B-cell receptor-mediated NFATc1 activation induces IL-10/STAT3/PD-L1 signaling in diffuse large B-cell lymphoma.

Knowledge of programmed death ligand 1 (PD-L1) expression and its regulation in B-cell lymphoma cells is limited. Investigating mechanisms that control PD-L1 expression in B-cell lymphoma cells might identify biomarkers that predict the efficacy of immunotherapy with anti-programmed death-1/PD-L1 antibodies. In addition, identification of mechanisms that regulate PD-L1 may identify molecules that can be targeted to improve the clinical efficacy of immune checkpoint inhibitors. In this study, we used proteomic approaches and patient-derived B-cell lymphoma cell lines to investigate mechanisms that regulate PD-L1 expression. We found that PD-L1 expression, particularly in nongerminal center B cell-derived diffuse large B-cell lymphoma (DLBCL), is controlled and regulated by several interactive signaling pathways, including the B-cell receptor (BCR) and JAK2/STAT3 signaling pathways. We found that that BCR-mediated NFATc1 activation upregulates IL-10 chemokine expression in PD-L1+ B-cell lymphoma cells. Released IL-10 activates the JAK2/STAT3 pathway, leading to STAT3-induced PD-L1 expression. IL-10 antagonist antibody abrogates IL-10/STAT3 signaling and PD-L1 protein expression. We also found that BCR pathway inhibition by BTK inhibitors (ibrutinib, acalabrutinib, and BGB-3111) blocks NFATc1 and STAT3 activation, thereby inhibiting IL-10 and PD-L1 expression. Finally, we validated the PD-L1 signaling network in 2 primary DLBCL cohorts consisting of 428 and 350 cases and showed significant correlations among IL-10, STAT3, and PD-L1. Thus, our findings reveal a complex signaling network regulating PD-L1 expression in B-cell lymphoma cells and suggest that PD-L1 expression can be modulated by small molecule inhibitors to potentiate immunotherapies.

The Role of Macrophage/B-Cell Interactions in the Pathophysiology of B-Cell Lymphomas.

Macrophages (MPs) are heterogeneous, multifunctional, myeloid-derived leukocytes that are part of the innate immune system, playing wide-ranging critical roles in basic biological activities, including maintenance of tissue homeostasis involving clearance of microbial pathogens. Tumor-associated MPs (TAMs) are MPs with defined specific M2 phenotypes now known to play central roles in the pathophysiology of a wide spectrum of malignant neoplasms. Also, TAMs are often intrinsic cellular components of the essential tumor microenvironment (TME). In concert with lymphoid-lineage B and T cells at various developmental stages, TAMs can mediate enhanced tumor progression, often leading to poor clinical prognosis, at least partly through secretion of chemokines, cytokines, and various active proteases shown to stimulate tumor growth, angiogenesis, metastasis, and immunosuppression. Researchers recently showed that TAMs express certain key checkpoint-associated proteins [e.g., programmed cell death protein 1 (PD-1), programmed cell death-ligand 1 (PD-L1)] that appear to be involved in T-cell activation and that these proteins are targets of other specific checkpoint-blocking immunotherapies (anti-PD-1/PD-L1) currently part of new therapeutic paradigms for chemotherapy-resistant neoplasms. Although much is known about the wide spectrum and flexibility of MPs under many normal and neoplastic conditions, relatively little is known about the increasingly important interactions between MPs and B-lymphoid cells, particularly in the TME in patients with aggressive B-cell non-Hodgkin lymphoma (NHL-B). Normal and neoplastic lymphoid and myeloid cell/MP lineages appear to share many primitive cellular characteristics as well as transcriptional factor interactions in human and animal ontogenic studies. Such cells are capable of ectopic transcription factor-induced lineage reprogramming or transdifferentiation from early myeloid/monocytic lineages to later induce B-cell lymphomagenesis in experimental in vivo murine systems. Close cellular interactions between endogenous clonal neoplastic B cells and related aberrant myeloid precursor cells/MPs appear to be important interactive components of aggressive NHL-B that we discuss herein in the larger context of the putative role of B-cell/MP cellular lineage interactions involved in NHL-B pathophysiology during ensuing lymphoma development.

