ALBERT

Description: Constitutive expression of the chimeric NPM/ALK fusion protein encoded by the t(2;5)(p23;q35) is a key oncogenic event in the majority of pediatric anaplastic large cell lymphomas (ALCL). To determine the pathogenetic mechanisms involved in NPM/ALK-mediated lymphomagenesis we employed a mass spectrometry (MS)-based proteomics approach to identify changes in protein expression caused by the overexpression of NPM/ALK. We identified many proteins which were downstream targets of the FRAP/mTOR pathway including ribosomal S6 kinase (1.6-fold), translational initiation factor eIF (4.8-fold), ribosomal protein L11 (4.8-fold), eukaryotic translation initiation factor 3 (3.2-fold), translation initiation factor IF-2 homolog (4.3-fold) and translation initiation factor eIF-2alpha kinase (3.4-fold). The FRAP/mTOR pathway plays a key role in the regulation of cell growth and proliferation and positively regulates translation and ribosome biogenesis and is selectively inhibited by rapamycin. To determine the feasibility of targeting the FRAP/mTOR pathway by rapamycin for treatment of pediatric ALCLs, we determined the prevalence of expression of key proteins in the FRAP/mTOR pathway in pediatric ALCLs and correlated its expression with that of the ALK protein. In addition we determined the in vitro effect of rapamycin on the viability of cell lines derived from t(2;5)-positive ALCLs. We used formalin-fixed paraffin-embedded tissues of ALK-positive ALCLs (n=18) obtained from the Children’s Oncology Group clinical trials (CCG5941 and ANHL0131) and determined the expression of phospho-mTOR, phospho-70S6Kinase and phospho-S6 ribosomal protein using immunohistochemistry. The effect of rapamycin on the viability of cell lines derived from t(2;5)-positive ALCLs was determined by MTT assay and cell cycle analysis. Western blot analysis was performed to determine the effect of rapamycin on cell cycle proteins and apoptosis. Immunohistochemical studies demonstrated diffuse strong nuclear expression of phospho-mTOR in 17/18 cases, and nuclear and cytoplasmic phospho-70S6kinase expression in 15/18 cases. In addition, cytoplasmic expression of phospho-S6 ribosomal protein was observed in 18/18 (100%) of cases. Importantly, the reactive lymphocytes demonstrated negligible expression of all three proteins. Furthermore, rapamycin potently decreased the viability of SUDHL-1 cells (30% reduction by 10nM at 48 hours) and resulted in G1 cell cycle arrest without induction of caspase-3 activity. Western blot analysis demonstrated a reduction in the level of phospho-p70S6Kinase as well as 4EBP-1 levels. Our studies demonstrate overexpression of many proteins in the FRAP/mTOR pathway in NPM/ALK-positive ALCLs. Our data indicate that the majority of pediatric ALCLs express proteins in the FRAP/mTOR pathway and are constitutively activated. Furthermore, our in vitro data support the use of rapamycin as a therapeutic agent in ALK-positive ALCLs.

Description: Constitutive expression of the chimeric NPM/ALK fusion protein encoded by the t(2;5)(p32;q35) is a key oncogenic event in the pathogenesis of most anaplastic large cell lymphomas (ALCLs). The proteomic network alterations produced by this aberration remain largely uncharacterized. Using a mass spectrometry (MS)–driven approach to identify changes in protein expression caused by the NPM/ALK fusion, we identified diverse NPM/ALK-induced changes affecting cell proliferation, ribosome synthesis, survival, apoptosis evasion, angiogenesis, and cytoarchitectural organization. MS-based findings were confirmed using Western blotting and/or immunostaining of NPM/ALK-transfected cells and ALK-deregulated lymphomas. A subset of the proteins distinguished NPM/ALK-positive ALCLs from NPM/ALK-negative ALCLs and Hodgkin lymphoma. The multiple NPM/ALK-deregulated pathways identified by MS analysis also predicted novel biologic effects of NPM/ALK expression. In this regard, we showed loss of cell adhesion as a consequence of NPM/ALK expression in a kinase-dependent manner, and sensitivity of NPM/ALK-positive ALCLs to inhibition of the RAS, p42/44ERK, and FRAP/mTOR signaling pathways. These findings reveal that the NPM/ALK alteration affects diverse cellular pathways, and provide novel insights into NPM/ALK-positive ALCL pathobiology. Our studies carry important implications for the use of MS-driven approaches for the elucidation of neoplastic pathobiology, the identification of novel diagnostic biomarkers, and pathogenetically relevant therapeutic targets.

