Anaplastic Large Cell Lymphoma Tumors Research Articles

Synergistic growth inhibition of anaplastic large cell lymphoma cells by combining cellular ALK gene silencing and a low dose of the kinase inhibitor U0126

Abnormal expression of anaplastic lymphoma kinase (ALK) gene is a key pathogenic factor for Anaplastic Large Cell Lymphoma (ALCL). To study the role of ALK, an inducible shRNA system was stably introduced into cultured human ALCL cells. Inducing shRNA expression in the generated cells resulted in cellular ALK gene silencing and led to inactivation of multiple signaling pathways and growth arrest. Interestingly, a combination of ALK gene silencing with U0126, a kinase inhibitor specific for the ERK1/2 pathway, resulted in an augmented reduction in cellular JunB expression. Functional studies indicated that combining ALK gene silencing with U0126 treatment provided a synergistic growth inhibition, which occurred faster and was more profound than with either treatment alone. This synergistic effect was also observed when measuring cell proliferation, apoptosis, and in vitro cell colony formation. Importantly, the combination of ALK gene silencing and U0126 had a prolonged inhibitory effect, preventing recovery of ALCL cell growth even after treatments were removed. Moreover, this synergistic inhibitory effect was confirmed in vivo using a mouse model with xenografted ALCL tumors. Our findings indicate that combining cellular ALK gene silencing with a low dose of U0126 may prove to be an effective and more specific therapeutic approach to treating ALCL.

Sonic Hedgehog Signaling Pathway Is Activated in ALK-Positive Anaplastic Large Cell Lymphoma

Deregulation of the sonic hedgehog (SHH) signaling pathway has been implicated in several cancers but has not been explored in T-cell lymphomas. Here, we report that the SHH/GLI1 signaling pathway is activated in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL). We show that SHH, but not its transcriptional effector GLI1, is amplified in ALK+ ALCL tumors and cell lines, and that SHH and GLI1 proteins are highly expressed in ALK+ ALCL tumors and cell lines. We also show that inhibition of SHH/GLI1 signaling with cyclopamine-KAAD, as well as silencing GLI1 gene expression by small interfering (si)RNA, decreased cell viability and clonogenicity of ALK+ ALCL cells. Transfection of wild-type or mutant NPM-ALK into 293T cells showed that only wild-type NPM-ALK increased GLI1 protein levels and activated SHH/GLI1 signaling as shown by increase of CCND2 mRNA levels. Inhibition of ALK tyrosine kinase and phosphatidylinositol 3-kinase (PI3K)/AKT or forced expression of pAKT down-regulated or up-regulated SHH/GLI1 signaling, respectively. Inhibition of GSK3beta in 293T cells also increased protein levels of GLI1. In conclusion, the SHH/GLI1 signaling pathway is activated in ALK+ ALCL. SHH/GLI1 activation is the result of SHH gene amplification and is further mediated by NPM-ALK through activation of PI3K/AKT and stabilization of GLI1 protein. There is a positive synergistic effect between the SHH/GLI1 and PI3K/AKT pathways that contributes to the lymphomagenic effect of NPM-ALK.

The helix‐loop‐helix protein Id2 is expressed differentially and induced by myc in T‐cell lymphomas

Inhibitor of DNA binding 2 (Id2), a helix-loop-helix protein of the inhibitor of differentiation (ID) family, is involved in hematopoiesis, mainly through interaction with the E-family of transcription factors. Recent studies have shown that Id2 is overexpressed in some types of B-cell lymphoma, including classical Hodgkin lymphoma. The authors of the current study hypothesized that Id2 also is overexpressed in T-cell lymphomas. By using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot analyses, high Id2 messenger RNA (mRNA) and protein levels, respectively, were detected in all 4 T-cell anaplastic large cell lymphoma (ALCL) cell lines that were tested. Immunohistochemistry results indicated that Id2 was expressed strongly in 21 of 27 (78%) ALCL tumors (79% anaplastic lymphoma kinase [ALK]-negative and 75% ALK-positive), in 5 of 10 (50%) extranodal natural killer/T (NK/T)-cell lymphomas of the nasal type, in 2 of 8 (25%) cutaneous ALCLs, in 2 of 9 (22%) enteropathy type T-cell lymphomas, in 1 of 5 (20%) peripheral T-cell lymphomas not otherwise specified, and in 1 of 8 (13%) T-cell prolymphocytic leukemias. Other types of T-cell lymphoma were negative for Id2. Because it is known that myc is expressed in ALCL, myc was inhibited selectively in 2 ALCL cell lines, resulting in a concentration-dependent decrease in Id2 mRNA and protein levels. Conversely, forced expression of myc in HEK 293T cells using an myc/green fluorescent protein adenoviral vector resulted in Id2 up-regulation. Taken together, the current data suggest that Id2 commonly is overexpressed in highly proliferative T-cell lymphomas, and its expression may result from transcriptional activation of myc in these tumors.

Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma

A t(2;5) chromosomal translocation resulting in expression of an oncogenic kinase fusion protein known as nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) has been implicated in the pathogenesis of anaplastic large-cell lymphoma (ALCL). PF-2341066 was recently identified as a p.o. bioavailable, small-molecule inhibitor of the catalytic activity of c-Met kinase and the NPM-ALK fusion protein. PF-2341066 also potently inhibited NPM-ALK phosphorylation in Karpas299 or SU-DHL-1 ALCL cells (mean IC(50) value, 24 nmol/L). In biochemical and cellular screens, PF-2341066 was shown to be selective for c-Met and ALK at pharmacologically relevant concentrations across a panel of >120 diverse kinases. PF-2341066 potently inhibited cell proliferation, which was associated with G(1)-S-phase cell cycle arrest and induction of apoptosis in ALK-positive ALCL cells (IC(50) values, approximately 30 nmol/L) but not ALK-negative lymphoma cells. The induction of apoptosis was confirmed using terminal deoxyribonucleotide transferase-mediated nick-end labeling and Annexin V staining (IC(50) values, 25-50 nmol/L). P.o. administration of PF-2341066 to severe combined immunodeficient-Beige mice bearing Karpas299 ALCL tumor xenografts resulted in dose-dependent antitumor efficacy with complete regression of all tumors at the 100 mg/kg/d dose within 15 days of initial compound administration. A strong correlation was observed between antitumor response and inhibition of NPM-ALK phosphorylation and induction of apoptosis in tumor tissue. In addition, inhibition of key NPM-ALK signaling mediators, including phospholipase C-gamma, signal transducers and activators of transcription 3, extracellular signal-regulated kinases, and Akt by PF-2341066 were observed at concentrations or dose levels, which correlated with inhibition of NPM-ALK phosphorylation and function. Collectively, these data illustrate the potential clinical utility of inhibitors of NPM-ALK in treatment of patients with ALK-positive ALCL.

Activation of Mammalian Target of Rapamycin Signaling Pathway Contributes to Tumor Cell Survival in Anaplastic Lymphoma Kinase–Positive Anaplastic Large Cell Lymphoma

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in aberrant expression of chimeric nucleophosmin-ALK. Previously, nucleophosmin-ALK has been shown to activate phosphatidylinositol 3-kinase (PI3K) and its downstream effector, the serine/threonine kinase AKT. In this study, we hypothesized that the mammalian target of rapamycin (mTOR) pathway, which functions downstream of AKT, mediates the oncogenic effects of activated PI3K/AKT in ALK+ ALCL. Here, we provide evidence that mTOR signaling phosphoproteins, including mTOR, eukaryotic initiation factor 4E-binding protein-1, p70S6K, and ribosomal protein S6, are highly phosphorylated in ALK+ ALCL cell lines and tumors. We also show that AKT activation contributes to mTOR phosphorylation, at least in part, as forced expression of constitutively active AKT by myristoylated AKT adenovirus results in increased phosphorylation of mTOR and its downstream effectors. Conversely, inhibition of AKT expression or activity results in decreased mTOR phosphorylation. In addition, pharmacologic inhibition of PI3K/AKT down-regulates the activation of the mTOR signaling pathway. We also show that inhibition of mTOR with rapamycin, as well as silencing mTOR gene product expression using mTOR-specific small interfering RNA, decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis in ALK+ ALCL cells. Cell cycle arrest was associated with modulation of G(1)-S-phase regulators, including the cyclin-dependent kinase inhibitors p21(waf1) and p27(kip1). Apoptosis following inhibition of mTOR expression or function was associated with down-regulation of antiapoptotic proteins, including c-FLIP, MCL-1, and BCL-2. These findings suggest that the mTOR pathway contributes to nucleophosmin-ALK/PI3K/AKT-mediated tumorigenesis and that inhibition of mTOR represents a potential therapeutic strategy in ALK+ ALCL.

