High BRE Expression in Pediatric MLL-Rearranged AML Is Associated with Favorable Outcome

High BRE expression in pediatric MLL-rearranged AML is associated with favorable outcome

BV Balgobind1, CM Zwaan1, D Reinhardt2, TJCM Arentsen-Peters1, IHIM Hollink1, V de Haas3, GJL Kaspers4, ESJM de Bont5, A Baruchel6, J Stary7, C Meyer8, R Marschalek8, U Creutzig9, ML den Boer1, R Pieters1 and MM van den Heuvel-Eibrink1

1Pediatric Oncology/Hematology, Erasmus MC-Sophia Children’s Hospital, Rotterdam, The Netherlands; 2AML-BFM Study Group, Pediatric Oncology/Hematology, Medical School Hannover, Hannover, Germany; 3Dutch Childhood Oncology Group (DCOG), The Hague, The Netherlands; 4Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands; 5Pediatric Oncology/Hematology, Beatrix Children’s Hospital, University Medical Center Groningen, Groningen, The Netherlands; 6Hematology, St Louis Hospital, Paris, France; 7Pediatric Hematology/Oncology, 2nd Medical School, Charles University, Prague, Czech Republic; 8Institute of Pharmaceutical Biology, ZAFES, Diagnostic Center of Acute Leukemias (DCAL), Frankfurt, Germany and 9Pediatric Hematology/Oncology, University Hospital Muenster, Muenster, Germany

Translocations involving the mixed lineage leukemia (MLL) gene.2 In AML, the most common 11q23 rearrangements are gene, localized at 11q23, frequently occur in pediatric acute t(9;11)(p22;q23)(MLL-AF9) (approximately 50% of cases), t(11;19) myeloid leukemia (AML). We recently reported differences (q23;p13.1)(MLL-ENL), t(11;19)(q23;p13.3)(MLL-ELL), t(6;11)(q27; in prognosis between the different translocation partners, 3,4 suggesting differences in biological background. To unravel q23)(MLL-AF6) and t(10;11)(p12;q23)(MLL-AF10). Most AML the latter, we used microarrays to generate gene expression samples with 11q23 rearrangements are morphologically classified profiles of 245 pediatric AML cases, including 53 MLL- as FAB (French–American British morphology classification)-M4 or rearranged cases. Thereby, we identified a specific gene FAB-M5.5 expression signature for t(9;11)(p22;q23), and identified BRE We recently showed by a large retrospective international (brain and reproductive organ expressed) to be discriminative collaborative study that t(1;11)(q21;q23)(MLL-AF1q) was asso- for t(9;11)(p22;q23) (Po0.001) when compared with other MLL subtypes. Patients with high BRE expression showed a ciated with a favorable outcome, whereas t(10;11)(p12;q23), significantly better 3-year relapse-free survival (pRFS) (80±13 t(10;11)(p11.2,q23)[MLL-ABI1] or t(6;11)(q27;q23) was asso- 6 vs 30±10%, P ¼ 0.02) within MLL-rearranged AML cases. ciated with a poor outcome. In some studies, t(9;11)(p22;q23) Moreover, multivariate analysis identified high BRE expression had been associated with a better prognosis, which may at least as an independent favorable prognostic factor within pediatric partially be due to enhanced sensitivity to different drugs.7–10 AML for RFS (HR ¼ 0.2, P ¼ 0.04). No significant differences We found that within the t(9;11)(p22;q23) cases, prognosis was were identified for 3-year event-free survival or for 3-year overall survival. Forced expression of BRE did not result related to the cell type from which the leukemia originated, as in altered cell proliferation, apoptosis or drug sensitivity, patients with FAB-M5 showed a significantly better outcome which could explain the favorable outcome. In conclusion, than those with other FAB types.6 These outcome differences overexpression of the BRE gene is predominantly found in between the various translocation partners may point at MLL-rearranged AML with t(9;11)(p22;q23). Although further differences in biological background. investigation for the role of BRE in leukemogenesis and In gene expression profiling studies in MLL-rearranged outcome is warranted, high BRE expression is an independent AML and acute lymphoblastic leukemia, MLL-rearranged cases prognostic factor for pRFS in pediatric AML. 11–14 15 Leukemia (2010) 24, 2048–2055; doi:10.1038/leu.2010.211; clustered together as a single entity. However, Stam et al. published online 23 September 2010 showed that, within MLL-rearranged infant acute lymphoblastic Keywords: MLL; BRE; pediatric AML leukemia, each type of MLL translocation is associated with a translocation-specific gene expression signature. In this study, we performed a supervised analysis of gene expression profiles in a large cohort of pediatric AML cases (n ¼ 245) to identify and Introduction analyze differentially expressed genes between the various MLL- rearranged AML cases stratified by translocation partners, to Acute myeloid leukemia (AML) is a heterogeneous disease. elucidate potential genes of interest that are related to the Currently, initial response to therapy and cytogenetic abnorm- observed differences in outcome. This led to the identification of alities are the main prognostic factors.1 Translocations involving high expression of a novel gene of interest related to chromosome 11q23 comprise 15–20% of all pediatric AML t(9;11)(p22;q23), that is, BRE (brain and reproductive organ cases. In more than 95% of the cases with 11q23 rearrange- expressed), which is further described in this paper. ments, the mixed lineage leukemia (MLL) gene is involved. The heterogeneity of MLL-rearranged AML is reflected by the identification of more than 60 different fusion partners of this Material and methods

