Systematic chemical and molecular profiling of MLL-rearranged infant acute lymphoblastic leukemia reveals efficacy of romidepsin.

To address the poor prognosis of mixed lineage leukemia (MLL)-rearranged infant acute lymphoblastic leukemia (iALL), we generated a panel of cell lines from primary patient samples and investigated cytotoxic responses to contemporary and novel Food and Drug Administration-approved chemotherapeutics. To characterize representation of primary disease within cell lines, molecular features were compared using RNA-sequencing and cytogenetics. High-throughput screening revealed variable efficacy of currently used drugs, however identified consistent efficacy of three novel drug classes: proteasome inhibitors, histone deacetylase inhibitors and cyclin-dependent kinase inhibitors. Gene expression of drug targets was highly reproducible comparing iALL cell lines to matched primary specimens. Histone deacetylase inhibitors, including romidepsin (ROM), enhanced the activity of a key component of iALL therapy, cytarabine (ARAC) in vitro and combined administration of ROM and ARAC to xenografted mice further reduced leukemia burden. Molecular studies showed that ROM reduces expression of cytidine deaminase, an enzyme involved in ARAC deactivation, and enhances the DNA damage-response to ARAC. In conclusion, we present a valuable resource for drug discovery, including the first systematic analysis of transcriptome reproducibility in vitro, and have identified ROM as a promising therapeutic for MLL-rearranged iALL.

Silencing of GATA3 defines a novel stem cell-like subgroup of ETP-ALL.

BACKGROUND: GATA3 is pivotal for the development of T lymphocytes. While its effects in later stages of T cell differentiation are well recognized, the role of GATA3 in the generation of early T cell precursors (ETP) has only recently been explored. As aberrant GATA3 mRNA expression has been linked to cancerogenesis, we investigated the role of GATA3 in early T cell precursor acute lymphoblastic leukemia (ETP-ALL). METHODS: We analyzed GATA3 mRNA expression by RT-PCR (n = 182) in adult patients with T-ALL. Of these, we identified 70 of 182 patients with ETP-ALL by immunophenotyping. DNA methylation was assessed genome wide (Illumina Infinium® HumanMethylation450 BeadChip platform) in 12 patients and GATA3-specifically by pyrosequencing in 70 patients with ETP-ALL. The mutational landscape of ETP-ALL with respect to GATA3 expression was investigated in 18 patients and validated by Sanger sequencing in 65 patients with ETP-ALL. Gene expression profiles (Affymetrix Human genome U133 Plus 2.0) of an independent cohort of adult T-ALL (n = 83) were used to identify ETP-ALL and investigate GATA3low and GATA3high expressing T-ALL patients. In addition, the ETP-ALL cell line PER-117 was investigated for cytotoxicity, apoptosis, GATA3 mRNA expression, DNA methylation, and global gene expression before and after treatment with decitabine. RESULTS: In our cohort of 70 ETP-ALL patients, 33 % (23/70) lacked GATA3 expression and were thus defined as GATA3low. DNA methylation analysis revealed a high degree of GATA3 CpG island methylation in GATA3low compared with GATA3high ETP-ALL patients (mean 46 vs. 21 %, p < 0.0001). Genome-wide expression profiling of GATA3low ETP-ALL exhibited enrichment of myeloid/lymphoid progenitor (MLP) and granulocyte/monocyte progenitor (GMP) genes, while T cell-specific signatures were downregulated compared to GATA3high ETP-ALL. Among others, FLT3 expression was upregulated and mutational analyses demonstrated a high rate (79 %) of FLT3 mutations. Hypomethylating agents induced reversal of GATA3 silencing, and gene expression profiling revealed downregulation of hematopoietic stem cell genes and upregulation of T cell differentiation. CONCLUSIONS: We propose GATA3low ETP-ALL as a novel stem cell-like leukemia with implications for the use of myeloid-derived therapies.

High expression of connective tissue growth factor accelerates dissemination of leukaemia.

