NFATC2 regulates Targets of MYC Signaling in MLL-AF9 AML

Abstract

Background: Acute myeloid leukemia (AML) arises due to an accumulation of genetic lesions within myeloid progenitors and oncogenic transformation is often characterised by disordered transcription. Recently the histone lysine demethylase KDM4A was shown to be essential for AML blast survival and self-renewal. shRNA knockdown (KD) of KDM4A led to downregulated expression of the transcription factor NFATC2 an MLL-AF9 AML model, suggesting that it is a key target of KDM4A oncogenic function. The Nuclear Factor of Activated T Cells (NFAT) family of transcription factors control cell cycle genes and self-renewal pathways in hematopoietic tissues and are well-defined as oncogenic regulators in various malignancies. NFATs have recently been attributed roles in the development of FLT3 ITD AML and resistance to tyrosine kinase inhibitors (TKIs) in myeloid leukemias but there is little evidence detailing the role(s) of NFATC2 specifically in AML. We hypothesized that NFATc2 activity is essential for the survival of AML cells and the oncogenic transcriptional networks within these.Aims: To determine if AML cells are dependent on NFATC2 for survival and to elucidate the transcriptional and binding targets of NFATc2 in AML.Methods: NFATC2 was depleted using shRNA KD in numerous cell line models of AML and putative transcriptional targets were elucidated using RNA-seq following KD. Binding targets of NFATc2 were determined using ChIP-seq. Transcriptomic targets of NFATc2 were validated using the Fluidigm Biomark multiplex PCR system and real time quantitative PCR.Results: KD of NFATC2 significantly impaired the colony forming capacity and expansion in liquid cultures of AML cell lines from diverse (cyto)genetic backgrounds. MLL-AF9/TP53 mut THP-1 cells showed reduced entry to the S-phase of the cell cycle and downregulation of cyclin D1 following NFATC2 depletion, suggesting that NFATC2 is critical for cell cycle progression in these cells. Overexpression of human NFATC2 in THP-1 led to an increased rate of cell growth.RNA-seq analysis of THP-1 cells with NFATC2 KD revealed >20 genes with deregulated expression (FDR<0.1), which have been validated using PCR methods. Overexpression of human NFATC2 resulted in significant deregulation of 9 of these genes (FDR<0.1), defining a subset of genes which may regulate the observed phenotype. Additionally, these top genes were not all differentially regulated in other MLL-AF9 AML cell lines MOLM-13 and NOMO-1 following NFATC2 KD. Finally, in THP-1, gene set enrichment analysis (GSEA) of sequencing results revealed that targets of MYC and calmodulin kinase STK33 were enriched within the genes perturbed by NFATC2 depletion. Targets of MYC signaling were validated by PCR in THP-1 but were not found to be deregulated in MOLM-13 following NFATC2 KD.ChIP-seq analysis of NFATc2 binding in THP-1 cells showed that >30% of NFATc2 targets were at promoter regions within 5kb of the transcription start site. Motif analysis of precipitated DNA fragments discovered two novel motifs which were enriched at NFATc2 binding sites (p<0.0001).Discussion: NFATC2 was found to be essential for expansion of AML cells in various cell line models. In the MLL-AF9 driven THP-1 model a number of putative transcriptional and genomic targets were defined, which include novel targets not previously described in AML pathogenesis and targets of MYC, an established oncogenic protein in AML. The differing expression profiles observed across AML cell lines of diverse (cyto)genetic backgrounds with NFATC2 KD suggest that the regulatory targets of NFATc2 vary depending on the cellular signaling landscape. Together with the finding that NFATC2 is indispensable for AML cell survival this study has elucidated novel roles(s) for NFATC2 in AML oncogenesis. DisclosuresMassett: Kymab Ltd: Current Employment. Huang: Janssen Pharmaceutical Companies of Johnson & Johnson (China): Current Employment.