Background: In translational cancer research, gene expression data is collected together with clinical data and genomic data arising from other chip based high throughput technologies. Software tools for the joint analysis of such high dimensional data sets together with clinical data are required.
Summary Core binding factor (CBF) leukaemias, characterized by either inv(16)(p13.1q22) or t(8;21)(q22;q22), constitute acute myeloid leukaemia (AML) subgroups with favourable prognosis. However, 40–50% of patients relapse, emphasizing the need for risk‐adapted treatment approaches. In this regard, studying secondary genetic aberrations, such as mutations of the KIT gene, is of great interest, particularly as they can be targeted by receptor tyrosine kinase inhibitors (TKI). However, so far little is known about the biology underlying KIT‐mutated CBF leukaemias. We analysed gene expression profiles of 83 CBF AML cases with known KIT mutation status in order to gain novel insights in KIT‐mutated CBF pathogenesis. KIT‐mutated cases were characterized by deregulation of genes belonging to the NFkB signalling complex suggesting impaired control of apoptosis. Notably, a subgroup of KIT wildtype cases was also characterized by the KIT mutation signature due to yet unknown aberrations. Our data suggest that this CBF leukaemia subgroup might profit from TKI therapy, however, the relevance of the KIT mutation‐associated signature remains to be validated prior to clinical implementation. Nevertheless, the existence of such a signature supports the notion of relevant biological differences in CBF leukaemia and might serve as diagnostic tool in the future.
The online version of this article has a Supplementary Appendix. BackgroundMicroRNAs are regulators of gene expression, which act mainly by decreasing mRNA levels of their multiple targets. Deregulated microRNA expression has been shown for acute myeloid leukemia, a disease also characterized by altered gene expression associated with distinct genomic aberrations such as nucleophosmin (NPM1) mutations. To shed further light on the role of deregulated microRNA and gene expression in cytogenetically normal acute myeloid leukemia with NPM1 mutation we performed an integrative analysis of microRNA and mRNA expression data sets. Design and MethodsBoth microRNA and gene expression profiles were investigated in samples from a cohort of adult cytogenetically normal acute myeloid leukemia patients (n=43; median age 46 years, range 23-60 years) with known NPM1 mutation status (n=23 mutated, n=20 wild-type) and the data were integratively analyzed. Putative microRNA-mRNA interactions were validated by quantitative reverse transcriptase polymerase chain reaction, western blotting and luciferase reporter assays. For selected microRNAs, sensitivity of microRNA-overexpressing cells to cytarabine treatment was tested by FACS viability and cell proliferation assays. ResultsOur integrative approach of analyzing both microRNA-and gene expression profiles in parallel resulted in a refined list of putative target genes affected by NPM1 mutation-associated microRNA deregulation. Of 177 putative microRNA -target mRNA interactions we identified and validated 77 novel candidates with known or potential involvement in leukemogenesis, such as IRF2-miR-20a, . Furthermore, our data showed that deregulated expression of tumor suppressor microRNAs, such as miR-29a and miR-30c, might contribute to sensitivity to cytarabine, which is observed in NPM1 mutated acute myeloid leukemia. ConclusionsOverall, our observations highlight that integrative data analysis approaches can improve insights into leukemia biology, and lead to the identification of novel microRNA -target gene interactions of potential relevance for acute myeloid leukemia treatment.Key words: microRNA, miRNA, gene expression profiling, GEP, acute myeloid leukemia, AML, NPM1 mutation.Citation: Russ AC, Sander S, Lück SC, Lang KM, Bauer M, Rücker FG, Kestler HA, Schlenk RF, Döhner H, Holzmann K, Döhner K, and Bullinger L. Integrative nucleophosmin mutation-associated microRNA and gene expression pattern analysis identifies novel microRNA -target gene interactions in acute myeloid leukemia.
