Successful Application of OPLS-DA for the Discrimination of Wild-Type and Mutated Cells in Acute Lymphoblastic Leukemia

Introduction Functional variants of the Methylenetetrahydrofolate reductase ( MTHFR) gene, the C677T and A1298C, have largely investigated in pharmacogenomics of Methotrexate (MTX) in acute lymphoblastic leukemia (ALL), yet the conclusions are inconsistent. In addition; most of these studies do not analyze haplotypes. Here, we investigate the MTHFR 677/1298 genotypes and the 677-1298 haplotype and characterize the MTX response in Northern African ALL patients. Methods Genomic DNA was extracted from whole venous from a total of 28 patients with ALL. Genotyping were carried out with restriction fragment length polymorphism (RFLP). A toxicity score (TS) is calculated for each patient and correlate to the haplotype. Results The allelic frequency of MTHFR 677T-1298C haplotype was 10.7% in ALL patients. According to the toxicity’s score (TS) there was no significant differences between haplotype groups (p = 0.79): TS was higher with wild type of MTHFR (TS = 3.43; SEM ± 0.85) followed by combined genotype (677T-1298C) (TS = 2.67; SEM ± 0.88) and isolated variant (C677T or A1298C) (TS = 2.64; SEM ± 0.92). Conclusion Despite the limitation of this study; our results suggest that the MTHFR 677T-1298C haplotype is common in ALL and may be a promising HD-MTX chemotherapy-related adverse effects biomarker.

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A novel PAX5-ELN fusion protein identified in B-cell acute lymphoblastic leukemia acts as a dominant negative on wild-type PAX5

Blood ◽

10.1182/blood-2006-05-025221 ◽

2006 ◽

Vol 109 (8) ◽

pp. 3417-3423 ◽

Cited By ~ 55

Author(s):

Marina Bousquet ◽

Cyril Broccardo ◽

Cathy Quelen ◽

Fabienne Meggetto ◽

Emilienne Kuhlein ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Quantitative Polymerase Chain Reaction ◽

Dominant Negative ◽

Leukemic Cells ◽

Dominant Negative Effect ◽

Wild Type ◽

Cell Acute Lymphoblastic Leukemia

Abstract We report a novel t(7;9)(q11;p13) translocation in 2 patients with B-cell acute lymphoblastic leukemia (B-ALL). By fluorescent in situ hybridization and 3′ rapid amplification of cDNA ends, we showed that the paired box domain of PAX5 was fused with the elastin (ELN) gene. After cloning the full-length cDNA of the chimeric gene, confocal microscopy of transfected NIH3T3 cells and Burkitt lymphoma cells (DG75) demonstrated that PAX5-ELN was localized in the nucleus. Chromatin immunoprecipitation clearly indicated that PAX5-ELN retained the capability to bind CD19 and BLK promoter sequences. To analyze the functions of the chimeric protein, HeLa cells were cotransfected with a luc-CD19 construct, pcDNA3-PAX5, and with increasing amounts of pcDNA3-PAX5-ELN. Thus, in vitro, PAX5-ELN was able to block CD19 transcription. Furthermore, real-time quantitative polymerase chain reaction (RQ-PCR) experiments showed that PAX5-ELN was able to affect the transcription of endogenous PAX5 target genes. Since PAX5 is essential for B-cell differentiation, this translocation may account for the blockage of leukemic cells at the pre–B-cell stage. The mechanism involved in this process appears to be, at least in part, through a dominant-negative effect of PAX5-ELN on the wild-type PAX5 in a setting ofPAX5 haploinsufficiency.

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MDM2 antagonist nutlin-3 is a potent inducer of apoptosis in pediatric acute lymphoblastic leukemia cells with wild-type p53 and overexpression of MDM2

Leukemia ◽

10.1038/leu.2008.11 ◽

2008 ◽

Vol 22 (4) ◽

pp. 730-739 ◽

Cited By ~ 76

Author(s):

L Gu ◽

N Zhu ◽

H W Findley ◽

M Zhou

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

Wild Type ◽

Potent Inducer ◽

Pediatric Acute Lymphoblastic Leukemia ◽

Mdm2 Antagonist ◽

Wild Type P53

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Pax5 Haploinsufficiency Cooperates with BCR-ABL1 to Induce Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v112.11.293.293 ◽

