of AML patients. AML patients with activated unfolded protein response present specific clinical characteristics and a more favorable course of the disease. The current paradigm on leukemogenesis indicates that leukemias are propagated by leukemic stem cells. The genomic events and pathways involved in the transformation of hematopoietic precursors into leukemic stem cells are increasingly clarified. Traditionally, research on the pathogenesis of acute myeloid leukemia (AML) has focused on the analysis of tumor suppressor genes or oncogenes, which regulate proliferation. Disruption of normal differentiation represents another hallmark of AML because leukemic blasts typically display a block in their normal differentiation process at a particular stage (1). Additional characteristics of leukemic stem cells may involve deregulation of cell death and self-renewal pathways. Another feature of some cancer stem cells is the activation of the unfolded protein response, which is triggered by the accumulation of misfolded proteins in the endoplasmic reticulum, leading to endoplasmic reticulum stress (2–4). Two key events are noteworthy during the unfolded protein response. First, the global protein synthesis is reduced, which is achieved by a reduction of the protein load entering the endoplasmic reticulum, and second, specific endoplasmic reticulum chaperone molecules are activated, thereby markedly increasing the capacity to handle misfolded proteins (2, 3). If homeostasis cannot be achieved after activation of the unfolded protein response, cell death is triggered (4, 5). Initial key events of the unfolded protein response comprise the activation of the inositol-requiring protein-1 (IRE1), of the activating transcription factor 6 (ATF6), and of the protein kinase RNA–like endoplasmic reticulum kinase (6). In particular, activation of Authors' Affiliations: Departments of Medical Oncology and Clinical Research and Internal Medicine and Clinical Research, University Hospital Bern and University of Bern, Bern, Switzerland Received 11/12/08; revised 1/30/09; accepted 3/6/09; published OnlineFirst 5/26/09. Grant support: A grant from the Swiss National Science Foundation SF 310000-109388 (T. Pabst). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: J.A. Schardt researched, D. Weber analyzed the data, M. Eyholzer researched, B.U. Mueller analyzed the data and wrote the article, and T. Pabst designed the research, analyzed the data, and wrote the article. The funding source had no role in the publication of these data. Requests for reprints: Thomas Pabst, Department of Medical Oncology, University Hospital, 3010 Bern, Switzerland. Phone: 41-31-632-8430; Fax: 41-31-632-3410; E-mail: [email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-2870 3834 Clin Cancer Res 2009;15(11) June 1, 2009 www.aacrjournals.org Research. on April 14, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from IRE1 induces the cleavage of the X-box–binding protein 1 (XBP1) mRNA, generating a spliced mRNA that encodes a potent novel transcriptional activator of downstream unfolded protein response target genes (7–10). Other prominent cellular mediators of the unfolded protein response besides XBP1 are calreticulin, CHOP, and GRP78 (2–4). The role of the unfolded protein response during leukemogenesis has not been investigated thus far. However, we previously reported that the endoplasmic reticulum chaperone calreticulin is specifically induced in AML cells expressing the AML1-MDS1-EVI1 protein (11), suggesting that the unfolded protein response can be activated in subtypes of AML patients. In this report, we screened a large collection of primary leukemic cells of all AML subtypes for the activation of the unfolded protein response. We found that activated unfolded protein response is observed in 16.2% of AML patients, as determined by the induction of the XBP1 spliced variant and increased expression of GRP78, CHOP, and calreticulin. AML patients with activated unfolded protein response had specific clinical characteristics, and an association was observed between activated unfolded protein response and deletion of (parts of) chromosome 7. Remarkably, the clinical course of AML patients with activated unfolded protein response was more favorable with lower relapse rate and better survival. These results suggest that assessing AML cells for the presence of the spliced variant of XBP1smRNA is a sensitive marker for activated unfolded protein response and that the unfolded protein response is activated in a considerable subgroup of AML patients. Patients, Materials, and Methods Patients. Source of malignant cells was Ficoll-separated mononucleated cells of bone marrow (82 patients) or peripheral blood (23 patients). All AML patients were consecutively diagnosed between 2005 and 2007 using standard morphology and immunophenotype markers at the Department of Oncology, University Hospital, Bern, Switzerland. Informed consent from all patients was obtained according to the Declaration of Helsinki, approved by decisions of the local ethics committee of Bern, Switzerland. Only patients fit for intensive treatment were included in this study, and they were all treated within the HOVONSAKK 30/00 protocol for patients ≤60 y or the HOVON-SAKK 30/01 protocol for patients >60 y. Treatment for patients consisted of cytarabine and an anthracycline in cycle 1 and of cytarabine in cycle 2 (and amsacrin for patients ≤60 y). Patients ≤60 y in complete remission after cycle 2 were randomly assigned to a third cycle of chemotherapy with etoposide and mitoxantrone or high-dose chemotherapy with busulfan and cyclophosphamide, followed by autologous transplant. Allogenous transplant was done in patients <<55 y of age and with intermediateor bad-risk cytogenetics. Immunophenotyping and cytogenetic analysis. A panel ofmonoclonal antibodies against myeloid lineage associated antigens (including Translational Relevance Here, we identified for the first time the activation of the unfolded protein response in a subset of patients with acute myeloid leukemia (AML) by assessing the spliced variant of the X-box–binding protein 1 (XBP1) mRNA, as well as the expression of calreticulin, GRP78, and CHOP mRNA. In particular, the PCR assay to detect the spliced variant of the XBP1 mRNA has the potential to be applied in clinical practice. Remarkably, we identified AML patients with activated unfolded protein response to have a favorable course of their disease. In addition, an association was observed between activated unfolded protein response and deletion of chromosome 7 among AML patients with bad-risk karyotype abnormalities. Finally, cell lines with activated unfolded protein response are especially prone to undergo apoptosis after treatment with the proteasome inhibitor bortezomib. Thus, our studies might provide a rationale for the use of bortezomib in AML patients presenting with an activated unfolded protein response and/or deletion of chromosome 7 in the leukemic cells at diagnosis. Fig. 1. A subgroup of AML patients is expressing XBP1s. A, the sensitivity of the PCR assay was determined using pcDNA3 plasmids encoding for human XBP1 WT (U) or the spliced (S) form of XBP1. Top, illustrates a competitive PCR with decreasing amounts of plasmid encoding for XBP1 WT and increasing amounts of plasmid encoding for the spliced form of XBP1. B, middle, a competitive PCR with constant amounts of XBP1 WT plasmid combined with increasing amounts of the spliced form of XBP1. The ratio observed with 30 ng of XBP1 WT plasmid and 20 ng of the spliced form of XBP1 in (A) and (B) was determined to separate AML patients with induced versus uninduced XBP1s mRNA accordingly. C, whereas only the unspliced form of XBP-1 mRNA is detectable in SAML patients (left, bottom), both the spliced and unspliced form are observed in S+ AML patients (right, bottom), as assessed by semiquantitative reverse transcriptionPCR. The expression of the ABL1 gene is presented as a control. 3835 Clin Cancer Res 2009;15(11) June 1, 2009 www.aacrjournals.org Unfolded Protein Response in Acute Myeloid Leukemia Research. on April 14, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from CD9, CD11b, CD13, CD14, CD15, CD33, glycophorin, andmyeloperoxidase), as well as lymphoid lineage–associated antigens (including CD2, CD3, CD7, CD10, CD19, CD22, CD79, and lineage nonspecific antigens, including HLA-DR, TdT, CD34, CD45, and CD56), was used to analyze the leukemic cells. The cutoff for a positive result of a particular marker