Differential regulation of cell death pathways by the microenvironment correlates with chemoresistance and survival in leukaemia

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.

Endoplasmic reticulum stress, unfolded protein response and autophagy contribute to resistance to glucocorticoid treatment in human acute lymphoblastic leukaemia cells

“…Various forms of autophagy triggered by distinct stimuli, such as ER stress, Ca 2+ homeostasis and ROS signalling, control T cell survival (61). Since ER stress and UPR contribute to inflammatory signalling by activating a number of stress-responsive kinases including ERK, p38 mitogen-activated protein kinase and JNK (62), which have been demonstrated to induce GR post-translational modifications that affect its transcriptional activity (51,63,64), it is possible that these mechanisms lead to resistance to GC-induced apoptosis in ALL by modulating the function of the GR (51,(62)(63)(64).…”

“…Total RNAs were isolated from 3x10 6 CEM-C7-14, CEM-C1-15 and MOLT4 cells using a High Pure RNA Isolation kit (Roche Molecular Systems, Inc.) and the concentration of the total RNA for each sample was measured using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Inc.). Complementary dNA was reverse-transcribed using a High-Capacity RNA-to-cDNA™ kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) at 37˚C for 1 h. QPCR was performed using GRP94, GRP78 and RPL19 (internal control) primers (Eurofins Scientific) mixed with SensiFAST SYBR ® No-ROX kit (Bioline) as previously described (50,51). The primer sequences were as follows: GRP78 forward, 5'-TGCCGTTCAAGGTGGTTG-3' and reverse, 5'-CCA AATAAGCCTCAGCGG-3'; GRP94 forward, 5'-CTGGGTCCA GCAGAAAAGAG-3' and reverse, 5'-CACTCCTTCCTTGGC AACAT-3'; RPL19 forward, 5'-ATGTATCACAGCCTGTAC CTG-3' and reverse, 5'-TTCTTGGTCTCTTCCTCCTTG-3' .…”

p53 modeling as a route to mesothelioma patients stratification and novel therapeutic identification

“…Total RNA was extracted from untreated or treated Mero-14 cells with etoposide (10 µM) or gemcitabine (1 µM) for 24 h [ 38 – 40 ] and subjected to The Affymetrix Human Genome U133 plus 2.0 Array (Eurofins, UK) as previously described [ 15 , 41 ]. The raw data were normalized by robust multi-array average (RMA) algorithm [ 42 ] and annotated.…”

Insight into glucocorticoid receptor signalling through interactome model analysis

“…Their varied effects allow for their clinical application in numerous diseases, particularly for their potent anti-inflammatory and immunosuppressive effects to treat diseases such as arthritis [ 4 , 5 ]. GCs are also prescribed for the treatment of lymphoid cancers, as they selectively induce cell death in leukocytes [ 6 – 8 ], highlighting the tissue specificity of their action and the need for further research into GC signalling.…”