Abstract
Glucocorticoids (GCs) and topoisomerase II inhibitors are used to treat acute lymphoblastic leukaemia (ALL) as they induce death in lymphoid cells through the glucocorticoid receptor (GR) and p53 respectively. Mechanisms underlying ALL cell death and the contribution of the bone marrow microenvironment to drug response/resistance remain unclear. The role of the microenvironment and the identification of chemoresistance determinants were studied by transcriptomic analysis in ALL cells treated with Dexamethasone (Dex), and Etoposide (Etop) grown in the presence or absence of bone marrow conditioned media (CM). The necroptotic (RIPK1) and the apoptotic (caspase-8/3) markers were downregulated by CM, whereas the inhibitory effects of chemotherapy on the autophagy marker Beclin-1 (BECN1) were reduced suggesting CM exerts cytoprotective effects. GCs upregulated the RIPK1 ubiquitinating factor BIRC3 (cIAP2), in GC-sensitive (CEM-C7-14) but not in resistant (CEM-C1-15) cells. In addition, CM selectively affected GR phosphorylation in a site and cell-specific manner. GR is recruited to RIPK1, BECN1 and BIRC3 promoters in the sensitive but not in the resistant cells with phosphorylated GR forms being generally less recruited in the presence of hormone. FACS analysis and caspase-8 assays demonstrated that CM promoted a pro-survival trend. High molecular weight proteins reacting with the RIPK1 antibody were modified upon incubation with the BIRC3 inhibitor AT406 in CEM-C7-14 cells suggesting that they represent ubiquitinated forms of RIPK1. Our data suggest that there is a correlation between microenvironment-induced ALL proliferation and altered response to chemotherapy.
Highlights
Leukaemia is a cancer characterised by aberrant proliferation of white blood cells and may be acute/chronic and myeloid/lymphoblastic
In order to establish the experimental conditions to analyse the effects of clinically used drugs and the microenvironment on acute lymphoblastic leukaemia (ALL) cells, effects of glucocorticoid Dex and Etop were analysed by assessing the viability of the resistant CEM-C1-15 and the sensitive CEM-C7-14 ALL cells to glucocorticoid and anthracycline induced apoptosis, as well as K562 chronic myelogenous leukemia (CML) cell lines, using trypan blue exclusion assay (Figs A and B in S1 File)
It was observed that treatment with 1μM Dex for 36hrs and 10μM Etoposide for 24hrs displayed optimal effect on ALL GC sensitive cells, whereas CML cells were largely GC-resistant
Summary
Leukaemia is a cancer characterised by aberrant proliferation of white blood cells and may be acute/chronic and myeloid/lymphoblastic. 80% of childhood ALL patients reach remission [1]. Topoisomerase II inhibitors and GCs are used to treat ALL [2]. Drug toxicity and chemoresistance are major challenges and the outcome for patients who fail therapy remains poor, increasing the necessity for more potent, less toxic therapies. GCs are used to treat ALL [3,4,5] as they induce leukocyte cell death through the glucocorticoid receptor (GR) [6]. GCs utilise mainly the intrinsic apoptotic pathway [9,10,11,12,13] modulating the gene expression of the pro-apoptotic BCL-2-interacting mediator of cell death (Bim) [14], as well as fine tuning the balance between NOXA and Mcl-1 [10]
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