Syk-dependent mTOR activation in follicular lymphoma cells

The mammalian target of rapamycin (mTOR) is emerging as a promising target for antitumor therapy. However, the mechanism that contributes to its regulation in B lymphomas remains unknown. This study shows that in follicular lymphoma (FL) cells, mTOR is active because the cells displayed rapamycin-sensitive phosphorylation of p70S6 kinase and 4E-BP1. Moreover, immunohistochemistry applied on lymph node tissue sections obtained from patients with FL revealed that, in most cases, p70S6 kinase was highly phosphorylated compared to normal tonsillar tissue. In FL cells, mTOR was under control of both phospholipase D (PLD) and phosphatidylinositol 3-kinase (PI3K). Moreover, we demonstrated that Syk plays a central role in mTOR activation because we found that both expression and activity are elevated compared to normal or chronic lymphocytic leukemia B cells. We also provide evidence that Syk operates through PLD- and PI3K-independent pathways. Finally, Syk inhibition by piceatannol or by siRNA plasmids resulted in a potent inhibition of mTOR activity in FL cells, as well as in mantle cell lymphoma, Burkitt lymphoma, and diffuse large B-cell lymphoma. These findings suggest that the Syk-mTOR pathway has a critical function in FL survival, and therefore, that Syk could be a promising new target for B-lymphoma therapy.     

New agents in follicular lymphoma

The follicular lymphomas are indolent diseases that are highly responsive to various combinations of standard chemotherapy drugs. Nevertheless, until recently, no regimen had improved patient outcome. The incorporation of effective and well-tolerated monoclonal antibodies, such as rituximab, into chemoimmunotherapeutic strategies provided the first evidence that survival of these patients could be prolonged. Nevertheless, follicular lymphoma remains incurable and characterized by recurrent relapses requiring additional treatment. An increasing number of effective drugs are now being evaluated either alone or in combinations including the chemotherapy drugs bendamustine and bortezomib. More targeted agents include monoclonal antibodies and their derivatives such as drug-antibody conjugates and small modular immunopharmaceuticals. Other agents inhibit various cellular pathways including those triggered by the B-cell receptor, including spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase, and other intracellular pathways such as the mammalian target of rapamycin (mTOR), PI3-kinase, and apoptosis, and drugs that target the tumor microenvironment, notably the immunomodulatory agent lenalidomide. The development of combinations of these agents should be based on scientific rationale with correlative studies to enhance our understanding of the mechanisms of action and resistance of the drugs and the biology of the tumor to further improve the outcome of patients with follicular lymphoma.     

Protein kinase Czeta mediated Raf-1/extracellular-regulated kinase activation by daunorubicin

In light of the emerging concept of a protective function of the mitogen-activated protein kinase (MAPK) pathway under stress conditions, we investigated the influence of the anthracycline daunorubicin (DNR) on MAPK signaling and its possible contribution to DNR-induced cytotoxicity. We show that DNR increased phosphorylation of extracellular-regulated kinases (ERKs) and stimulated activities of both Raf-1 and extracellular-regulated kinase 1 (ERK1) within 10 to 30 minutes in U937 cells. ERK1 stimulation was completely blocked by either the mitogen-induced extracellular kinase (MEK) inhibitor PD98059 or the Raf-1 inhibitor 8-bromo-cAMP (cyclic adenosine monophosphate). However, only partial inhibition of Raf-1 and ERK1 stimulation was observed with the antioxidant N-acetylcysteine (N-Ac). Moreover, the xanthogenate compound D609 that inhibits DNR-induced phosphatidylcholine (PC) hydrolysis and subsequent diacylglycerol (DAG) production, as well as wortmannin that blocks phosphoinositide-3 kinase (PI3K) stimulation, only partially inhibited Raf-1 and ERK1 stimulation. We also observed that DNR stimulated protein kinase C zeta (PKCzeta), an atypical PKC isoform, and that both D609 and wortmannin significantly inhibited DNR-triggered PKCzeta activation. Finally, we found that the expression of PKCzeta kinase-defective mutant resulted in the abrogation of DNR-induced ERK phosphorylation. Altogether, these results demonstrate that DNR activates the classical Raf-1/MEK/ERK pathway and that Raf-1 activation is mediated through complex signaling pathways that involve at least 2 contributors: PC-derived DAG and PI3K products that converge toward PKCzeta. Moreover, we show that both Raf-1 and MEK inhibitors, as well as PKCzeta inhibition, sensitized cells to DNR-induced cytotoxicity.     

