- Open Access
Influence of wild-type MLL on glucocorticoid sensitivity and response to DNA-damage in pediatric acute lymphoblastic leukemia
© Beesley et al; licensee BioMed Central Ltd. 2010
Received: 11 January 2010
Accepted: 28 October 2010
Published: 28 October 2010
Rearrangement of the mixed-lineage leukemia gene (MLL) is found in 80% of infant acute lymphoblastic leukemia (ALL) and is associated with poor prognosis and resistance to glucocorticoids (GCs). We have recently observed that GC resistance in T-ALL cell lines is associated with a proliferative metabolism and reduced expression of MLL. In this study we have further explored the relationship between MLL status and GC sensitivity.
Negative correlation of MLL expression with GC resistance in 15 T-ALL cell lines was confirmed by quantitative RT-PCR. The absence of MLL-rearrangements suggested that this relationship represented expression of wild-type MLL. Analysis of MLL expression patterns revealed a negative relationship with cellular metabolism, proliferation and anti-apoptotic transcriptional networks. In silico analysis of published data demonstrated that reduced levels of MLL mRNA are associated with relapse and prednisolone resistance in T-ALL patients and adverse clinical outcome in children with MLL-rearranged ALL. RNAi knockdown of MLL expression in T-ALL cell lines significantly increased resistance to dexamethasone and gamma irradiation indicating an important role for wild-type MLL in the control of cellular apoptosis.
The data suggests that reduced expression of wild-type MLL can contribute to GC resistance in ALL patients both with and without MLL-translocations.
Among pediatric subtypes of acute lymphoblastic leukemia (ALL), infants and those with T-lineage ALL are particularly resistant to glucocorticoids (GCs), one of the most important classes of drug for this disease . Rearrangement of the mixed lineage leukemia gene (MLL) gene affects 80% of ALL in infants and is associated with a particularly poor prognosis [2, 3]. MLL is located at 11q23 and encodes a histone methyltransferase that through its regulation of HOX genes is essential for normal mammalian development and hematopoiesis . A unique feature of the MLL locus is that it is subject to an extremely wide variety of rearrangements, including translocations with >50 partner genes on various chromosomes, as well as deletions, inversions, internal duplications and gene amplifications [4–6]. There are conflicting reports on the relative GC responses of patients with different MLL translocations [7, 8], but those with t(4;11) translocations appear particularly resistant [3, 8, 9]. The biological basis for the documented GC resistance of patients with MLL-disease has not been explored but has generally been assumed to be due to the oncogenic effects of translocated MLL fusion proteins.
Despite the clinical importance of GCs for the treatment of ALL, detailed knowledge about the transduction pathways leading to GC-induced apoptosis in lymphoid tissues remains limited . Recently we performed transcriptional profiling of a panel of T-ALL cell lines and reported that GC resistance was associated with a proliferative metabolism . We also observed that GC resistance profiles were significantly correlated with reduced expression of MLL. In this study we have further investigated the relationship between MLL expression and GC sensitivity in T-ALL and provide evidence that it is the wild-type expression of the gene, rather than the effect of translocations, that appears to be critical for determining a resistant phenotype. This novel finding may help to explain why GC-resistance is a common feature of most patients with MLL-disease despite the wide variety of possible gene rearrangements
Cell lines and drug sensitivity profiling
The cell line panel has been previously described and comprised nine T-ALL lines derived in our own laboratory from pediatric ALL bone marrow specimens (PER cell lines), plus six additional T-ALL cell lines obtained from external sources [12, 13]. Cell lines were grown in RPMI-1640 supplemented with 2 mM L-glutamine, 10 nM 2-mercaptoethanol and 10-20% heat-inactivated fetal calf serum. The media for PER-cell lines contained additional non-essential amino acids and pyruvate, whilst 300 units/ml interleukin-2 is required for growth of PER-427 and PER-487. The sensitivity of the T-ALL cell lines to methylprednisolone (MPRED) and dexamethasone (DEX) has been previously published  and was measured using the MTT assay with drugs incubated over four days. The IC50 (drug concentration that inhibits cell growth by 50%) was used as the measure of drug resistance.
