Glucocorticoid resistance in childhood leukaemia: mechanisms and modulation (2003)

Glucocorticoids (GCs) are a central component in contemporary paediatric acute lymphoblastic leukaemia treatment protocols. GC resistance has an adverse impact on clinical outcome in childhood leukaemia. Mechanisms of GC resistance have been described in cell line models, but have largely remained elusive in clinical specimens. GCs exert their cytotoxic effect through the induction of apoptosis. Recent advances in knowledge about the apoptotic pathway have opened a whole new field of drug resistance research and identified a large number of potential targets for enhancing GC action. It is of importance, however, to realize that lymphoid tumours are among the few cell types that can respond to GC exposure by initiating their apoptotic programme (in several non-lymphoid tumours GCs induce proliferation or inhibit apoptosis). Very little is known about the GC-mediated gene-regulatory events preceding the apoptotic cascade. The main challenge facing today's researchers in steroid resistance research is to unravel this part of the GC-induced apoptotic pathway. This review summarizes the efforts to elucidate the molecular mechanisms of GC-induced apoptosis and resistance, with particular reference to childhood leukaemia. Several strategies are also discussed for modulating or circumventing this type of drug resistance. The cytolytic effects of glucocorticoids (GCs) on thymocytes were first described in 1936 (Selye, 1936). GCs were first used in the treatment of childhood leukaemia in the late 1940s and early 1950s. Consecutive trials showed that, as single agents, GCs induced partial and complete responses in up to 80% of children suffering from newly diagnosed acute lymphoblastic leukaemia [initial (i)ALL]. In relapsed ALL (rALL) and acute myeloid leukaemia (AML) patients, however, response rates were much lower (35% and 20% respectively) (reviewed by Kaspers et al, 1994a). Today GCs are an integral part of ALL treatment protocols. Resistance to GC-induced cell kill (determined both in vivo and in vitro) has been identified as a major adverse prognostic factor in iALL. The Berlin–Frankfurt–Münster (BFM) study group showed a worse treatment outcome for patients who responded poorly to a 7-d regimen of prednisolone (PRD) monotherapy in the ALL-BFM-83 study, despite the subsequent use of usually effective combination chemotherapy (Riehm et al, 1987). Compared with PRD poor responders, defined by a cut-off point of more than 1 × 109 leukaemic cells/l in the peripheral blood after 7 d PRD, good responders had a significantly higher probability of achieving complete remission (99%vs 92%) and a higher probability of event-free survival at 3 years (75%vs 43%). The prognostic significance of the in vivo PRD response was retained in the ALL-BFM-86 and -90 trials (Fig 1), even though, thanks to improvements in treatment, the overall probability of event-free survival at 5 years increased from 64% to 78% (Schrappe et al, 2000). Our group (Fig 2; Kaspers et al, 1997) and Hongo et al (1997) reported significant correlations between in vitro GC resistance, determined with the total cell kill methyl-thiazol-tetrazolium (MTT) assay, and clinical outcome in childhood iALL. Prognostic significance of the in vivo PRD response in studies ALL-BFM 83 (A), ALL-BFM 86 (B) and ALL-BFM 90 (C). pEFS = probability of event-free survival. The lower curves represent the cumulative incidence of central nervous system relapses among PRD good and poor responders. In ALL-BFM 83 and ALL-BFM 86, but not in ALL-BFM 90, PRD poor responders had significantly more CNS relapses than PRD good responders [adapted from Schrappe et al (2000), with permission of the author and the Nature Publishing Group (Basingstoke, UK)]. Relationship between in vitro drug resistance and probability of disease-free survival (pDFS) in newly diagnosed childhood ALL, for prednisolone. The numbers in the figures along the X-axis indicate the patients at risk at the different time-points [adapted from Kaspers, G.J.L., Veerman, A.J.P., Pieters, R., Van Zantwijk, C.H., Smets, L.A., Van Wering, E.R., & Van der Does-van den Berg, A. (1997). In vitro cellular drug resistance and prognosis in newly diagnosed childhood acute lymphoblastic leukaemia. Blood, 90, 2723–2729. Copyright American Society of Hematology, used by permission]. GCs also form an integral part of rALL, and some AML treatment protocols. However, in line with the lower response rate to GC monotherapy in vivo, our laboratory demonstrated that these leukaemias were > 75-fold more resistant to GCs in vitro (Klumper et al, 