Entinostat,a novel histone deacetylase inhibitor is active in B‐cell lymphoma and enhances the anti‐tumour activity of rituximab and chemotherapy agents

Histone deacetylases (HDACs) inhibitors are active in T‐cell lymphoma and are undergoing pre‐clinical and clinical testing in other neoplasms. Entinostat is an orally bioavailable class I HDAC inhibitor with a long half‐life, which is under evaluation in haematological and solid tumour malignancies. To define the activity and biological effects of entinostat in B‐cell lymphoma we studied its anti‐tumour activity in several rituximab‐sensitive or ‐resistant pre‐clinical models. We demonstrated that entinostat is active in rituximab‐sensitive cell lines (RSCL), rituximab‐resistant cell lines (RRCL) and primary tumour cells isolated from lymphoma patients (n = 36). Entinostat exposure decreased Bcl‐XL (BCL2L1) levels and induced apoptosis in cells. In RSCL and RRCL, entinostat induced p21 (CDKN1A) expression leading to G1 cell cycle arrest and exhibited additive effects when combined with bortezomib or cytarabine. Caspase inhibition diminished entinostat activity in some primary tumour cells suggesting that entinostat has dual mechanisms‐of‐action. In addition, entinostat increased the expression of CD20 and adhesion molecules. Perhaps related to these effects, we observed a synergistic activity between entinostat and rituximab in a lymphoma‐bearing severe combined immunodeficiency (SCID) mouse model. Our data suggests that entinostat is an active HDAC inhibitor that potentiates rituximab activity in vivo and supports its further clinical development in B‐cell lymphoma.     

Variation in gene expression patterns in follicular lymphoma and the response to rituximab

Analysis of the patterns of gene expression in follicular lymphomas from 24 patients suggested that two groups of tumors might be distinguished. All patients, whose biopsies were obtained before any treatment, were treated with rituximab, a monoclonal antibody directed against the B cell antigen, CD20. Gene expression patterns in the tumors that subsequently failed to respond to rituximab appeared more similar to those of normal lymphoid tissues than to gene expression patterns of tumors from rituximab responders. These findings suggest the possibility that the response of follicular lymphoma to rituximab treatment may be predicted from the gene expression pattern of tumors.     

Multiple signaling pathways promote B lymphocyte stimulator dependent B-cell growth and survival

We investigated the mechanism by which B lymphocyte stimulator (BLyS)/BAFF, a tumor necrosis factor superfamily ligand, promotes B-cell survival and resistance to atrophy. BLyS stimulation activates 2 independent signaling pathways, Akt/mTOR and Pim 2, associated with cell growth and survival. BLyS blocks the cell volume loss (atrophy) that freshly isolated B cells normally undergo when maintained in vitro while concurrently increasing glycolytic activity and overall metabolism. This atrophy resistance requires Akt/mTOR. We used a genetic approach to resolve the contributions of Akt/mTOR and Pim kinase pathways to BLyS-mediated survival. Pim 2-deficient B cells are readily protected from death by BLyS stimulation, but this protection is completely abrogated by treatment with the mTOR inhibitor rapamycin. Furthermore, rapamycin treatment in vivo significantly reduces both follicular and marginal zone B cells in Pim-deficient but not healthy hosts. BLyS-dependent survival requires the antiapoptotic protein Mcl-1. Mcl-1 protein levels rise and fall in response to BLyS addition and withdrawal, respectively, and conditional deletion of the Mcl-1 gene renders B cells refractory to BLyS-mediated protection. Because BlyS is required for the normal homeostasis of all B cells, these data suggest a therapeutic strategy simultaneously inhibiting mTOR and Pim 2 could target pathogenic B cells.     