2008 ◽

Vol 112 (11) ◽

pp. 293-293 ◽

Cited By ~ 9

Author(s):

ChristoPher B Miller ◽

Charles G Mullighan ◽

Xiaoping Su ◽

Jing Ma ◽

Michael Wang ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Point Mutation ◽

Copy Number ◽

Lymphoblastic Leukemia ◽

Wild Type ◽

Lymphoid Development ◽

B Lymphoid

Abstract Genes regulating B lymphoid development are somatically mutated in over 40% of B-progenitor acute lymphoblastic leukemia (ALL) cases, with the most common targets being the transcription factors PAX5, IKZF1 (encoding Ikaros), and EBF1. Notably, BCR-ABL1 ALL is characterized by a high frequency of mutations of IKZF1 (85%), PAX5 (55%), and CDKN2A/B (encoding INK4/ARF, 55%), suggesting that these lesions cooperate with BCR-ABL1 in lymphoid leukemogenesis. To examine cooperativity between Pax5 haploinsufficiency and BCR-ABL1, we transplanted Pax5+/+ and Pax5+/− bone marrow cells transduced with MSCV-GFP-IRES-p185 BCR-ABL1 retrovirus into lethally irradiated wild-type C57BL6 recipient mice. Mice transplanted with BCR-ABL1 transduced Pax5+/− marrow developed B progenitor cell ALL with significantly higher penetrance and decreased latency when compared to animals transplanted with BCR-ABL1 transduced Pax5+/+ marrow (median survival 36 vs. 60 days, P=0.0003). The latency of tumor onset was further decreased in the presence of Arf haploinsufficiency (Pax5+/+Arf+/+ 60 days, Pax5+/−Arf+/+ 36 days, Pax5+/−Arf+/− 21 days, P<0.0001). All leukemias were of B cell lineage and were transplantable to secondary recipients. In addition, Southern blot analysis revealed the Pax5+/−Arf+/+ leukemias to be monoclonal, where as the Pax5+/−Arf+/− leukemias were oligoclonal. Importantly, the Pax5+/− leukemias exhibited a more immature B cell immunophenotype than Pax5 wild type leukemias. Moreover, a proportion of the Pax5+/− leukemias (19%) exhibited a very immature early pro B cell immunophenotype (Cd19−, Bp1−), suggesting the possibility of acquired lesions in other key regulators of normal B cell differentiation. To explore this possibility and to identify the total complement of genetic lesions required to generate overt leukemia, we performed genome-wide copy number analysis on 30 murine leukemias (15 Pax5+/+, 15 Pax5+/−) using a custom CGH microarray (Agilent) that interrogated 477,000 autosomal loci, including 18,000 probes covering 20 genes encoding B lymphoid transcription factors and genes targeted by recurring copy number abnormalities (CNAs) in human BCR-ABL1 ALL (Bcl11a, Cdkn2a, Ebf1, Ikzf1, Ikzf2, Ikzf3, Il7r, Lef1, Mdm2, Mef2c, Myb, Pax5, Pten, Rb1, Sfpi1, Sox4, Stat5a, Tcf3, Tcf4, and Trp53). This analysis identified focal recurring CNAs in multiple genes including Cdkn2a/b, Ebf1, Ikzf1, Ikzf2, Ikzf3, and Pax5, each of which is a target of mutation in human B-ALL. Overall, there were on average 3.5 CNAs in Pax5+/+ leukemias versus 0.7 CNAs in Pax5+/− leukemias. Genomic resequencing was also performed on Pax5 and revealed three missense mutations in the DNA binding paired domain (R38H, P80R and G85R), one of which (P80R) is the most common PAX5 point mutation in human B-ALL. All three point mutations are predicted to impair DNA binding of Pax5. Interestingly, the majority of the pro-B cell leukemias that arose in the Pax5+/−Arf+/+ animals were found to harbor mutations (CNAs or point mutation) of the retained Pax5 allele, consistent with the immature immunophenotype. To further explore the relationship between our murine model and human BCR-ABL1 ALL, we performed gene expression profiling of Pax5+/+ and Pax5+/− leukemias and compared their signatures to those of human BCR-ABL1 ALL and stage-specific murine B lymphoid developmental signatures using gene set enrichment analysis (GSEA). This analysis identified significant similarity between murine and human BCR-ABL1 leukemias, thus providing further evidence that this model closely recapitulates human BCR-ABL1 ALL. Notably, Pax5+/− leukemias, or Pax5+/+ leukemias that acquired additional mutations of B-lymphoid regulators exhibited a less mature gene expression profile than leukemias lacking B-lymphoid regulatory mutations. These data indicate that loss of Pax5 contributes to leukemogenesis, that additional genomic alterations in genes regulating B lymphoid development and cell cycle regulators/tumor suppressors (Arf) are frequent events in BCR-ABL1 acute lymphoblastic leukemia, and that these lesions result in impaired B-lymphoid maturation in B-ALL. The genetic complexity of BCR-ABL1 ALL is likely to have important therapeutic implications for this poor prognosis subtype of leukemia.

