ALBERT

Description: The Ets related gene, ERG, encodes a transcription factor with a vital role in hematopoiesis. Recent findings have shown that ERG knockout mice require a minimum of one functional allele to ensure embryonic blood development and adult stem cell maintenance. Moreover, it was earlier reported that enforced expression of ERG induced oncogenic transformation in 3T3 cells. Overexpression of ERG, observed in a subset of acute T-lymphoblastic and acute myeloid leukemia patients, was associated with an inferior outcome. However, the impact of ERG contributing to this unfavourable phenotype has yet to be determined, as downstream targets of ERG in leukemia remain unknown. Herein, we conducted a genome-wide analysis of ERG target genes in T-lymphoblastic leukemia. Chromatin immunoprecipitation-on-chip array (ChIP-on-chip) analyses were performed using two ERG specific antibodies for the enrichment of ERG-bound DNA templates in T-lymphoblastic leukemia cells (Jurkat) with input DNA or IgG precipitated DNA as controls. Enriched DNA templates and control DNA were differentially labelled and co-hybridized to high resolution promoter chip arrays with 50–75mer probes (770,000) representing 29,000 annotated human transcripts (NimbleGen). Based on two independent ChIP-on-chip assays, bioinformatic analysis (ACME) yielded statistically significant enriched peaks (using a sliding window of 1000 bp, and a P-value 〈 0.0001) identifying promoter regions of 365 potential ERG target genes. From these genes, clustering by functional annotation was performed using the DAVID database and subsequently genes related to leukemia were further selected for quantitative PCR validation. The design of promoter primers included the highly conserved ETS GGAA DNA binding site. Genes with greater than two-fold enrichment (ERG ChIP versus control) included WNT2 (17-fold), OLIG2 (14-fold), WNT11 (7-fold), CCND1 (5-fold), WNT9A (4-fold), CD7 (3-fold), EPO (3-fold), ERBB4 (3-fold), RPBJL (3-fold), TRADD (3-fold), PIWIL1 (2-fold), TNFRSF25 (2-fold), TWIST1 (2-fold), and HDAC4 (2-fold). Interestingly, enriched target genes involved in developmental processes (WNT2, WNT9A, WNT11, TWIST1, PIWIL1, ERBB4, and OLIG2) have shown oncogenic potential when mutated or overexpressed. Thus, we hypothesize that overexpression of ERG may contribute to T-cell leukemogenesis by the deregulation of these oncogenic targets. Further disclosure of ERG directed downstream pathways may contribute to the design of specific treatment strategies (such as WNT inhibitors) with particular effectiveness in ERG deregulated leukemia.

Description: The tyrosine kinase inhibitor STI571 is a promising agent for the treatment of advanced Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL), but resistance develops rapidly in most patients after an initial response. To identify mechanisms of resistance to STI571, 30 complementary DNAs (including 9 matched samples) obtained from the bone marrow of individuals with Ph+ ALL were analyzed by direct sequencing of a 714–base pair region of ABL encoding for the adenosine triphosphate (ATP)–binding site and the kinase activation loop. A single point mutation was found at nucleotide 1127 (GI6382056) resulting in Glu255Lys. This mutation occurred in 6 of 9 patients (67%) following their treatment with STI571 but not in the samples from patients before beginning treatment with STI571. Glu255Lys is within the motif important for forming the pocket of the ATP-binding site in ABL and it is highly conserved across species. In conclusion, Ph+ ALL samples resistant to STI571 have a unique mutation Glu255Lys of BCR-ABL.

Description: C/EBPδ belongs to the family of highly conserved CCAAT/enhancer binding protein (C/EBP) transcription factors. Members of this family play a critical role in the regulation of mitotic growth arrest and differentiation in numerous cell types. To examine the consequences of C/EPBδ expression, we transfected C/EPBδ into CML myeloid leukemia (KCL22, K562), prostate (LNCaP, PC3, DU145), and breast (MCF-7, T47D, MDA-MB-231) cancer cell lines. C/EBPδ expression resulted in a proliferative arrest and an increase in apoptosis of the myeloid leukemia cells, as well as the prostate cells LNCaP and PC3, and the breast cells MCF-7 and T47D. In contrast, DU145 prostate and MDA-MB-231 breast cancer cells were not inhibited by C/EBPδ, indicating that the biologically properties of C/EBPδ depend upon its cellular context. We further studied the molecular mechanisms underlying the affect of C/EPBδ expression in CML leukemic cells. Myeloid differentiation of KCL22 and K562 blast cells as shown by morphologic changes and induction of secondary specific granule genes, occurred within 4 days of inducing expression of C/EBPδ. Furthermore, expression of C/EBPδ was associated with downregulation of c-Myc and cyclin E, and upregulation of the forkhead transcription factor FoxO1a (FKHR) and the cyclin-dependent kinase inhibitor p27Kip1. In addition, microarray analysis showed that C/EBPδ mRNA is upregulated during granulocytic differentiation of normal CD34+ bone marrow cells, suggesting that C/EBPδ is involved in lineage-specific differentiation. Taken together, these results show that expression of C/EBPδ in BCR-ABL-positive CML cells in blast crisis, is sufficient for neutrophil differentiation and suggest that ectopic induction of C/EBPδ in the blastic phase of CML, as well as in certain cases of prostate and breast cancers, may hold promising therapeutic potential.