Activation of the PI3K/AKT/mTOR pathway in diffuse large B cell lymphoma: clinical significance and inhibitory effect of rituximab

Diffuse large B cell lymphoma (DLBCL) represents the most common subtype of non-Hodgkin lymphoma and accounts for approximately 30 % of newly diagnosed lymphoid neoplasms in Western countries, and 40–50 % in China. A better understanding of the biology of DLBCL is needed for the development of potential therapeutic agents that target specific intracellular pathways. In this study, expression of the important components of the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway and their clinical significance were investigated in 73 DLBCL cases. The effect of rituximab alone or combined with the PI3K/AKT/mTOR pathway inhibitor rapamycin was further evaluated in the DLBCL cell lines. A total of 73 patients were identified, including 45 men and 28 women aged 18 to 78 years (median age 50 years). Of these patients, p-AKT was positive in 40 cases (54.8 %), p-p70S6K in 34 cases (46.6 %), and p-4E-BP1 in 33 cases (45.2 %). Activation of the PI3K/AKT/mTOR pathway was related to poor disease outcome in DLBCL patients treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) but not in those treated with rituximab-CHOP. Rituximab combined with rapamycin synergically downregulated the PI3K/AKT/mTOR signaling pathway. Western blot analysis revealed a baseline activation status of the PI3K/AKT/mTOR pathway in DLBCL cell lines, with high levels of p-AKT, p-mTOR, in addition to downstream molecules p-p70S6K and p-4E-BP1. The results indicate that the PI3K/AKT/mTOR pathway is a potentially important signaling route and an unfavorable prognostic factor for DLBCL. Patients with PI3K/AKT/mTOR activation experience a more rapidly deteriorating clinical course with poor treatment response and decreased survival time. Addition of rituximab could downregulate PI3K/AKT/mTOR activation, reversing its negative effect on chemotherapy-treated patients. In addition, our results indicate that the combination of rituximab and inhibition of the activated PI3K/AKT/mTOR pathway could be a promising target for DLBCL therapeutic intervention in the future.     

Protein kinase Czeta is required for oleic acid-induced secretion of glucagon-like peptide-1 by intestinal endocrine L cells

Long-chain, monounsaturated fatty acids (FAs) stimulate secretion of the incretin hormone, glucagon-like peptide-1 (GLP-1) from the intestinal L cell. Because the atypical protein kinase C (PKC), PKCzeta, is involved in FA signaling in many cells, the role of PKCzeta in FA-induced GLP-1 secretion was investigated, using the murine GLUTag L cell line and primary rat intestinal L cells. GLUTag cells expressed mRNA for several PKC isoforms, including PKCzeta, and PKCzeta protein was localized throughout the cytoplasm in GLUTag and primary L cells as well as normal mouse and rat L cells. Treatment with oleic acid (150-1000 microm) for 2 h increased GLP-1 secretion (P < 0.001), and this was abrogated by the PKCzeta inhibitor ZI (P < 0.05) and PKCzeta small interfering RNA transfection (P < 0.05) but not inhibition of classical/novel PKC isoforms. Although most PKCzeta was localized in the particulate compartment of GLUTag cells, oleate treatment did not alter PKCzeta levels or activity in this cell fraction. GLUTag cells expressed mRNA for the Gq-coupled FA receptor GPR120; however, oleic acid did not induce any changes in Akt, MAPK, or calcium, and pretreatment with LY294002 and PD98059 to inhibit phosphatidylinositol 3-kinase and MAPK, respectively, did not prevent the effects of oleic acid. Finally, GLUTag cells also released GLP-1 in response to arachidonic acid (P < 0.001) but were not affected by other long-chain FAs. These findings demonstrate that PKCzeta is required for oleic acid-induced GLP-1 secretion. This enzyme may therefore serve as a therapeutic target to enhance GLP-1 release in type 2 diabetes.     