To improve treatment of acute lymphoblastic leukaemia (ALL), a better understanding of disease development is needed to tailor new therapies. Connective tissue growth factor (CTGF/CCN2) is highly expressed in leukaemia cells from the majority of paediatric patients with B-lineage ALL (pre-B ALL). CTGF is a matricellular protein and plays a role in aggressive cancers. Here we have genetically engineered leukaemia cells to modulate CTGF expression levels. Elevated CTGF levels accelerated disease dissemination and reduced survival in NOD/SCID mice. In vitro studies showed that CTGF protein induces stromal cell proliferation, promotes adhesion of leukaemia cells to stromal cells and leads to overexpression of genes associated with cell cycle and synthesis of extracellular matrix (ECM). Corresponding data from our leukaemia xenograft models demonstrated that CTGF leads to increased proliferation of non-leukaemia cells and deposition of ECM in the bone marrow. We document for the first time a functional role of CTGF in altering disease progression in a lymphoid malignancy. The findings provide support for targeting the bone marrow microenvironment in aggressive forms of leukaemia.

A novel BRD4-NUT fusion in an undifferentiated sinonasal tumor highlights alternative splicing as a contributing oncogenic factor in NUT midline carcinoma.

NUT midline carcinoma (NMC) is a fatal cancer that arises in various tissues along the upper midline of the body. The defining molecular feature of NMC is a chromosomal translocation that joins (in the majority of cases) the nuclear testis gene NUT (NUTM1) to the bromodomain protein family member 4 (BRD4) and thereby creating a fusion oncogene that disrupts cellular differentiation and drives the disease. In this study, we report the case of an adolescent NMC patient presenting with severe facial pain, proptosis and visual impairment due to a mass arising from the ethmoid sinus that invaded the right orbit and frontal lobe. Treatment involved radical resection, including exenteration of the affected eye with the view to consolidate treatment with radiation therapy; however, the patient experienced rapid tumor progression and passed away 79 days post resection. Molecular analysis of the tumor tissue identified a novel in-frame BRD4-NUT transcript, with BRD4 exon 15 fused to the last 124 nucleotides of NUT exon 2 (BRD4-NUT ex15:ex2Δnt1-585). The partial deletion of NUT exon 2 was attributed to a mid-exonic genomic breakpoint and the subsequent activation of a cryptic splice site further downstream within the exon. Inhibition of the canonical 3' acceptor splice site of NUT intron 1 in cell lines expressing the most common NMC fusion transcripts (PER-403, BRD4-NUT ex11:ex2; PER-624, BRD4-NUT ex15:ex2) induced alternative splicing from the same cryptic splice site as identified in the patient. Detection of low levels of an in-frame BRD4-NUT ex11:ex2Δnt1-585 transcript in PER-403 confirmed endogenous splicing from this alternative exon 2 splice site. Although further studies are necessary to assess the clinical relevance of the increasing number of variant fusions described in NMC, the findings presented in this case identify alternative splicing as a mechanism that contributes to this pathogenic complexity.

Novel CT domain-encoding splice forms of CTGF/CCN2 are expressed in B-lineage acute lymphoblastic leukaemia.

INTRODUCTION: Connective tissue growth factor (CTGF/CCN2) has been shown previously to be aberrantly expressed in a high proportion of paediatric precursor B cell acute lymphoblastic leukaemia (pre-B ALL), suggesting a potential oncogenic role in this tumour type. We therefore assessed CTGF mRNA transcript diversity in B-lineage ALL using primary patient specimens and cell lines. METHODS: CTGF mRNA expression was evaluated by quantitative real-time PCR and Northern blotting. We performed a structural analysis of CTGF mRNA by nested reverse-transcriptase PCR and examined CTGF protein diversity by immunoblotting. RESULTS: Northern blot analysis of pre-B ALL cell lines revealed short CTGF transcripts that were expressed in association with the active phase of cellular growth. Structural analysis confirmed the synthesis of several novel CTGF mRNA isoforms in B-lineage ALL cell lines that were uniformly characterised by the retention of the coding sequence for the C-terminal (CT) domain. One of these novel spliceforms was expressed in a majority (70%) of primary pre-B ALL patient specimens positive for canonical CTGF mRNA. Evidence that these alternative transcripts have coding potential was provided by cryptic CTGF proteins of predicted size detected by immunoblotting. CONCLUSION: This study identifies for the first time alternative splicing of the CTGF gene and shows that a short CTGF splice variant associated with cell proliferation is expressed in most cases of primary CTGF-positive pre-B ALL. This novel variant encoding only the CT domain may play a role in pre-B ALL tumorigenesis and/or progression.