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Cryptic Truncating Rearrangements of the Erythropoietin Receptor in Ph-like Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v124.21.128.128 ◽

2014 ◽

Vol 124 (21) ◽

pp. 128-128

Author(s):

Ilaria Iacobucci ◽

Kathryn G. Roberts ◽

Yongjin Li ◽

Debbie Payne-Turner ◽

Marcus Valentine ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cells ◽

Ex Vivo ◽

Fetal Liver ◽

Lymphoblastic Leukemia ◽

Erythropoietin Receptor ◽

Leukemic Cells ◽

Wild Type ◽

Fetal Liver Cells

Abstract Introduction: BCR-ABL1-like, or Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), is characterized by a gene expression profile similar to BCR-ABL1-positive ALL, with a broad range of genetic alterations activating cytokine receptor and kinase signaling and poor outcome. We previously reported a rearrangement of EPOR, encoding the erythropoietin receptor, into the immunoglobulin heavy chain locus (IGH). The aims of this study were to define the frequency and genomic architecture of EPOR rearrangements in B-ALL and to examine their role in kinase signaling and lymphoid transformation. Methods: Whole genome and/or transcriptome sequencing was performed on 154 Ph-like ALL cases. Sanger sequencing and fluorescent in situ hybridization were used to confirm and map the EPOR rearrangements. Wild-type or EPOR rearranged alleles were expressed in interleukin-3 (IL-3)-dependent mouse hematopoietic Ba/F3 cells and interleukin-7 (IL-7)-dependent pre-B cells harboring alterations of Arf and/or the dominant negative IKZF1 allele IK6 observed in EPOR-rearranged ALL. Proliferation and signaling were examined in the absence or presence of erythropoietin (EPO). EPOR expression and signaling in cell lines and primary leukemic cells were examined by immunofluorescence, flow cytometry and immunoblotting. Epor-/- fetal liver cells were transduced with empty vector, EPOR wild-type or rearranged alleles and used for erythroid colony forming unit (CFU-E) and erythroid burst-forming unit (BFU-E) assays. Luciferase-marked xenografts of human EPOR-rearranged ALL were established in NOD-SCID-IL2R gamma (NSG) null mice, and signaling, EPO-dependent proliferation and sensitivity to the JAK inhibitor ruxolitinib were assessed ex vivo and in vivo. Results: Eight cases (5.2% of Ph-like ALL) harbored rearrangements of EPOR into either the IGH or immunoglobulin kappa light chain (IGK) loci with two consequences: i) inversion and insertion of EPOR 5’ untranscribed region into the the promoter and enhancer region of IGH/IGK; ii) truncation of the last coding exon of EPOR. Such rearrangements resulted in overexpression of a C-terminal truncated receptor that retained the phosphorylation site required for STAT5 activation, but lacked multiple intracytoplasmic tyrosine residues whose phosphorylation is required for normal negative regulation of the receptor. Notably, the locations of the truncation sites overlap with those arising from inherited mutations in primary familial congenital polycythemia, in which frameshift and nonsense mutations truncate the receptor. A real-time quantitative PCR assay was established to provide a diagnostic tool and to confirm that primary leukemia cells with these EPOR rearrangements overexpress N-terminal exons but lack expression of C-terminal truncated exon eight. The truncated alleles were expressed at higher levels than wild-type EPOR in IL-3-dependent Ba/F3 and IL-7-dependent Arf-/- mouse pre-B cells, and sustained cell proliferation and increased STAT5 phosphorylation following stimulation with exogenous EPO. Expression of wild-type or truncated EPOR in Epor-/- fetal liver cells promoted erythroid differentiation with formation of CFU-E and BFU-E colonies, indicating that truncated receptors sustain erythroid development. Xenografted EPOR-rearranged leukemic cells exhibited high levels of mutant EPOR on the cell surface, constitutive STAT5 phosphorylation and sensitivity to the JAK2 inhibitor ruxolitinib ex vivo and in vivo. Conclusions: We have identified a subset of Ph-like ALL cases characterized by rearrangements of truncated EPOR into the IGH/IGK chain loci. This represents an entirely new mechanism of EPOR deregulation and unexpectedly implicates EPOR signaling as an important factor influencing B-lymphoid malignancies that are amenable to JAK-STAT5 inhibition. Clinical trials testing ruxolitinib in ALL patients with EPOR rearrangements are warranted. Disclosures No relevant conflicts of interest to declare.