1995; Zwaan et al, 2000). This may at least partly explain their relatively low cure rate (Henze et al, 1991; Stevens et al, 1998). During the past decades, extensive studies have been performed in the attempt to elucidate the molecular mechanisms of GC-induced cell lysis and GC resistance. This review includes an overview of the results obtained to date. In the first section of this review, background information is given about glucocorticoid hormones, the glucocorticoid receptor and the apoptotic pathway. In the second part, the molecular events in GC-induced apoptosis are linked to GC resistance, and the third section discusses several strategies for modulating GC resistance. The family of steroid hormones comprises four major types: the progestins, androgens, oestrogens and corticosteroids. These lipophilic compounds are all derived from cholesterol but differ structurally and functionally. The corticosteroids can be subdivided into mineralocorticoids, which regulate electrolyte and fluid balance, and GCs, which have a wide range of biological activities, including inhibition of the immune and inflammatory responses and induction of apoptosis in lymphoid cells. The predominant naturally occurring GC in humans is cortisol. Mainly because of the severe side-effects resulting from the mineralocorticoid properties of cortisol, synthetic GCs were developed with enhanced GC and decreased mineralocorticoid action. In Table I, several corticosteroid hormones are summarized with their relative potency and mineralocortiocoid action. There is no clear explanation for the differences in potency of the various steroids. The affinity for the glucocorticoid receptor (GR) does not appear to be a major determinant (Ponec et al, 1986). Other factors, such as differences in the stability of the GR–GC complex (Ichii et al, 1984) or interaction between the GC nested in the GR and transcriptional machinery at the postreceptor level have been proposed as possible explanations (Zeelen, 1990). Traditionally PRD has been used in the treatment of childhood ALL. More recently, several clinical trials showed that treatment results with dexamethasone (DXM) as part of combination chemotherapy for ALL were better than those with PRD, despite the use of ‘equivalent’ dosage (DXM was assumed to be seven times more potent than PRD) (Jones et al, 1991; Veerman et al, 1996; Gaynon et al, 2000). These results were explained by the more favourable pharmacokinetics of DXM. However, Kaspers et al (1996) demonstrated that DXM was 16 times more potent than PRD in killing ALL cells, which is much higher than the originally assumed factor of seven. In current ALL treatment protocols, DXM is used in six to 10 times lower dosages than PRD, which may thus result in stronger cytolytic effects. This was disputed by Ito et al (1996), who showed that, using their stroma-supported cell culture system, DXM was only five to six times as potent as PRD in killing ALL cells. The glucocorticoid receptor alpha (GRα), the predominantly occurring glucocorticoid receptor, is a ligand-dependent transcription factor. In its inactive form, the GRα is located in the cytoplasm in association with several proteins (receptor-associated proteins or RAPs) that increase GR affinity for GCs and anchor the receptor in the cytoplasm (Miyata & Yahara, 1991; Prima et al, 2000). Within the GR, three domains can be determined: the N-terminal part or modulating domain that is involved in modulation of gene transcription (Wright et al, 1993); the DNA binding domain that is involved in DNA binding, homo- and heterodimerization (Tao et al, 2001) and gene transcription enhancement (Miesfeld et al, 1987); and the ligand binding domain at the carboxyl terminus that controls the activity of the receptor as a whole through its interaction with heat shock proteins and cyclophilins (inactive receptor) or with GCs and co-activators (active receptor). The GR non-covalently binds GCs with high specificity and affinity [dissociation constant (Kd) 2–20 nmol/l]. In its unbound form, the ligand binding domain inhibits DNA binding (Gehring & Segnitz, 1988). In addition, the ligand binding domain contains a nuclear localization signal and binding sites for heat shock protein 90 (Hsp 90). Structurally and functionally different receptors can be formed by alternative splicing of the GR mRNA. The human GR primary mRNA transcript consists of nine exons. Alternative splicing of exon 9 results in an mRNA sequence coding for the glucocorticoid receptor beta (GRβ). This receptor is predominantly localized in the nucleus, and is capable of binding and inactivating the activated GRα (dominant negative