Short term exposure to elevated levels of leptin reduces proximal tubule cell metabolic activity

Leptin plays a pathophysiological role in the kidney, however, its acute effects on the proximal tubule cells (PTCs) are unknown. In opossum kidney (OK) cells in vitro, Western blot analysis identified that exposure to leptin increases the phosphorylation of the mitogen-activated protein kinase (MAPK) p44/42and the mammalian target of rapamycin (mTOR). Importantly leptin (0.05, 0.10, 0.25 and 0.50 μg/ml) significantly reduced the metabolic activity of PTCs, and significantly decreased protein content per cell. Investigation of the role of p44/42 and mTOR on metabolic activity and protein content per cell, demonstrated that in the presence of MAPK inhibitor U0126 and mTOR inhibitor Ku-63794, that the mTOR pathway is responsible for the reduction in PTC metabolic activity in response to leptin. However, p44/42 and mTOR play no role the reduced protein content per cell in OKs exposed to leptin. Therefore, leptin modulates metabolic activity in PTCs via an mTOR regulated pathway.     

B cell-targeted therapy in autoimmune disease: rationale, mechanisms, and clinical application

B cells play a critical role in the pathogenesis of rheumatoid arthritis (RA) and other autoimmune diseases. Recently, a number of biologic agents that target B cells have been tested as therapies for these conditions. These agents either deplete B cells, by targeting cell-surface antigens such as CD20, or block B cell function, for example by inhibiting the activity of B cell survival factors such as BLyS. Of this group of agents, the first in clinical use has been rituximab, a chimeric monoclonal antibody that depletes B cells by binding to the CD20 cell-surface antigen. Initially introduced as a treatment for non-Hodgkin's lymphoma, rituximab is now approved for the treatment of RA. In this review we explore the rationale behind B cell-targeted therapy, highlight the results of clinical trials with rituximab in RA and other autoimmune diseases, and describe other emerging therapies directed at B cells.     

Follicular lymphoma: time for a re-think?

Follicular lymphoma (FL) is a malignancy of follicle centre B cells that have at least a partially follicular pattern, and is the commonest type of indolent Non-Hodgkin's lymphoma. Except in the subset of patients with localized disease, FL should still be regarded as an incurable malignancy with a relentless relapsing/remitting course. However, the provocative new data covered by this review (including anti-CD20 antibody therapy, BCL-2, radioimmunotherapy, new chemotherapeutic agents and anti-idiotype vaccination), provides much cause for excitement and guarded optimism. Rituximab represents a novel treatment approach for a variety of disease settings, with a proven excellent efficacy and toxicity profile. Long-term data is required to establish whether its use translates into survival benefit. As the clinical activity of rituximab and other new therapeutic approaches becomes established, it will be important to determine how best to integrate these results into the standard care of patients with follicular lymphoma.     

In vitro mechanisms of action of rituximab on primary non-Hodgkin lymphomas

To assess the sensitivity of primary non-Hodgkin lymphoma cells to rituximab-mediated cytotoxicity, we compared the potency of several rituximab-mediated killing mechanisms on fresh lymphoma cells. All lymphoma cells tested were equally sensitive to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-mediated phagocytosis of tumor cells, and rituximab-induced apoptosis. However, they were differentially lysed by complement-dependent cytotoxicity (CDC). We found that taking into account both CD20 and complement regulatory protein expression on tumor cells could predict CDC sensitivity in vitro. Importantly, the sensitivity of lymphoma cells to CDC was consistent with the reported different clinical response rates of lymphomas: rituximab induced high CDC killing of follicular lymphoma cells, whereas mantle cell lymphoma and diffuse large cell lymphoma cells were moderately sensible to CDC, and small lymphocytic lymphoma cells were almost all resistant. We propose that CDC is a determinant mechanism of rituximab-induced killing in vivo. Poor sensitivity to CDC in vitro might predict a poor clinical response, whereas high sensitivity to CDC would only indicate a likelihood of response to rituximab treatment.     

Radioimmunotherapy for B-cell non-Hodgkin lymphoma

Radioimmunotherapy (RIT) combines the targeting advantage of a monoclonal antibody with the radiosensitivity of non-Hodgkin lymphoma (NHL) cells. There are now two radioimmunoconjugates (RICs) - ibritumomab tiuxetan (Zevalin) and tositumomab (Bexxar) - that are approved by the FDA in the US for relapsed low-grade or follicular B-cell NHL. Both agents target the CD20 antigen on B-cell lymphoma cells. In relapsed disease, single doses of RIT produce an 80% overall response rate, with approximately 20% of patients achieving durable responses. RIT is very well tolerated and is delivered on an outpatient basis over 1 week. The only significant toxicity is reversible myelosuppression. Both RIT agents have demonstrated high anti-tumor activity in patients who are refractory to rituximab. Current trials are testing RIT as initial therapy with rituximab maintenance, as adjuvant therapy after chemotherapy, or in high-dose protocols with stem-cell support.     