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DNA-thioguanine nucleotide as a treatment marker in acute lymphoblastic leukemia patients with NUDT15 variant genotypes

PLoS ONE ◽

10.1371/journal.pone.0245667 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0245667

Author(s):

Hee Young Ju ◽

Ji Won Lee ◽

Hee Won Cho ◽

Ju Kyung Hyun ◽

Youngeun Ma ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Wild Type ◽

Spectrometry Method ◽

Chromatography Tandem Mass Spectrometry ◽

Individual Variations ◽

Significant Difference ◽

Liquid Chromatography Tandem Mass ◽

Thioguanine Nucleotide ◽

Wbc Count

Background Large inter-individual variations in drug metabolism pose a challenge in determining 6-mercaptopurine (6MP) doses. As the last product of 6MP metabolism, DNA-thioguanine nucleotide (DNA-TGN) could reflect the efficacy of 6MP, especially in patients harboring variants in the 6MP metabolism pathway. The aim of this study was to investigate the clinical significance of DNA-TGN monitoring in Korean pediatric acute lymphoblastic leukemia (ALL) patients, focusing on the NUDT15 genotype. Methods The subjects of this study were patients who underwent ALL treatment with 6MP. Tests for the NUDT15 and TPMT genotypes were performed, and prospective DNA-TGN and erythrocyte TGN samples were collected after two weeks or more of 6MP treatment. DNA-TGN was quantified using the liquid chromatography-tandem mass spectrometry method. Results A total of 471 DNA-TGN measurements in 71 patients were analyzed, which ranged from 1.0 to 903.1 fmol thioguanine/μg DNA. The 6MP intensity demonstrated a significant relationship with DNA-TGN concentration (P<0.001). Patients harboring NUDT15 variants were treated with a lower dose of 6MP (P<0.001); however, there was no significant difference in DNA-TGN concentration when compared to patients carrying wild-type NUDT15 (P = 0.261). These patients also presented higher variation in DNA-TGN levels (P = 0.002) and DNA-TGN/6MP intensity (P = 0.019) compared to patients carrying wild-type NUDT15. DNA-TGN concentration did not show a significant correlation with WBC count (P = 0.093). Conclusions Patients harboring NUDT15 variants demonstrated similar DNA-TGN concentrations even at low doses of 6MP and showed high variability in DNA-TGN. Particularly in patients with NUDT15 variants who need a reduced 6MP dose, DNA-TGN could be applied as a useful marker to monitor the therapeutic effect of 6MP.

