Jette Ford

Divergent mechanisms of glucocorticoid resistance in experimental models of pediatric acute lymphoblastic leukemia.

Cell line models of glucocorticoid resistance in childhood acute lymphoblastic leukemia (ALL) almost invariably exhibit altered glucocorticoid receptor (GR) function. However, these findings are incongruous with those using specimens derived directly from leukemia patients, in which GR alterations are rarely found. Consequently, mechanisms of glucocorticoid resistance in the clinical setting remain largely unresolved. We present a novel paradigm of glucocorticoid resistance in childhood ALL, in which patient biopsies have been directly established as continuous xenografts in immune-deficient mice, without prior in vitro culture. We show that the GRs from six highly dexamethasone-resistant xenografts (in vitro IC(50) >10 micromol/L) exhibit no defects in ligand-induced nuclear translocation and binding to a consensus glucocorticoid response element (GRE). This finding contrasts with five commonly used leukemia cell lines, all of which exhibited defective GRE binding. Moreover, whereas the GRs of dexamethasone-resistant xenografts were transcriptionally active, as assessed by the ability to induce the glucocorticoid-induced leucine zipper (GILZ) gene, resistance was associated with failure to induce the bim gene, which encodes a proapoptotic BH3-only protein. Furthermore, the receptor tyrosine kinase inhibitor, SU11657, completely reversed dexamethasone resistance in a xenograft expressing functional GR, indicating that pharmacologic reversal of glucocorticoid resistance in childhood ALL is achievable.

The gene expression signature of relapse in paediatric acute lymphoblastic leukaemia: implications for mechanisms of therapy failure

Despite significant improvements in the treatment of childhood acute lymphoblastic leukaemia (ALL), the prognosis for relapsing patients remains poor. The aim of this study was to generate a transcriptional profile of relapsed ALL to increase our understanding of the mechanisms involved in therapy failure. RNA was extracted from 11 pairs of cryopreserved pre‐B ALL bone marrow specimens taken from the same patients at diagnosis and relapse, and analysed using HG‐U133A microarrays. Relapse specimens overexpressed genes that are involved with cell growth and proliferation, in keeping with their aggressive phenotype. When tested in 72 independent specimens of pre‐B ALL and T‐ALL, the identified genes could successfully differentiate between diagnosis and relapse in either lineage, indicating the existence of relapse mechanisms common to both. These genes have functions relevant for oncogenesis, drug resistance and metastasis, but are not related to classical multidrug‐resistance pathways. Increased expression of the top‐ranked gene (BSG) at diagnosis was significantly associated with adverse outcome. Several chromosomal loci, including 19p13, were identified as potential hotspots for aberrant gene expression in relapsed ALL. Our results provide evidence for a link between drug resistance and the microenvironment that has previously only been considered in the context of solid tumour biology.

Altered glucose metabolism in childhood pre-B acute lymphoblastic leukaemia.

The cells of solid tumours are known to have an altered metabolism, with high rates of glucose uptake and glycolysis, which results in the excessive production of lactate. To date there has been no definitive research documenting metabolic changes in acute lymphoblastic leukaemia (ALL) cells. In order to investigate whether ALL cells have an altered metabolism, we initially compared the transcriptional profiles of 22 specimens from paediatric patients diagnosed with ALL to five CD34+ specimens isolated from bone marrow, which was verified in an independent cohort of 101 specimens. Profiling revealed the upregulation of genes facilitating glycolysis in the ALL specimens compared to the CD34+ specimens, while those involved in the tricarboxylic acid cycle were downregulated. Functional studies supported the microarray findings threefold: (1) higher expression of the glucose transport protein glucose transporter 1 in ALL compared to CD34+ specimens, (2) the excessive production of lactate in ALL cell lines and (3) sensitivity of ALL cell lines to the glycolysis inhibitor 2-deoxy-D-glucose. While metabolic alterations have been well documented in solid tumours, this is the first study to provide direct evidence for the existence of metabolic changes in the leukaemic cells of ALL patients. The finding offers new options for targeted therapy for ALL patients.