Strategic Therapeutic Targeting to Overcome Venetoclax Resistance in Aggressive B-cell Lymphomas.

Purpose: B-cell lymphoma-2 (BCL-2), an antiapoptotic protein often dysregulated in B-cell lymphomas, promotes cell survival and provides protection from stress. A recent phase I first-in-human study of the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma showed an overall response rate of 44%. These promising clinical results prompted our examination of the biological effects and mechanism of action underlying venetoclax activity in aggressive B-cell lymphoma, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL).Experimental Design: MCL and DLBCL cell lines, primary patient samples, and in vivo patient-derived xenograft (PDX) models were utilized to examine venetoclax efficacy. Furthermore, the mechanisms underlying venetoclax response and the development of venetoclax resistance were evaluated using proteomics analysis and Western blotting.Results: Potential biomarkers linked to venetoclax activity and targeted combination therapies that can augment venetoclax response were identified. We demonstrate that DLBCL and MCL cell lines, primary patient samples, and PDX mouse models expressing high BCL-2 levels are extremely sensitive to venetoclax treatment. Proteomics studies showed that venetoclax substantially alters the expression levels and phosphorylation status of key proteins involved in cellular processes, including the DNA damage response, cell metabolism, cell growth/survival, and apoptosis. Short- and long-term exposure to venetoclax inhibited PTEN expression, leading to enhanced AKT pathway activation and concomitant susceptibility to PI3K/AKT inhibition. Intrinsic venetoclax-resistant cells possess high AKT activation and are highly sensitive to PI3K/AKT inhibition.Conclusions: These findings demonstrate the on-target effect of venetoclax and offer potential mechanisms to overcome acquired and intrinsic venetoclax resistance through PI3K/AKT inhibition. Clin Cancer Res; 24(16); 3967-80. ©2018 AACR.

Preclinical efficacy and biological effects of the oral proteasome inhibitor ixazomib in diffuse large B-cell lymphoma.

Despite advances in deciphering the molecular pathogenesis of diffuse large B-cell lymphoma (DLBCL), patients with relapsed/refractory disease, particularly those with adverse genetic features (e.g., mutated p53 or double hit lymphoma (DHL)) have very poor prognoses, and effective therapies are lacking. In this study we examined the preclinical efficacy and associated biological effects of the first oral proteasome inhibitor, ixazomib, in DLBCL in vitro and in vivo models. We demonstrated that ixazomib exhibited anti-tumor activities in 28 representative DLBCL cell lines, 10 primary DLBCL samples, and a DHL xenotransplant mouse model, at clinically achievable drug concentrations. Ixazomib sensitivity in DLBCL cells is correlated with immunoproteasomal activity; stimulating lymphoma cells with interferon gamma induced immunoproteasome activity and sensitized these cells to ixazomib. In addition, we showed that ixazomib induces apoptosis and the DNA damage response pathway, through activation of the checkpoint kinase 2 (CHK2). Hence, pharmacological inhibition of CHK2 enhances the anti-tumor activity of ixazomib in DLBCL cells. Our results indicate that ixazomib is an effective proteasome inhibitor active in DLBCL, including DHL, and its combination with a CHK2 inhibitor offers a potentially more robust therapeutic regimen for treatment-resistant DLBCL.

Targeting the hexosamine biosynthetic pathway and O-linked N-acetylglucosamine cycling for therapeutic and imaging capabilities in diffuse large B-cell lymphoma.