C-FLIP confers resistance to FAS-mediated apoptosis in anaplastic large-cell lymphoma

AbstractWe hypothesized that inhibition of the FAS-mediated apoptosis pathway by FLICE-like inhibitory protein (c-FLIP) may contribute to oncogenesis in ALK+ anaplastic large-cell lymphoma (ALCL). Treatment with increasing concentrations of CH-11 (CD95/FAS agonistic antibody) had no effect on cell viability of 2 ALK+ ALCL cell lines, Karpas 299 and SU-DHL1, each expressing high levels of c-FLIP. However, inhibition of endogenous c-FLIP expression by specific c-FLIP siRNA in Karpas 299 and SU-DHL1 cells treated with CH-11 resulted in FAS-mediated cell death associated with increased annexin V binding, apoptotic morphology, and cleavage of caspase-8. In 26 ALK+ ALCL tumors, assessed for expression of DISC-associated proteins, CD95/FAS and c-FLIP were commonly expressed, in 23 (92%) of 25 and 21 (91%) of 23 tumors, respectively. By contrast, CD95L/FASL was expressed in only 3 (12%) of 26 ALCL tumors, although it was strongly expressed by surrounding small reactive lymphocytes. Our findings suggest that overexpression of c-FLIP protects ALK+ ALCL cells from death-receptor-induced apoptosis and may contribute to ALCL pathogenesis. (Blood. 2006;107:2544-2547)

Reappraisal of BCL3 as a Molecular Marker of Anaplastic Large Cell Lymphoma

The BCL3 gene was initially discovered through its involvement in a recurring translocation, t(14;19)(q32;q13), which is found in some patients with B-cell chronic lymphocytic leukemia (B-CLL). The translocation leads to the juxtaposition of BCL3 to the immunoglobulin heavy chain gene locus, resulting in high-level expression of the BCL3 transcript. The Bcl-3 protein includes 7 tandem copies of the ankyrin repeat element in the central domain, a structure that is characteristic of the IkappaB family of inhibitors of the nuclear factor kappaB transcription factors. Anaplastic large cell lymphoma (ALCL) is a subtype of aggressive non-Hodgkin's lymphoma that is characterized by expression of CD30 and the NPM/ALK chimeric protein, which is generated by t(2;5)(p23;q35). We compared the gene expression profiles of ALCL with those of another CD30+ neoplasm, Hodgkin's disease (HD), and found that BCL3 is expressed at higher levels in ALCL than in HD. A comparison by real-time polymerase chain reaction assay revealed that t(2;5)+ ALCL expresses a high level of BCL3 messenger RNA relative to the levels expressed in other hematologic tumors, and the level in ALCL is comparable to or even higher than that in t(14;19)+ B-CLL. An immunohistochemical analysis of ALCL tumor tissues showed that the lymphoma cells exhibited strong nuclear staining by a monoclonal antibody against Bcl-3. We suggest that Bcl-3 sequestrates the (p50)2 homodimer to the nucleus and that the kappaB sites are occupied by the (p50)2/Bcl-3 ternary complex. Future studies should identify the relationships among the 3 independent molecules (ie, NPM/ALK, CD30, and Bcl-3) that are activated in t(2;5)+ ALCL.

CJun Is Phosphorylated at Ser73 and Contributes to Cell Cycle Progression in Anaplastic Large Cell Lymphoma.

Anaplastic Large Cell Lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) or variant translocations resulting in overexpression of anaplastic lymphoma kinase (ALK). cJun is a member of the activator protein-1 (AP-1) family, which is a group of transcription factors that control cell proliferation, differentiation, growth and apoptosis. The activity of cJun can be regulated by phosphorylation at serine 73 (Ser73) and serine 63 (Ser63) residues of the N-terminal domain. It is believed that cJun promotes cell cycle progression, in part, through downregulation of the cyclin-dependent kinase inhibitor p21. Previous studies have shown high AP-1 activity and cJun overexpression in Hodgkin lymphoma and ALCL (Mathas et al, EMBO J 2002; 21:4104). In this study, we assessed for expression of cJun and its Ser73- and Ser63-phosphorylated forms in two ALK+ (Karpas 299 and SU-DHL-1) and one ALK- (Mac2A) ALCL cell lines by western blot analysis, and in 31 ALCL tumors (15 ALK+, 16 ALK-) by immunohistochemistry using tissue microarrays and specific antibodies. To examine the role of cJun in cell survival and proliferation in our in vitro system, ALCL cells were transiently transfected with small interfering RNA (siRNA) specific for cJun. Cell viability, proliferation of viable cells and cell cycle progression from G1 to S-phase were assessed by trypan blue exclusion, MTS and BrdU assays, respectively. All three ALCL cell lines expressed total cJun and Ser73-phosphorylated cJun (Ser73p-cJun) at a high level, whereas Ser63-phosphorylated cJun was expressed at a low level. In addition, all 31 ALCL tumors expressed total cJun in most neoplastic cells. Ser73p-cJun was also detected in all ALCL tumors at a variable level with the percentage of Ser73p-cJun-positive tumor cells ranging from 5% to 95%. By contrast, Ser63p-cJun was detected rarely in tumor cells. Transient transfection of ALCL cells with specific siRNA resulted in almost complete silencing of total cJun expression and absence of Ser73p-cJun expression, which was associated with decreased cell viability and a substantial (40%) decrease of cell growth. cJun silencing also resulted in cell cycle arrest as shown by decreased S-phase fraction. These cell cycle changes were associated with a marked increase of p21 levels and downregulation of cyclin D2 and D3. In conclusion, cJun is highly phosphorylated at serine 73 in ALCL cell lines and tumors and may contribute to cell cycle progression. Targeting cJun expression or phosphorylation using gene therapy approaches may represent a novel therapeutic strategy for patients with ALCL.