Glucocorticoid resistance in T-lineage acute lymphoblastic leukaemia is associated with a proliferative metabolism

Glucocorticoids (GCs) are among the most important drugs for acute lymphoblastic leukaemia (ALL), yet despite their clinical importance, the exact mechanisms involved in GC cytotoxicity and the development of resistance remain uncertain. We examined the baseline profile of a panel of T-ALL cell lines to determine factors that contribute to GC resistance without prior drug selection. Transcriptional profiling indicated GC resistance in T-ALL is associated with a proliferative phenotype involving upregulation of glycolysis, oxidative phosphorylation, cholesterol biosynthesis and glutamate metabolism, increased growth rates and activation of PI3K/AKT/mTOR and MYC signalling pathways. Importantly, the presence of these transcriptional signatures in primary ALL specimens significantly predicted patient outcome. We conclude that in lymphocytes the activation of bioenergetic pathways required for proliferation may suppress the apoptotic potential and offset the metabolic crisis initiated by GC signalling. It is likely that the link between GC resistance and proliferation in T-ALL has not been fully appreciated to date because such effects would be masked in the context of current multiagent therapies. The data also provide the first evidence that altered expression of wild-type MLL may contribute to GC-resistant phenotypes. Our findings warrant the continued development of selective metabolic inhibitors for the treatment of ALL.

High expression of connective tissue growth factor in pre-B acute lymphoblastic leukaemia

In recent years microarrays have been used extensively to characterize gene expression in acute lymphoblastic leukaemia (ALL). Few studies, however, have analysed normal haematopoietic cell populations to identify altered gene expression in ALL. We used oligonucleotide microarrays to compare the gene expression profile of paediatric precursor‐B (pre‐B) ALL specimens with two control cell populations, normal CD34+ and CD19+IgM− cells, to focus on genes linked to leukemogenesis. A set of eight genes was identified with a ninefold higher average expression in ALL specimens compared with control cells. All of these genes were significantly deregulated in an independent cohort of 101 ALL specimens. One gene, connective tissue growth factor (CTGF, also known as CCN2), had exceptionally high expression, which was confirmed in three independent leukaemia studies. Further analysis of CTGF expression in ALL revealed exclusive expression in B‐lineage, not T‐lineage, ALL. Within B‐lineage ALL approximately 75% of specimens were consistently positive for CTGF expression, however, specimens containing the E2A‐PBX1 translocation showed low or no expression. Protein studies using Western blot analysis demonstrated the presence of CTGF in ALL cell‐conditioned media. These findings indicate that CTGF is secreted by pre‐B ALL cells and may play a role in the pathophysiology of this disease.

In vitro cytotoxicity of nelarabine, clofarabine and flavopiridol in paediatric acute lymphoblastic leukaemia

The in vitro efficacies of three new drugs – clofarabine (CLOF), nelarabine (NEL) and flavopiridol (FP) – were assessed in a panel of acute lymphoblastic leukaemia (ALL) cell lines. The 50% inhibitory concentration (IC50) for CLOF across all lines was 188‐fold lower than that of NEL. B‐lineage, but not T‐lineage lines, were >7‐fold more sensitive to CLOF than cytosine arabinoside (ARAC). NEL IC50 was 25‐fold and 113‐fold higher than ARAC in T‐ and B‐lineage, respectively. T‐ALL cells were eightfold more sensitive to NEL than B‐lineage but there was considerable overlap. FP was more potent in vitro than glucocorticoids and thiopurines and at doses that recent phase I experience predicts will translate into clinical efficacy. Potential cross‐resistance of CLOF, NEL and FP was observed with many front‐line ALL therapeutics but not methotrexate or thiopurines. Methotrexate sensitivity was inversely related to that of NEL and FP. Whilst NEL was particularly effective in T‐ALL, a subset of patients with B‐lineage ALL might also be sensitive. CLOF appeared to be marginally more effective in B‐lineage than T‐ALL and has a distinct resistance profile that may prove useful in combination with other compounds. FP should be widely effective in ALL if sufficient plasma levels can be achieved clinically.

Prediction of relapse in paediatric pre-B acute lymphoblastic leukaemia using a three-gene risk index

Despite high cure rates 25% of children with acute lymphoblastic leukaemia (ALL) relapse and have dismal outcome. Crucially, many are currently stratified as standard risk (SR) and additional markers to improve patient stratification are required. Here we have used diagnostic bone marrow specimens from 101 children with pre‐B ALL to examine the use of gene expression profiles (GEP) as predictors of long‐term clinical outcome. Patients were divided into two cohorts for model development and validation based on availability of specimen material. Initially, GEP from 55 patients with sufficient material were analysed using HG‐U133A microarrays, identifying an 18‐gene classifier (GC) that was more predictive of outcome than conventional prognostic parameters. After feature selection and validation of expression levels by quantitative reverse transcription polymerase chain reaction (qRT‐PCR), a three‐gene qRT‐PCR risk index [glutamine synthetase (GLUL), ornithine decarboxylase antizyme inhibitor (AZIN), immunoglobulin J chain (IGJ)] was developed that predicted outcome with an accuracy of 89% in the array cohort and 87% in the independent validation cohort. The data demonstrate the feasibity of using GEP to improve risk stratification in childhood ALL. This is particularly important for the identification of patients destined to relapse despite their current stratification as SR, as more intensive front‐line treatment options for these individuals are already available.

