scholarly journals HMGB1 Interacts with the MLL-AF4 Fusion Complex to Regulate Pro-Leukemic Gene Transcription in Infant Acute Lymphoblastic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2602-2602
Author(s):  
Liana M Toia ◽  
Erica Lynne Braverman ◽  
Jinno Antonio Magno ◽  
Jessica C. Shand
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Protein Expression ◽  
Western Blot ◽  
Mrna Expression ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Mll Gene ◽  
Gel Shift

Abstract Acute lymphoblastic leukemia (ALL) in infants carries a poor prognosis and is characterized by cytogenetic rearrangements producing abnormal MLL fusion genes. Clinically effective targeting of the MLL fusion heterocomplex remains challenging, and therapeutic options remain limited. We have observed that the reduced isoform of HMGB1, a chromatin architectural protein that stabilizes DNA and facilitates transcription, is selectively over-expressed in the nuclei of infant MLL-ALL cells. In this study, we generated an HMGB1 siRNA knockdown in primary MLL-ALL cells from 3 infants to test our hypothesis that HMGB1-MLL interactions regulate pro-leukemic gene expression and represent a rational therapeutic target. CD19-selected leukemic blasts were isolated from the cryopreserved bone marrow or peripheral blood specimens of 3 infants with cytogenetically confirmed MLL-AF4 rearrangements. HMGB1 knockdown was confirmed by comparing HMGB1 mRNA and protein expression, by qPCR and Western Blot, in cells transfected with HMGB1 vs. control sequence siRNA. First, determined whether HMGB1 knockdown affected expression of the MLL fusion gene itself, by comparing MLL-AF4 mRNA and protein levels 72 hours after siRNA transfection. HMGB1 knockdown produced a 2.8 (± 0.55)- fold decrease in MLL-AF4 mRNA expression by qPCR (p<0.05), with a corresponding decrease in MLL-AF4 fusion protein expression by Western Blot, in each of the 3 specimens. Next, we determined whether HMGB1 binds functionally relevant regions of the MLL gene. We developed an electrophoretic mobility assay (EMSA) to compare the mobility of lysates from control vs. HMGB1 siRNA-treated infant MLL-ALL cells when mixed with biotinylated oligonucleotides spanning the transcriptionally active domains of MLL1. In each of 3 primary infant MLL-ALL cells, we detected a consistent gel-shift pattern on SDS-PAGE, in wild-type and control siRNA lysates, with oligonucleotides spanning exons 6-9- where many MLL-AF4 fusions occur. The gel-shift was completely abrogated in HMGB1 siRNA lysates. We then compared the expression of MLL target genes involved in leukemic transformation, by qPCR, in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. We observed a significant (p<0.01) reduction in expression of MEIS1 (5.8 ± 2.2-fold decrease), HOXA7 (4.3 ± 0.4-fold decrease) and HOXA9 (3.7 ± 1.5-fold decrease) in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. These data confirmed a role for HMGB1 in MLL gene/target gene regulation at the DNA level. Finally, we considered whether HMGB1, as a scaffold protein, could interact directly with the MLL fusion heterocomplex at the protein level. We immunoprecipitated HMGB1 from the nuclear fraction of wild-type primary infant MLL-ALL cells (n=3 patients), then probed the pull-down for N-terminal MLL (MLLn), C-terminal MLL (MLLc), the MLLn-AF4 fusion, the MLLn-ENL fusion, and the MLL-associated histone 3 methyltransferase DOT1L. MLLn and MLLn-AF4 were strongly detected in all HMGB1 immunoprecipitates. Individual and sequential co-immunoprecipitation of HMGB1 with MLL-AF4 and DOT1L in revealed loss of known complex formation between MLL-AF4 and DOT1L following HMGB1 knockdown. This was accompanied by a 3.4 (± 0.9)-fold decrease in DOT1L mRNA expression (p<0.001) by qPCR and a complete loss of histone 3k79me2 protein expression by Western blot. Taken together, these data suggest a central role for the fully reduced isoform of HMGB1, found in high abundance in infant ALL nuclei, in the formation of the MLL-AF4 transcription complex- including for the stable recruitment of DOT1L and H3K79me2, and in the regulation of MLL target genes such as HOXA9 and MEIS1. We are currently conducting chromatin immunoprecipitation and sequencing studies to identify methylation marks, particularly at H3K79me2, impacted by HMGB1 knockdown in infant ALL cells. We hope these studies will directly inform the development of small molecule inhibitors that specifically disrupt the binding sites and capacities of HMGB1 with MLL, which could synergize with the effects of methyltransferase inhibitors to more completely silence leukemic gene expression in infant ALL and improve the prognosis of this devastating disease. Disclosures No relevant conflicts of interest to declare.