The hexosamine biosynthetic pathway (HBP) requires two key nutrients glucose and glutamine for O-linked N-acetylglucosamine (O-GlcNAc) cycling, a post-translational protein modification that adds GlcNAc to nuclear and cytoplasmic proteins. Increased GlcNAc has been linked to regulatory factors involved in cancer cell growth and survival. However, the biological significance of GlcNAc in diffuse large B-cell lymphoma (DLBCL) is not well defined. This study is the first to show that both the substrate and the endpoint O-GlcNAc transferase (OGT) enzyme of the HBP were highly expressed in DLBCL cell lines and in patient tumors compared with normal B-lymphocytes. Notably, high OGT mRNA levels were associated with poor survival of DLBCL patients. Targeting OGT via small interference RNA in DLBCL cells inhibited activation of GlcNAc, nuclear factor kappa B (NF-κB), and nuclear factor of activated T-cells 1 (NFATc1), as well as cell growth. Depleting both glucose and glutamine in DLBCL cells or treating them with an HBP inhibitor (azaserine) diminished O-GlcNAc protein substrate, inhibited constitutive NF-κB and NFATc1 activation, and induced G0/G1 cell-cycle arrest and apoptosis. Replenishing glucose-and glutamine-deprived DLBCL cells with a synthetic glucose analog (ethylenedicysteine-N-acetylglucosamine [ECG]) reversed these phenotypes. Finally, we showed in both in vitro and in vivo murine models that DLBCL cells easily take up radiolabeled technetium-99m-ECG conjugate. These findings suggest that targeting the HBP has therapeutic relevance for DLBCL and underscores the imaging potential of the glucosamine analog ECG in DLBCL.

Establishment and characterization of a novel MYC/BCL2 "double-hit" diffuse large B cell lymphoma cell line, RC.

BACKGROUND: Diffuse large B cell lymphoma (DLBCL) is the most common type of lymphoid malignancy worldwide. Approximately 5 % of cases of DLBCL are so-called double-hit lymphomas (DHL), defined by a chromosomal translocation or rearrangement involving MYC/8q24.2 in combination with another recurrent breakpoint, usually BCL2/18q21.3. Patients with MYC/BCL2 DHL are resistant to standard front-line therapy, and currently, there is no consensus for a therapeutic strategy to treat these patients. Lack of clinically relevant or validated human experimental DHL models of any type that would improve our understanding of the biologic basis of MYC/BCL2 DHL pathophysiology continues to hamper identification of valid therapeutic targets. We describe a unique MYC/BCL2 DHL cell line with morphologic features of DLBCL that we have established, designated as RC. METHODS: We used tissue culture techniques to establish the RC cell line from primary DLBCL cells. We also utilized molecular and cellular biological techniques including flow cytometry, polymerase chain reaction (PCR), DNA fingerprinting, reverse-phase protein array, conventional cytogenetics, and fluorescence in situ hybridization (FISH) analysis to characterize the RC cell line. NSG-severe combined immunodeficiency (SCID) mice were utilized as a model for xeno-transplantation of RC cells. RESULTS: RC cells had the following immunophenotype: positive for CD10, CD19, CD20, CD22, CD38, CD43, CD44, and CD79b and negative for CD3, CD4, CD5, CD8, CD11c, CD14, CD30, CD56, and CD200, which was identical to the primary tumor cells. Conventional cytogenetic analysis showed a t(2;8)(p12;q24.2) and t(14;18)(q32;q21.3), corresponding to MYC and BCL2 gene rearrangements, respectively. DNA fingerprinting authenticated the RC cell line to be of the same clone as the primary tumor cells. In addition, RC cells were established in SCID mice as an in vivo model for translational therapeutics studies. Proteomic analysis showed activation of the mTOR signaling pathway in RC cells that can be targeted with an mTOR inhibitor. CONCLUSION: The data presented confirm the validity of the RC cell line as a representative model of MYC/BCL2 DHL that will be useful for both in vitro and in vivo studies of DHL pathogenesis and therapeutics.

Involvement of tumor-associated macrophage activation in vitro during development of a novel mantle cell lymphoma cell line, PF-1, derived from a typical patient with relapsed disease.

Human mantle cell lymphoma (MCL) cell lines are scarce and have been only sporadically described and validated, and only a few have been thoroughly molecularly or genetically characterized. We describe here the successful establishment of a new MCL line, PF-1, with typical MCL characteristics. Culturing primary MCL cells in vitro initially gave rise to an essential generative microenvironment "niche" involving macrophages required for MCL growth, and eventually produced the PF-1 MCL cell line. Our analysis revealed that PF-1 is morphologically and genotypically nearly identical to the original tumor cells. The PF-1 MCL cell line that we have developed will be useful for in vitro and in vivo studies of MCL pathogenesis and therapeutics.

In vitro and in vivo therapeutic efficacy of carfilzomib in mantle cell lymphoma: targeting the immunoproteasome.