Description: The oncogenic ETS transcription factor ERG is involved in various cellular pathways including developmental regulation, proliferation, and differentiation. In hematopoiesis ERG plays a specific role during normal T-cell differentiation showing high expression levels in stem cells and down regulation in the progenitor compartment. In this regard, it is intriguing that aberrant expression of ERG was found in a subset of patients with acute T-lymphoblastic leukemia (T-ALL) and was associated with an inferior outcome. Furthermore, high level ERG expression was of adverse prognostic significance in patients with newly diagnosed acute myeloid leukemia (AML), thus highlighting ERG’s potential role in myeloid as well as T-lineage leukemogenesis. ERG3 (NM_182918) and ERG2 (NM_004449) represent the main isoforms and show abundant expression in myeloid and lymphoid hematopoietic progenitor cells. The expression pattern of specific ERG isoforms in acute leukemias has yet to be investigated. To further elucidate the nature of aberrant ERG expression we have determined the existence and transcriptional regulation of ERG isoforms in pretreatment bone marrow samples of adult T-ALL (n=21) and AML (n=20) patients as well as in normal CD34+ hematopoietic cells of healthy volunteers (n=5). 5′RACE revealed the presence of a new ERG isoform (ERG3Δex12) characterized by expression of exon 5 and absence of exon 12. Expression of ERG3Δex12 was verified by RT-PCR in AML, T-ALL, and CD34+ cells. In addition, real-time RT-PCR showed concomitant expression of the two main isoforms ERG2 and ERG3 in AML and normal CD34+ cells. In contrast, T-ALL patients lacked expression of ERG isoforms harboring exon 4 (ERG2). Promoter analyses of ERG2 and ERG3 revealed the presence of two CpG islands in the ERG2 promoter region, whereas no CpG island was predicted in the ERG3 promoter. Bisulfit conversion of genomic DNA and sequencing of cloned PCR products revealed a significantly higher degree of methylation of CpG island 2 in T-ALL samples (median: 86.4%, range: 16.0 – 98.8%) as compared to AML (median: 38.1%, range: 10.9 – 60.7%; P-value=0.0002 - two sided T-test). As for CpG island 1, CD34+ cells had the lowest rate of methylation in CpG island 2 (median: 7.7%, range: 2.4 – 20.7%). Thus, the differential expression of ERG isoforms is mediated by epigenetic silencing of exon 4 containing transcripts in T-ALL. In conclusion, the identification of the new ERG isoform (ERG3Δex12) suggests the association with different partners as the central exons, including exon 12, guide the interaction with different proteins. Furthermore, the distinct expression of specific ERG transcripts controlled by methylation adds to the complexity of ERG directed downstream pathways in different leukemic subtypes.

Description: An imbalance between cellular apoptosis and survival may be critical for the pathogenesis of lymphoma. Therefore, the gene expression pattern in lymph node preparations from patients with mantle cell lymphoma (MCL) was compared to the pattern in nonmalignant hyperplastic lymph nodes (HLs). Oligonucleotide microarray analysis was performed comparing 5 MCLs to 4 HLs using high-density microarrays. The expression data were analyzed using Genespring software. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction using the RNA extracted from 16 MCL and 12 HL samples. The focus was on 42 genes that were at least 3-fold down-regulated in MCL; in addition to the B-cell leukemia 2 (BCL2) system other apoptotic pathways were altered in MCL. The FAS-associated via death domain (FADD) gene that acts downstream of the FAS cascade as a key gene to induce apoptosis was more than 10-fold down-regulated in MCL. Furthermore, the death-associated protein 6(DAXX) gene, the caspase 2 (CASP2) gene, and the RIPK1 domain containing adapter with death domain(RAIDD) gene, which are key genes in other proapoptotic pathways, were also decreased in the MCL samples. The suggestion is made that in addition to the known overexpression of cyclin D1, which drives entry into the cell cycle, disturbances of pathways associated with apoptosis contribute to the development of MCL.