Aktuelle Therapiestrategien beim Mantelzelllymphom

Mantle cell lymphoma is a subtype of B?cell lymphoma with a mostly aggressive behavior and poor long-term prognosis. The choice of therapy depends on the age, performance status and risk profile of the patient. Randomized trials have confirmed the superiority of a dose-intensified induction therapy containing cytarabine followed by autologous stem cell transplantation in the first-line treatment of younger patients with a good general condition. Elderly patients benefit from a rituximab maintenance therapy after immunochemotherapy. Novel targeted therapies of the B?cell receptor pathway with the Bruton’s tyrosine kinase inhibitor ibrutinib and the mechanistic target of rapamycin (mTOR) antagonist temsirolimus as well as immunomodulatory drugs (lenalidomide) have shown promising results in relapsed disease. The proteasome inhibitor bortezomib has been approved for first-line treatment in combination with conventional chemotherapy.     

Phase Ib trial of the PI3K/mTOR inhibitor voxtalisib (SAR245409) in combination with chemoimmunotherapy in patients with relapsed or refractory B‐cell malignancies

This phase Ib, dose‐escalation study investigated the maximum tolerated dose (MTD), recommended phase II dose (RP2D), safety, pharmacokinetics (PK) and preliminary efficacy of the pan‐class I phosphoinositide 3‐kinase (PI3K) and mechanistic target of rapamycin (mTOR) inhibitor voxtalisib [30 or 50 mg twice daily (BID)], in combination with rituximab (voxtalisib+rituximab) or rituximab plus bendamustine (voxtalisib+rituximab+bendamustine), in relapsed or refractory indolent B‐cell non‐Hodgkin lymphoma (NHL), mantle cell lymphoma and chronic lymphocytic leukaemia (CLL). MTD and RP2D of voxtalisib were determined using a 3 + 3 dose‐escalation design. Adverse events (AEs), plasma PK and disease response were recorded. Thirty‐seven patients were enrolled. The RP2D of voxtalisib in combination with rituximab or rituximab+bendamustine was 50 mg BID. Four patients experienced a total of five dose‐limiting toxicities. The most frequent AEs were nausea (45·9%), fatigue (37·8%) headache (32·4%) and pyrexia (32·4%). The most frequent grade ≥3 AEs were neutropenia (27·0%), thrombocytopenia (24·3%), anaemia (16·2%) and febrile neutropenia (10·8%). Voxtalisib PK parameters were not affected by co‐administration with rituximab or rituximab+bendamustine. Of 35 efficacy‐evaluable patients, four (11·4%) achieved complete response and 13 (37·1%) achieved partial response. Voxtalisib, in combination with rituximab or rituximab+bendamustine, demonstrated an acceptable safety profile and encouraging anti‐tumour activity in relapsed or refractory B‐cell malignancies.     

MALT1 and the API2-MALT1 fusion act between CD40 and IKK and confer NF-kappa B-dependent proliferative advantage and resistance against FAS-induced cell death in B cells