Mantle cell lymphoma (MCL) remains incurable due to its inevitable pattern of relapse after treatment with current existing therapies. However, the promise of a cure for MCL lies in the burgeoning area of novel agents. In this study, we elucidated the therapeutic effect and mechanism of carfilzomib, a novel long-acting second-generation proteasome inhibitor, in MCL cells. We found that carfilzomib induced growth inhibition and apoptosis in both established MCL cell lines and freshly isolated primary MCL cells in a dose-dependent manner. In contrast, carfilzomib was less toxic to normal peripheral blood mononuclear cells from healthy individuals. The carfilzomib-induced apoptosis of MCL cells was mediated by the activation of JNK, Bcl-2, and mitochondria-related pathways. In addition, carfilzomib inhibited the growth and survival signaling pathways NF-κB and STAT3. Interestingly, we discovered that expression of immunoproteasome (i-proteasome) subunits is required for the anti-MCL activity of carfilzomib in MCL cells. In MCL-bearing SCID mice/primary MCL-bearing SCID-hu mice, intravenous administration of 5 mg/kg carfilzomib on days 1 and 2 for 5 weeks slowed/abrogated tumor growth and significantly prolonged survival. Our preclinical data show that carfilzomib is a promising, potentially less toxic treatment for MCL. Furthermore, an intact i-proteasome, especially LMP2, appears to be necessary for its anti-MCL activity, suggesting that i-proteasome could serve as a biomarker for identifying patients who will benefit from carfilzomib.

Managing lymphoma with non-FDG radiotracers: current clinical and preclinical applications.

Nuclear medicine imaging modalities such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) have played a prominent role in lymphoma management. PET with [(18)F]Fluoro-2-deoxy-D-glucose (FDG) is the most commonly used tool for lymphoma imaging. However, FDG-PET has several limitations that give the false positive or false negative diagnosis of lymphoma. Therefore, development of new radiotracers with higher sensitivity, specificity, and different uptake mechanism is in great demand in the management of lymphoma. This paper reviews non-FDG radiopharmaceuticals that have been applied for PET and SPECT imaging in patients with different types of lymphoma, with attention to diagnosis, staging, therapy response assessment, and surveillance for disease relapse. In addition, we introduce three radiolabeled anti-CD20 antibodies for radioimmunotherapy, which is another important arm for lymphoma treatment and management. Finally, the relatively promising radiotracers that are currently under preclinical development are also discussed in this paper.

Novel selective inhibitors of nuclear export CRM1 antagonists for therapy in mantle cell lymphoma.

Overexpression of the cellular nuclear exportin 1, more commonly called chromosomal region maintenance 1 (CRM1), has been associated with malignant progression and mortality. Therefore, activation of nuclear export can play a significant etiologic role in some forms of human neoplasia and serve as a novel target for the treatment of these cancers. Mantle cell lymphoma (MCL) is an aggressive histotype of B-cell non-Hodgkin lymphoma that remains incurable. The objective of this study was to investigate the functional significance of CRM1 in MCL by evaluating the therapeutic efficacy of CRM1 inhibition in MCL in vitro and in vivo. Our results showed that CRM1 is highly expressed in MCL cells and is involved in regulating growth and survival mechanisms through the critical nuclear factor-κB survival pathway, which is independent of p53 status. Inhibition of CRM1 by two novel selective inhibitors of nuclear export (SINE), KPT-185 and KPT-276, in MCL cells resulted in significant growth inhibition and apoptosis induction. KPT-185 also induced CRM1 accumulation in the nucleus, resulting in CRM1 degradation by the proteasome. Oral administration of KPT-276 significantly suppressed tumor growth in an MCL-bearing severe combined immunodeficient mouse model, without severe toxicity. Our data suggest that SINE CRM1 antagonists are a potential novel therapy for patients with MCL, particular in relapsed/refractory disease.