Correspondence: Dr MM van den Heuvel-Eibrink, Erasmus MC-Sophia Patients Children’s Hospital, Department of Pediatric Oncology/Hematology, Viably frozen diagnostic bone marrow or peripheral blood Room: Sp 2456, Dr Molewaterplein 60, P.O. Box 2060, 3000 CB samples from 237 de novo and 8 secondary pediatric AML Rotterdam, The Netherlands. E-mail: [email protected] patients, including 53 pediatric MLL-rearranged AML cases, Received 18 March 2010; revised 11 July 2010; accepted 5 August were provided by the Dutch Childhood Oncology Group 2010; published online 23 September 2010 (DCOG), the AML ‘Berlin–Frankfurt–Mu¨nster’ Study Group High BRE expression in pediatric MLL-rearranged AML BV Balgobind et al 2049 (AML-BFM-SG), the Czech Pediatric Hematology Group and the with GAPDH as the housekeeping gene.31 Primer and probe St Louis Hospital in Paris, France. Informed consent was sequences are described in Supplementary Table S1. obtained after Institutional Review Board approval according to local law and regulations. Each study group performed a central morphology review. In addition, the collaborative study Protein extraction and western blot analysis groups also provided data on the clinical follow-up of these In 11 of the 53 MLL-rearranged AML samples, material for patients. Survival analysis was restricted to the subset of 205 protein extraction and western blot analysis was available. Western blots were probed with mouse anti-BRE (kindly pediatric AML patients who were treated according to the BFM 32 and Dutch pediatric AML protocols (studies AML-BFM 98, AML- provided by Dr Y.L. Chui ). Further details on protein BFM 04, DCOG-BFM 87, DCOG 92/94 and DCOG 97). Details extraction and western blot are described in Supplementary of the treatment protocols included in the survival analysis and Material and Methods. overall outcome data have been previously published, with the 16–18 exception of study AML-BFM 2004, which is ongoing. Cell culture, transfection and cell-cycle proliferation Leukemic cells were isolated by sucrose density centrifuga- Different cell lines with an MLL rearrangement, that is, tion and non-leukemic cells were eliminated as previously Monomac-1, THP1, NOMO1, MV4;11 and ML2 (DSMZ, described.19 All processed samples contained more than 80% Braunschweig, Germany), were tested for BRE expression. leukemic cells, as determined morphologically using cytospins However, none of them showed overexpression of BRE.Asa stained with May–Gru¨nwald–Giemsa (Merck, Darmstadt, cell line model was not available, the Monomac-1 cell line was Germany). Subsequently, a minimum of 5 Â 106 leukemic cells transfected to overexpress BRE, and cell viability, transfection were lysed in Trizol reagent (Gibco BRL, Life Technologies, efficiency and cell-cycle proliferation were measured as Breda, The Netherlands). Genomic DNA and total RNA were described in Supplementary Material and Methods. isolated according to the manufacturer’s protocol, with minor modifications.20 In vitro drug resistance assay In vitro drug resistance for the different cytostatic agents Cytogenetic and molecular analysis as described in Supplementary Material and Methods was Leukemic samples were routinely investigated for MLL rearran- determined using the 4-day 3-(4,5-dimethylthiazol-2-yl)-2, gements by standard chromosome-banding analysis and/or 33 5-diphenyltetrazolium bromide assay as described previously. fluorescent in situ hybridization. If necessary, RT–PCR was performed for the common translocations MLL-AF9, MLL-AF10, MLL-AF6, MLL-ENL and MLL-ELL (primers are described