A pre-clinical model of resistance to induction therapy in pediatric acute lymphoblastic leukemia.

Relapse and acquired drug resistance in T-cell acute lymphoblastic leukemia (T-ALL) remains a significant clinical problem. This study was designed to establish a preclinical model of resistance to induction therapy in childhood T-ALL to examine the emergence of drug resistance and identify novel therapies. Patient-derived T-ALL xenografts in immune-deficient (non-obese diabetic/severe combined immunodeficient) mice were exposed to a four-drug combination of vincristine, dexamethasone (DEX), L-asparaginase and daunorubicin (VXLD). 'Relapse' xenografts were characterized by responses to drugs, changes in gene expression profiles and Connectivity Map (CMap) prediction of strategies to reverse drug resistance. Two of four xenografts developed ex vivo and in vivo drug resistance. Both resistant lines showed altered lipid and cholesterol metabolism, yet they had a distinct drug resistance pattern. CMap analyses reinforced these features, identifying the cholesterol pathway inhibitor simvastatin (SVT) as a potential therapy to overcome resistance. Combined ex vivo with DEX, SVT was significantly synergistic, yet when administered in vivo with VXLD it did not delay leukemia progression. Synergy of SVT with established chemotherapy may depend on higher drug doses than are tolerable in this model. Taken together, we have developed a clinically relevant in vivo model of T-ALL suitable to examine the emergence of drug resistance and to identify novel therapies.

The evolution of clinical trials for infant acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) in infants has a significantly inferior outcome in comparison with older children. Despite initial improvements in survival of infants with ALL since establishment of the first pediatric cooperative group ALL trials, the poor outcome has plateaued in recent years. Historically, infants were treated on risk-adapted childhood ALL protocols. These studies were pivotal in identifying the need for infant-specific protocols, delineating prognostic categories and the requirement for a more unified approach between study groups to overcome limitations in accrual because of low incidence. This subsequently led to the development of collaborative infant-specific studies. Landmark outcomes have included the elimination of cranial radiotherapy following the discovery of intrathecal and high-dose systemic therapy as a superior and effective treatment strategy for central nervous system disease prophylaxis, with improved neurodevelopmental outcome. Universal prospective identification of independent adverse prognostic factors, including presence of a mixed lineage leukemia rearrangement and young age, has established the basis for risk stratification within current trials. The infant-specific trials have defined limits to which conventional chemotherapeutic agents can be intensified to optimize the balance between treatment efficacy and toxicity. Despite variations in therapeutic intensity, there has been no recent improvement in survival due to the equilibrium between relapse and toxicity. Ultimately, to improve the outcome for infants with ALL, key areas still to be addressed include identification and adaptation of novel prognostic markers and innovative therapies, establishing the role of hematopoietic stem cell transplantation in first complete remission, treatment strategies for relapsed/refractory disease and monitoring and timely intervention of late effects in survivors. This would be best achieved through a single unified international trial.

Comparative drug screening in NUT midline carcinoma.

BACKGROUND: The NUT midline carcinoma (NMC) is a rare but fatal cancer for which systematic testing of therapy options has never been performed. METHODS: On the basis of disease biology, we compared the efficacy of the CDK9 inhibitor flavopiridol (FP) with a panel of anticancer agents in NMC cell lines and mouse xenografts. RESULTS: In vitro anthracyclines, topoisomerase inhibitors, and microtubule poisons were among the most cytotoxic drug classes for NMC cells, while efficacy of the bromodomain inhibitor JQ1 varied considerably between lines carrying different BRD4 (bromodomain-containing protein 4)-NUT (nuclear protein in testis) translocations. Efficacy of FP was comparable to vincristine and doxorubicin, drugs that have been previously used in NMC patients. All three compounds showed significantly better activity than etoposide and vorinostat, agents that have also been used in NMC patients. Statins and antimetabolites demonstrated intermediate single-agent efficacy. In vivo, vincristine significantly inhibited tumour growth in two different NMC xenografts. Flavopiridol in vivo was significantly effective in one of the two NMC xenograft lines, demonstrating the biological heterogeneity of this disease. CONCLUSIONS: These results demonstrate that FP may be of benefit to a subset of patients with NMC, and warrant a continued emphasis on microtubule inhibitors, anthracyclines, and topoisomerase inhibitors as effective drug classes in this disease.