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IKZF1 Status As an Independent Prognostic Marker in Adult Common B-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v124.21.1075.1075 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1075-1075

Author(s):

Qiumei Yao ◽

Kaiyan Liu ◽

Bing Jiang ◽

Yanrong Liu ◽

Qian Jiang ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Prognostic Marker ◽

Lymphoblastic Leukemia ◽

Wild Type ◽

Survival Curves ◽

Free Survival ◽

Pcr Multiplex ◽

In The Wild ◽

Group 5

Abstract Previous studies have suggested that IKZF1 gene deletions are associated with an adverse prognosis in acute lymphoblastic leukemia (ALL). However, the prognostic value of IKZF1 deletions in adult common B-cell ALL (Com-B ALL) has not been well-defined, especially in the context of different post-remission therapies. The objectives of this study were to evaluate the prognostic role of IKZF1 deletions in adult Com-B ALL. Overall, 162 untreated adult Com-B ALL patients were recruited between April 2006 and April 2013. Patients received one or two cycles of induction therapy and, if in remission, were allowed to select either ongoing systemic chemotherapy or hematopoietic stem cell transplantation (HSCT). BCR-ABL positive patients were additionally treated with tyrosine kinase inhibitors (TKIs). Deletions in the IKZF1 gene were detected using multiplex RQ-PCR, multiplex fluorescent PCR, multiplex ligation-dependent probe amplification (MLPA) and sequence analysis. High-risk (HR) was defined as having any of the following factors: central nervous system involvement at diagnosis; age ¡Ý50 years; WBC count ¡Ý30x109/L; hypodiploidy; mixed lineage leukemia (MLL) gene rearrangements; BCR-ABL rearrangements; t(1;19) translocations; or a complicated karyotype (¡Ý5 chromosomal abnormalities); failure to achieve remission after two cycles of induction therapy. The study end points included relapse-free survival (RFS), disease-free survival (DFS) and overall survival (OS), which was analyzed using the Kaplan-Meier method. The cumulative incidence of relapse (CIR) was estimated, adjusting for competing risks of death, and compared by Gray's test. IKZF1 deletions were detected in 79 (48.8%) out of 162 adult Com-B ALL patients. Patients with an IKZF1 deletion (n=79) had a significantly inferior prognosis than those with wild-type IKZF1 (n=83) (Fig.1). The prognosis of patients with IKZF1 deletions with or without the BCR-ABL rearrangements was significantly inferior compared to those of patients with neither IKZF1 deletions nor BCR-ABL rearrangements. The power of IKZF1 deletion status as a prognostic factor remained in both non-HR group (5-yr OS: 52.5%¡À13.1% for IKZF1 deletions (n=25) vs. 72.2%¡À8.8% for wild-type IKZF1 (n=42), P=0.048) and HR group (5-yr OS: 36.7%¡À8.2% for IKZF1 deletions (n=54) vs. 69.3%¡À8.3% for wild-type IKZF1 (n=41), P=0.015). Among patients who received chemotherapy as post-remission therapy (n=52), IKZF1 deletions were associated with an unfavorable OS (P=0.003). In this group, the median OS time for IKZF1-deletion patients (n=28) was 10.0¡À1.0 months, while the median OS time for IKZF1-wild-type carriers (n=24) was not achieved. Among those that received HSCT as post-remission therapy (n=94), IKZF1 status did not affect the prognosis. For patients with IKZF1 deletions, HSCT (n=40) was significantly superior to chemotherapy (n=28) as a post-remission therapy. In the wild-type IKZF1 HR group, patients that received HSCT (n=24) achieved a longer DFS and RFS while the OS did not significantly differ from that of patients who received chemotherapy alone (n=12). In the wild-type IKZF1 non-HR group, there was no significant difference between patients that received HSCT (n=30) and those who received chemotherapy (n=12) as a post-remission therapy. Comparing survival curves of HSCT to chemotherapy are shown in Fig.2. Multivariate analysis confirmed the negative impact of IKZF1 deletions on OS (RR 2.3; 95% CI, 1.3-4.3; P=0.007), DFS (RR 1.8; 95% CI, 1.1-3.0; P=0.029), RFS (RR 2.1; 95% CI, 1.3-3.9; P=0.018) and favorable impact of HSCT on OS (RR 0.2; 95% CI, 1.0-0.5; P=0.000), DFS (RR 0.2; 95% CI, 0.1-0.4; P=0.000), RFS (RR 0.2; 95% CI, 0.1-0.4; P=0.000). These results indicated that IKZF1 status served as an independent negative prognostic marker in adult Com-B ALL and surpassed conventional HR features in the era of TKIs. Patients may benefit from therapy stratified by IKZF1 deletions and conventional HR features. Fig. 1 Fig. 1. Survival curves related to status of IKZF1 in Com-B-ALL cohort. Abbreviation: IKZF1 WT, IKZF1 wide type; IKZF1 DEL, IKZF1 deletion. Fig. 2 Fig. 2. Comparing survival curves of HSCT to chemotherapy in IKZF1-deleted group, wild-type IKZF1 HR group and wild-type IKZF1 non-HR group. Disclosures No relevant conflicts of interest to declare.