Gene Expression Profiling
Briefly, RNA was extracted from cell lines in exponential growth phase and hybridized to Affymetrix HG-U133A microarrays [11, 14]. Microarray data were normalized using robust multi-array analysis (RMA) and all passed quality control criteria for noise, background, absent/present calls, and 3'/5' signal ratios for ACTB and GAPDH. To interrogate the biological pathways represented by MLL expression profiles we used Gene Set Enrichment Analysis (GSEA) . The median value of the five MLL probe sets present on the HG-U133A was calculated for each cell line, and correlated across the panel against all other probe sets on the array using Pearson's correlation as metric (GSEA v2.0, May 2006, 10,000 permutations). GSEA examines ranked lists of genes for enrichment of biological pathways contained within four different databases: C1 (genomic loci), C2 (curated biological pathways), C3 (genes with common regulatory motifs), and C4 (computational gene networks). Since not all genes within a given biological pathway are expected to be regulated in the same direction, rankings were performed using absolute correlation values as previously described . Published microarray data used for in silico analysis [14, 16–18] was downloaded from publicly available depositories or authors' websites.
Real-time quantitative RT-PCR
Real-time quantitative RT-PCR (qRT-PCR) was performed on total RNA from cell lines in accordance with standard Applied Biosystems protocols (Foster City, CA) and in accordance with our published methods . All experiments were run in duplicates on an ABI 7700 sequence detector and data normalized to expression of beta-actin (ACTB). Primers and probe for MLL and GILZ qRT-PCR were purchased from Applied Biosystems (ABI Assays on Demand); the MLL assay targeted exons 30-31 (Refseq NM_005933).
RNAi knockdown of MLL expression
Three pSM2 retroviral RNAi vectors for MLL (V2HS_196843, V2HS_198375, V2HS_214961) and a non-silencing (NS) control vector were obtained from Open Biosystems (Huntsville, USA). For optimal mammalian expression, shRNA inserts were subcloned with EcoRI and XhoI into MSCV-LMP (MSCV/LTRmiR30-PIGΔRI, a generous gift from Prof. Scott Lowe, Cold Spring Harbour Laboratory ), which contains GFP and puromycin selection cassettes and drives miR30-shRNA expression using the retroviral 5'LTR. V2HS_198375 (MLL198) was found to suppress MLL expression most efficiently in transient transfection experiments and was used for subsequent experiments. The retroviral packaging cell line PA317 (selected in HAT medium) was transfected with linearised miR30-shRNA plasmid DNA (for both NS control and MLL198) using Lipofectamine, and GFP-positive cells were selected with puromycin. Stably transfected retroviral-producing PA317 cell lines were γ-irradiated (30 Gy) and incubated at 37°C overnight before co-culture with PER-117 cells for 48 hours. Retrovirally infected PER-117 cells were subsequently removed and selected with puromycin to generate cell lines stably expressing shRNA for MLL (MLL-KD) or the NS control (MLL-Scr). Efficiency of RNAi knockdown for MLL was assessed both by qRT-PCR as described above, and by immunoblot of nuclear protein extracted from cell lines in log-phase growth using standard methods. Antibodies used were mouse anti-MLLC/HRX, clone 9-12 (Upstate Cell Signaling Solutions, Temecula, CA), which detects the C-terminal proteolytic fragment of MLL (~180 kDa), and mouse anti-human β-actin as loading control (Pan Actin Ab-5 (ACTN05) NeoMarkers, Fremont CA). Densitometric quantitation of protein bands from multiple extractions taken at independent time points and from different cell-line stocks was performed using ImageJ software http://rsbweb.nih.gov/ij/, with MLL expression normalized to β-actin loading.
Cell growth and viability were measured using the Vi-CELL XR Viable Cell Analyzer (Beckman Coulter). Cells in exponential growth phase were seeded at 5 × 105 ml-1 in a 96-well plate in the presence or absence of dexamethasone (10 μg/ml - 258 μg/ml, Mayne Pharma Pty Ltd, VIC, Australia), 0.025 μg/ml cytarabine (ARAC; Pharmacia Pty Ltd, NSW, Australia), 0.01 μg/ml methotrexate (MTX; David Bull Laboratories), or 1 Gy gamma-irradiation, and incubated for two days at 37°C before measuring cell survival. Each drug concentration or condition was tested in triplicate and data were normalised to values obtained from untreated cells. For metabolic assays, cells in exponential growth were seeded at 5 × 105 ml-1 in fresh media and incubated for two days at 37°C before harvesting supernatants. Glucose and lactate supernatant concentrations were measured using the Amplex Red kit (Invitrogen, Australia), substituting lactate oxidase (Sigma, Australia) as required. For assessment of GILZ induction, MLL-KD and MLL-Scr cells in exponential growth were incubated with 1 μM dexamethasone (Mayne Pharma Pty Ltd, VIC, Australia) for four hours prior to RNA extraction and measurement by qRT-PCR.