The most frequently recurring translocations in mucosa-associated lymphoid tissue (MALT) B-cell non-Hodgkin lymphoma, t(11;18)(q21;q21) and t(14;18)(q32; q21), lead to formation of an API2-MALT1 fusion or IgH-mediated MALT1 overexpression. Various approaches have implicated these proteins in nuclear factor kappaB (NF-kappa B) signaling, but this has not been shown experimentally in human B cells. Immunohistochemistry showed that MALT1 is predominantly expressed in normal and malignant germinal center B cells, corresponding to the differentiation stage of MALT lymphoma. We expressed MALT1 and apoptosis inhibitor-2 API2/MALT1 in human B-cell lymphoma BJAB cells and found both transgenes in membrane lipid rafts along with endogenous MALT1 and 2 binding partners involved in NF-kappa B signaling, B-cell lymphoma 10 (BCL10) and CARMA1 (caspase recruitment domain [CARD]-containing membrane-associated guanylate kinase [MAGUK] 1). API2-MALT1 and exogenous MALT1 increased constitutive NF-kappa B activity and enhanced I kappa B kinase (IKK) activation induced by CD40 stimulation. Both transgenes protected BJAB cells from FAS (CD95)-induced death, consistent with increases in NF-kappa B cytoprotective target gene expression, and increased their proliferation rate. Expression of a dominant-negative I kappa B alpha mutant showed that these survival and proliferative advantages are dependent on elevated constitutive NF-kappa B activity. Our findings support a model in which NF-kappa B signaling, once activated in a CD40-dependent immune response, is maintained and enhanced through deregulation of MALT1 or formation of an API2-MALT1 fusion.     

Rituximab antiproliferative effect in B-lymphoma cells is associated with acid-sphingomyelinase activation in raft microdomains

Rituximab is a chimeric human immunoglobulin G1 (IgG1) anti-CD20 monoclonal antibody with significant activity against CD20+ malignant B cells. Rituximab is currently used with success in the treatment of B-cell-derived lymphoid neoplasias either alone or in combination with chemotherapy. However, the predominant mechanism by which rituximab exerts its antitumor properties in vivo remains unknown. In the present study, we demonstrate that in Daudi and RL B-lymphoma cells, rituximab (without cross-linking) used at the saturating dose of 10 microg/mL induced moderate accumulation in G1 phase, growth inhibition, and significant loss in clonogenic potential. However, in these cells, rituximab induced no apoptosis. Furthermore, we observed that treatment with rituximab resulted in a rapid and transient increase in acid-sphingomyelinase (A-SMase) activity and concomitant cellular ceramide (CER) generation in raft microdomains. We also observed that rituximab-treated cells externalized both A-SMase and CER that colocalized with the CD20 receptor. Finally, we present evidence that rituximab-induced growth inhibition may be mediated through a CER-triggered signaling pathway, leading to the induction of cell cycle-dependent kinase inhibitors such as p27Kip1 through a mitogen-activated protein kinase (MAPK)-dependent mechanism.     

The chimeric anti-CD20 antibody rituximab induces apoptosis in B-cell chronic lymphocytic leukemia cells through a p38 mitogen activated protein-kinase-dependent mechanism

Antibodies against CD20 can activate complement and induce antibody-dependent cellular cytotoxicity (ADCC) in B lymphocytes. In B-cell lines, such antibodies also induce apoptosis. In this study, the expression and function of CD20 on B-cell chronic lymphocytic leukemia (B-CLL) cells were analyzed. Flow cytometric analysis demonstrated that B-CLL cells express CD20 with a fluorescence intensity that is significantly weaker than that of normal CD5(+) and CD5(-) B cells and that of malignant CD5(-) low-grade non-Hodgkin lymphoma cells. A small population of cells from healthy donors that have an expression pattern of CD5 and CD20 identical to that of B-CLL cells were identified, and this population was confirmed to be of T lineage, not B lineage. Culture of freshly isolated B-CLL cells in the presence of the chimeric anti-CD20 antibody rituximab and a cross-linking F(ab)(2) fragment, resulted in dose- and time-dependent induction of apoptosis. The induction of apoptosis occurred under conditions in which the influence of complement activation and ADCC was negligible. Cross-linking of rituximab induced strong and sustained phosphorylation of the 3 mitogen activated protein (MAP) kinases c-Jun NH2-terminal protein kinase, extracellular signal-regulated kinase, and p38. Introduction of the p38 inhibitor SB203580 into the system completely blocked signaling downstream of p38, as evidenced by the absence of MAPKAP K2 activity, and significantly reduced the degree of anti-CD20-induced apoptosis. These results demonstrate that cross-linking of rituximab bound to CD20 on freshly isolated B-CLL cells induces apoptosis through a signaling pathway that is dependent on p38 MAP-kinase activation.