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NUDT15 genetic variants are related to thiopurine-induced neutropenia in Thai children with acute lymphoblastic leukemia

Pharmacogenomics ◽

10.2217/pgs-2019-0177 ◽

2020 ◽

Vol 21 (6) ◽

pp. 403-410

Author(s):

Apichaya Puangpetch ◽

Rawiporn Tiyasirichokchai ◽

Samart Pakakasama ◽

Supaporn Wiwattanakul ◽

Usanarat Anurathapan ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Maintenance Therapy ◽

Therapy Group ◽

Lymphoblastic Leukemia ◽

Wild Type ◽

Homozygous Variant ◽

Heterozygous Variant ◽

Group 3 ◽

Group 2 ◽

Group 1

Aim: 6-Mercaptopurine (6MP) is key to the treatment of acute lymphoblastic leukemia (ALL) as part of maintenance therapy. NUDT15 was identified as a novel thiopurine regulator conferring 6MP sensitivity. The aim of this study was to evaluate the influence of NUDT15 variants on 6MP-induced neutropenia in Thai children with ALL. Materials & methodology: Genotyping of NUDT15 (c.415C>T; rs116855232) and c.36_37insGGAGTC; rs554405994) was performed by Sanger sequencing in 100 patients with ALL. Patients were classified into wild-type (group 1), heterozygous variant (group 2) and homozygous variant (group 3). Clinical and laboratory features during the first 6 months of maintenance therapy were investigated. Therapy-induced neutropenia was observed in 31 patients during the weeks 1–8 (early myelotoxicity), while therapy-induced neutropenia was observed in 47 patients during the weeks 9–24 (late myelotoxicity). Results: There were 85 wild-type patients, 14 heterozygous variant patients and one homozygous variant patient. NUDT15 variants were associated with neutropenia as compared with wild-type (odds ratio: 17.862; 95% CI: 4.198–75.992, padj = 9.5 × 10-5). Multivariate analysis showed that the low-risk group was associated with neutropenia (p = 0.014) in the first 8 weeks of 6MP therapy. Group 2 and group 3 patients had significantly lower absolute neutrophil counts compared with group 1. The adjusted dose during the first 6 months of maintenance therapy with NUDT15 genotype group 1, 2 and 3 were 50, 36.6 and 12.5 mg/m2/day, respectively. Conclusion: Taken together, our results indicate NUDT15 variants may cause neutropenia, and the 6MP dosage should be considered in patients according to the NUDT15 variants to inform personalized 6MP therapy.

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ERG Deletions Define a Novel Subtype of B-Progenitor Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v110.11.691.691 ◽

2007 ◽

Vol 110 (11) ◽

pp. 691-691 ◽

Cited By ~ 10

Author(s):