The therapeutic effects of rGel/BLyS fusion toxin in in vitro and in vivo models of mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an incurable, aggressive histo-type of B-cell non-Hodgkin lymphoma associated with both high relapsed rates and relatively short survival. Because MCL over-expresses receptors for B lymphocyte stimulator (BLyS) and displays constitutively active NF-κB, agents targeting these pathways may be of therapeutic relevance in this disease. To investigate the potential clinical use of the rGel/BLyS fusion toxin in combination with bortezomib, we evaluated this fusion toxin for its ability to inhibit MCL growth in severe combined immunodeficiency (SCID) xenograft model. Compared with PBS-treated mice, mice treated with this fusion toxin prolonged both median (84 days vs. 125 days) and overall survival (0% vs. 40%) (p=0.0027). Compared with bortezomib alone-treated mice, mice treated with rGel/BLyS plus bortezomib showed significantly increased median (91 days vs. 158 days) and overall survival (0% vs. 20%) (p=0.0127). Histopathologic analysis of peritoneal intestinal mesentery from MCL-SCID mice showed no demonstrable microscopic lymphomatous involvement at 225 days after treatment with rGel/BLyS. Combination treatment resulted in a synergistic growth inhibition, down-regulation of NF-κB DNA-binding activity, inhibition of cyclin D1, Bcl-x(L), p-Akt, Akt, p-mTOR, and p-Bad, up-regulation of Bax, and induction of cellular apoptosis. Our findings demonstrate that rGel/BLyS is an effective therapeutic agent for both primary and salvage treatment of aggressive MCL expressing constitutively active NF-κB and BLyS receptors and may be an excellent candidate for clinical development.

Over-expression of Thioredoxin-1 mediates growth, survival, and chemoresistance and is a druggable target in diffuse large B-cell lymphoma.

Diffuse Large B cell lymphomas (DLBCL) are the most prevalent of the non-Hodgkin lymphomas and are currently initially treated fairly successfully, but frequently relapse as refractory disease, resulting in poor salvage therapy options and short survival. The greatest challenge in improving survival of DLBCL patients is overcoming chemo-resistance, whose basis is poorly understood. Among the potential mediators of DLBCL chemo-resistance is the thioredxoin (Trx) family, primarily because Trx family members play critical roles in the regulation of cellular redox homeostasis, and recent studies have indicated that dysregulated redox homeostasis also plays a key role in chemoresistance. In this study, we showed that most of the DLBCL-derived cell lines and primary DLBCL cells express higher basal levels of Trx-1 than normal B cells and that Trx-1 expression level is associated with decreased patients survival. Our functional studies showed that inhibition of Trx-1 by small interfering RNA or a Trx-1 inhibitor (PX-12) inhibited DLBCL cell growth, clonogenicity, and also sensitized DLBCL cells to doxorubicin-induced cell growth inhibition in vitro. These results indicate that Trx-1 plays a key role in cell growth and survival, as well as chemoresistance, and is a potential target to overcome drug resistance in relapsed/refractory DLBCL.

The microenvironment in mantle cell lymphoma: cellular and molecular pathways and emerging targeted therapies.

There is growing evidence suggesting that cross talk between mantle cell lymphoma (MCL) cells and stromal cells in tissue microenvironments, such as the bone marrow and secondary lymphoid organs, causes disease progression by promoting lymphoma cell survival, growth, and drug resistance. Conceivably, while conventional treatment eliminates the bulk of MCL cells, residual lymphoma cells may lurk in protective tissue niches, where they receive signals from accessory cells that promote survival and drug-resistance, thereby paving the way for residual disease and relapses. Based on this concept, the lymphoma microenvironment has become a growing area of current research, and initial clinical trials targeting cross talk between MCL cells and their microenvironment are showing promising early results. In this review, we summarize key cellular and molecular interactions between MCL cells and their microenvironment, and update new clinical developments in this area.

Constitutive BR3 receptor signaling in diffuse, large B-cell lymphomas stabilizes nuclear factor-κB-inducing kinase while activating both canonical and alternative nuclear factor-κB pathways.