activity) (Bamberger et al, 1995). More recently, two other variants have been described: glucocorticoid receptor P (GR-P) and glucocorticoid receptor gamma (GRγ). GR-P is encoded by exons 2–7 and a part of intron 7. The GR-P is thought to augment the activity of GRα (De Lange et al, 2001). GRγ is the result of the retention of 3 base pairs between exon 3 and 4 due to the use of an alternative splice donor site. Consequently, an mRNA sequence is produced, coding for an additional amino acid (arginine) located in the region between the two zinc fingers of the DNA binding domain. GRγ is a ligand-dependent transcription factor with reduced transactivating activity (Rivers et al, 1999). The process of GC-induced apoptosis can be subdivided into three stages: an initiation stage that involves GR activation and GR-mediated gene regulation, a decision stage that involves the counterbalancing influence of prosurvival and pro-apoptotic factors at the mitochondrial level, and the execution stage, which involves caspase and endonuclease activation (Fig 3). As a number of reviews have recently been published, which give a detailed description of the mitochondrial pathway and execution of apoptosis (Adams & Cory, 1998; Deveraux & Reed, 1999; Green, 2000), this section will focus on receptor activation and GR-mediated gene transcription regulation. Schematic representation of GC-induced apoptosis. 1. GCs (_) enter the cell through passive diffusion. 2. The glucocorticoid receptor (GR) resides in the cytoplasm in its inactive form as part of a heteromeric complex consisting of the receptor itself and several receptor-associated proteins (RAP): two molecules of heat shock protein (Hsp) 90, one molecule each of and and an of the and the binding of the receptor from the proteins The GR to the The GR binds to DNA and the transcription of The gene transcription can result in a 7. by the is and activation of The caspase which the of caspase activity through caspase caspase activation the cellular is resulting in the of apoptosis. GCs enter the cell through passive by their relatively and lipophilic et al, 1987). through the cell the steroid molecules to the GR binding, the receptor from its The occurring after ligand binding and domains for nuclear DNA binding and a process receptor the activated receptor to the In the cell nucleus, the activated receptor binds as a to the glucocorticoid or as to other transcription factors or nuclear the transcription by these factors & are DNA by the GR, usually located in of glucocorticoid results in increased gene transcription & or decreased gene transcription et al, 1987). It is a of which of these gene events is for GC-induced apoptosis. This may to be of However, have recently been described that or gene transcription et al, et al, This may possible to the activity of GCs from some of the other such as steroid and and of are among the few cell types that their apoptotic programme GC is no explanation for this of cells. efforts have been to attempt to the that the apoptotic after GC on the apoptosis et al, 1998; 2000), or to by et al, et al, of these have identified the for GC-induced apoptosis. Very recently the DNA have obtained to have identified that were in GC-induced apoptosis et al, 2001). of these was the GR which was Several of the other GC coding for in were It was that the of the GR in of that induced a with cell survival. the study of of resistant variants the past decades, the of resistance was to be the of low or of the GR et al in their study of DXM resistant of five lymphoid cell that of these variants were receptor as defined by their to that the were not to these the GR or the affinity of the receptor for the was including to the GR have identified that were to because of the of GR or an receptor, but one with reduced binding activity due to a in the binding domain & et al, & et al, 1995). of the GR, may influence receptor affinity to GC resistance et al, 1988). The receptor in GC resistant variants is the nuclear receptor & These receptors to the with low and DNA with lower than the receptor (Gehring & et al, 1990). These receptors are the result of in one of the nuclear localization cell line are in drug resistance may not mechanisms of resistance for several obtained ALL in vitro and the of paediatric ALL patients be this a cell line derived from an ALL a of the only the of that in vitro is the of in vitro may induce biological large number of studies about the between GR and the clinical response to GC or prognosis in childhood leukaemia were performed in the and early a detailed of these to the review of Kaspers et al Several the between GR and