Dysregulation of c-MYC in leukemic microenvironment via kinases in acute myeloid leukemia
Introduction
Acute myeloid leukemia (AML) cells manipulate their surrounding microenvironment, particularly bone marrow mesenchymal stromal cells (MSCs), to support their survival and growth, leading to a high relapse rate and poor prognosis with treatments. The deregulation of c-MYC is implicated as one of the genes in AML pathogenesis that regulates cell proliferation, differentiation, and apoptosis. The purpose of the current study is to investigate the role of c-MYC in modulating AML-MSC interactions and promoting leukemic cell survival and growth and to identify the mechanism of c-MYC deregulation in AML.
Methods
To recreate the microenvironment of AML cells, we generated conditioned medium by culturing HS-5 cells, human marrow stromal cells, for three days and mixing it with DMEM medium. Cytokines secreted by HS-5 cells were determined using Human cytokine ELISA plate assay. For the current project, we utilized 6 AML cell line models, including HEL, MOLM-14, NB4, MV-4-11, MOLM-13, Kasumi-1. To mimic bone marrow microenvironment, AML cells were cultured in conditioned medium. RNA-sequencing analysis of the AML cells cultured in conditioned medium vs. standard medium identified differentially expressed genes in AML cells cultured in the conditioned medium. These genes were confirmed via RT-PCR and Western blot analysis for their expression, and the function of the genes were confirmed by gene silencing. The copy number of MYC gene was assessed by TaqMan Copy Number assay.
Results
Various cytokines, especially IL-6, IL-8, and VEGF were significantly higher in conditioned medium relative to standard culture medium. To identify pathways or genes that are potentially affected by the cytokines, we performed RNA sequencing and found significant upregulation of MYC, STAT3, CK2, GSK3A and AKT expression in AML cells cultured in conditioned medium, and these genes were confirmed by RT-PCR or Western blotting. Since c-MYC is one of the bad prognostic factors in AML, we investigated the c-MYC activation pathway via DNA-PKcs and OCT4, which was identified as upstream regulators of c-MYC in our previous study. To rule out higher c-MYC due to genomic amplification, we measured the copy number of MYC gene in 6 AML cell lines and found that none of the AML cell lines have MYC amplification. These data indicate that higher c-MYC is due to transcriptional activation or inhibition of c-MYC degradation. Silencing DNA-PKcs and STAT3 using siRNA did not affect the expression of c-MYC, suggesting that other mechanisms, such as epigenetic changes or other kinases identified by RNA-sequencing and immunoblotting, may contribute to the increase of c-MYC in AML.
Conclusion
AML cell lines cultured in conditioned medium with human marrow stromal cells have different genetic characteristics relative to those cultured in standard medium, possibly due to the cytokines released by the MSCs. The genes include c-MYC which is one of the poor prognostic factors in AML. Our study implicates two important points in AML: 1) The utilization of MSC-conditioned medium for in vitro AML models may better represent the clinical disease, and 2) the investigation of c-MYC activation pathway that include kinases may lead to identification of new therapeutic targets in the treatment of AML with higher c-MYC.