TYK2-STAT1 Pathway Positively Regulates BCL2 Gene Expression in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1470-1470
Author(s):  
Takaomi Sanda ◽  
Jeffrey W Tyner ◽  
Alejandro Gutierrez ◽  
Vu N Ngo ◽  
Jason M Glover ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Protein Expression ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bcl2 Protein ◽  
Bcl2 Expression

Abstract Abstract 1470 To discover oncogenic pathways that are characteristically deregulated in T-cell acute lymphoblastic leukemia (T-ALL), we performed RNA interference screens both in T-ALL cell lines and primary specimens. We found that the JAK tyrosine kinase family member, TYK2, and its downstream effector, STAT1, are each required for the survival of T-ALL cells. To identify the effector molecules downstream of the TYK2-STAT1 pathway in T-ALL, we analyzed global gene expression profiles in TYK2-dependent T-ALL cell lines after silencing of TYK2 or STAT1. As expected, gene set enrichment analysis revealed that genes downregulated by TYK2 knockdown were generally also downregulated by knockdown of STAT1. Importantly, we found that expression of the anti-apoptotic gene BCL2 was significantly downregulated after silencing of both TYK2 and STAT1. Analysis by quantitative PCR of additional T-ALL cell lines revealed that silencing of TYK2 resulted in significant reductions of BCL2 mRNA expression in multiple TYK2-dependent cell lines. Expression of the wild-type but not the kinase-dead TYK2 protein was sufficient to rescue BCL2 protein expression and to prevent apoptosis after knockdown of endogenous TYK2, indicating that the tyrosine kinase activity of TYK2 is required for BCL2 upregulation. Similarly, expression of the shRNA-resistant wild-type STAT1A protein partially rescued BCL2 protein expression and prevented apoptosis, while a variant of STAT1A (Y701F) that is incapable of becoming phosphorylated on a requisite tyrosine residue did not rescue BCL2 levels. Taken together, our findings indicate that aberrant activation of a TYK2-STAT1 pathway upregulates BCL2 expression in T-ALL cells, and that the T-ALL cells develop pathway dependence, in that they require these sustained high levels BCL2 expression for survival. Disclosures: No relevant conflicts of interest to declare.

Lack of Protein Kinase C Alpha Is Associated with Poor Prognosis in Pediatric T-Lineage Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 744-744
Author(s):  
Gloria Milani ◽  
Benedetta Accordi ◽  
Marco Giordan ◽  
Silvia Bresolin ◽  
Luisa Galla ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase C ◽  
Acute Lymphoblastic Leukemia ◽  
Protein Expression ◽  
Western Blot ◽  
Cell Lines ◽  
Gene Expression Analysis ◽  
Pediatric Patients ◽  
Lymphoblastic Leukemia ◽  
Kinase C

