sirolimus and Lymphoma--B-Cell

To summarize recent advances that contribute to our understanding of the pathobiology of Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD), the host immune response to virally infected B cells, and the molecular basis for the effects of mammalian target of rapamycin inhibitors on EBV+ B-cell lymphomas.. Cytogenetic and genomic analyses support the concept that the underlying biology of EBV-associated PTLD is complex. Transplant recipients can generate and maintain significant populations of EBV-specific CD8+ memory T cells but the function of these cells may be impaired. EBV invokes multiple strategies to subvert and evade the host immune response. The phosphoinositide-3 kinase/Akt/mammalian target of rapamycin signal transduction pathway is a nexus for growth and survival signals in PTLD-associated EBV+ B-cell lymphomas.. Multiple factors influence the development of EBV-associated PTLD including the host immune response to EBV, virally induced effects on the infected cell and the host immune system, and the type and intensity of immunosuppression.

Topics: Animals; CD8-Positive T-Lymphocytes; Epstein-Barr Virus Infections; Graft Survival; Herpesvirus 4, Human; Humans; Immunologic Memory; Immunosuppressive Agents; Lymphoma, B-Cell; Lymphoproliferative Disorders; Organ Transplantation; Protein Kinases; Signal Transduction; Sirolimus; T-Lymphocytes, Regulatory; TOR Serine-Threonine Kinases

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Bendamustine Hydrochloride; Boronic Acids; Bortezomib; Brentuximab Vedotin; Diphtheria Toxin; Drug Discovery; Everolimus; Humans; Hydroxamic Acids; Immunoconjugates; Indoles; Inotuzumab Ozogamicin; Interleukin-2; Lenalidomide; Lymphoma; Lymphoma, B-Cell; Nitrogen Mustard Compounds; Purine Nucleosides; Pyrazines; Pyrimidinones; Recombinant Fusion Proteins; Sirolimus; Thalidomide; Vorinostat

Topics: Allografts; Animals; B-Lymphocytes; Epstein-Barr Virus Infections; Female; Graft Rejection; Graft Survival; Heart Transplantation; Heterocyclic Compounds, 3-Ring; Humans; Inhibitory Concentration 50; Lymphoma, B-Cell; Lymphoproliferative Disorders; Male; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Organ Transplantation; Phosphoinositide-3 Kinase Inhibitors; Postoperative Complications; Proto-Oncogene Proteins c-akt; Purines; Quinazolinones; Sirolimus; TOR Serine-Threonine Kinases

B cell activating factor from the TNF family (BAFF) stimulates B-cell proliferation and survival, but excessive BAFF promotes the development of aggressive B cells leading to malignant and autoimmune diseases. Recently, we have reported that rapamycin, a macrocyclic lactone, attenuates human soluble BAFF (hsBAFF)-stimulated B-cell proliferation/survival by suppressing mTOR-mediated PP2A-Erk1/2 signaling pathway. Here, we show that the inhibitory effect of rapamycin on hsBAFF-promoted B cell proliferation/survival is also related to blocking hsBAFF-stimulated phosphorylation of Akt, S6K1, and 4E-BP1, as well as expression of survivin in normal and B-lymphoid (Raji and Daudi) cells. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor or rictor enhanced rapamycin's suppression of hsBAFF-induced survivin expression and proliferation/viability in B cells. Also, PP242, an mTORC1/2 kinase inhibitor, repressed survivin expression, and cell proliferation/viability more potently than rapamycin (mTORC1 inhibitor) in B cells in response to hsBAFF. Of interest, ectopic expression of constitutively active Akt (myr-Akt) or constitutively active S6K1 (S6K1-ca), or downregulation of 4E-BP1 conferred resistance to rapamycin's attenuation of hsBAFF-induced survivin expression and B-cell proliferation/viability, whereas overexpression of dominant negative Akt (dn-Akt) or constitutively hypophosphorylated 4E-BP1 (4EBP1-5A), or downregulation of S6K1, or co-treatment with Akt inhibitor potentiated the inhibitory effects of rapamycin. The findings indicate that rapamycin attenuates excessive hsBAFF-induced cell proliferation/survival via blocking mTORC1/2 signaling in normal and neoplastic B-lymphoid cells. Our data underscore that rapamycin may be a potential agent for preventing excessive BAFF-evoked aggressive B-cell malignancies and autoimmune diseases.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; B-Cell Activating Factor; B-Lymphocytes; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Lymphoma, B-Cell; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Phosphoproteins; Phosphorylation; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection

Activated regulatory T cells (Tregs) suppress proliferation and differentiation of normal B cells. In our study, allogeneic polyclonal CD4 (+) CD25 (+) Tregs and CD4 (+) CD25 (+) CD127(lo)Tregs expanded in vitro in the presence of rapamycin and low dose IL-2 suppressed proliferation of 11 out of 12 established lymphoma B-cell lines. The effect of expanded CD4 (+) CD25 (+) Tregs on survival of freshly isolated lymphoma B cells maintained in culture with soluble multimeric CD40L and IL-4 was variable across lymphoma entities. The survival of freshly isolated follicular lymphoma cells usually decreased in cocultures with CD4 (+) CD25 (+) Tregs. Treg effect on chronic lymphocytic leukemia/small lymphocytic lymphoma cells ranged from suppression to help in individual patients. CD4 (+) CD25 (+) Tregs or CD4 (+) CD25 (+) CD127(lo)Tregs expanded ex vivo with rapamycin could be used to suppress regrowth of residual lymphoma after autologous hematopoietic cell transplantation (HCT), and to counteract both graft-versus-host disease and lymphoma re-growth after allogeneic HCT in select patients with lymphoma susceptible to the regulation by Tregs.

Topics: Adult; Aged; Aged, 80 and over; B-Lymphocytes; Biopsy, Fine-Needle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Coculture Techniques; Female; Flow Cytometry; Forkhead Transcription Factors; Humans; Interleukin-2; Leukemia, Lymphocytic, Chronic, B-Cell; Lymph Nodes; Lymphocyte Activation; Lymphoma, B-Cell; Male; Middle Aged; Primary Cell Culture; Sirolimus; T-Lymphocytes, Regulatory

Posttransplant lymphoproliferative disorder (PTLD) continues to be a devastating and potentially life-threatening complication in organ transplant recipients. PTLD is associated with EBV infection and can result in malignant B cell lymphomas. Here we demonstrate that the PI3K/Akt/mTOR pathway is highly activated in EBV+ B cell lymphoma lines derived from patients with PTLD. Treatment with the mTORC1 inhibitor Rapamycin (RAPA) partially inhibited the proliferation of EBV+ B cell lines. Resistance to RAPA treatment correlated with high levels of Akt phosphorylation. An mTORC1/2 inhibitor and a PI3K/mTOR dual inhibitor suppressed Akt phosphorylation and showed a greater anti-proliferative effect on EBV+ B lymphoma lines compared to RAPA. EBV+ B cell lymphoma lines expressed high levels of PI3Kδ. We demonstrate that PI3Kδ is responsible for Akt activation in EBV+ B cell lymphomas, and that selective inhibition of PI3Kδ by either siRNA, or a small molecule inhibitor, augmented the anti-proliferative effect of RAPA on EBV+ B cell lymphomas. These results suggest that PI3Kδ is a novel, potential therapeutic target for the treatment of EBV-associated PTLD and that combined blockade of PI3Kδ and mTOR provides increased efficacy in inhibiting proliferation of EBV+ B cell lymphomas.

Topics: Blotting, Western; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Epstein-Barr Virus Infections; Herpesvirus 4, Human; Humans; Imidazoles; Lymphoma, B-Cell; Lymphoproliferative Disorders; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Organ Transplantation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Postoperative Complications; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Sirolimus; TOR Serine-Threonine Kinases

The application of kinase inhibitors in cancer treatment is growing rapidly. However, methods for monitoring the effectiveness of the inhibitors are still poorly developed and currently rely mainly on the tracking of changes in the tumor volume, a rather late and relatively insensitive marker of the therapeutic response. In contrast, MRS can detect changes in cell metabolism and has the potential to provide early and patient-specific markers of drug activity. Using human B-cell lymphoma models and MRS, we have demonstrated that the inhibition of the mTOR signaling pathway can be detected in malignant cells in vitro and noninvasively in vivo by the measurement of lactate levels. An mTOR inhibitor, rapamycin, suppressed lactic acid production in lymphoma cell line cultures and also diminished steady-state lactate levels in xenotransplants. The inhibition was time dependent and was first detectable 8 h after drug administration in cell cultures. In xenotransplants, 2 days of rapamycin treatment produced significant changes in lactic acid concentration in the tumor measured in vivo, which were followed by tumor growth arrest and tumor volume regression. The rapamycin-induced changes in lactate production were strongly correlated with the inhibition of expression of hexokinase II, the key enzyme in the glycolytic pathway. These studies suggest that MRS or (18) F-fluorodeoxyglucose positron emission tomography (FDG PET) detection of changes in glucose metabolism may represent effective noninvasive methods for the monitoring of mTOR targeting therapy in lymphomas and other malignancies. Furthermore, the measurement of glucose metabolic inhibition by MRS or FDG PET imaging may also prove to be effective in monitoring the efficacy of other kinase inhibitors given that the rapamycin-sensitive mTOR lies downstream of many oncogenic signaling pathways.