Description: Anaplastic large-cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35), resulting in aberrant expression of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). We show that in 293T and Jurkat cells, forced expression of active NPM-ALK, but not kinase-dead mutant NPM-ALK (210K〉R), induced JNK and cJun phosphorylation, and this was linked to a dramatic increase in AP-1 transcriptional activity. Conversely, inhibition of ALK activity in NPM-ALK+ ALCL cells resulted in a concentration-dependent dephosphorylation of JNK and cJun and decreased AP-1 DNA-binding. In addition, JNK physically binds NPM-ALK and is highly activated in cultured and primary NPM-ALK+ ALCL cells. cJun phosphorylation in NPM-ALK+ ALCL cells is mediated by JNKs, as shown by selective knocking down of JNK1 and JNK2 genes using siRNA. Inhibition of JNK activity using SP600125 decreased cJun phosphorylation and AP-1 transcriptional activity and this was associated with decreased cell proliferation and G2/M cell-cycle arrest in a dose-dependent manner. Silencing of the cJun gene by siRNA led to a decreased S-phase cell-cycle fraction associated with upregulation of p21 and downregulation of cyclin D3 and cyclin A. Taken together, these findings reveal a novel function of NPM-ALK, phosphorylation and activation of JNK and cJun, which may contribute to uncontrolled cell-cycle progression and oncogenesis.

Description: Abstract 2922 Poster Board II-898 Introduction: Lymphomas derived from mature lymphocytes in children and adolescents are predominantly aggressive B-cell non-Hodgkin lymphomas (B-NHL), including Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). Differences in the clinical course between pediatric and adult aggressive B-NHL suggest distinct pathogenetic mechanisms. This study sought to identify both shared and unique genetic alterations between gene expression-defined pediatric and adult cases of BL and DLBCL. Patients and Methods: Gene expression profiling (GEP) was done on 45 BL and 18 DLBCL specimens from patients 18 years of age or younger, and 38 BL and 106 DLBCL from adult patients. Pediatric specimens were collected from the Cooperative Human Tissue Network (CHTN) pediatric NHL repository through the Children's Oncology Group (COG) and adult specimens were collected from the Nebraska Lymphoma Study Group Registry and Tissue Bank through the Lymphoma/Leukemia Molecular Profiling Project (LLMPP). Previously-published gene signatures were used to classify lymphomas molecularly into mBL and mDLBCL groups (Dave et al., NEJM, 2006). The mDLBCL tumors were further classified into activated B-cell-like (ABC), germinal center B-cell-like (GCB), and primary mediastinal B-cell lymphoma (PMBL) subtypes (Rosenwald et al., JEM, 2003). High resolution array comparative genomic hybridization (aCGH) was done on a subset of the pediatric cases using the 250K NspI Human Mapping Array (Affymetrix) to detect DNA copy number alterations (CNA). Results: Molecular classification of the pediatric cases resulted in a 20% reclassification rate for cases with a morphologic diagnosis of BL or DLBCL. Among the 63 pediatric cases, there were 38 mBL (3 of which were DLBCL by morphology), 23 mDLBCL (9 of which were BL by morphology) and two cases which were unclassifiable by the molecular gene signatures. Comparison of the GEP profiles for adult and pediatric mBL failed to identify pathways that differed significantly; however high resolution aCGH analysis revealed a number of abnormalities in the pediatric cases not previously reported in BL, including gains of 3q21, 11q13 and 16p11. A predominance of the GCB to ABC subtype (3:1) was found among pediatric mDLBCL patients. Two cases with mediastinal tumors were classified as PMBL molecularly. Both PMBL cases were female and carried copy number gains of the Rel/BCL11A locus. Comparison of adult and pediatric GCB mDLBCL gene expression revealed enrichment in B-cell surface molecules and markers of antigen-dependent B-cell activation in the adult cases. aCGH analysis identified abnormalities that were both shared (+12q15, +19q13, -6q) between adult and pediatric mDLBCL and unique (-4p14, -19q13.32, +16p11.2) to the pediatric cases. Correlation of DNA copy number and gene expression revealed potential candidate genes for these loci. Conclusions: Pediatric BL and DLBCL classified by morphology were reclassified molecularly in a significant fraction of cases. Although pediatric BL and DLBCL are