title: Investigating the roles of NFATC2 in acute myeloid leukaemia
Creators: name: Patterson, Shaun David
Publisher: name: University of Glasgow
Year: 2022

description: Acute myeloid leukaemia (AML) is a malignancy affecting the myeloid lineage of haemopoietic cells and has poor patient outcomes. Chemotherapy is poorly tolerated in a high proportion of patients and targeted therapies have limited impact at present. Additionally, relapse and/or resistance to existing therapy are common. High (cyto)genetic heterogeneity within and between AML patients make the development of effective, long-term targeted therapies highly challenging. As such, there is an unmet clinical need for the identification of novel oncogenic mechanisms and pathways which can be targeted therapeutically, in order to improve the clinical outcomes for larger groups of AML patients. Previously, the histone lysine demethylase KDM4A was identified as a putative ‘master regulator’ of leukaemogenic signalling in models of AML, which was not essential for healthy haemopoietic cell survival. One of the key targets of KDM4A identified in AML was the nuclear factor of activated T cells (cytoplasmic) 2 (NFATC2). The wider NFAT family of transcription factors (TFs) has been attributed roles in normal myelopoiesis, in which it is thought to regulate elements of the cell cycle and differentiation, which are key processes that become deregulated in AML. NFATs are also characterised as contributing to oncogenesis and drug resistance in myeloid leukaemias, however much of the evidence focuses on NFATC1. In light of the identification of the KDM4A-NFATC2 axis in AML, it was hypothesised that NFATC2 is essential for the oncogenic function and survival of AML cells. Prior to testing this hypothesis, NFATC2 was first characterised in cell line models of AML. Using established compounds, it was found that the MLL-AF9 TP53mut THP-1 AML cell line was sensitive to depletion/inhibition of calcium and calcineurin, which are both upstream regulators of NFATs in T cells. Next, shRNA knockdown (KD) of NFATC2 led to the loss of colony-forming capacity in a number of AML cell lines, highlighting that numerous subtypes of AML cells are dependent on NFATC2. In addition, increased apoptosis and cell cycle arrest were individually observed in some of these AML models, but not others, indicating that the mechanisms affected by NFATC2 depletion are dependent on the (cyto)genetic landscape. Global transcriptome profiling of THP-1 cells with NFATC2 KD identified a list of deregulated genes, of which a subset was validated in several other cell lines with NFATC2 KD. These included genes involved in intracellular transport and membrane protein function. Enrichment analyses also highlighted targets of oncogene MYC and serine/threonine kinase 33 (STK33) as enriched in the genes perturbed by NFATC2 depletion. Chromatin immunoprecipitation sequencing (ChIP-Seq) found NFATc2 gene binding targets to be enriched with a c-Myc DNA consensus binding sequence. Additionally, a number of novel NFATc2 DNA binding motifs were identified. The expression of NFATC2 was found to stratify patient outcomes in the TARGET-AML dataset, from paediatric AML patients. Of the genes identified by sequencing analyses as putative NFATC2/NFATc2 targets, the expression levels of 13 genes were found to be prognostic for patients in the TARGET-AML dataset, also. Together these data have shown that NFATC2 is essential for the survival of multiple AML cell lines and that it likely regulates elements of the cell cycle and/or apoptosis, depending on the cellular context. Newly-identified transcriptional and binding targets suggest that the oncogene MYC cooperates with NFATC2 and it could be hypothesised that they maintain an oncogenic transcriptional program together in THP-1 cells. These findings require translation into patient cells, which is challenging given the lack of NFATc2-specific inhibitors available. However, findings from open-source patient datasets indicate that NFATC2 and its targets have a significant role to play in clinical outcome and so warrant further investigation to elucidate some of the cellular mechanisms involved.