MLL mRNA Expression and GC resistance in T-ALL Cell Lines
Our analysis of the microarray data revealed that GC resistance was significantly correlated with reduced expression of MLL . To confirm this correlation we used qRT-PCR to measure MLL mRNA expression across the panel, using a probe targeting the 3' end of the MLL coding region. Expression levels measured by qRT-PCR were highly correlated with resistance to both GCs (Figure 1A; correlation vs. dexamethasone IC50 -0.849 (p < 0.0001), methylprednisolone IC50 -0.851 (p < 0.0001)). Whilst translocations of the MLL gene are prevalent in infant ALL they are infrequent in T-ALL [8, 9, 22], suggesting that the observed correlation reflected expression of the wild-type gene. Indeed, T-ALL cell line karyotypes indicated no abnormalities at the 11q23 MLL-locus , a conclusion confirmed by Southern Blot for all 15 cell lines (data not shown). On the HG-U133A microarray there are five independent probes for MLL, and these span the entire length of the gene, encompassing both sides of the major break region (MBR) that is involved in almost all translocation events (Figure 1B). Across the 15 T-ALL cell lines correlation of MLL mRNA expression and GC resistance was significant for all five probe sets (median probe significance DEX p = 0.0025, MPRED p < 0.0001) indicating no discrepancy in expression between the 5' and 3' regions of the gene. Based on these data we conclude that the observed correlation with GC sensitivity in T-ALL cell lines is related to expression levels of wild-type MLL rather than MLL-translocation products.
Biological features of MLL expression in T-ALL
Top ranked GSEA gene sets from the C2 database (curated pathways) associated with MLL expression profiles in T-ALL cell lines.
Description of Biological Pathway
Electron transport chain
Glycolysis and gluconeogenesis
Down-regulated in response to leucine starvation
RNA transcription reactome
Amino-acyl tRNA biosynthesis
Genes upregulated by curcumin, transcription inhibitor
Genes upregulated by curcumin, transcription inhibitor
Genes up-regulated by myc
Down-regulated in response to rapamycin
Proteasomal pathway genes
Down-regulated in response to glutamine starvation
Krebs (TCA) cycle genes
Top ranked GSEA gene sets from the C4 database (computed gene networks) associated with MLL expression profiles in T-ALL cell lines.
Description of Network Hub Genes and Associated Functions
Peroxiredoxin 3 - MYC-mediated proliferation, glucose responses
Superoxide dismutase 1 - mitochondria, oxidative metabolism
MAP2K2 - ERK, JNK, p38, NFkB, and apoptosis pathways
Protein tyrosine phosphatase, cell growth, differentiation, metabolism
RAS oncogene family - cell cycle, mitotic spindle regulation
Guanine monphosphate synthetase - purine synthesis, cell cycle
DEAF1 or supressin, inhibitor of proliferation
Enhancer of rudimentary homolog - cell cycle regulator
Nucleostemin - cell cycle progression in stem cells, links with p53
Glutathione peroxidase 4 - cellular antioxidant defence
Apoptosis antagonizing transcription factor
EIF3S2 - eukaryotic translation initiation factor
ATX1 antioxidant protein 1 homolog - antioxidant defense
Etoposide induced mRNA - early p53 response gene
Proteasome 26S subunit, ATPase
RAS oncogene family - cell cycle, mitotic spindle regulation
Ras-associated protein - exocytosis, actin organisation
Uracil-DNA glycosylase - base-excision DNA repair pathway
Fibrillarin - component of snRNP synthesis of ribosomal RNA
Myc-associated factor X - transcriptional regulator
MLL-Translocation Partner Genes Correlate with MLL Expression
MLL Translocation Partner Genes Significantly Correlated with GC IC50 in T-ALL Cell Lines.