treated similarly, defining homogeneous molecular entities will be relevant for developing new therapies and future clinical trials. In general, pediatric cases have a more favorable outcome relative to adult patients. However, it is unclear whether this is due to the ability of children to tolerate very intensive therapies or whether distinct pathogenetic mechanisms modulate the disease course. Higher B-cell receptor signaling in adult relative to pediatric GCB DLBCL may be relevant to the outcome. The identification of previously undetected chromosomal alterations unique to the pediatric cases also suggests distinct pathogenetic mechanisms. Elucidation of the underlying genes may provide insight into factors which modulate outcome and could provide novel therapeutic targets with reduced toxicity. Disclosures: Gascoyne: Roche Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Background: NK cells are characterized by absent CD3 but expression of CD56dim (90%, cytotoxic) and CD56bright (10%, mediator). NK cells may contribute to the immaturity in cord blood innate and adaptive immunity, and play an important role in the GVL effect post CBT. However, little is known regarding the NKR signaling pathways in CB vs PB CD56dim NK cells and its relationship to the cytotoxic activity. We previously demonstrated the ability to ex-vivo expand CB into NK subsets with profound NK in-vitro and in-vivo cytotoxic activity (Ayello/Cairo BBMT 2006). We further observed that there were 33 and 37 proteins over and under expressed by proteomic expression profiling studies of CB vs PB CD56dim (Shereck/Cairo, ASH 2007; ASPHO 2007; AACR 2007). The differential protein expressions included NKG2A, IP3R type 3, NCR3, MAPKAPK5, Notch 2, PLEK, and NF-X1 using both immunophenotype and proteomic profiling studies. Objectives: To understand the importance of NKR signaling pathways in mediating the differential protein expression and thus in regulating the NK cytotoxic activities in CB vs PB CD56dim, we compared the genomic expression pattern in CB vs PB CD56dim. Methods: For CD56dim isolation, first, NK cells were isolated indirectly by magnetic separation from non-NK cells. Second, the pre-enriched NK cells (CD56+/CD3−) from CB and PB were directly labeled with CD16 (FCGR3) MicroBeads, and the CD56+ CD16+ NK cells (CD56dim) were eluted after removing the column from the magnetic field (Miltenyi). Purity of CD56dim NK cells were then examined by flow cytometry (BD FACScan). For genomic studies, total RNA was isolated and reverse transcribed to cDNA using T7-Oligo (dT) primer. cRNA was Biotin-labeled by in vitro transcription. Fragmented biotin-labeled cRNA was hybridized to GeneChip U133A_2 in GCOS-operated Fluidics Station 450, and then scanned by GeneChip Scanner 3000 (Affymetrix). Data were analyzed using Agilent GeneSpring. Signal intensities were compared using one way ANOVA and Welch Test for statistical analysis. Results: There were 193 and 222 genes over and under expressed at the genomic level between CB vs PB CD56dim NK cells, respectively. CB vs PB CD56dim significantly overexpressed NKG2A (2.14F), CD16b (2.46F), KIR2D (2.13F), NKp44 (NCR2; 2.62F), PBX1 (4.29F), ENPEP (3.93F). There was no significant difference in NKR gene expression of CD16a, CD161, NKG2C, and NKp46 in CB vs PB CD56dim. CB vs PB CD56dim underexpressed the following NK genes: IP3R (1.32F), MAPKAPK5 (1.77F), NCR3 (1.24F), ACACB (3.23F), BBS1 (2.00F). Conclusion: CB vs PB CD56dim overexpressed NKG2A, CD16b, KIR2D, and NKp44 genes compared to only NKG2A was overexpressed at the protein level. These results suggest that NKR protein product levels in CB CD56dim may be directly regulated at the translation level, but not the transcription level. The discrepancy of IP3R, ENPEP, PBX1, and MAPKAPK5 gene expression suggest the involvements of IP3 and calcium ions in NKR signaling pathways. Since the Notch2, PLEK, and NF-X1 gene expression patterns were not increased, the augmented protein levels may result from the regulation of protein translation. The potential regulators of this process may include PBX1, ENPEP, ACACB, and BBS1 though the roles of these regulators need to be defined. We conclude that genomic differences between CB vs PB CD56dim may play an important role in regulating NKR signaling pathway, and thus contribute to disparate cytotoxic activity between CB vs PB and suggest a possible explanation for immaturity of cord blood innate and adaptive immunity.