in Additional statistical analysis Supplementary Table S1). Of the 53 cases, 21 harbored Statistical analysis was performed with SPSS 15.0 (SPSS Inc., a t(9;11)(p22;q23), 16 a t(10;11)(p12;q23) and 5 a t(6;11) Chicago, IL, USA). Different variables were compared with the (q27;q23). The remaining 11 cases were confirmed with long- w2-test, t-statistics test or the Mann–Whitney U-test. Probabilities distance-inverse–PCR to have another translocation partner and of overall survival (pOS), event-free survival (pEFS, events: no were considered as MLL others.21 NPM1, CEBPA, WT1, NRAS, CR, relapse, secondary malignancy or death from any cause) KRAS, PTPN11, CKIT and FLT3 hotspot mutational screening and relapse-free survival (pRFS, events: relapse) were estimated was performed as previously described.22–26 Overexpression of by the method of Kaplan and Meier. The Cox proportional EVI1 was previously established by gene expression profiling hazards model analysis was applied to determine the associa- and real-time quantitative (RQ)–PCR.27 tion of overexpression of BRE with pOS, pEFS and pRFS adjusted for prognostic factors as described for pediatric AML (white blood cell count 450 Â 109/l, age 410 years, favorable Microarray-based gene expression profiling karyotype, that is, t(8;21), inv(16) and t(15;17), and t(9;11) Integrity of total RNA was checked using the Agilent 2100 Bio- (p22;q23) ). All tests were two-tailed and a P-value of less than analyzer (Agilent, Santa Clara, CA, USA). Complementary DNA 0.05 was considered significant. and biotinylated complementary RNA were synthesized, hybri- dized and processed on the Affymetrix Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, USA) according to Results the manufacturer’s guidelines. Data acquisition was performed using expresso (Bioconductor package Affy, www.bioconductor. High BRE expression in t(9;11)(p22;q23) org) and probe-set intensities were normalized using variance From the microarray-based gene expression profiles of 245 stabilization normalization (VSN) (Bioconductor package VSN) in pediatric AML cases, the 21 t(9;11)(p22;q23) cases were com- the statistical data analysis environment R, version 2.2.0.28,29 pared with the 32 other MLL-rearranged cases, and a specific To find a gene expression signature characteristic for the gene expression signature for t(9;11)(p22;q23) was identified t(9;11)(p22;q23) group, which were mostly FAB-M5 cases, an (Figure 1). Interestingly, 5 of the top 50 most discriminative empirical Bayes linear regression model was used to compare probe sets for the t(9;11)(p22;q23) group were probe sets for the samples from this group with all other samples.30 Moderated BRE gene (Supplementary Table S2). The VSN-normalized mean T-statistics P-values were corrected for multiple testing using average intensity of three of these probe sets (205550_s_at, the false discovery rate method defined by Benjamini and 211566_x_at and 212645_x_at) was 3.7-fold higher for Hochberg. t(9;11)(p22;q23) compared with the other MLL-rearranged AML cases (Po0.001) (Figure 2a). In the total cohort of pediatric AML cases (n ¼ 245), high expression of BRE was only identified RQ–PCR for BRE in MLL-rearranged AML cases, and in one infant AML harboring In 45 of the 53 MLL-rearranged AML samples, the RNA expression a t(8;16)(p11;p13) (Figure 2b). could be validated by RQ–PCR. The relative expression of BRE The gene expression data were validated with RQ–PCR in 45 was calculated using the comparative cycle time (DCt) method, of 53 MLL-rearranged AML cases. A correlation between the