clinical response to GCs or that a lower number of receptors cell was with poor treatment response et al, et al, et al, et al, et al, but this was not et al, large between receptor in poor and good responders was et al, In addition, the cut-off of GR used in the different studies to with poor and good prognosis by et al to 16 by et al improvements in treatment protocols, the prognostic of GR et al, studies of GR at and at in childhood leukaemia no significant between and patients & 1986). In addition, of the GR were in paediatric AML and ALL despite the that the is resistant to GC-induced cell lysis et al, These have researchers to that clinical resistance to GCs is due to more in receptor properties or due to in the apoptotic pathway. affinity as determined by have been in of the clinical studies described However, reported a significant with treatment response et al, et al, et al, et al, & et al, et al, More recently, Van den et al a on the of the GR ligand interaction in several cell in binding affinity between and resistant of the line were at but not at that differences in binding affinity between resistant and paediatric leukaemia may have been as are usually performed at et al, et al, et al, & et al, or the primary may have been performed at in et al, et al, 1987). In with that differences in binding between and resistant childhood leukaemia are at of the ligand binding as described by den et al, were to that, from lower GR ALL and AML relatively resistant to GC-induced cell had receptors with reduced affinity at et al, of the activated receptor to the does not appear to be a of resistance in childhood lymphoblastic leukaemia. and demonstrated that was no between the of nuclear of the activated GR and the clinical response to PRD in ALL. of glucocorticoid resistance is the of the of the activated receptor with or with other transcription factors can be by clinical that is capable of GC of has been obtained in showed that in and peripheral blood from patients with was higher than from patients with or et al, 1999). only one study has been GRα and in paediatric leukaemia et al showed that in a leukaemia relatively resistant to GC-induced cell decreased were However, the number of patients was ALL and correlations with in vivo or in vitro treatment responses were not can that other splice variants by GRα and because of its reduced transactivating may be involved in the GC in childhood leukaemia. However, these splice variants to GC or resistance in clinical is have been identified in the coding and of the GR the use of glucocorticoids in the treatment of GR for the of glucocorticoid resistance in human have only been described in to & 1991; & is known about which are for GC-induced apoptosis even about the factors that which GC are However, some can be with to the between gene transcription and GC resistance. et al described a cell line in which of DNA was with the of glucocorticoid resistance. results that in the of involved in GC-induced cell lysis and thus the of GC resistance. a lymphoid cell line resistant to GC-induced apoptosis with a were to induce DNA and GC et al, In paediatric partial of GC resistance by of in only one of 16 acute lymphoblastic leukaemia et al, 2001). and proposed the on their that of the GR is a for GC-induced apoptosis the section on GC-induced cell that the response of GR to GCs in to GC-induced apoptosis and GR in that are may be by alternative The GR gene has several in the and et al, et al, of these to be used in of which are to the of studies to elucidate the human of this is used in cells. In addition, the mechanisms alternative to be and have also been in GC resistance et al, The of the has been linked to the of apoptosis through the mitochondrial pathway et al, In addition, has been that can GC hormones, thus GC and action. In with these et al showed that with in vitro drug resistance among other in ALL et al reported a significantly worse prognosis in of in iALL. the or activity of these compounds can be et al, 1995). acid inhibits by the binding site. by the inhibition of a in the However, et al not a significant between and PRD resistance. In addition, a study performed in our laboratory showed that acid and not in vitro GC resistance et al, 2001). Several demonstrated in cell line that or increased were with resistance to GCs & Reed, et al, et al, proteins have also been in childhood but no correlations with treatment response or in vitro GC resistance were et al, et al, 1996; et al, 1999). However, increased or increased were in a that is resistant to GC-induced cell kill et al, et al, 2000). The more recently described of apoptosis have been linked