Description: TCL1 is a proto-oncogene whose deregulation has been implicated in the pathogenesis of T- and B-cell lymphoproliferative disorders. TCL1 expression is predominantly observed in developing B lymphocytes, whereas its overexpression in T cells is associated with mature T-cell lymphoproliferation in transgenic mice. Although recent studies indicate that dysregulated expression of TCL1 is important in mature B-cell transformation, very little is currently known about the function or interactions of TCL1, specifically in the B-cell context. In this study, we hypothesized that identification of proteins that interact with TCL1 may facilitate our understanding of the functional properties of TCL1. Proteomic analysis provides an opportunity to carry out functional studies of protein-protein interactions and characterization of functional interactomes. Using a functional proteomic approach, we determined the identity of proteins that interact with TCL1 by co-immunoprecipitation with anti-TCL1 antibody followed by liquid chromatography (LC), electrospray ionization (ESI) and tandem mass spectrometry (MS/MS). Immunoprecipitates of the TCL1 expressing SUDHL-16 B-cell lymphoma derived cell line were compared to that of hyperimmune rabbit immunoglobulin and a cell line that does not express TCL1 (SUDHL-1) by 1-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Silver-stained protein bands from both immunocomplexes were excised and analyzed by ESI-LC-MS/MS. Proteins which were unique to the TCL1 immunocomplex included transcription factors (Jun B), phosphatases (protein phosphatase 2), ubiquitin-associated proteins (ubiquitin activating enzyme), cell adhesion proteins (ADP ribosylation factor 3) and proteins associated with apoptosis regulation (peroxidoxin 1 and scaffold attachment factor). Analysis of subcellular fractions demonstrated that TCL1 and complexes were localized to both the nuclear and cytoplasmic compartments. Importantly, known TCL1 interactors such as histone 3 were uniquely identified in the TCL1 complex. Proteins identified by MS were confirmed by western blotting and reciprocal immunoprecipitation. This study reveals novel TCL1 interactors and indicates that the protein may exert diverse cellular effects impacting apoptosis, cell survival, cytoskeletal organization and cell adhesion. Our interactome analysis provides unique insights into the cellular function of TCL1, a protein whose deregulation is increasingly implicated in the pathogenesis of a variety of hematopoietic neoplasms.

Description: Background: The progress of childhood BL and DLBCL has improved dramatically in the past three decades; however, patients with a 13q-deletion have a significantly poorer outcome (Cairo/Patte et al Blood, 2007 and Patte/Cairo et al Blood, 2007; Poirel/Cairo et al Leukemia 2008). DLEU1, a potential tumor suppressor gene, is located within the 13q-deletion. DLEU1 was reported to be a key gene in the Burkitt classifier genes (Dave/Staudt et al NEJM, 2006) and c-myc binds to the promoter region of DLEU1. DLEU1-network proteins include, among others, E3 ubiquitin-protein ligase (UBR1), Tubulin beta-2C (TUBB2C) and RASSF1A. We previously demonstrated that UBR1, TUBB2C, and RASSF1A, were differentially expressed in BL vs DLBCL patients and cell lines by global gene profiles and real time RT-PCR studies (Day/Cairo et al AACR 2008; Day/Cairo et al ICML 2008). We further demonstrated decreased expression of UBR1 (33.2±4.5% reduction compared to control (p

Description: Abstract 1928 Poster Board I-951 Background: Burkitt lymphoma (BL) represents approximately 40% of all childhood or adolescent NHL (Cairo et al., Blood, 2007). Following a multivariate analysis for known prognostic factors, we have identified a subgroup of BL patients with a 13q deletion who had a significantly poorer outcome despite aggressive short, intensive multiagent chemotherapy (Poirel/Cairo et al Leukemia 2009). More recently, we identified in a subset analysis that children with BL and a 13q14.3 deletion by FISH analysis have a significant inferior OS (Nelson/Cairo/Perkins/Sanger et al BJHaem, In Press). DLEU1, a gene within the Burkitt classifier genes as reported by Dave/Staudt et al. NEJM, 2006, is located within the region of 13q14.3. In addition, DLEU1 is recognized to interact with c-Myc, histone acetylase (HTATIP), tumor antigen p53, histone-lysine N-methyltransferase (SETDB1), Tubulin beta-2C (TUBB2C), RASSF1A, and E3 ubiquitin-protein ligase (UBR1). We have previously reported that DLEU1 may in part function as a potential