Novel BRD4-NUT fusion isoforms increase the pathogenic complexity in NUT midline carcinoma.

Nuclear protein in testis (NUT)-midline carcinoma (NMC) is a rare, aggressive disease typically presenting with a single t(15;19) translocation that results in the generation of a bromodomain-containing protein 4 (BRD4)-NUT fusion. PER-624 is a cell line generated from an NMC patient with an unusually complex karyotype that gave no initial indication of the involvement of the NUT locus. Analysis of PER-624 next-generation transcriptome sequencing (RNA-Seq) using the algorithm FusionFinder identified a novel transcript in which Exon 15 of BRD4 was fused to Exon 2 of NUT, therefore differing from all published NMC fusion transcripts. The three additional exons contained in the PER-624 fusion encode a series of polyproline repeats, with one predicted to form a helix. In the NMC cell line PER-403, we identified the 'standard' NMC fusion and two novel isoforms. Knockdown by small interfering RNA in either cell line resulted in decreased proliferation, increased cell size and expression of cytokeratins consistent with epithelial differentiation. These data demonstrate that the novel BRD4-NUT fusion in PER-624 encodes a functional protein that is central to the oncogenic mechanism in these cells. Genomic PCR indicated that in both PER-624 and PER-403, the translocation fuses an intron of BRD4 to a region upstream of the NUT coding sequence. Thus, the generation of BRD4-NUT fusion transcripts through post-translocation RNA-splicing appears to be a common feature of these carcinomas that has not previously been appreciated, with the mechanism facilitating the expression of alternative isoforms of the fusion. Finally, ectopic expression of wild-type NUT, a protein normally restricted to the testis, could be demonstrated in PER-403, indicating additional pathways for aberrant cell signaling in NMC. This study contributes to our understanding of the genetic diversity of NMC, an important step towards finding therapeutic targets for a disease that is refractory to current treatments.

MYCN sensitizes neuroblastoma to the MDM2-p53 antagonists Nutlin-3 and MI-63.

MYCN amplification is a major biomarker of poor prognosis, occurring in 25-30% of neuroblastomas. MYCN has contradictory roles in promoting cell growth and sensitizing cells to apoptosis. We have recently shown that p53 is a direct transcriptional target of MYCN in neuroblastoma and that p53-mediated apoptosis may be an important mechanism of MYCN-induced apoptosis. Although p53 mutations are rare in neuroblastoma at diagnosis, the p53/MDM2/p14(ARF) pathway is often inactivated through MDM2 amplification or p14(ARF) inactivation. We hypothesized that reactivation of p53 by inhibition of its negative regulator MDM2, using the MDM2-p53 antagonists Nutlin-3 and MI-63, will result in p53-mediated growth arrest and apoptosis especially in MYCN-amplified cells. Using the SHEP Tet21N MYCN-regulatable system, MYCN(-) cells were more resistant to both Nutlin-3 and MI-63 mediated growth inhibition and apoptosis compared with MYCN(+) cells and siRNA-mediated knockdown of MYCN in four MYCN-amplified cell lines resulted in decreased p53 expression and activation, as well as decreased levels of apoptosis following treatment with MDM2-p53 antagonists. In a panel of 18 neuroblastoma cell lines treated with Nutlin-3 and MI-63, the subset amplified for MYCN had a significantly lower mean GI(50) value (50% growth inhibition) and increased caspase 3/7 activity compared with the non-MYCN-amplified group of cell lines, but p53 mutant cell lines were resistant to the antagonists regardless of MYCN status. We conclude that amplification or overexpression of MYCN sensitizes neuroblastoma cell lines with wild-type p53 to MDM2-p53 antagonists and that these compounds may therefore be particularly effective in treating high-risk MYCN-amplified disease.