Core-binding factor (CBF) leukemias, characterized by translocations t(8;21) or inv(16)/t(16;16) targeting the CBF, constitute acute myeloid leukemia (AML) subgroups with favorable prognosis. However, about 40% of patients relapse and the current classification system does not fully reflect this clinical heterogeneity. Previously, gene expression profiling (GEP) revealed two distinct CBF leukemia subgroups displaying significant outcome differences and identified apoptotic signaling, MAPKinase signaling and chemotherapy-resistance mechanisms among the most significant differentially regulated pathways. We now tested different inhibitors of the respective pathways in a cell line model (six cell lines reflecting the CBF subgroup-specific gene expression alterations), and found apoptotic signaling to be differentiating between the CBF subgroup models. In accordance, primary samples from newly diagnosed CBF AML patients (n ¼ 23) also showed differential sensitivity to in vitro treatment with a Smac mimetic such as BV6, an antagonist of inhibitor of apoptosis (IAP) proteins, and ABT-737, a BCL2 inhibitor. Furthermore, GEP revealed the BV6-resistant cases to resemble the previously identified unfavorable CBF subgroup. Thus, our current findings show deregulated IAP expression and apoptotic signaling to differentiate clinically relevant CBF subgroups, which were independent of known molecular markers, thereby providing a starting point for novel therapeutic approaches.
363 MicroRNAs (miRs) have been shown to control a wide range of biological functions such as differentiation, proliferation and apoptosis, either by translational repression, mRNA cleavage or miR mediated decay of the respective target mRNA. Deregulated miR expression has been associated with various human cancers, including acute myeloid leukemia (AML), a disease characterized by the accumulation of acquired genetic alterations in hematopoietic progenitor cells that lead to altered self-renewal, proliferation and differentiation. Mutations of the nucleophosmin (NPM1) gene could be identified as the most common genetic alteration in AML, mainly occurring in cytogenetically normal karyotype (CN-AML) cases. Furthermore, while NPM1 mutated cases show a favorable prognosis (in the absence of FLT3-ITD) and have been shown to possess a distinct gene expression profile (GEP), so far the biology underlying this aberration has still not been fully understood. In previous work, we profiled the miR expression in a cohort of 91 AML cases comprising all major cytogenetic and molecular genetic subgroups. Significance Analysis of Microarrays (SAM) revealed a distinct miR-signature associated with NPM1 mutation (NPM1mut) in CN-AML as also shown by other groups: 66 miRs were differentially expressed in NPM1mut compared to NPM1 wild-type (NPM1wt) cases. The vast majority of these miRs was strongly upregulated in NPM1mut CN-AML, whereas only few miRs were downregulated compared to NPM1wt cases. Therefore, overexpression of a distinct set of miRs seems to be an important characteristic of NPM1mut CN-AML, and the resulting deregulated expression of target genes of these NPM1mut signature miRs might contribute to leukemogenesis. To identify putative target genes of NPM1mut-associated miRs, we performed an integrative analysis of miR-expression and NPM1mut-related gene expression data in our cohort. First, we generated target gene lists for the core 33 overexpressed miRs of the NPM1mut signature by using the miRGator database. This resulted in a theoretical NPM1mut associated GEP. Then, a comparison of the theoretical with the measured NPM1mut GEP was performed in order to find putative targets whose mRNA levels are directly affected by the respective miRs. This approach revealed several promising candidate genes with known implication in tumorigenesis and/or leukemogenesis like APP, CCND1, IRF2, BCL2L1, MLL and KIT. Interestingly, these genes are putative targets of not only one, but several miRs (4 to 15) of the NPM1mut signature, thereby pointing towards a synergistic effect of these miRs. Validation of individual miR-target gene relations was carried out by qRT-PCR in cell lines transfected with the respective miR mimics, supplemented by Western Blot and 3'UTR-luciferase-reporter assays. This validation was successful, not only for already known miR-target gene connections, but also for novel candidates including e.g. CCND1, a cell cycle regulator, and interferon regulatory factor-2 (IRF2). IRF2 is known to show dysregulated expression in the majority of AML cases and has recently been described to be essential for preserving the self-renewal and multilineage differentiation capacity of hematopoietic stem cells (Sato et al., Nat Med 2009). Thus, our approach of combining miR expression information and GEP in NPM1mut CN-AML led to the identification of promising target genes with potential implication in leukemogenesis. Additional functional analyses of relevant miRs and target genes are currently in progress to further illuminate the mechanism of NPM1mut AML pathogenesis. Disclosures: No relevant conflicts of interest to declare.
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