Activation of mTOR Signaling Pathway Contributes to Tumoral Cell Survival in ALK-Positive Anaplastic Large Cell Lymphoma.

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in aberrant expression of nucleophosmin (NPM)-ALK. Previously, NPM-ALK has been shown to activate phosphatidylinositol 3-kinase (PI3K) and its downstream effector, the serine/threonine kinase AKT. Recently, we have shown that mTOR signaling proteins are activated in ALK-positive ALCL tumors and that mTOR activation depends, at least in part, on activation of AKT (Lab Invest 2005; 85: 255A). In this study, we investigate the biological effects of inhibition of mTOR on two ALK-positive ALCL cell lines, Karpas 299 and SU-DHL1. For this purpose, we used rapamycin to inhibit mTOR-raptor complex and mTOR-specific small interfering RNA (siRNA) to silence the endogenous mtor gene. Treatment with rapamycin, resulted in a marked concentration-dependent decrease of phosphorylated (p)-mTOR, and its downstream targets, p-p70S6K, p-S6K, p-4E-BP1 and total eIF4E. Similarly, silencing the expression of mtor resulted in a decrease in the activation/phosphorylation level of these proteins as well as in the level of p-AKT. Both treatments induced apoptosis and cell cycle arrest in both ALK-positive ALCL cell lines as demonstrated by trypan blue exclusion, annexin V staining, BrdU incorporation, and cell cycle studies. There was a concentration-dependent decrease in the anti-apoptotic proteins BCL-2, BCL-XL, MCL-1 and c-FLIP (L and S) with increasing concentrations of rapamycin or after mTOR siRNA treatment. The cyclin dependent kinase inhibitors p21waf1 and p27kip1 and underphosphorylated (Un-p)-RB protein were upregulated, after treatment with rapamycin or after mTOR siRNA treatment. In conclusion, we provide evidence that inhibition of mTOR induces cell cycle arrest and apoptosis in ALK-positive ALCL cells. The decrease of p-AKT by silencing mtor suggests that mTOR is necessary to activate AKT in ALK-positive ALCL, and thus, mTOR can function as a feedback signal activity of its own pathway.

Jak3 activation is significantly associated with ALK expression in anaplastic large cell lymphoma

Janus kinase 3 (Jak3) is a tyrosine kinase that activates signal transducer and activator of transcription 3 (Stat3) in response to cytokine stimulation. Stat3 is an oncogene. In previous studies of anaplastic large cell lymphoma (ALCL), we showed that inhibition of Jak3 down-regulates activated/phosphorylated Stat3 (pStat3), decreases anaplastic lymphoma kinase (ALK) enzymatic activity, and induces cell-cycle arrest and apoptosis in ALK-positive ALCL. These findings implicate Jak3 as playing a significant role in the pathogenesis of ALK-positive ALCL; most likely via Stat3 and ALK activation. To assess this possibility, we used immunohistochemical staining to evaluate the frequency of expression of Jak3 and its activated/phosphorylated form (pJak3) in 48 systemic ALCL tumors included in a tissue microarray. pJak3 was detected in 17 (81%) of 21 ALK-positive tumors, compared with 3 (11%) of 27 ALK-negative tumors (P < .0001, Fisher exact test). pStat3 was present in 12 (86%) of 14 ALK-positive tumors and in 10 (40%) of 25 ALK-negative tumors assessed (P = .0078). Of 12 ALK-positive/pStat3-positive tumors, 8 (67%) expressed pJak3, but none of 10 ALK-negative/pStat3-positive tumors expressed pJak3. We conclude that Jak3 activation is predominantly restricted to ALK-positive ALCL tumors. Most likely, Jak3 collaborates with ALK in activating Stat3, leading to cell survival, cell-cycle progression, and tumor growth. In contrast, the mechanism of Stat3 activation in ALK-negative ALCL tumors appears to be independent of Jak3.