Leukemia High BRE expression in pediatric MLL-rearranged AML BV Balgobind et al 2050

Figure 1 Hierarchical clustering based on the top 50 most discriminative genes for t(9;11)(p22;q23). Hierarchical clustering of 53 MLL-rearranged AML cases based on the top 50 most discriminative genes showed for t(9;11)(p22;q23) after supervised analysis (Supplementary Table S2).

log-transformed gene expression profiling data and the probe sets within the MLL-rearranged AML cases, a distinction log-transformed RQ–PCR data was found (r2 ¼ 0.6, Po0.001) was made into a group with high BRE expression (4712 AU) (Supplementary Figure S1). The median relative BRE expression and a group with low expression (o712 AU). to GAPDH for t(9;11)(p22;q23) was 56% compared with 8% for Within the total cohort of pediatric AML, high BRE expre- the other MLL-rearranged AML cases (Po0.001) (Figure 2c). ssion was found in 19 patients, including 18 of 53 with In a previous pediatric AML study by Ross et al., gene MLL-rearranged AML, and one case with a t(8;16)(p11;p13). In expression profiling was performed with the Affymetrix Human all, 17 of the 19 cases with a high BRE expression were FAB-M5. Genome U133A microarray, which had one probe set The remaining two cases were FAB-M4 and an unknown FAB representing the BRE gene. We reanalyzed their MLL-rearranged type (Po0.001). In addition, patients with a high BRE expression AML cases (n ¼ 23) for validation purposes and confirmed were significantly younger than those with a low BRE expression that high BRE expression was predominantly found in the (2.8 vs 9.8 years, P ¼ 0.007). None of the cases with high BRE t(9;11)(p22;q23) cases (Supplementary Figure S2). expression had an FLT3-ITD (P ¼ 0.03) (Table 1a). However, the differences in morphology, age and FLT3-ITD were determined Clinical and genetic characteristics of patients by the significantly high frequency of MLL-rearranged AML in with high BRE expression patients with high BRE expression. On the basis of the VSN-normalized mean average intensity Within the MLL-rearranged cases, the group with high BRE (712 AU) of the three most significant differentially expressed expression consisted of 15 of 21 with t(9;11)(p22;q23) and 3 of

Leukemia High BRE expression in pediatric MLL-rearranged AML BV Balgobind et al 2051

Figure 2 Expression of BRE in pediatric AML. BRE þ was predominantly found in t(9;11)(p22;q23). Using gene expression proﬁling, 18 MLL- rearranged AML cases showed an expression higher than the mean average expression and were considered BRE þ (a). In the remaining cohort of 245 pediatric AML cases also, one case with a t(8;16)(p11;p13) showed high expression of BRE (b). RQ–PCR conﬁrmed the gene expression data (c). Protein analysis in 10 MLL-rearranged AML cases did not show differences in expression (d).

Table 1a Clinical characteristics of patients with high expression of BRE within pediatric AML

No. of BREÀ patients (%) No. of BRE+ patients (%) P-value

Sex (N ¼ 245) 0.822a Male 125 (55) 10 (53) Female 101 (45) 9 (47)

Age (median, range, years, N ¼ 245) 9.8 (0–18.0) 2.8 (0.3–17.7) 0.007b WBC Â 109/l (median, range, N ¼ 219) 40.2 (0.0–483) 57.6 (5.9–469) 0.077b

FAB (N ¼ 245) o0.001a M0 14 (6) 0 (0) M1 28 (12) 0 (0) M2 54 (24) 0 (0) M3 20 (9) 0 (0) M4 55 (24) 1 (5) M5 36 (16) 17 (90) M6 3 (1) 0 (0) M7 8 (4) 0 (0) Other/unknown 8 (4) 1 (5)

Cytogenetics (N ¼ 245) o0.001a MLL-rearrangements 35 (16) 18 (95) t(8;21) 28 (12) 0 (0) inv(16) 27 (12) 0 (0) t(15;17) 18 (8) 0 (0) CN-AML 41 (18) 0 (0) AML-other/unknown 77 (34) 1 (5)c

EVI1-overexpression (N ¼ 245) 30 (13) 1 (5) 0.482a WT1 mutations (N ¼ 238) 21 (10) 1 (5) 1.000a CEBPA mutations (N ¼ 236) 16 (7) 0 (0) 0.376a NPM1 mutations (N ¼ 240) 17 (8) 0 (0) 0.374a FLT3-ITD (N ¼ 245) 46 (20) 0 (0) 0.029a CKIT mutations (N ¼ 245) 17 (8) 1 (5) 1.000a N/K-RAS mutations (N ¼ 243) 45 (20) 3 (16) 0.773a PTPN11 mutations (N ¼ 243) 3 (1) 1 (5) 0.279a aw2-test. bMann–Whitney-U test. cThis case harbored a t(8;16)(p11;p13).

16 with t(10;11)(p12;q23). No differences for age, sex, white high BRE expression showed an inverse correlation with EVI1 blood cell count and FAB morphology, nor for mutations in overexpression (P ¼ 0.02) (Table 1b). In fact, four of the ﬁve WT1, FLT3-ITD, CKIT, N/K-RAS, were found between cases patients with EVI1 overexpression and a t(9;11)(p22;q23) had with high and low BRE expression. In contrast, cases with the lowest expression of BRE.