Aberrant nuclear factor κB (NF-κB) signaling has been found to be of particular importance in diffuse, large B-cell lymphoma (DLBCL) cell survival and proliferation. Although the canonical NF-κB signaling pathway has been studied in some detail, activation of the alternative NF-κB pathway in DLBCL is not well characterized. Important insights into the regulation of the alternative NF-κB pathway in B lymphocytes has recently revealed the regulatory importance of the survival kinase NIK (NF-κB-inducing kinase) in genetically engineered murine models. Our studies demonstrate that both the canonical and alternative NF-κB pathways are constitutively activated in DLBCL. We also demonstrate that NIK kinase aberrantly accumulates in DLBCL cells due to constitutive activation of B-cell activation factor (BAFF)-R (BR3) through interaction with autochthonous B-lymphocyte stimulator (BLyS) ligand in DLBCL cells. Activation of BR3 in DLBCL induces recruitment and degradation of tumor necrosis factor receptor-associated factor 3, which results in NIK kinase accumulation, IκBα phosphorylation, and NF-κB p100 processing, thereby resulting in continuous activation of both NF-κB pathways in DLBCL cells, leading to autonomous lymphoma cell growth and survival. These results further elucidate mechanisms involved in abnormal NF-κB activation in DLBCL, and should contribute to better future therapeutic approaches for patients with DLBCL.

Degrasyn potentiates the antitumor effects of bortezomib in mantle cell lymphoma cells in vitro and in vivo: therapeutic implications.

Mantle cell lymphoma (MCL) is an aggressive histotype of B-cell non-Hodgkin lymphoma that has increased in incidence over the past few decades and is incurable, usually poorly responsive to standard chemotherapy combinations, and associated with poor prognoses. Discovering new therapeutic agents with low toxicity that produce better outcomes in patients with MCL is an ongoing challenge. Recent studies showed that degrasyn, a novel small-molecule inhibitor of the Janus kinase/signal transducer and activation of transcription (JAK/STAT) pathway, exerts antitumor activity in lymphoid tumors by inhibiting key growth and survival signaling (JAK/STAT) pathways. In the present study, we found that treatment of both typical and blastoid-variant MCL cells with degrasyn in combination with bortezomib resulted in synergistic growth inhibition and apoptosis induction in vitro. The apoptosis in these cells was correlated with the downregulation of constitutive NF-kappaB and phosphorylated STAT3 activation, leading to the inhibition of c-Myc, cyclin D1, and bcl-2 protein expression and the upregulation of bax protein expression. In vivo, degrasyn and bortezomib interacted to synergistically prevent tumor development and prolong survival durations in a xenotransplant severe combined immunodeficient mouse model of MCL. These findings suggest that agents such as degrasyn that can pharmacologically target constitutively expressed NF-kappaB and STAT3 in MCL cells may be useful therapeutic agents for MCL when administered together with bortezomib.

An epigenetic chromatin remodeling role for NFATc1 in transcriptional regulation of growth and survival genes in diffuse large B-cell lymphomas.

The nuclear factor of activated T cells (NFAT) family of transcription factors functions as integrators of multiple signaling pathways by binding to chromatin in combination with other transcription factors and coactivators to regulate genes central for cell growth and survival in hematopoietic cells. Recent experimental evidence has implicated the calcineurin/NFAT signaling pathway in the pathogenesis of various malignancies, including diffuse large B-cell lymphoma (DLBCL). However, the molecular mechanism(s) underlying NFATc1 regulation of genes controlling lymphoma cell growth and survival is still unclear. In this study, we demonstrate that the transcription factor NFATc1 regulates gene expression in DLBCL cells through a chromatin remodeling mechanism that involves recruitment of the SWItch/Sucrose NonFermentable chromatin remodeling complex ATPase enzyme SMARCA4 (also known as Brahma-related gene 1) to NFATc1 targeted gene promoters. The NFATc1/Brahma-related gene 1 complex induces promoter DNase I hypersensitive sites and recruits other transcription factors to the active chromatin site to regulate gene transcription. Targeting NFATc1 with specific small hairpin RNA inhibits DNase I hypersensitive site formation and down-regulates target gene expression. Our data support a novel epigenetic control mechanism for the transcriptional regulation of growth and survival genes by NFATc1 in the pathophysiology of DLBCL and suggests that targeting NFATc1 could potentially have therapeutic value.

Side population of a murine mantle cell lymphoma model contains tumour-initiating cells responsible for lymphoma maintenance and dissemination.