Acetyl-CoA carboxylase alpha
Guanine monphosphate synthetase
Decapping enzyme, scavenger
Elongation factor RNA pol II
LIM domain containing preferred translocation partner in lipoma
Acetyl-CoA carboxylase alpha
MLL (trithorax homolog, Drosophila); translocated to, 10
Microtubule-associated protein, RP/EB family, member 1
CREB binding protein (Rubinstein-Taybi syndrome)
MLL (trithorax homolog, Drosophila); translocated to, 10
Growth arrest-specific 7
Growth arrest-specific 7
Growth arrest-specific 7
Growth arrest-specific 7
E1A binding protein p300
Formin binding protein 1
Rho GTPase activating protein 26
Cas-Br-M (murine) ecotropic retroviral transforming sequence
E1A binding protein p300
Reduced MLL Expression in T-ALL Patients is Associated with GC Resistance and Relapse
Relevance of MLL Expression Level in Patients with MLL-Disease
MLL Knockdown Increases Resistance to GC Exposure and DNA Damage
To assess the effects of MLL knockdown on cell metabolism we compared rates of glucose consumption and lactate production between the two cell lines. Consistent with an increased rate of proliferation MLL-KD cells demonstrated an increased rate of glucose consumption compared to control cells. This was accompanied by a decreased rate of lactate production, resulting in a significant drop in the lactate production:glucose consumption ratio in MLL-KD cells (Figure 5C). Finally, since MLL is known to be a master transcriptional regulator we assessed whether the GC resistant phenotype of MLL-KD cells might represent transcriptional suppression of GC response elements by measuring the induction of GILZ, a well-characterized GC-response gene, following incubation with dexamethasone. There was no significant difference in the induction of GILZ mRNA between MLL-KD and MLL-Scr cell lines following a 4 hour incubation with dexamethasone (Figure 5D), indicating that GC-transcriptional responses in MLL-KD cells appeared to be intact.
Although there are conflicting reports of the effect of MLL-rearrangements on steroid resistance [7, 8], it is clear that all infants with MLL-rearrangements have significantly worse prognosis than those with non-rearranged MLL regardless of the type of translocation involved [3, 27]. However, the present study has been conducted using T-ALL cell lines without MLL-translocations and provides evidence that in the absence of such translocations cellular GC sensitivity is related to the level of expression of wild-type MLL. One interpretation of this data is that alterations in MLL support the proliferative phenotype that we have previously associated with GC resistance . In lymphocytes, GCs are thought to trigger a metabolic crisis that ultimately leads to apoptosis . In addition to suppressing apoptotic potential through the modulation of mitochondrial energetics, up-regulation of biosynthetic and metabolic pathways to support proliferation may therefore confer GC resistance by offsetting the adverse metabolic consequences of GC signalling . MLL has recently been shown to be important for the control of cell proliferation but the mechanism is complex, involving a bimodal pattern of expression throughout the cell cycle . In our experiments, suppression of MLL was associated with a small increase in proliferation and glucose consumption but decreased lactate production, indicating a shift away from aerobic glycolysis to alternative pathways, such as oxidative phosphorylation or the pentose-phosphate shunt. Besides energy production, these pathways are essential for the synthesis of macromolecules, nucleotides and nucleic acids required for proliferation .
In addition to elevated GC resistance, knockdown of MLL expression was associated with increased resistance to gamma-irradiation indicating an unexpected protection from the effects of DNA-damage. Recently it has been demonstrated that the MLL family of H3K4 methyltransferases are critical components of an E2F1-signalling pathway that mediates links cell cycle control to DNA damage responses, and that their knockdown attenuates the apoptotic response to adriamycin . This highlights the tumor suppressor role of these proteins and is consistent with the protection from DNA-damage we have observed following MLL-knockdown in T-ALL cell lines. In contrast however, no protective effect of MLL-knockdown was seen for ARAC or MTX in the present study. Whilst one might expect that suppression of DNA-damage response pathways should increase resistance to both of these agents, it is interesting to note that, unlike GCs, elevated resistance to neither of these drugs is associated with MLL-rearrangement [32, 33]; infants in fact are known to be generally more sensitive to ARAC [8, 32]. There may therefore be some unexplained insult specificity in the role of MLL in mediating responses to DNA-damage.
Across the T-ALL cell lines there was a 35-fold variation in the level of MLL-expression. Surprisingly the mechanisms controlling expression of wild-type MLL have not been extensively studied, with most work focusing on the downstream effects of the gene and its various fusion products. However the putative MLL promoter has binding motifs for a large number of transcription factors, including SREBF1 (sterol regulatory element binding transcription factor) and MYC. MYC is a pivotal player in the control of cell cycle and apoptosis , is one of the known downstream targets of GC signaling in lymphocytes , and has been reported to be up-regulated in MLL-disease . MLL expression is also likely to be subject to miRNA control, with numerous miRNA binding sites predicted to reside in the MLL 3'UTR. Although downstream effects of MLL or MLL-translocations on miRNA expression has been reported by a number of groups, to our knowledge only one recent study has reported the upstream miRNA regulation of MLL itself . In that study ectopic expression of mirR-221 and miR128 was shown to affect levels of MLL, MLL-fusions and GC sensitivity in ALL cell lines , consistent with the hypothesis that levels of MLL expression are important for GC resistance. It remains to be seen whether the observed effects of miRNA ectopic expression on GC sensitivity were due to effects on MLL-fusion proteins or endogenous wild-type MLL and the hierarchy for these mechanisms therefore remains to be untangled.