tumor suppressor gene. When DLEU1 is down regulated by a DLEU1 siRNA, the spontaneous apoptotic rate was decreased with concomitant significantly reduced levels of UBR1 and TUBB2C gene expression (Day/Cairo et al SIOP 2008). In gene array profiling studies, we also found that the expression levels of RASSF1, ERG, UBR1, and TUBB2C were significantly higher in BL than in DLBCL (Day/Cairo et al, AACR 2008). Objective: In this study, we sought to examine the percent of apoptosis induced by CY and/or rituximab in DLEU1 siRNA transfected BL cells. Methods: Ramos BL cell lines were transiently transfected (24 hrs) with DLEU1 siRNA as previously described (5'-AUACUUGGCAUGAAUGAACUUAUGU-3' and 3'-UAUGAACCGUACUUACUUGAAUACA-5') (Day/Cairo SIOP 2008). Stealth RNAi whose GC content is similar to that of this DLEU1 siRNA was used as negative control. The siRNA transfected cells were then treated with CY (0, 89.5, 895, 8950 nM) and/or rituximab (0, 4, 40, 400 mg/mL) for additional 4 hrs. Cells were evaluated for percent apoptosis using Annexin V-FITC and Propidium Iodide followed by FACS using BD LSRII. Statistics was conducted by one-way ANOVA followed by Dunnett multiple comparisons test. Results: There was a significant reduction in apoptosis in the CY treated BL transfected DLEU1 siRNA vs mock control cells (89.5 nM CY: 10.26±0.23% reduction, p

Description: Follicular lymphoma (FL) is the most common form of low-grade non-Hodgkin lymphoma in the western hemisphere. The vast majority of cases are incurable and transformation to diffuse large B-cell lymphoma (DLBCL) is an important cause of death. The molecular and biologic mechanisms underlying FL transformation are largely uncharacterized. In this study, we utilized a global quantitative proteomics approach for the identification of differentially expressed proteins associated with follicular lymphoma transformation. Five matched pairs of clonally identical cases of follicular lymphoma and their transformed counterparts (DLBCL) arising in the same individual were utilized. Quantitative analysis of differentially expressed proteins was performed by isotope-coded affinity tagging (ICAT™) followed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS). Equivalent quantities of total cell lysates obtained from the FLs and the DLBCLs were ICAT™ labeled, and subjected to avidin affinity chromatography. Offline fractions were collected, digested with trypsin, and analyzed by automated reverse phase nanospray LC-MS/MS. Our proteomic studies revealed upregulation of the IGF-1R (3–5 fold) in the transformed lymphomas. Western blot analysis using an antibody to the a-subunit of IGF-1R revealed overexpression in the transformed lymphoma samples as compared to their preceding FL counterparts (discovery set). Similarly, IGF-1R upregulation was demonstrated in an additional independent set of 6/7 DLBCL samples as compared to their preceding FL counterparts. Immunohistochemical studies were performed on formalin-fixed paraffin-embedded tissue sections of 15 matched pairs of FL and their transformed DLBCL counterparts. The neoplastic cells of FL demonstrated negligible levels of IGF-1R whereas in 5/15 cases, the neoplastic cells of DLBCL demonstrated strong cytoplasmic and membranous expression of IGF-1R. We carried out studies to determine the functional role of IGF-1R in the survival of lymphoma cells in vitro. Blocking antibodies to IGF-1R caused a significant reduction of cell viability in all three transformed FL cell lines (SUDHL-4, OCI-LY1, Karpas 4224) as determined by MTT assays. In contrast, antibodies against EGFR, EphA and Frizzled 8 protein did not affect the cell viability of any of the transformed FL cell lines, indicating specificity. Furthermore, knockdown of IGF-1R expression in SUDHL-4 cells by RNA interference resulted in significant reduction in cell viability whereas the control “scramble” siRNA or EGFR siRNA did not have an effect. Cell cycle analysis of the IGF-1R siRNA transfected cells indicated an increase in cells undergoing apoptosis relative to control cells. We utilized a synthetic tyrphostin compound (AG1024) which selectively inhibits the IGF-1R tyrosine kinase activity to determine the effects of pharmacologic inhibition of IGF-1R on the viability of transformed FL cells. Inhibition of IGF-1R resulted in inhibition of cell viability with IC50 of 22mM. This study, for the first time, reveals the role of deregulated expression of IGF-1R in transformed FL and provides a rational basis for the use of IGF-1R blocking agents in the therapy of these neoplasms.