Topics: Animals; Down-Regulation; Glycolysis; Lactic Acid; Lymphoma, B-Cell; Magnetic Resonance Spectroscopy; Mice; Mice, SCID; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Adenosine monophosphate-activated protein kinase (AMPK) acts as a major sensor of cellular energy status in cancers and is critically involved in cell sensitivity to anticancer agents. Here, we showed that AMPK was inactivated in lymphoma and related to the upregulation of the mammalian target of rapamycin (mTOR) pathway. AMPK activator metformin potentially inhibited the growth of B- and T-lymphoma cells. Strong antitumor effect was also observed on primary lymphoma cells while sparing normal hematopoiesis ex vivo. Metformin-induced AMPK activation was associated with the inhibition of the mTOR signaling without involving AKT. Moreover, lymphoma cell response to the chemotherapeutic agent doxorubicin and mTOR inhibitor temsirolimus was significantly enhanced when co-treated with metformin. Pharmacologic and molecular knock-down of AMPK attenuated metformin-mediated lymphoma cell growth inhibition and drug sensitization. In vivo, metformin induced AMPK activation, mTOR inhibition and remarkably blocked tumor growth in murine lymphoma xenografts. Of note, metformin was equally effective when given orally. Combined treatment of oral metformin with doxorubicin or temsirolimus triggered lymphoma cell autophagy and functioned more efficiently than either agent alone. Taken together, these data provided first evidence for the growth-inhibitory and drug-sensitizing effect of metformin on lymphoma. Selectively targeting mTOR pathway through AMPK activation may thus represent a promising new strategy to improve treatment of lymphoma patients.

Topics: AMP-Activated Protein Kinase Kinases; Animals; Antineoplastic Agents; Autophagy; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Drug Synergism; Enzyme Activation; Humans; Lymphoma, B-Cell; Lymphoma, T-Cell; Metformin; Mice; Protein Kinase Inhibitors; Protein Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Previous studies have shown that rapamycin can inhibit the growth of several different types of human tumor cells in vitro. In certain cases, it can reverse the phenotype of multidrug resistant (MDR) cells. However, there is limited information concerning its effect on P-glycoprotein (P-gp), a pump that is responsible for chemoresistance in many MDR cells. We investigated the effect of rapamycin on both P-gp function and the MDR phenotype in four cell lines. One cell line was also xenografted into SCID mice to determine whether rapamycin would chemosensitize the cells in vivo. Because rapamycin targets the mammalian target of rapamycin (mTOR) pathway, we also used our cells to confirm that rapamycin modified the expression of mTOR and effectively suppressed the phosphorylation of two downstream effector molecules in the mTOR pathway, S6K1, and 4E-BP1. We demonstrated that it inhibited the growth of the three cell lines in vitro and one in vivo showing that it modulated both the expression and function of P-gp and chemosensitized the three cell lines as effectively as verapamil.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Lymphoma, B-Cell; Mice; Mice, SCID; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases

Posttransplant lymphoproliferative disease (PTLD) is a serious complication of solid organ and bone marrow transplantation and is closely associated with Epstein-Barr virus (EBV) infection. We have previously shown that rapamycin (RAPA) directly inhibits the in vitro and in vivo proliferation of EBV-infected B lymphoblastoid cell lines (SLCL), derived from patients with PTLD, by arresting cells in the G1 phase of the cell cycle. The aim of this study is to elucidate the mechanism by which RAPA causes cell cycle arrest in EBV+ B cells.. SLCL were cultured without or with RAPA (10 ng/ml) and G1-associated cell cycle proteins were analyzed by immunoblot and densitometric analysis. CDK complexes were immunoprecipitated and incubated with retinoblastoma protein (Rb) substrate. Kinase activity of the complex was determined by Western blot with anti-phospho-Rb antibodies.. We show that RAPA decreased both Cyclin D2 and Cyclin D3 protein levels. Furthermore, RAPA decreased the protein levels of cyclin dependent kinase 4 (CDK4) and increased the expression of the CDK inhibitor p27. In contrast, expression of the CDK inhibitor p21 was markedly inhibited by RAPA in the SLCL. Finally, in vitro kinase assays revealed that downstream hyperphosphorylation of Rb by CDK complexes was also decreased by RAPA.. The results presented here elucidate key targets of RAPA-induced cell cycle arrest, provide insight into the growth pathways of EBV+ B-cell lymphomas, and demonstrate the potential for RAPA as a therapeutic option in the treatment of PTLD and other EBV+ lymphomas.

Topics: Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Herpesvirus 4, Human; Humans; Lymphoma, B-Cell; Phosphorylation; Retinoblastoma Protein; Sirolimus

Posttransplant lymphoproliferative disease remains a serious morbidity. Herein we have reported a case of complete regression of a biopsy-proven B-cell lymphoma that occurred in the posttransplant period. A 48-year-old man received a living donor renal transplant for end-stage renal disease due to undetermined etiology. His initial immunosuppression consisted of corticosteroid, mycophenolate mofetil, and cyclosporin. The patient developed severe pneumonia within the first 2 months after transplantation due to Acineotobacter, fungus, and cytomegalovirus infections. He experienced a complete recovery and was discharged for regional follow-up. Four months after discharge, he was referred again because of presence of two nodules on his trunk. A biopsy of the nodules revealed B-cell lymphoma. Cyclosporin was stopped and he was converted to sirolimus. The lesions regressed progressively and completely within 7 weeks. The patient remains well without clinical relapses at 19 months after conversion. Renal functions remained stable. We postulated that the antincoplastic properties of sirolimus may have played an active part in the positive outcome.

Topics: Biopsy; Cyclosporine; Humans; Immunosuppressive Agents; Kidney Transplantation; Lymphoma, B-Cell; Male; Middle Aged; Neoplasm Regression, Spontaneous; Sirolimus; Skin Neoplasms

The inhibition of mTOR is a target for anticancer drugs in posttransplant malignancies. The influence of conversion to sirolimus after malignancy diagnosis was investigated on patient and renal allograft survivals. The 20 renal allograft recipients (4 women, 16 men) of ages 26 to 73 years (mean, 59 years) developed malignancies within 6 to 172 months (mean, 53 months) after transplantation. Three patients developed posttransplant lymphoproliferative disease (PTLD); four, Kaposi sarcoma, three, lung cancer; two, malignant melanoma; two, breast cancer; two, renal cell carcinoma; one, Merkel cell carcinoma; one, cutaneous T-cell lymphoma; one, larynx cancer; and one, gingival cancer. After tumor diagnosis, calcineurin inhibitors, azathioprine, or mycophenolate mofetil (MMF) were discontinued abruptly and sirolimus introduced (2 mg/d; target trough level, 4.0 to 8.0 ng/mL). Prednisone was maintained. The observation time of sirolimus therapy was 4 to 48 months (mean, 14 months). Two patients with PTLD (large B-cell lymphoma) and four with Kaposi sarcoma had full regressions. Eleven patients (larynx cancer, melanoma, breast cancer, T-cell lymphoma, renal cell carcinoma, Merkel cell carcinoma, and skin lymphoma) in addition to sirolimus therapy, underwent oncologic treatment, namely, surgery and/or chemotherapy. Six patients died from disseminated malignancy 4 to 9 months after conversion. One patient with T-cell lymphoma lost his graft; in the remaining patients, serum creatinine level was stable. In conclusion, Conversion to sirolimus resulted in regression of large B-cell lymphoma and Kaposi sarcoma. In patients with advanced or disseminated malignancy, the tumors progressed. Graft function was preserved after conversion to sirolimus.