Interactions between acute lymphoblastic leukemia and bone marrow stromal cells influence response to therapy

The cure rate for pediatric patients with B precursor acute lymphoblastic leukemia (pre-B ALL) is steadily improving, however relapses do occur despite initial response to therapy. To identify links between drug resistance and gene deregulation we used oligonucleotide microarray technology and determined in 184 pre-B ALL specimen genes differentially expressed compared to normal CD34(+) specimens. We identified 20 signature genes including CTGF, BMP-2, CXCR4 and IL7R, documented to regulate interactions in the bone marrow. We recorded remarkably similar levels of expression in three independent patient cohorts, and found distinct patterns in cytogenetically defined subgroups of pre-B ALL. The canonical pathways that were affected are involved in inter- and intra-cellular communication, regulating signaling within the microenvironment. We tested experimentally whether interaction with stromal cells conferred protection to four drugs used in current ALL therapy, and demonstrated that bone marrow stromal cells significantly influenced resistance to vincristine and cytosine arabinoside. Compounds designed to block the identified cellular interactions within the bone marrow microenvironment are expected to mobilise the leukemic cells and make them more accessible to contemporary antileukemic agents. The data provide novel insight into the pathobiology of ALL and indicate new therapeutic targets for patients with ALL.

A microarray model system identifies potential new target genes of the proto-oncogene HOX11

HOX11 is a homeobox gene originally identified at a chromosomal breakpoint in T‐cell acute lymphoblastic leukemia (T‐ALL). It is one of the most frequently deregulated genes in T‐ALL, although the precise role of HOX11 in leukemogenesis as well as in normal development remains obscure. To gain more insight into the functional role of HOX11, we utilized a microarray model system to characterize the gene expression network that it directs. Using one of our T‐ALL cell lines that had been stably transfected to express HOX11 and high‐density oligonucleotide HG‐U95A arrays, we identified a large number of differentially expressed genes in response to the enforced expression of HOX11. We focused on examining genes found to be up‐regulated according to the microarray analysis and selected three putative target genes, NFKB2, SMARCD3, and NR4A3, for further investigation. We could not only confirm the up‐regulation of NR4A3 by an independent method in all clones expressing HOX11, but luciferase reporter assays demonstrated that the effect that HOX11 exerted on the proximal promoter of NR4A3 was dependent on the presence of an intact homeodomain, providing support for the idea that HOX11 manifests its regulatory function via its action as a transcription factor.

A new pineoblastoma cell line, PER-480, with der(10)t(10;17), der(16)t(1;16), and enhanced MYC expression in the absence of gene amplification

Pineoblastoma is a rare, but highly malignant tumor of the central nervous system (CNS) in children and is classified as a central primitive neuroectodermal tumor (PNET). Despite notable recent advances in understanding the molecular genetic basis of malignancies, the pathogenesis of PNETs remains enigmatic. There is scant information on the cytogenetics of PNETs arising in the pineal gland and the only three reported cases did not show any common aberrations. Here we report the establishment and characterization of a new pineoblastoma cell line, PER-480. The biopsy material and the cell line were characterized using light and electron microscopy and immunohistochemical analyses. The cell line was examined for expression of cell surface markers using a panel of monoclonal antibodies and by cytogenetic analysis. MYC family genes were studied at the DNA, RNA, and protein level. Cell line PER-480 showed neuronal differentiation and the karyotype demonstrated two abnormalities, a der(10)t(10;17) and a der(16)t(1;16). An intriguing finding is that all three pineoblastoma cell lines established in our laboratory, PER-452, PER-453, and PER-480, showed enhanced expression but not amplification of a member of the MYC family of proto-oncogenes. Cell line PER-480 reported here will be useful for the further investigation of the molecular genetic basis of central PNETs.