Leukemia High BRE expression in pediatric MLL-rearranged AML BV Balgobind et al 2052 Table 1b Clinical characteristics of patients with high expression of BRE within MLL-rearranged AML

No. of BREÀ patients (%) No. of BRE+ patients (%) P-value

Sex (N ¼ 53) 0.268a Male 23 (66) 9 (50) Female 12 (34) 9 (50)

Age (median, range, years, N ¼ 53) 5.8 (0–18.0) 2.8 (0.3–17.7) 0.778b WBC Â 109/l (median, range, N ¼ 49) 68.3 (1.1–443) 42.3 (5.9–469) 0.737b

FAB 0.192a M0 3 (9) 0 (0) M1 2 (6) 0 (0) M2 1 (3) 0 (0) M3 0 (0) 0 (0) M4 6 (17) 1 (6) M5 21 (60) 16 (89) M6 0 (0) 0 (0) M7 2 (6) 0 (0) Other/unknown 0 (0) 1 (6)

MLL-rearrrangement o0.001a t(9;11)(p22;q23) 6 (17) 15 (83) t(10;11)(p12;q23) 13 (37) 3 (17) t(6;11)(q27;q23) 5 (14) 0 (0) t(11;19)(q23;p13) 5 (14) 0 (0) MLL-other 6 (17) 0 (0)

EVI1-overexpression (N ¼ 53) 13 (37) 1 (6) 0.020a WT1 mutations (N ¼ 52) 1 (3) 1 (6) 1.000a FLT3-ITD (N ¼ 53) 2 (0) 0 (0) 0.543a CKIT mutations (N ¼ 52) 0 (0) 1 (6) 0.346a N/K-RAS mutations (N ¼ 52) 11 (32) 3 (17) 0.329a PTPN11 mutations (N ¼ 53) 0 (0) 1 (6) 0.340a aw2-test. bMann–Whitney-U test.

Within the patients with a t(9;11)(p22;q23), 13 of 15 patients P ¼ 0.04), but not for EFS (HR 0.4, P ¼ 0.06) and OS (HR 0.5, with a high BRE expression had a FAB-M5, which was P ¼ 0.29) (Table 2). not significantly different compared with those with a low BRE expression (P ¼ 0.31). Within the patients with a t(10;11) (p12;q23), numbers were too small to identify significant Functional assays after transfection correlations between cases with high and low BRE expression. Functional studies were performed to find an explanation for the association of high BRE expression and favorable outcome. To explore a possible role for BRE in cell proliferation, the Favorable disease-free survival in pediatric AML Monomac-1 cell line, harboring a t(9;11)(p22;q23), was patients with high BRE expression transiently transfected with pLNCX-BRE by means of electro- Within the total cohort of pediatric AML, follow-up data were poration. However, a maximum transfection efficiency of only available for 205 patients, including 17 cases with high BRE 25–30% was achieved with pLNCX-EGFP. At different time expression. No differences were found for 3-year pEFS between points, an increased expression of BRE on messenger (m) RNA cases with high and low BRE expression (53±12 vs 43±4%, was measured (Supplementary Figure 3A), but not evidently on P ¼ 0.61) and for 3-year pOS (64±12 vs 62±4%, P ¼ 0.77). protein level (Supplementary Figure 3B), which could be However, the cases with a high BRE expression had a higher explained by the low transfection efficiency. This could explain 3-year pRFS as compared with cases with a low expression why we did not detect significant differences in cell-cycle (82±12 vs 52±4%, P ¼ 0.01) (Figure 3a). proliferation between BRE-transfected cells and those trans- Within the MLL-rearranged cases, high BRE expression fected with an empty vector (Supplementary Figure 3C), nor an suggested a better 3-year pEFS compared with those with a apoptotic effect of BRE (Supplementary Figure 3D). In addition, low BRE expression (50±13 vs 21±8%, P ¼ 0.12) and a better in vitro drug sensitivity for cells overexpressing BRE showed no 3-year pOS (62±13 vs 32±9%, P ¼ 0.19), but the results were significant differences in drug sensitivity compared with cell not statistically significant. However, again, cases with high BRE transfected with an empty vector or non-transfected cell line expression had a significantly better 3-year pDFS compared (Supplementary Table 3). with cases with a low expression (80±13 vs 32±11%, P ¼ 0.03) (Figure 3b). Multivariate analysis within the total cohort of pediatric AML, BRE protein expression in MLL-rearranged AML including prognostic factors such as age, white blood cell To identify a correlation between protein and mRNA expression count, favorable karyotype and t(9;11)(p22;q23), showed levels, we performed western blot analysis on 11 MLL-rear- that next to favorable karyotype, high BRE expression was an ranged AML cases, for whom protein was available in our cell independent favorable prognostic factor for RFS (HR 0.2, bank. No differences in BRE protein expression were found