Topics: Adult; Aged; Antibiotics, Antineoplastic; Female; Humans; Immunosuppressive Agents; Kidney Transplantation; Lymphoma, B-Cell; Lymphoproliferative Disorders; Male; Middle Aged; Neoplasms; Postoperative Complications; Retrospective Studies; Sarcoma, Kaposi; Sirolimus; Transplantation, Homologous

Diffuse large B-cell lymphoma (DLBCL) is a common lymphoma entity. Although a significant amount of DLBCL patients can be cured with modern chemotherapeutic regimens, a substantial proportion of patients die because of progressive disease. Therefore, new therapeutic strategies are clearly needed. Inhibitors of mTOR [mammalian target of rapamycin (Rap)] represent a new class of antiproliferative drugs with applications as immunosuppressive and anticancer agents. Extensive safety data exist on the mTOR inhibitor RAD001, which is already approved as an immunosuppressant in organ transplant recipients. Rap and RAD001 inhibited cell cycle progression in DLBCL cells by inducing a G1 arrest without inducing apoptosis. Phosphorylation of the main targets of mTOR, p70 s6 kinase and 4-EBP-1 was reduced in cells cultured in the presence of RAD001. Cell cycle arrest was accompanied by reduced phosphorylation of the retinoblastoma protein (RB) as well as reduced expression of cyclin D3 and A in all cell lines. Although the effect of the chemotherapeutic agent vincristine (vin) was not enhanced by RAD001, rituximab-induced cytotoxicity was augmented in the rituximab-sensitive cell lines. mTOR inhibition is a promising therapeutic strategy in DLBCL by inducing a G1 arrest and augments rituximab-induced cytotoxicity. Therefore, combination of these drugs might be an interesting new therapeutic approach in DLBCL patients.

Topics: Adaptor Proteins, Signal Transducing; Annexin A5; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Electrophoretic Mobility Shift Assay; Everolimus; Flow Cytometry; G1 Phase; Humans; Immunosuppressive Agents; In Situ Nick-End Labeling; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Non-Hodgkin; Oncogene Protein v-akt; Phosphoproteins; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; PTEN Phosphohydrolase; Retinoblastoma Protein; Ribosomal Protein S6 Kinases, 70-kDa; Rituximab; Sirolimus; TOR Serine-Threonine Kinases

Posttransplant lymphoproliferative disorders are a group of lymphoid proliferations and lymphomas that develop as a consequence of immunosuppression in recipients of solid organ or bone marrow allografts. We describe an unusual oral presentation of posttransplant Epstein-Barr virus-associated diffuse large B-cell lymphoma in a 45-year-old woman after pancreatic transplant.

Topics: Diagnosis, Differential; Epstein-Barr Virus Infections; Female; Herpesvirus 4, Human; Humans; Immunosuppressive Agents; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Middle Aged; Mouth Neoplasms; Oral Ulcer; Pancreas Transplantation; RNA, Viral; Sirolimus; Tongue Diseases

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Survival; Drug Resistance, Neoplasm; Eukaryotic Initiation Factor-4E; Lymphoma, B-Cell; Mice; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Evading apoptosis is considered to be a hallmark of cancer, because mutations in apoptotic regulators invariably accompany tumorigenesis. Many chemotherapeutic agents induce apoptosis, and so disruption of apoptosis during tumour evolution can promote drug resistance. For example, Akt is an apoptotic regulator that is activated in many cancers and may promote drug resistance in vitro. Nevertheless, how Akt disables apoptosis and its contribution to clinical drug resistance are unclear. Using a murine lymphoma model, we show that Akt promotes tumorigenesis and drug resistance by disrupting apoptosis, and that disruption of Akt signalling using the mTOR inhibitor rapamycin reverses chemoresistance in lymphomas expressing Akt, but not in those with other apoptotic defects. eIF4E, a translational regulator that acts downstream of Akt and mTOR, recapitulates Akt's action in tumorigenesis and drug resistance, but is unable to confer sensitivity to rapamycin and chemotherapy. These results establish Akt signalling through mTOR and eIF4E as an important mechanism of oncogenesis and drug resistance in vivo, and reveal how targeting apoptotic programmes can restore drug sensitivity in a genotype-dependent manner.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Cell Division; Cell Survival; Disease Progression; Drug Resistance, Neoplasm; Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factor-4G; Female; Lymphoma, B-Cell; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53