Stimulation of CD30 in anaplastic large cell lymphoma leads to production of nuclear factor‐κB p52, which is associated with hyperphosphorylated Bcl‐3

Anaplastic large cell lymphoma (ALCL) and Hodgkin lymphoma (HL) express CD30 at high levels, but stimulation of this molecule has been reported to induce contradictory effects. To elucidate the molecular mechanism of CD30-mediated apoptosis of ALCL, we compared the gene expression profiles of t(2;5)(p23;q35)-positive ALCL with those of HL altered by CD30 agonistic stimulation. The results showed that BCL3, the high-level expression of which in ALCL was previously reported, was further upregulated in response to CD30 stimulation, along with several pro-apoptotic genes. Bcl-3 protein was present as an intermediate phospho-form in the resting-state ALCL, becoming hyperphosphorylated (Bcl-3P) upon stimulation. We next found that the stimulation promoted de novo synthesis of the nuclear factor (NF)-kappaB2/p100 precursor as well as processing to p52, and a series of immunoprecipitation and western blotting analyses consistently showed association of Bcl-3P with p52 in CD30-stimulated ALCL. An electrophoretic mobility shift assay revealed the induction of kappaB binding activity of the p52 homodimer, and nuclear colocalization of Bcl-3 and p52 was demonstrated in anaplastic lymphoma kinase-positive ALCL tumor tissues by immunohistochemistry. As Bcl-3 can act as an anti-repressor or transactivator or both, we propose that the (p52)2/Bcl-3P ternary complex, which is specifically induced in CD30-stimulated ALCL, can modulate expression of apoptosis-related genes regulated by NF-kappaB, thereby accounting for CD30-mediated apoptosis of ALCL.

Differential expression of cyclin D3 in ALK+ and ALK− anaplastic large cell lymphoma

As defined in the World Health Organization classification, anaplastic large cell lymphoma (ALCL) is a distinct type of non-Hodgkin lymphoma of T/null cell lineage, a subset of which is associated with translocations involving 2p23 resulting in expression of anaplastic lymphoma kinase (ALK). The most common translocation, the t(2;5)(p23;q35), results in expression of nucleophosmin (NPM)-ALK. NPM-ALK has been shown to activate signal transducer and activator of transcription (STAT) 3, a transcriptional regulator of cyclin D3. In this study, we assessed cyclin D3 expression in 2 ALK+ ALCL cell lines (Karpas 299 and SU-DHL1) and 1 ALK- ALCL cell line (Mac2A) by Western blot analysis. We also assessed cyclin D3 expression in 52 ALCL tumors (32 ALK+, 20 ALK-) by immunohistochemistry using tissue microarrays. These results were compared with phosphorylated (activated) STAT3 (pSTAT3) expression. Both ALK+ ALCL cell lines, but not the ALK- ALCL cell line, expressed cyclin D3 and pSTAT3. Cyclin D3 was expressed in 25 (78%) of 32 ALK+ ALCL tumors and in 4 (20%) of 20 ALK- ALCL tumors (P < .001, Fisher exact test ). In ALK+ ALCL tumors, the mean percentage of cyclin D3-positive tumor cells was 40.6% compared with 5.1% in ALK- ALCL tumors (P < .001, Mann-Whitney U test). The percentages of cyclin D3-positive and pSTAT3-positive tumor cells were positively correlated (Spearman R = 0.35, P = .036). We conclude that cyclin D3 is differentially expressed in ALK+ and ALK- ALCL and that high expression levels of cyclin D3 in ALK+ ALCL may be attributable to STAT3 activation.

P53 gene mutations are uncommon but p53 is commonly expressed in anaplastic large-cell lymphoma

Anaplastic large-cell lymphoma (ALCL), as defined in the World Health Organization, is a heterogeneous category in which a subset of cases is associated with the t(2;5)(p23;q35) or variant translocations resulting in overexpression of anaplastic lymphoma kinase (ALK). p53 has not been assessed in currently defined subsets of ALCL tumors. In this study, we assessed ALK+ and ALK- ALCL tumors for p53 gene alterations using PCR, single-strand conformation polymorphism and direct sequencing methods. We also immunohistochemically assessed ALCL tumors for p53 expression. Three of 36 (8%) ALCL tumors (1/14 ALK+, 2/22 ALK-) with adequate DNA showed p53 gene mutations. By contrast, p53 was overexpressed in 36 of 55 (65%) ALCL tumors (16 ALK+, 20 ALK-). p21, a target of p53, was expressed in 15 of 31 (48%) ALCL tumors including seven of 15 (47%) p53-positive tumors. p21 expression in a subset of ALCL suggests the presence of functional p53 protein. Apoptotic rate was significantly higher in p53-positive than p53-negative tumors (mean 2.78 vs 0.91%, P = 0.0003). We conclude that the p53 gene is rarely mutated in ALK+ and ALK- ALCL tumors. Nevertheless, wild-type p53 gene product is commonly overexpressed in ALCL and may be functional in a subset of these tumors.