Leukemia High BRE expression in pediatric MLL-rearranged AML BV Balgobind et al 2053 between t(9;11)(p22;q23) (n ¼ 6) and other MLL-rearranged t(6;11)(q27;q23) were found to have a unfavorable prognosis. AML cases (n ¼ 5) (Figure 2d). The BRE-transfected Monomac- This diversity in outcome indicates differences in leukemogen- 1 cell line was used as positive control (Supplementary esis within MLL-rearranged AML. However, to date, the factors Figure 3B), indicating the specificity of the antibody against BRE. that have a role in these biological differences are largely unknown. In this study, we performed gene expression profiling on a large cohort of pediatric AML samples, with the aim to Discussion detect such differences in biology. Supervised clustering analysis identified a specific gene expression signature for Pediatric AML is a heterogeneous disease, and currently, t(9;11)(p22;q23), the most common translocation partner in response to therapy and cytogenetic abnormalities are the main MLL-rearranged AML. High expression of the BRE gene was prognostic factors. Interestingly, for the MLL-rearranged group, one of the strongest components of this signature. High BRE we previously showed in a large international retrospective expression was not restricted to t(9;11)(p22;q23) only; study that prognosis mainly depends on the translocation 6 three cases with a t(10;11)(p12;q23) and one case with a partner of MLL. AML patients with t(1;11)(q21;p23) and t(8;16)(p11;p13) also showed high BRE expression. Interestingly, t(9;11)(p22;q23) with FAB-M5 were found to have a favorable t(8;16)(p11;q23) has been linked to MLL-rearranged AML on the prognosis, whereas patients with t(10;11)(p12;q23) and basis of gene expression profiles in adult AML.34 This particular t(8;16)(p11;p13) case occurred in an infant with a FAB-M5 AML. None of the other pediatric AML cases showed a high BRE expression, indicating that particularly MLL fusions may lead to high BRE expression and hence have a role in leukemogenesis in AML. Interestingly, high BRE expression was not found in t(9;11)(p22;q23) (n ¼ 10) or in other MLL-rearranged precursor B or infant acute lymphoblastic leukemia (n ¼ 71) cases in two large GEP studies conducted at our center, indicating its specific role in AML.15,35 Recently, overexpression of BRE has been described in hepatocellular and esophageal carcinomas.32,36 However, to date BRE has never been associated with hematological malignancies. This study shows that BRE has a role in pediatric AML and mainly in patients with t(9;11)(p22;q23). Moreover, high BRE expression was an independent favorable prognostic factor because of a reduced relapse rate in remission. To explain the observed difference in relapse risk, we examined the effect of BRE expression in vitro on altered cell proliferation, apoptosis and drug sensitivity in AML. The BRE protein is mainly localized in the cytoplasm, but nuclear localization has also been reported as a subunit of the holoenzyme complex BRCC, which contains BRCA1, BRCA2, RAD51 and BARD1.37 BRCC enhances cellular survival after DNA damage by ubiquitin E3 ligase activity. Interestingly, association of BRE to this complex further enhances the E3 ligase activity. After death-receptor stimulation an increased binding of cytosolic BRE to ubiquitinated proteins was found, suggesting that BRE has an important role in post-translational modification of proteins. Intriguingly, BRE transcription has been shown to be downregulated after DNA damage and retinoic acid treatment.38 Li et al.39 showed that BRE is a death receptor- Figure 3 RFS outcome for BRE expression in pediatric AML. Survival analysis showed that high expression of BRE was related to a better associated antiapoptotic protein, inhibiting the mitochondrial 3-year pRFS in all pediatric AML cases (a), and in the MLL-rearranged apoptotic pathway. We could not find evidence AML cases (b). that overexpression of BRE influenced apoptosis in an