Charles G. Mullighan ◽

Christopher B. Miller ◽

Xiaoping Su ◽

Ina Radtke ◽

James Dalton ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

Gene Expression Profile ◽

Expression Profile ◽

Lymphoblastic Leukemia ◽

Luciferase Reporter ◽

The Novel ◽

Wild Type ◽

Cytogenetic Abnormalities ◽

Novel Gene

Abstract In a previous gene expression profiling study of acute lymphoblastic leukemia (ALL), we identified a novel subtype of B-progenitor ALL (4.9% of 284 cases) with a unique gene expression profile, aberrant expression of CD2 and the absence of recurring cytogenetic abnormalities (Cancer Cell2002;1:133). Efforts to identify rearrangement or mutation of many of the top-ranked genes in the novel expression signature failed to identify a causative lesion. To further investigate the genetic basis of this subtype, we performed integrated genomic analysis of 277 ALL cases. Affymetrix 250k Sty and Nsp SNP microarrays were used in all cases, and Affymetrix U133A gene expression profiles were obtained on 183 of the cases. Unsupervised clustering of gene expression data identified 16 cases of the novel subtype, including all of the 14 previously defined cases. Remarkably, focal mono-allelic deletions of the ETS family member ERG (v-ets erythroblastosis virus E26 oncogene homolog) were detected by genome-wide copy number analysis in 11/16 (69%) of the novel cases, but not in any other ALL subtype. Extensive analysis failed to reveal evidence of translocations involving the altered ERG allele, indicating that these are intragenic deletions limited to ERG. The ERG deletions involved a subset of exons (ERG isoform 1 exons 3–7 or 3–9) resulting in the expression of internally deleted ERG transcripts with altered reading frames predicted to produce a prematurely truncated N-terminal protein fragment. However, using an alternative translational start site 5′ to exon 10, the transcripts also encode a ∼28kDa C-terminal ERG fragment that contains the entire C-terminal ETS DNA-binding and transactivation domains, but lacks all N-terminal domains. Importantly, western blot analysis of primary leukemic blasts revealed expression of only the 28kDa C-terminal ERG protein, along with full length ERG expressed from the retained wild type allele. Remarkably, the C-terminal ERG protein was also detected in 4 of 5 novel ALL cases that lacked detectable ERG deletions, but not in any other ALL subtype. In luciferase reporter assays, this aberrant ERG protein acts as a competitive inhibitor of wild type ERG. Analysis of a second cohort of 35 B-progenitor ALL cases lacking recurring cytogenetic abnormalities identified two cases with ERG deletions and a third expressing the aberrant ERG protein, all of which had the novel gene expression profile. Sequencing of ERG in 252 ALL cases identified only one case with an ERG mutation that resulted in a frameshift in the ETS domain. This case did not share the novel signature nor express the aberrant C-terminal ERG protein. Finally, in an analysis of 37 acute leukemia cell lines, the B-progenitor ALL line NALM-6 was found to harbor a focal, internally truncating ERG deletion, expressed the aberrant ERG protein, and shared the novel gene expression profile, thus identifying it as a model of this novel ALL subtype. These data establish focal ERG deletions as the genetic lesion underlying a novel subtype of ALL, and have expanded the genetic mechanisms that lead to the dysregulation of ERG from chromosomal translocations that result in enhanced transcriptional activity, to deletions that generate dominant negative forms of the transcription factor.

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The MDM2 Antagonist Nutlin-3 Is a Potent Inducer of Apoptosis in Pediatric Acute Lymphoblastic Leukemia Cells with Wild-Type p53 and Overexpression of MDM2.

Blood ◽

10.1182/blood.v110.11.4277.4277 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4277-4277

Author(s):

Lubing Gu ◽

Ningxi Zhu ◽

Harry W. Findley ◽

Muxiang Zhou

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Cell Lines ◽

Cytotoxic Effect ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Wild Type ◽

Potent Inducer ◽

Pediatric Acute Lymphoblastic Leukemia ◽

Mdm2 Antagonist ◽

Mdm2 Expression

In pediatric acute lymphoblastic leukemia (ALL), overexpression of MDM2 by leukemic cells is typically associated with a wild-type (wt) p53 phenotype and chemoresistance. A recently-developed small-molecule antagonist of MDM2, nutlin-3, inhibits the MDM2-p53 interaction, resulting in induction of p53 activity and apoptosis. In the present study, we evaluated the cytotoxic effect of nutlin-3 on ALL cells with different p53 status and MDM2 expression, using 18 cell lines and 30 primary leukemia samples. We found that both ALL cell lines and primary ALL samples with wt-p53 are sensitive to nutlin-3. No cytotoxic effect of nutlin-3 was detected in ALL cells with either p53-mutant or null phenotypes. In wt-p53 ALL cells, there was a significant positive correlation between MDM2 expression levels and sensitivity to nutlin-3. Nutlin-3-induced cell death was mediated by p53-induced activation of pro-apoptotic proteins and by p53-induced repression of the anti-apoptotic protein survivin. Because p53 function is inhibited by MDM2 in chemoresistant, MDM2-overexpressing ALL cells, potent killing of these cells by nutlin-3 suggests that this agent may be a novel therapeutic for refractory ALL.

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