Abstract Abstract 744 Background: T-lineage Acute Lymphoblastic Leukemia (T-ALL) accounts for about 15% of pediatric ALL. Although the outcome of T-ALL has improved with current therapies, T-ALL pediatric patients remain at high risk for early relapse. It is therefore very important to identify new prognostic biomarkers and to develop new therapeutic approaches for these patients. More specific and less toxic therapies might be developed through the identification of molecular targets, such as aberrantly activated phosphoproteins, that offer the possibility to use specific kinase inhibitors. In this research we first explored the phosphoproteomic profile of pediatric T-ALL patients at diagnosis, through Reverse Phase Protein Arrays (RPPA), and we further investigated the origin and the functional significance of Protein Kinase C alpha (PKCα) downregulation in patients that are liable to relapse. Methods: We analyzed 98 pediatric T-ALL patients at diagnosis using RPPA. This innovative approach allows to profile phosphorylation modifications and to characterize the activation state of cellular protein networks. The whole proteome of each patient was immobilized on nitrocellulose coated glass slides and each slide was stained with one of 53 different antibodies included in the study. Protein expression/activation was compared between patients subgroups defined by clinical/molecular features through statistical analyses. Gene expression analysis was performed by means of HG-U133 Plus 2.0 array. Permission for this study was obtained following the tenets of the Declaration of Helsinki. A set of commercially available human T-ALL cell lines was studied by Western Blot and cells were treated with Ro-32-0432, a commercial PKC inhibitor. Results: The phosphoproteomic profile of T-ALL patients was compared between patients subgroups at diagnosis. Comparison between relapsed vs non relapsed patients samples revealed the downregulation of PKCα(S657) in relapsed patients (t test with BH multiplicity corrections p=0.02) and Relapse Free Survival analysis through Kaplan-Meier estimates showed that patients with low PKCα(S657) have a high probability to relapse. Notably, 54.5% of patients with low PKCα(S657) relapsed whereas no relapses were observed among patients with high PKCα activity. Multivariate analysis showed no correlation between PKCα(S657) levels and other parameters currently used for clinical characterization of T-ALL patients. Differences in PKCα(S657) between relapsed and non relapsed patients were confirmed by Western Blot in two independent sets of T-ALL patients. Analysis of PKCα total protein form revealed concordance between total protein expression and PKCα(S657) levels. Furthermore, gene expression analysis revealed a significant difference in PKCα gene expression levels, comparable with levels of protein expression among T-ALL patients studied by RPPA. Gene expression data were validated by Sybr Green Real-Time Quantitative PCR. Moreover, gene pathway analysis in a larger cohort of T-ALL pediatric patients, divided by high and low PKCα mRNA levels, revealed the phosphatidylinositol signalling pathway as the most important deregulated pathway. Of note, this pathway is involved in the production of DAG and Ca2+, the PKCα cofactors that play an essential role in mediating its translocation from the cytosol to the plasmatic membrane and subsequent activation. PKCα and PKCα(S657) were then analysed by Western Blot in five T leukemic cell lines (DND41, P12 ICHIKAWA, MOLT3, TALL1 and CEM). These cell lines were treated with a PKCα inhibitor and PKCα inhibition effects were studied by the means of MTT assay and Trypan Blue cell count. An increase in cell proliferation was observed in each treated cell line 9 hours after PKCα inhibition. Conclusions: PKCα is differentially expressed in T-ALL pediatric patients. Low levels of PKCα(S657) are associated with a higher probability to relapse and downregulation or absence of PCKα seems to be related to a more aggressive T-ALL phenotype. Ongoing studies are needed to better define its role as a new prognostic marker. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2006 ◽  
Vol 109 (8) ◽  
pp. 3417-3423 ◽  
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.

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

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 293-293 ◽  
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&lt;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.

High CD45 (PTPRC) Expression Is Associated with An Overall Poor Outcome in Childhood Acute Lymphoblastic Leukemia Treated on the ALL-BFM 2000 Protocol and Exerts An Especially Pronounced Effect in Intermediate Risk Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 742-742
Author(s):  
Gunnar Cario ◽  
Rita Mitlohner ◽  
Martin Zimmermann ◽  
Renja Romey ◽  
Peter Rhein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
Protein Expression ◽  
Lymphoblastic Leukemia ◽  
Treatment Resistance ◽  
Intermediate Risk ◽  
Prognostic Relevance ◽  
Risk Patients ◽  
Low Expression