CDNA Microarray Analysis of Genes Associated with NPM-ALK Overexpression in Anaplastic Large Cell Lymphomas.

Constitutive expression of the NPM-ALK tyrosine kinase is a key oncogenic event in the development of anaplastic large cell lymphoma (ALCL) harboring the t(2;5)(p23;q35). The identity and function of a limited number of downstream molecules and pathways important for the oncogenic activity of NPM-ALK are known. However, the global molecular and cellular consequences of NPM-ALK overexpression are largely unknown. In this study, we used cDNA microarrays composed of 9200 unique sequences to compare the expression profiles of pediatric (age <19) NPM-ALK+ ALCLs (n=14) obtained from the Cooperative Human Tissue Network (CHTN) with pediatric NPM-ALK- large cell lymphomas (LCL) (n=10). In addition, to directly determine the transcriptional effects of NPM-ALK overexpression, we analyzed Jurkat cells transfected with a plasmid containing the NPM-ALK chimeric gene (Jurkat/NPM-ALK). The ectopic, forced overexpression of NPM-ALK was confirmed by reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis, which showed that the transfected cells express comparable levels of NPM-ALK to that observed in the ALCL-derived cell lines SUDHL-1 and Karpas 299. The transcriptional profile of Jurkat/NPM-ALK cells was compared to that of Jurkat/LacZ cells. Microarray analysis of NPM-ALK+ ALCL tumors demonstrated 156 genes and ESTs which were differentially expressed by greater than 1.5-fold using a cut-off of p <0.05 compared to NPM-ALK- LCLs. P-values were obtained using the unequal variance t-test. Of these, 62 genes were overexpressed and 94 genes were underexpressed in the NPM-ALK+ ALCLs compared with NPM-ALK- LCLs. Comparison of the transcription profiles of Jurkat/NPM-ALK cells and Jurkat/LacZ cells showed 530 genes (341 upregulated and 189 downregulated) that were differentially expressed by greater than 2-fold. We identified genes that belong to diverse cellular functional groups such as cytokine receptors, kinases, phosphatases, and genes involved in ubiquitin regulation, adhesion and invasion, apoptosis, proliferation and T-cell activation. A subset of these genes were also expressed in NPM-ALK+ ALCL tumors. Genes that were consistently overexpressed in NPM-ALK+ ALCLs compared with NPM-ALK- LCLs included TIMP-1 (p< 0.001), IL-2Rγ (p<0.034) IL-1R2 (p<0.01) and CYP1B1 (p<0.00077). Real-time quantitative RT-PCR was performed to validate a subset of the differentially expressed genes in pediatric ALCL tissue samples as well as the transfected Jurkat cells. Additionally, immunohistochemical analysis of tissue microarrays consisting of 17 cases of ALK+ ALCLs not included in the cDNA microarray analysis was also performed to validate the cDNA microarray findings. This study reveals for the first time, the global transcriptional consequences of NPM-ALK overexpression and provides further insights into the multiple pathogenetic cellular pathways influenced by transformation in NPM-ALK+ ALCL.

Suppressor of cytokine signaling 3 expression in anaplastic large cell lymphoma.

Using a cDNA microarray, we found that suppressor of cytokine signaling 3 (SOCS3) is highly expressed in anaplastic lymphoma kinase (ALK)+ anaplastic large cell lymphoma (ALCL) cell lines. As SOCS3 is induced by activated signal transducer and activator of transcription 3 (STAT3), and ALK activates STAT3, we hypothesized that SOCS3 may play a role in ALK+ ALCL pathogenesis via the Janus kinase 3 (JAK3)-STAT3 pathway. Using ALCL cell lines, we show by coimmunoprecipitation experiments that SOCS3 physically binds with JAK3 in vitro, and that JAK3 inhibition by WHI-P154 downregulates SOCS3 expression. Western blot analysis confirmed expression of SOCS3 and also showed coexpression of phosphorylated (activated) STAT3 (pSTAT3). Direct sequencing of the SOCS3 gene showed no mutations or alternative splicing. In ALCL tumors that were assessed by immunohistochemistry, nine of 12 (75%) ALK+ tumors were SOCS3 positive and eight (67%) coexpressed pSTAT3. In comparison, 18 of 25 (72%) ALK-- tumors were SOCS3 positive and seven (28%) coexpressed pSTAT3. These results show that SOCS3 is overexpressed in ALCL, attributable to JAK3-STAT3 activation and likely related to ALK in ALK+ tumors. However, SOCS3 is also expressed in tumors that lack STAT3 and ALK suggesting alternative mechanisms of upregulation.