EBV-infected B-cell lymphomas are a potentially life-threatening complication in bone marrow and solid organ transplant recipients. Immunosuppressive drugs required to prevent allograft rejection also impair anti-EBV T-cell immunity, thereby increasing the risk of EBV-associated disease. Here we demonstrate that the immunosuppressant rapamycin (RAPA) has a strong antiproliferative effect in vitro on B-cell lines derived from organ transplant recipients with EBV-associated posttransplant lymphoproliferative disorder (PTLD). Furthermore, RAPA significantly inhibits or delays the growth of solid tumors established from EBV-infected B-cell lines in a xenogeneic mouse model of PTLD. RAPA acts via cell cycle arrest, induction of apoptosis, and, most importantly, inhibition of interleukin 10 secretion, a necessary autocrine growth factor. The reduced interleukin 10 production is accompanied by corresponding decreases in the constitutive activation of the growth-promoting transcription factors signal transducer and activator of transcription 1 and 3. Thus, RAPA can limit B-cell lymphoma growth while simultaneously providing immunosuppression to prevent graft rejection in patients who are otherwise at risk for EBV-associated PTLD. Moreover, these findings may have application to other EBV-associated malignancies.

Topics: Animals; Antibiotics, Antineoplastic; Cell Division; DNA-Binding Proteins; Enzyme Activation; Epstein-Barr Virus Infections; Herpesvirus 4, Human; Humans; Immunosuppressive Agents; Interleukin-10; Janus Kinase 1; Lymphoma, B-Cell; Male; Mice; Protein-Tyrosine Kinases; Signal Transduction; Sirolimus; STAT3 Transcription Factor; Trans-Activators; Transplantation; Xenograft Model Antitumor Assays

RAFT1/FRAP/mTOR is a key regulator of cell growth and division and the mammalian target of rapamycin, an immunosuppressive and anticancer drug. Rapamycin deprivation and nutrient deprivation have similar effects on the activity of S6 kinase 1 (S6K1) and 4E-BP1, two downstream effectors of RAFT1, but the relationship between nutrient- and rapamycin-sensitive pathways is unknown. Using transcriptional profiling, we show that, in human BJAB B-lymphoma cells and murine CTLL-2 T lymphocytes, rapamycin treatment affects the expression of many genes involved in nutrient and protein metabolism. The rapamycin-induced transcriptional profile is distinct from those induced by glucose, glutamine, or leucine deprivation but is most similar to that induced by amino acid deprivation. In particular, rapamycin treatment and amino acid deprivation up-regulate genes involved in nutrient catabolism and energy production and down-regulate genes participating in lipid and nucleotide synthesis and in protein synthesis, turnover, and folding. Surprisingly, however, rapamycin had effects opposite from those of amino acid starvation on the expression of a large group of genes involved in the synthesis, transport, and use of amino acids. Supported by measurements of nutrient use, the data suggest that RAFT1 is an energy and nutrient sensor and that rapamycin mimics a signal generated by the starvation of amino acids but that the signal is unlikely to be the absence of amino acids themselves. These observations underscore the importance of metabolism in controlling lymphocyte proliferation and offer a novel explanation for immunosuppression by rapamycin.

Topics: Amino Acids; Animals; B-Lymphocytes; Carrier Proteins; Cell Line; Down-Regulation; Gene Expression; Gene Expression Profiling; Glucose; Humans; Immunosuppressive Agents; Lymphoma, B-Cell; Mice; Phosphotransferases (Alcohol Group Acceptor); Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Starvation; T-Lymphocytes; TOR Serine-Threonine Kinases; Up-Regulation

The immunosuppressant rapamycin, an immunophilin-binding antibiotic, has been studied in follicular B-cell lymphoma lines that express the highest level of the BCL-2 protein. The growth rate of human follicular B-cell lymphoma lines was slowed more efficiently than that of other human B-cell lines or non-B-cell lines. This effect was dependent on the arrest of cells in the G(1) phase; the number of apoptotic cells was not increased. Rapamycin inhibited apoptosis or caspase activation induced by cytotoxic drugs, whereas caspase activation by doxorubicin was not inhibited. The increase in the cellular concentration of BCL-2 protein was related to its concentration in the steady state and was unrelated to the amount of bcl-2 mRNA. The increase of BCL-2 level in the cells rather than its level in the steady state may be important for drug resistance. The biochemical target of rapamycin, the mTOR kinase, may be a candidate sensitising agent for chemotherapy. This effect of rapamycin shows that G(1) arrest and protection from apoptosis are combined events susceptible to regulation by pharmacological means.