Abstract Abstract 742 Further improvement of outcome in childhood acute lymphoblastic leukemia (ALL) could be achieved by identifying additional high-risk (HR) patients who then may benefit from an intensified treatment. In trial ALL-BFM 2000, the HR group was defined by inadequate initial response to induction treatment [poor prednisone response on treatment day eight (PPR), non remission on treatment day 33, and/or a high load of minimal residual disease (MRD, ≥10E-3) after 12 weeks of treatment (TP2)] and/or by positive cytogenetics for a t(4;11) or t(9;22). No MRD already on treatment day 33 defined standard risk (SR) patients, a measurable MRD at a low level characterized the intermediate risk (IR) group. Of importance, the majority of relapses occurred within this heterogeneous group of patients. In order to identify potential new stratification markers we earlier compared gene expression profiles of MRD resistance (HR) and sensitive (SR) ALL in a case-control setting (Cario et al, Blood 2005). Subsequently, we aimed at confirming the potential prognostic relevance of genes identified and their respective proteins in representative study populations. CD45 (also PTPRC, protein-tyrosine phosphatase, receptor-type, C) was one of these candidate genes. In order to assess its prognostic relevance, CD45 gene expression was first analyzed by quantifiable RT-PCR in a set of 555 precursor B-ALL (pB-ALL); its protein expression subsequently in 422 pB-ALL patients by flow cytometry. About one third of patients were included in both study sets. Normalization of protein expression was done by assessing the density of surface expression relative to its density on normal lymphocytes. The 90th percentile was used as a cut-off to distinguish a CD45-high from a CD45-low expression group in both analyses. In gene expression analysis we observed a significant association of a high CD45 expression with a high white blood cell count at diagnosis (WBC) (P = 0.0004), NCI-HR (P = 0.03) as well as presence of the MLL-AF4 rearrangement (P < 0.0001). Moreover, a high CD45 expression was associated with in-vivo treatment resistance as defined by MRD (P = 0.0025). Analyzing CD45 protein expression confirmed the association of a high expression with a high WBC (P < 0.0001), NCI-HR (P = 0.0002) as well as presence of the MLL-AF4 rearrangement (P < 0.0001). Moreover, although the association to treatment resistance was lower (P = 0.055), patients with a high CD45 expression had a significantly worse 5-years EFS probability of 62±8% compared to 82±2% for those in the low-expression group (P=0.002). Focussing on the IR group, patients with a high CD45 expression had a very poor outcome (EFS 45±15%) as compared to those with a low expression (EFS 86±3%, P < 0.0001). This effect was mainly related to a higher cumulative relapse incidence (55±16% vs. 13±3%, P < 0.0001). Of interest, no significant differences in EFS were seen in HR patients. Based on our results, consideration of CD45 protein expression may serve as additional stratification tool in BFM-based protocols to further refine true non-high-risk patients with a low risk of relapse by identifying additional patients at high relapse risk. Of importance, in view of the fact that CD45 expression was not prognostic in the high-risk group, patients with a high CD45 expression currently treated on non high risk arms, may potentially benefit from an intensified treatment in the HR arm. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 691-691 ◽  
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.

A Novel Approach for Analyzing Gene Expression Profiles Defines Two Distinct Subgroups of t(4;11) Positive Infant Acute Lymphoblastic Leukemia Patients.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4282-4282
Author(s):  
Ronald W. Stam ◽  
Stefan Bohlander ◽  
Renate Kirschner-Schwabe ◽  
Susan Arentsen-Peters ◽  
Rob Pieters ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Relational Database ◽  
Target Genes ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Biological Properties ◽  
Database Program ◽  
Infant Acute Lymphoblastic Leukemia

Abstract Gene expression profiles (GEPs) are becoming increasingly more important for diagnostic procedures, allowing clinical predictions including treatment response and outcome for various types of cancer. However, established gene signatures not always appear helpful in understanding underlying disease mechanisms. Therefore, we explored an alternative approach for analyzing GEPs of a group of t(4;11) positive infant acute lymphoblastic leukemia patients (n=15), a highly aggressive type of MLL rearranged leukemia. We developed a method that uses a relational database program in combination with a normalization approach and specific discriminators. For normalization, every GEP of a given t(4;11) positive ALL sample was compared with 3 GEPs of normal bone marrow aspirates derived from healthy donors. Using the GeneChip Analysis Suite 5.0 program for single comparison analysis, the resulting gene lists were then compared with each other and only consistently identified genes (present on all three gene lists) were used for further analysis. This noise reduction decreased the amount of potentially deregulated target genes to about 30–40%. The remaining gene lists represented highly significant target genes that were then incorporated into a relational database program using specific discriminators. These discriminators were: upregulation of HOXA9 and HOXA10, presence/absence of the AF4-MLL fusion transcripts in addition to the MLL-AF4 fusion, and the localization of the genomic breakpoint within the MLL gene. This pilot study led to promising results, surprisingly classifying individual t(4;11) positive ALL patients into two distinct subgroups. Both subgroups share about 80 target genes, but also display particular sets of subgroup specific target genes. Importantly, these identified target genes can directly be linked to biological properties of t(4;11) positive leukemia cells, and therefore, allow important novel insights into this aggressive type of leukemia in infants.