Selective inhibition of STAT3 induces apoptosis and G(1) cell cycle arrest in ALK-positive anaplastic large cell lymphoma.

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is an aberrant fusion gene product expressed in a subset of cases of anaplastic large cell lymphoma (ALCL). It has been shown that NPM-ALK binds to and activates signal transducer and activator of transcription 3 (STAT3) in vitro, and that STAT3 is constitutively active in ALK(+) ALCL cell lines and tumors. In view of the oncogenic potential of STAT3, we further examined its biological significance in ALCL using two ALK(+) ALCL cell lines (Karpas 299 and SU-DHL-1) and an adenoviral vector that carries dominant-negative STAT3 (AdSTAT3DN). Infection by AdSTAT3DN led to the expression of STAT3DN in both ALK(+) ALCL cell lines at a similar efficiency. Subcellular fractionation studies showed that a significant proportion of the expressed STAT3DN protein translocated to the nucleus, despite the fact that STAT3DN has a mutation at residue 705(tyrosine --> phenylalanine), a site that is believed to be crucial for STAT3 activation and nuclear translocation. Introduction of STAT3DN induced apoptosis and G(1) cell cycle arrest. Western blot studies showed that expression of STAT3DN resulted in caspase-3 cleavage, downregulation of Bcl-2, Bcl-xL, cyclin D3, survivin, Mcl-1, c-Myc and suppressor of cytokine signaling 3. These results support the concept that STAT3 activation is pathogenetically important in ALCL cells by deregulating the expression of multiple target proteins that are involved in the control of apoptosis and cell cycle progression.

Retinoblastoma Protein Is Frequently Absent or Phosphorylated in Anaplastic Large-Cell Lymphoma

The possible role of retinoblastoma protein (Rb) in the pathogenesis of anaplastic large-cell lymphoma (ALCL) is unknown. We investigated Rb protein expression, both total (phosphorylated and underphosphorylated) and active (underphosphorylated), in four anaplastic lymphoma kinase (ALK)-positive ALCL cell lines (Karpas 299, JB-6, SU-DHL1, and SR-786) by Western blot analysis, and in 67 ALCL tumors (30 ALK-positive, 37 ALK-negative) using immunohistochemical methods. We also used fluorescence in situ hybridization and polymerase chain reaction methods to assess for loss of heterozygosity of the rb gene. The findings were correlated with apoptotic rate assessed by the terminal dUTP nick-end labeling assay. Immunoblots showed high total Rb levels in Karpas 299, SU-DHL1 and SR-786 and relatively lower levels in and JB-6. Underphosphorylated Rb was negative or expressed at low levels in all cell lines. In ALCL tumors, total Rb was detected in 44 (66%) and absent in 23 (34%). The mean apoptotic rate was 3.2% in Rb-negative tumors compared with 2.7%, 2.2%, and 1.2% in tumors with <10%, 10 to 50%, and >50% Rb-positive cells, respectively (P = 0.2, Kruskall-Wallis test). In a subset of 25 total Rb-positive tumors we assessed for underphosphorylated Rb, which was detected in 12 tumors. The detection of only total Rb in the remaining 13 tumors suggests that Rb was phosphorylated. Fluorescence in situ hybridization showed allelic loss of the rb gene in 10 (40%) of 25 tumors analyzed and was significantly associated with absence of Rb expression (P = 0.003). Similar results were obtained for loss of heterozygosity of the 13q14 locus. Five-year progression-free survival for patients with Rb-negative ALCL was 89.4% compared with 47.7% for patients with total Rb-positive ALCL (P = 0.006, log-rank test). Similar trends for progression-free survival held true for patients with ALK-positive and ALK-negative tumors analyzed separately. In conclusion, Rb is absent or phosphorylated in most ALCL cell lines and tumors and absence of Rb expression is associated with better clinical outcome in patients with ALCL.

Survivin expression predicts poorer prognosis in anaplastic large-cell lymphoma.