How do the present findings, performed in T-ALL with no MLL-translocations, relate to patients with MLL-disease? Although loss-of heterozygosity (LOH) at the MLL locus has been reported to be a relatively frequent event in childhood ALL, consistent with a potential role as a tumor suppressor , this is not the case in patients with MLL-disease where one wild-type copy of MLL appears to be retained [38–40]. This indicates that allele loss and MLL-translocation are mutually exclusive oncogenic events, but little focus has been given to the regulation of the remaining wild-type allele following translocation. However Whitman et al have recently demonstrated that in myeloid leukemia MLL partial tandem duplications (PTD) are associated with silencing of the wild-type MLL copy through an autoregulatory mechanism involving altered methylation . Interestingly, in one MLL-PTD patient wild-type MLL was expressed at diagnosis but absent at relapse, suggesting a correlation with disease progression. Wild-type MLL expression could be re-induced in primary blasts with the use of DNA methyltransferase (DNMT) or histone deacetylase (HDAC) inhibitors, or suppression of the MLL-PTD transcript, and was associated with increased apoptotic sensitivity and reduced colony-forming capability. Other workers have recently demonstrated down-regulation of wild-type MLL in myeloid leukemia patients with different types of rearranged-MLL  suggesting that it may be a common feature of MLL-related leukemia.
Based on the evidence presented we hypothesize that GC resistance in patients with MLL-disease may partly result from decreased expression and tumor suppressive effects of wild-type MLL, either through a gene-dosage effect following the functional loss of one allele via translocation, auto-regulation from the MLL-fusion protein, or altered miRNA/transcription factor signaling. This would help to explain why GC-resistance is a common feature of most patients with MLL-disease despite the wide variety of possible gene rearrangements. Amplifications of the MLL gene do occur but are much more rare. To our knowledge only one report exists where such a patient has been tested for ex vivo GC sensitivity  - in that small study a single patient with MLL amplification demonstrated GC sensitivity whilst all patients with MLL deletions or rearrangements demonstrated GC resistance, observations entirely consistent with our hypothesis.
We do not propose that the MLL-translocation event itself is without oncogenic effects since this has been clearly demonstrated by other workers, but rather that our data may help to explain the poor-response to therapy in this disease. Neither do our findings negate the possibility that MLL-fusion proteins themselves may have additional effects upon apoptotic sensitivity. Indeed, recent experiments have shown that multiple MLL-fusion proteins inhibit p53 and confer resistance to DNA damage . However, it is important to note that in these experiments fusion protein constructs were ectopically expressed into cell lines containing wild-type MLL. In view of the evidence discussed here it would be important to know whether expression of endogenous MLL was altered during these experiments and whether this contributed to the observed anti-apoptotic effects. Increased resistance to DNA damage-induced apoptosis has been proposed as a phenotype of MLL-disease that explains the short latency associated with disease emergence . It is possible that this effect could originate from the loss of tumor suppressor function of the wild-type MLL as well as from direct anti-apoptotic effects of the fusion protein.
During the preparation of this manuscript Liu et al  published a report describing a role for wild-type MLL in the maintenance of genome integrity through the regulation of the S-phase cell cycle checkpoint. DNA synthesis in cells deficient in wild-type MLL was found to be resistant to ionizing radiation and a range of DNA-damaging agents, supporting a role for wild-type MLL in the mediation of cellular DNA damage responses . Under this model, MLL-fusion proteins acted as dominant negative mutants to abrogate the ATR-mediated stabilization of wild-type MLL reported to occur in response to DNA damage. The findings are in keeping with those from the present study and support our conclusion that reduced levels of wild-type MLL can contribute to increased cellular resistance even in the absence of an MLL-translocation event.
Conflicts of interests
The authors declare that they have no competing interests.
The authors would like to thank Prof Michael Garlepp (Curtin University of Technology School of Pharmacy) and the patients and parents with whom this study is connected. This research was funded by the Children's Leukaemia and Cancer Research Foundation, and the Cancer Council of Western Australia.
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