IKZF1 and 22q11.22 Deletions and PDGFRA Gains Are Associated with Poor Outcome in Down Syndrome Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 289-289 ◽  
Author(s):  
Karen R Rabin ◽  
Clinton C Mason ◽  
Sivashankarappa Gurusiddappa ◽  
Hon-Chiu Eastwood Leung ◽  
Debra J. Morrison ◽  
...  
Keyword(s):  
Gene Expression ◽  
Down Syndrome ◽  
Acute Lymphoblastic Leukemia ◽  
Expression Profiling ◽  
Copy Number ◽  
Poor Prognosis ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Improve Risk Stratification ◽  
The Poor

Abstract Abstract 289 Children with Down syndrome (DS) have an increased risk of developing acute lymphoblastic leukemia (ALL), and consistently demonstrate poorer outcomes due to higher rates of both relapse and treatment-related mortality compared to other children with ALL. The biology of ALL in DS is unique, with lower frequency of the classic cytogenetic lesions generally observed in childhood ALL, and increased frequency of JAK2 mutations and CRLF2 rearrangements, which are not clearly associated with adverse prognosis in DS-ALL. In order to improve risk stratification and identify potential novel therapeutic targets in this vulnerable population, we analyzed 90 DS-ALL cases for prognostically significant copy number abnormalities (CNAs) in a collaborative cohort from the Children's Oncology Group (n=30), St. Jude Children's Research Hospital (n=22), AIEOP (n=16), Texas Children's Cancer Center (n=10), UKALL 2003 (n=6) and an archival UK sample (n=1), and Utah's Primary Children's Medical Center (n=5). Copy number profiling was performed using 500K, 6.0, CytoScan HD, and OncoScan FFPE Express arrays (Affymetrix), and Human CNV370-Duo arrays (Illumina). Gene expression profiling was performed using U133 Plus2.0 arrays (Affymetrix). Copy number was analyzed with Nexus Copy Number (BioDiscovery, Inc.) and gene expression was analyzed with Partek Genomics Suite (Partek, Inc.) and Gene Set Enrichment Analysis (Broad Institute). Deletions of a focal region on 22q11.22 (present in 28.9% of cases, similar to the incidence previously reported in a non-DS cohort [Mangum et al, ASH 2011:741]), and deletions of IKZF1 (present in 20.0% of cases), were significantly associated with poor event-free survival (EFS) (5-year EFS was 88.6 ± 6.3% in wild-type cases [n=31], 68.1 ± 13.3% in cases with deletion of 22q11.22 only [n=15], and 60.0 ± 21.9% in those with deletion of IKZF1 only [n=5]; combined deletion [n=6] was associated with an even poorer EFS (33.3 ± 19.3%, p<0.0001, Figure 1A). Patients were observed to have focal deletions on 22q11.22 spanning up to 235 kb; the most common recurring shared region is just under 10 kb in length, and does not contain known coding genes. Increased number of copies of a 14 kb region in the platelet-derived growth factor receptor alpha gene (PDGFRA), located at 4q12, occurred in 35.5% of cases performed on microarrays containing evaluable probes in this region, and were significantly associated with poor outcome (4-year EFS 87.5 ± 8.3% in wild-type cases [n=20] and 33.9 ± 17.6% in PDGFRA gain cases [n=11], p = 0.004, Figure 1B). Five cases contained 3 copies of PDGFRA, and six cases had at least 4 copies, with no correlation between outcome and number of copies gained. PDGFRA rearrangements and point mutations have been reported in other malignancies, but copy number gain is a novel mechanism of alteration. Cases with a focal 22q11.22 deletion were associated with an increased frequency of CRLF2 rearrangement (75% versus 47%, Fisher's exact p=0.028). No other differences in age, initial WBC, or CRLF2 rearrangement in cases with versus without the focal 22q11.22 deletion, IKZF1 deletion, or PDGFRA gain were present, nor were there significant differences in incidence of focal PDGFRA gains in cases with versus without IKZF1 or 22q11.22 deletions. Gene expression profiling in 27 evaluable cases demonstrated upregulation of genes with kinase activity in 12 cases with either focal 22q11.22 or IKZF1 deletions. This study represents one of the largest collaborative cohorts of DS-ALL for genomic profiling, confirms the poor prognosis associated with IKZF1 deletion (Buitenkamp et al., Leukemia 2012), and identifies two additional genomic loci strongly associated with poor prognosis. If validated in additional DS-ALL cohorts, these findings suggest key lesions that contribute to the poor outcomes observed in this population. These specific genomic changes may improve risk stratification of treatment in children with DS and ALL, and may lead to enhanced sensitivity to tyrosine kinase inhibitors in the estimated 60% of cases bearing one or more of these three lesions. Disclosures: No relevant conflicts of interest to declare.

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