Survivin, a member of the inhibitor of apoptosis (IAP) family, is not detected in normal adult tissues but is overexpressed in various cancers, including some types of lymphoma. The frequency and prognostic significance of survivin expression in anaplastic large-cell lymphoma (ALCL) is unknown. We assessed for survivin expression in 62 ALCL tumors (30 anaplastic lymphoma kinase [ALK]-positive and 32 ALK-negative) obtained before doxorubicin-based chemotherapy. Given that survivin is a target of the STAT3 signaling pathway and STAT3 is activated in ALCL, survivin expression was also correlated with STAT3 activation. Survivin was expressed in 34 tumors (55%) and did not correlate with ALK. A significant association between survivin expression and STAT3 activation was observed (P =.007, Fisher's exact test). For the ALK-positive group, the 5-year failure-free survival (FFS) was 34% for patients with survivin-positive ALCL compared with 100% for patients with survivin-negative ALCL (P =.009, log-rank test). For the ALK-negative group, the 5-year FFS was 46% for patients with survivin-positive tumors compared with 89% for patients with survivin-negative tumors (P =.03, log-rank test). Overall survival was similarly worse for patients with survivin-positive tumors in both the ALK-positive and ALK-negative groups. Furthermore, multivariate analysis confirmed the independent prognostic value of survivin expression, along with age older than 60 years and Ann Arbor stage III or IV. Survivin is expressed in approximately half of ALCL tumors and independently predicts unfavorable clinical outcome. Modulation of survivin expression or function may provide a novel target for experimental therapy in patients with ALCL.

Caspase-3 activation in systemic anaplastic large-cell lymphoma

Anaplastic large-cell lymphoma (ALCL), as currently defined, includes a subset of tumors that have abnormalities of chromosome 2p23 (alk gene) resulting in overexpression of anaplastic lymphoma kinase (ALK). We have previously shown differences in apoptotic rate and expression of apoptosis-related proteins between ALK-positive and ALK-negative ALCL. In this study, we assessed for activated caspase-3 (aC-3), an executioner of apoptotic cell death, in ALCL cell lines and tumors. We used the Karpas 299 and SU-DHL-1 cell lines, and the caspase inhibitors Boc-D-FMK and DEVD-FMK to investigate the role of caspase-3 activation in tumor cell death after treatment with doxorubicin. Cell viability and apoptosis were assessed by trypan blue and Annexin-V methods. A caspase-3 assay was used to evaluate caspase-3 enzymatic activity. Caspase-3 activity was significantly increased in Karpas-299 and SU-DHL-1 cells treated with doxorubicin, but remained as low as control levels with addition of Boc-D-FMK or DEVD-FMK. Expression of aC-3 was also assessed immunohistochemically in 57 ALCL tumors. The mean percentage of aC-3 positive tumor cells was 3.2% in ALK-positive ALCL compared with 1.2% in ALK-negative ALCL (P=0.0003, Mann–Whitney test), and inversely correlated with BCL-2 expression (P=0.01, Mann–Whitney test). aC-3 expression did not correlate with patient outcome in either the ALK-positive or ALK-negative ALCL groups. In conclusion, doxorubicin-induced cell death of ALK-positive ALCL cells involves caspase-3 activation in vitro. aC-3 levels correlate with ALK expression in ALCL tumors.

Caspase-3 activation in systemic anaplastic large-cell lymphoma

Anaplastic large-cell lymphoma (ALCL), as currently defined, includes a subset of tumors that have abnormalities of chromosome 2p23 (alk gene) resulting in overexpression of anaplastic lymphoma kinase (ALK). We have previously shown differences in apoptotic rate and expression of apoptosis-related proteins between ALK-positive and ALK-negative ALCL. In this study, we assessed for activated caspase-3 (aC-3), an executioner of apoptotic cell death, in ALCL cell lines and tumors. We used the Karpas 299 and SU-DHL-1 cell lines, and the caspase inhibitors Boc-D-FMK and DEVD-FMK to investigate the role of caspase-3 activation in tumor cell death after treatment with doxorubicin. Cell viability and apoptosis were assessed by trypan blue and Annexin-V methods. A caspase-3 assay was used to evaluate caspase-3 enzymatic activity. Caspase-3 activity was significantly increased in Karpas-299 and SU-DHL-1 cells treated with doxorubicin, but remained as low as control levels with addition of Boc-D-FMK or DEVD-FMK. Expression of aC-3 was also assessed immunohistochemically in 57 ALCL tumors. The mean percentage of aC-3 positive tumor cells was 3.2% in ALK-positive ALCL compared with 1.2% in ALK-negative ALCL (P=0.0003, Mann–Whitney test), and inversely correlated with BCL-2 expression (P=0.01, Mann–Whitney test). aC-3 expression did not correlate with patient outcome in either the ALK-positive or ALK-negative ALCL groups. In conclusion, doxorubicin-induced cell death of ALK-positive ALCL cells involves caspase-3 activation in vitro. aC-3 levels correlate with ALK expression in ALCL tumors.