Description: The transcription factor Wilms tumor protein (WT) 1 belongs to a new generation of tumor antigens, which are essential for tumor cell proliferation. WT1 is highly expressed in AML and in MDS upon appearance of blasts. A phase II trial of vaccination with the HLA-A2-restricted WT1.126–134 peptide was performed in patients with AML and MDS and overexpression of WT1 to determine immunogenicity and clinical activity. Patients received vaccinations with 0.2 mg WT1.126–134 peptide (day 3), 62.5 mcg dendritic cell-stimulating adjuvant GM-CSF (days 1–4) and 1 mg T helper protein keyhole limpet hemocyanin (day 3). The initial 13 patients were to receive 4 biweekly and subsequent 4-weekly vaccinations, the subsequent 13 patients were continuously vaccinated biweekly. Vaccination was continued in absence of overt disease progression. WT1 levels were assessed by quantitative RT-PCR and WT1-specific T cell responses by tetramer analyses and cytokine flow cytometry. Response assessment following IWG-MDS criteria was used, capturing stable disease and hematologic improvement. A duration of 8 weeks was required for stable disease. Enrolment was completed in June 2006 with 24 patients with AML and 2 with MDS (RAEB). Of the 24 AML patients, 16 had 〉 5% marrow blasts at study onset (8 without prior chemotherapy, 4 with disease persistence following chemotherapy, 4 with PR), and 8 were in CR at high risk for relapse. A median of 10 (range 4 – 23) vaccinations was administered with 8 patients currently still under treatment. No significant toxicity occurred. To date, 22 patients are evaluable for clinical response. Overall, 8/16 patients with 〉 5% marrow blasts at study onset displayed clinical efficacy of vaccine treatment (SD or better). One AML patient achieved CR for 12 months after brief initial progression, and 7 patients had disease stabilization (2, 2+, 3, 3, 6, 10+, 14 months). One of these patients with RAEBII had a major response of neutrophils and platelets, and one AML patient had initial progression and subsequent transient complete clearance of peripheral blasts. WT1 transcripts as molecular disease marker decreased at least 3-fold (range 3-fold - 〉50-fold, median 〉10-fold) in 12 of 20 currently evaluated patients, including all 8 patients with evidence of clinical efficacy and 4 of 5 AML patients vaccinated in CR. The generation of a WT1-specific T cell response in peripheral blood and bone marrow was detected in 12 of 16 evaluated patients including all 6 of these 16 patients with evidence for clinical efficacy. This study shows that WT1 vaccination has promising antileukemia activity. A multicenter comparative WT1 vaccination study in CR patients at high risk of relapse is currently initiated.

Description: The NOTCH1 gene encodes a transmembrane receptor important for the T-lineage commitment. Activating NOTCH1 mutations had been found in approximately 56% of pediatric T-lymphoblastic leukemia (T-ALL) samples and were associated with a favourable outcome. Here the frequency and the association of NOTCH1 mutations to clinical features, as well as molecular markers were analyzed for the first time in a large cohort of adult T-ALL patients. Genomic DNA of pretreatment samples of 126 adult T-ALL patients enrolled on the GMALL protocols 05/93 and 06/99 were analyzed by direct sequencing for the presence of NOTCH1 mutations in the heterodimerization domain (HD-C, HD-N), the polypeptide enriched in proline, glutamate, serine and threonine (PEST; divided into PEST-1 and PEST-2) domain, and the transactivation domain (TAD). In addition, leukemic blasts were molecularly characterized for mRNA expression of HOX11, HOX11L2, ERG, and BAALC by real-time RT-PCR. Immunophenotyping was performed differentiating T-ALL into three subtypes (early, thymic, mature). NOTCH1 mutations were identified in 72 (57%) of the 126 patients including mutations in the HD (n=56), in the TAD (n=5), and in the PEST (n=20) domain. Eleven patients had more than one mutation, including 3 patients with HD and TAD, and 6 patients with coexisting HD and PEST mutations. T-ALL patients carrying mutations revealed similar clinical characteristics as compared to NOTCH1 wild type cases with respect to age, sex, CNS involvement, white blood count. Moreover, no differences were seen in the response to induction therapy or survival between NOTCH1 wild type and mutated cases (in the overall groups as well as in subgroups analyses). In the expression pattern of HOX11, HOX11L2, BAALC, or ERG there were no significant differences between the two NOTCH1 groups. Patients with mutated NOTCH1 showed a higher frequency of a thymic immunophenotype (65% vs. 39% for unmutated cases; P=0.013). Furthermore, a significant association was observed between the expression of specific surface antigens and the NOTCH1 genotype: NOTCH1 mutated cases were significantly associated with the expression of CD10 (P

Description: Gene patterns of expression in purified CD34+ bone marrow cells from 7 patients with low-risk myelodysplastic syndrome (MDS) and 4 patients with high-risk MDS were compared with expression data from CD34+ bone marrow cells from 4 healthy control subjects. CD34+ cells were isolated by magnetic cell separation, and high-density oligonucleotide microarray analysis was performed. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction. Class membership prediction analysis selected 11 genes. Using the expression profile of these genes, we were able to discriminate patients with low-risk from patients with high-risk MDS and both patient groups from the control group by hierarchical clustering (Spearman confidence). The power of these 11 genes was verified by applying the algorithm to an unknown test set containing expression data from 8 additional patients with MDS (3 at low risk, 5 at high risk). Patients at low risk could be distinguished from those at high risk by clustering analysis. In low-risk MDS, we found that the retinoic-acid–induced gene (RAI3), the radiation-inducible, immediate-early response gene (IEX1), and the stress-induced phosphoprotein 1 (STIP1) were down-regulated. These data suggest that CD34+cells from patients with low-risk MDS lack defensive proteins, resulting in their susceptibility to cell damage. In summary, we propose that gene expression profiling may have clinical relevance for risk evaluation in MDS at the time of initial diagnosis. Furthermore, this study provides evidence that in MDS, hematopoietic stem cells accumulate defects that prevent normal hematopoiesis.