Abstract A38: Gene expression profile of the Wnt signaling pathway in mesenchymal stromal cells from acute myeloid leukemia

Abstract

Abstract Acute myeloid leukemia (AML) is a heterogeneous hematologic disease characterized by proliferation and accumulation of myeloid precursors in the bone marrow, decrease in apoptosis level, and differentiation arrest of these cells. Although there are several studies in the area, events related to the beginning of disease as well as its progression are still unknown. It is believed that malignant transformation in normal hematopoietic stem cells (HSC) can give rise to a leukemic stem cell (LSC) and this transformation could be related to changes in mesenchymal stromal cells (hMSC) signaling. Previous studies showed that mesenchymal stromal cells from acute myeloid leukemia patients (hMSC-AML) have a common molecular signature, compared to healthy donors' hMSCs (hMSC-HD), and these differentially expressed genes could be related to malignant transformation. Among the 55 differentially expressed genes, BMP4 has its expression decreased in hMSC-AML, and this decrease could be regulated by the Wnt pathway. In this context, the aim of this work was to evaluate the gene expression profile of the Wnt pathway in hMSC-AML. To address this hypothesis, we used a PCR Array assay approach to evaluate the gene expression profile of hMSC-AML compared to hMSC-HD. For this purpose, bone marrow samples from healthy donors (HD) and patients diagnosed with AML previously classified into subtypes were placed in culture. For PCR array we used 4 samples from different subtypes of AML and compared with two pools of HD. After the 3rd passage, total RNA from MSCs cultures was obtained using RNeasy Mini Kit according to the manufacturer's instructions (Qiagen). After that, the RNA was processed according to the Human WNT Signaling Pathway RT2 ProfilerTM PCR Array (Qiagen) according to the manufacturer's protocols. Data were analyzed using GeneGlobal data analysis center (Qiagen) and differentially expressed genes with ≥ 1.5 fold-change was used as a criterion to define overexpression or downregulation. To confirm the results we performed real-time PCR with a large number of patients with different subtypes. The pathway analysis and related processes were obtained using the MetaCoreTM software. To confirm the protein levels of some differentially expressed genes, Western blot analysis was performed. The results obtained in PCR array assay showed changes in the gene expression profile of the Wnt pathway when comparing hMSC-AML to hMSC-HD. Among the 84 genes presented on the PCR Array, 26 genes were found to be differentially expressed. To confirm the results, real-time PCR with some genes was performed (KREMEN1, NKD1, and PRICKLE1- overexpressed and TCF7, LEF, and PORCN downregulated). The results confirmed that the WNT pathway is altered in all hMSC-AML from AML patients instead of subtypes. TCF7 and LEF are transcription factors, and the decrease of their expression could be related to the BMP4 regulation. To verify if these proteins are differentially expressed in hMSC-AML compared to hMSC-HD, we also performed Western blot analysis. The results confirmed a decrease in these transcription factors in hMSC-AML. Moreover, we performed in silico analysis and identified four consensus sites Tcf/Lef in 2kb from BMP4 promoter region, suggesting that they could regulate BMP4. Altogether, these results suggest that the WNT signaling pathway is changed in hMSC-AML from AML patients instead of subtypes and this pathway regulation could be related to the decrease of BMP4 in hMSC-AML. Citation Format: Pedro Azevedo, Nathalia Oliveira, Eliana Abdelhay, Renata Binato. Gene expression profile of the Wnt signaling pathway in mesenchymal stromal cells from acute myeloid leukemia [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A38.