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.

BRD-NUT oncoproteins: a family of closely related nuclear proteins that block epithelial differentiation and maintain the growth of carcinoma cells.

An unusual group of carcinomas, here termed nuclear protein in testis (NUT) midline carcinomas (NMC), are characterized by translocations that involve NUT, a novel gene on chromosome 15. In about 2/3rds of cases, NUT is fused to BRD4 on chromosome 19. Using a candidate gene approach, we identified two NMCs harboring novel rearrangements that result in the fusion of NUT to BRD3 on chromosome 9. The BRD3-NUT fusion gene encodes a protein composed of two tandem chromatin-binding bromodomains, an extra-terminal domain, a bipartite nuclear localization sequence, and almost the entirety of NUT that is highly homologous to BRD4-NUT. The function of NUT is unknown, but here we show that NUT contains nuclear localization and export sequences that promote nuclear-cytoplasmic shuttling via a leptomycin-sensitive pathway. In contrast, BRD3-NUT and BRD4-NUT are strictly nuclear, implying that the BRD moiety retains NUT in the nucleus via interactions with chromatin. Consistent with this idea, FRAP studies show that BRD4, BRD4-NUT and BRD3-NUT have significantly slower rates of lateral nuclear diffusion than that of NUT. To investigate the functional role of BRD-NUT fusion proteins in NMCs, we investigated the effects of siRNA-induced BRD3-NUT and BRD4-NUT withdrawal. Silencing of these proteins in NMC cell lines resulted in squamous differentiation and cell cycle arrest. Together, these data suggest that BRD-NUT fusion proteins contribute to carcinogenesis by associating with chromatin and interfering with epithelial differentiation.

Authenticity and drug resistance in a panel of acute lymphoblastic leukaemia cell lines.

Cell lines are important models for drug resistance in acute lymphoblastic leukaemia (ALL), but are often criticised as being unrepresentative of primary disease. There are also doubts regarding the authenticity of many lines. We have characterised a panel of ALL cell lines for growth and drug resistance and compared data with that published for primary patient specimens. In contrast to the convention that cell lines are highly proliferative, those established in our laboratory grow at rates similar to estimates of leukaemic cells in vivo (doubling time 53-442 h). Authenticity was confirmed by genetic fingerprinting, which also demonstrated the potential stability of long-term cultures. In vitro glucocorticoid resistance correlated well with that measured ex vivo, but all lines were significantly more sensitive to vincristine than primary specimens. Sensitivity to methotrexate was inversely correlated to that of glucocorticoids and L-asparaginase, indicating possible reciprocity in resistance mechanisms. A cell line identified as highly methotrexate resistant (IC50 > 8000-fold higher than other lines) was derived from a patient receiving escalating doses of the drug, indicating in vivo selection of resistance as a cause of relapse. Many of these lines are suitable as models to study naturally occurring resistance phenotypes in paediatric ALL.

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.

The nuclear oncoprotein TLX1/HOX11 associates with pericentromeric satellite 2 DNA in leukemic T-cells.

TLX1/HOX11, a DNA-binding homeodomain protein, was originally identified by virtue of its aberrant expression in T-cell leukemia and subsequently found to be crucial for normal spleen development. The precise mechanism of TLX1 function remains poorly understood, although it is known that it can act as both a transcriptional activator and repressor and can downregulate the Aldh1a1 gene in embryonic mouse spleen. Using a whole-genome PCR approach, we show here that TLX1 protein directly interacts with pericentromeric human satellite 2 DNA sequences. Such DNA is known to localize to heterochromatin, which among other roles has been implicated in gene silencing. The interaction was confirmed in vitro and in vivo by gel retardation and chromatin immunoprecipitation assays involving satellite 2 DNA, which contained sequences resembling TLX1 binding sites. Using immunofluorescence microscopy, TLX1 demonstrated a punctate pattern of staining in the nuclei of leukemic T-cells (ALL-SIL). Double labelling indicated that TLX1 colocalized with the centromeric protein CENP-B, demonstrating that the TLX1 foci corresponded to clusters of centromeric DNA. The novel interaction of TLX1 with constitutive heterochromatin adds an additional level of complexity to the intracellular functions of this transcriptional regulator and may have relevance to its roles in transcriptional repression and T-cell immortalization.