Table 2 Multivariate analyses for event free survival (EFS), relapse free survival (RFS) and overall survival (OS)

EFS RFS OS

HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value

BRE+ 0.4 (0.1–1.0) 0.064 0.2 (0.0–0.9) 0.036 0.5 (0.2–1.7) 0.287 t(9;11)(q22;q23) 1.6 (0.6–4.2) 0.358 1.6 (0.4–6.6) 0.527 1.6 (0.5–4.7) 0.417 Favorable karyotypea 0.3 (0.2–0.6) o0.001 0.3 (0.2–0.6) o0.001 0.2 (0.1–0.5) o0.001 WBC 450b 1.0 (0.6–1.7) 0.890 0.9 (0.5–1.7) 0.858 1.1 (0.6–2.1) 0.659 Age 410c 0.9 (0.6–1.3) 0.578 0.8 (0.5–1.2) 0.264 0.9 (0.5–1.4) 0.545 aincluding t(8;21), inv(16) and t(15;17). bWBC ¼ White blood cell count above 50 Â 109/l. cChildren older than 10 years.

Leukemia High BRE expression in pediatric MLL-rearranged AML BV Balgobind et al 2054 MLL-rearranged AML with the current cell line studies. This may Therefore, high BRE expression seems to be part of a favorable be because of the fact that only a transfection rate of 30% was signature in MLL-rearranged AML. achieved despite rigorous efforts. Alternatively, other mechan- In conclusion, our study shows that overexpression of BRE isms could have a role, and the role in apoptosis may be tissue is predominantly found in MLL-rearranged AML with a dependent. Alternative approaches, such as stable transduction t(9;11)(p22;q23). Moreover, high BRE expression is an with a selectable vector and manipulation of primary hemato- independent favorable prognostic factor because of a reduced poietic progenitors, could elucidate this. relapse rate in remission in pediatric AML. So far, we could Previous studies demonstrated that overexpression of BRE not elucidate the exact underlying mechanism. Further enhances tumor growth, rather than initiating tumor formation research is warranted to explore the role of BRE in AML and in vivo.40 However, BRE overexpression did not lead to cell to identify the link between MLL-AF9 and the transcription proliferation in vitro.41 Tang et al.42 showed conflicting data, of BRE. that is, a decreased in vitro cell proliferation by overexpression of BRE and upregulation of p53, prohibitin and proteins involved in the nuclear factor-kB signaling. In an MLL-rear- Conflict of interest ranged AML cell line, we could not show such a role for BRE in cell proliferation. It is conceivable that stable transfection cell The authors declare no conflict of interest. line models or in vivo models could give more insight into whether BRE overexpression influences proliferation or apopto- Acknowledgements sis of AML cells similar to what was previously shown in hepatocellular carcinoma models. We thank Dr Yiu-Loon Chui, Department of Chemical Pathology Using in vitro assays, we were unable to determine that BRE and Sir YK Pao Center for Cancer, Prince of Wales Hospital, The overexpression was related to higher drug sensitivity, although Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, the low transfection rate could have influenced these results. 39 China, for providing the BRE antibody. Furthermore, we thank Li et al. showed that knockdown of endogenous BRE has little, Dr E Hulleman for her input in the transfection experiments. This if any, modulatory effect on apoptosis induced by etoposide in work was funded by the NWO ‘Netherlands Organization a contrast to tumor necrosis factor- -induced apoptosis. This for Scientfic Research’ (BVB) and KOCR ‘Kinder-Oncologisch seems to be in line with our results, as etoposide-induced Centrum Rotterdam’ (BVB, IHIM). apoptosis was not influenced by overexpression of BRE in AML. As we could not explain the observed difference Author contributions in relapse risk in our series, further investigation is required BVB designed and performed research and wrote the paper; to elucidate the role of BRE overexpression in the STJCMP and IHIM performed research. CM and RM performed leukemogenesis of t(9;11)(p22;q23). MLL-fusion proteins are LDI–PCR to identify the MLL rearrangements. VH, GJK, ESJMB, involved in inappropriate transcriptional activation, and a DR, UC, AB, and JS and JT made this research possible by specific role for MLL-AF9 in the transcription of BRE is collecting patient samples and characteristics in their own study most likely. groups and providing additional information; MMH-E, CMZ In this study, protein analysis of BRE did not correlate with and RP designed and supervised research and wrote the paper. mRNA expression and differences in outcome were, therefore, only related to mRNA expression levels. 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