'Cancer stem cells' or 'tumour initiating cells' in B-cell non-Hodgkin lymphomas have not been demonstrated, although some studies focused on other cancer types suggest that such populations exist and represent tumour cells resistant to therapy and involved in relapse. These cells may also represent a putative neoplastic 'cell of origin' in lymphomas, but there is little substantive data to support this suggestion. Using cell lines derived from a recently established murine IL-14alphax c-Myc double transgenic/mantle cell lymphoma-blastoid variant model, heretofore referred to as DTG cell lines, we identified a subset of cells within the side population (SP) with features of 'tumour-initiating cells'. These features include higher expression of ABCG2 and BCL-2, longer telomere length, greater self-renewal ability and higher in vitro clonogenic and in vivo tumorigenic capacities compared with non-SP. In addition, in vitro viability studies demonstrated that the non-SP lymphoma subpopulation has a limited lifespan in comparison with the SP fraction. Syngenic transplant studies showed that non-SP derived tumours, in comparison to the SP-derived tumours, exhibit greater necrosis/apoptosis and less systemic dissemination capability. In conclusion, our data support the interpretation that the DTG SP fraction contains a cell population highly capable of tumour maintenance and systemic dissemination and lends support to the concept that 'tumour-initiating cells' occur in lymphomas.

IGF-IR tyrosine kinase interacts with NPM-ALK oncogene to induce survival of T-cell ALK+ anaplastic large-cell lymphoma cells.

Type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase plays important roles in the pathogenesis of several malignancies. Although it promotes the growth of stimulated hematopoietic cells, a direct role of IGF-IR in malignant lymphoma has not been identified. Anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma (ALK(+) ALCL) is a unique type of T-cell lymphoma. Approximately 85% of ALK(+) ALCL cases harbor the translocation t(2;5)(p23;q35), which generates the chimeric oncogene NPM-ALK. In the present study, we explored a possible role of IGF-IR in ALK(+) ALCL. Our results demonstrate that IGF-IR and IGF-I are widely expressed in ALK(+) ALCL cell lines and primary tumors. Importantly, we identified novel reciprocal functional interactions between IGF-IR and NPM-ALK. Antagonism of IGF-IR decreased the viability, induced apoptosis and cell-cycle arrest, and decreased proliferation and colony formation of ALK(+) ALCL cell lines. These effects could be explained by alterations of cell survival regulatory proteins downstream of IGF-IR signaling. Our findings improve current understanding of the biology of IGF-IR and NPM-ALK and have significant therapeutic implications as they identify IGF-IR signaling as a potential therapeutic target in ALK(+) ALCL and possibly other types of malignant lymphoma.

BAFF-R promotes cell proliferation and survival through interaction with IKKbeta and NF-kappaB/c-Rel in the nucleus of normal and neoplastic B-lymphoid cells.

BLyS and its major receptor BAFF-R have been shown to be critical for development and homeostasis of normal B lymphocytes, and for cell growth and survival of neoplastic B lymphocytes, but the biologic mechanisms of this ligand/receptor-derived intracellular signaling pathway(s) have not been completely defined. We have discovered that the BAFF-R protein was present in the cell nucleus, in addition to its integral presence in the plasma membrane and cytoplasm, in both normal and neoplastic B cells. BAFF-R interacted with histone H3 and IKKbeta in the cell nucleus, enhancing histone H3 phosphorylation through IKKbeta. Nuclear BAFF-R was also associated with NF-kappaB/c-Rel and bound to NF-kappaB targeted promoters including BLyS, CD154, Bcl-xL, IL-8, and Bfl-1/A1, promoting the transcription of these genes. These observations suggested that in addition to activating NF-kappaB pathways in the plasma membrane, BAFF-R also promotes normal B-cell and B-cell non-Hodgkin lymphoma (NHL-B) survival and proliferation by functioning as a transcriptional regulator through a chromatin remodeling mechanism(s) and NF-kappaB association. Our studies provide an expanded conceptual view of the BAFF-R signaling, which should contribute a better understanding of the physiologic mechanisms involved in normal B-cell survival and growth, as well as in the pathophysiology of aggressive B-cell malignancies and autoimmune diseases.