to resistance a large number of apoptotic including pro-apoptotic family various or and et al, 1998). obtained from the of were relatively resistant to GC-induced apoptosis both in vitro and in vivo et al, 2001). In addition, has been that of caspase and is for GC-induced cell lysis in thymocytes & 1999; et al, 2000). In this may be of that the of the proteins are by the et al, 1998). The activity of this transcription factor can be by the activated GR through interaction and by induction of the of et al, 1995; et al, no studies have been that the between activity and GC resistance in childhood leukaemia. is an of the and is used in its form as a in the and treatment of tumours is also a of et al, 1990). et al demonstrated that GC resistance in resistant cell The of GC-induced cell lysis was by a two to increase in affinity of receptors for binding and an increase in binding In with these our group has that GC-induced cell lysis in a number of resistant ALL et al, 2001). particular is the synthetic steroid This capable of binding to the GR with two is times more potent than DXM in lysis of et al, & 1990). was to induce cell lysis in several lymphoblastic leukaemia cell by binding and receptors et al, et al, no is on the clinical use of in and the pathway have been linked and to to apoptosis in human lymphoid leukaemic et al, 1998). This has opened the of using of the pathway to the cytolytic effects of glucocorticoids & et al, 1998). In one study, was that a of not only increased the rate of apoptotic cell in the relatively but also GC in the resistant derived from the human acute lymphoblastic leukaemia cell line et al, 1998). In several and lymphoblastic leukaemia cell and in of activity has been linked to GC et al, et al, 1999). Several including and are activated by In line with has been that activation resistant to a number of apoptosis including glucocorticoids et al, et al, 1999). the use of such as and reduced and leukaemic and to the of glucocorticoids et al, 1998; 2001). The of the apoptotic pathway has identified a large number of targets for The protein has been induce apoptosis and cellular drug resistance a large number of cytotoxic in various cell et al, et al, et al, 1995; et al, et al showed that in a cell line had no effect on cell but induced cell kill with DXM. of the protein in a cell line the to all et al, Alternative for modulating activity have been et al (2000), using a on the of a molecule = capable of binding and inactivating was to induce apoptosis in cells. agents, the and apoptosis by et al, 1999). However, has not been that of these can GC-induced cell no have been treatment in childhood ALL. The of prognostic significance of and the of significant correlations with GC resistance (in vitro or in in childhood reduced the in this type of However, this of does not that treatment has no impact on drug treatment can the activity of glucocorticoids to be More recently, have been described for and et al, et al, et al, et al described between and DXM in killing leukaemia cell activation of caspase 3 is a but no with this is in of has only been described in cell et al, 2000). The of the an protein that binds and inhibits has new for the of molecules et al, 2000). to extensive research in cell line models, much of the GC-induced apoptotic pathway and possible mechanisms of resistance have been on have not determined significant of GC resistance. This may partly be to the that these studies a study on results from is and to on mechanisms of resistance in More that are in cell line studies of a large number of and cell This is not possible with obtained specimens. the from studies on cell have with a wide range of potential targets for enhancing the cytolytic activity of including strategies that receptor that with the GR DNA or lower the apoptotic of these strategies have to be on paediatric leukaemia little is known about the gene events preceding GC-induced apoptosis. In this is to realize that lymphoid are among the few cell types that respond to GC exposure by initiating their apoptotic programme have been reported to inhibit apoptosis et al, 2001) or even induce cell proliferation & & et al, et al, Kaspers et al, This that the decision or not to GC exposure is at the level the apoptotic is one that not only in resistant tumours myeloid but also in lymphoblastic leukaemic cells, resistance results from the that the activated GR the transcription of a different of than in cells. It of major to the GC those involved in and elucidate the mechanisms that which GC are or the mechanisms the responses may be the to the of treatment that are to leukaemia to GC-induced apoptosis.