Expression profiles of acute lymphoblastic and myeloblastic leukemias with ALL-1 rearrangements.

The ALL-1 gene is directly involved in 5-10% of acute lymphoblastic leukemias (ALLs) and acute myeloid leukemias (AMLs) by fusion to other genes or through internal rearrangements. DNA microarrays were used to determine expression profiles of ALLs and AMLs with ALL-1 rearrangements. These profiles distinguish those tumors from other ALLs and AMLs. The expression patterns of ALL-1-associated tumors, in particular ALLs, involve oncogenes, tumor suppressors, antiapoptotic genes, drug-resistance genes, etc., and correlate with the aggressive nature of the tumors. The genes whose expression differentiates between ALLs with and without ALL-1 rearrangement were further divided into several groups, enabling separation of ALL-1-associated ALLs into two subclasses. One of the groups included 43 genes that exhibited expression profiles closely linked to ALLs with ALL-1 rearrangements. Further, there were evident differences between the expression profiles of AMLs in which ALL-1 had undergone fusion to other genes and AMLs with partial duplication of ALL-1. The extensive analysis described here pinpointed genes that might have a direct role in pathogenesis.

Expression of HOX11 in childhood T-lineage acute lymphoblastic leukaemia can occur in the absence of cytogenetic aberration at 10q24: a study from the Children's Cancer Group (CCG).

Clonal genetic aberrations in tumour cells provide critical information for the development of new diagnostic and therapeutic strategies for patients. In paediatric T-cell acute lymphoblastic leukaemia (T-ALL) chromosomal translocations are present in 30-35% of cases. HOX11 and the closely related HOX11L2 genes play a key role in T-ALL. HOX11 is aberrantly activated by either of the two chromosomal translocations, t(7;10) and t(10;14). In this study, HOX11 expression levels were measured by real-time quantitative reverse-transcriptase polymerase chain reaction. We show that leukaemic blasts from 15/76 (19.7%) paediatric T-ALL patients expressed the HOX11 gene at high level and 22/76 (28.9%) at low level, yet the reported frequency for chromosomal rearrangement of 10q24 is 4-7%. Direct cytogenetic analysis revealed that only 2/16 specimens that showed HOX11 expression exhibited abnor-malities at 10q24. These results confirm and extend our previously published findings, and implicate mechanisms other than gross chromosomal translocations for the deregulation of HOX11. Analysis of clinical outcome for the whole study group showed a trend for better outcome for patients with leukaemic blasts expressing HOX11 at high level. A statistically significant difference in clinical outcome was found in a subgroup of 20 patients treated for high-risk disease on CCG-1901 from the Children's Cancer Group, where HOX11 expression in leukaemic blasts conferred a prognostic advantage (P=0.01).

A complex containing PBX2 contributes to activation of the proto-oncogene HOX11.

Ectopic expression of the homeobox gene HOX11 is associated with a significant proportion of childhood T-cell acute lymphoblastic leukaemias (T-ALLs). We hypothesise that one mechanism of gene deregulation involves overcoming the silencing mechanism(s) of gene expression present in normal cells. Here, we describe a search for trans-acting factors that control transcriptional activity from a distal 5' region of the HOX11 promoter. We have identified a region of this promoter which contributes significantly to HOX11 activation and two distinct regulatory elements are involved. First, a PBX2 Regulatory Element PRE-1048 has been identified which contains a novel DNA-binding sequence and mediates significant activation of the HOX11 gene in K562 cells. This is the first report of a homeobox gene being specifically regulated by PBX2 and the second report of a vertebrate homeobox target gene of a PBX protein. The PREP1 protein was also shown to be part of the PRE-1048-binding complex. The other regulatory element we describe here RE-1019 contains little sequence conservation to known transcription control elements. It appears that this element is a novel sequence that binds an as yet unidentified factor, mediating significant activation of the HOX11 gene in K562 cells. This is the first detailed report of elements that mediate regulation of the proto-oncogene HOX11.