Topics: Antibiotics, Antineoplastic; Apoptosis; Caspases; Cell Cycle; Cell Division; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Immunosuppressive Agents; Lymphoma, B-Cell; Neoplasm Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; RNA, Neoplasm; Sirolimus; Tumor Cells, Cultured

Extravasation is a critical process for the physiological lymphocyte traffic as well as the hematogenous spread of malignant hemopoietic cells. Here we report that abrogation of calcineurin activity leads to in vitro transendothelial migration and in vivo infiltration of human lymphoma Nalm-6 cells, which are associated with the abrogation of the VLA-4/VCAM-1 mediated pathway. Rapamycin, which can antagonize FK506 but not CsA to inhibit calcineurin, abrogates FK-506 mediated but not CsA mediated inhibition of in vitro transendothelial migration. FK506 may exert its potent immunosuppressive action partly by inhibiting VLA-4/VCAM-1 mediated transendothelial migration or insinuation of lymphoid cells to tissues.

Topics: Animals; B-Lymphocytes; Bone Marrow Cells; Calcineurin; Cell Adhesion; Cell Movement; Central Nervous System; Endothelium; Humans; Immunosuppressive Agents; Integrin alpha4beta1; Integrins; Lymphoma, B-Cell; Mice; Mice, SCID; Polyenes; Receptors, Lymphocyte Homing; Sirolimus; Spleen; Tacrolimus; Tumor Cells, Cultured; Vascular Cell Adhesion Molecule-1

Rapamycin, a potent immunosuppressive drug that prevents rejection of organ transplants in many animals, caused profound growth inhibition in an immature B cell lymphoma, BKS-2, at very low concentrations (2 ng/ml). Similar growth inhibition was also observed in a series of B cell lymphomas (i.e., L1.2, NFS.1.1, and WEHI-279) as well as in thymoma cells. The cell death induced by rapamycin in BKS-2 lymphoma was found to be via programmed cell death, or apoptosis. In contrast to rapamycin, neither FK506 nor CsA affected the normal growth of these cells. FK506, but not CsA antagonized the effect of rapamycin and rescued the BKS-2 cells from undergoing apoptosis. Further, suboptimal concentrations of anti-IgM antibodies and rapamycin acted synergistically in causing the growth inhibition of BKS-2 cells and this inhibitory effect was also completely reversed by FK506. Thus, rapamycin appeared to inhibit lymphoma growth by binding to FK506 binding protein. These results indicate that rapamycin should be evaluated as an effective immunosuppressive therapeutic agent to prevent the incidence of lymphoma after transplantations.

Topics: Animals; Antibiotics, Antineoplastic; Antibodies, Anti-Idiotypic; Apoptosis; B-Lymphocytes; Cell Division; Drug Screening Assays, Antitumor; Immunosuppressive Agents; Lymphoma, B-Cell; Mice; Mice, Inbred CBA; Polyenes; Sirolimus; Tacrolimus; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured

The WEHI-231 B lymphoma cell line expresses the phenotype of immature B cells. Cross-linking of surface IgM induces programmed cell death (PCD) with typical features of apoptosis demonstrated by the decrease of cell DNA content, chromatin condensation, and nuclear fragmentation. Activation of protein kinase C (PKC) by phorbol esters was reported to protect WEHI-231 cells against apoptosis induced by ligation of antigen receptors. It was therefore hypothesized that PCD could result from a defect in PKC response with an imbalance in the phosphoinositide pathway in favor of Ca2+ mobilization. In support of this hypothesis, we show here that apoptosis can be readily triggered by the calcium ionophore ionomycin. Furthermore, pretreatment of cells with cyclosporin A or FK506 which inhibit selectively the phosphoprotein calcineurin, a calcium-and calmodulin-dependent serine/threonine phosphatase, protects WEHI-231 cells against apoptosis induced by ionomycin or ligation of surface IgM. Unlike phorbol esters, cyclosporin A did not impair the rise of intracellular Ca2+ induced by cross-linking of antigen receptors. Altogether, the data indicate that the phosphorylation status of yet undefined key cellular substrates controls the cellular response to calcium-dependent apoptotic signals in this B cell lymphoma.

Topics: Animals; Apoptosis; Calcium; Cross-Linking Reagents; Cyclosporine; Immunoglobulin M; Immunosuppressive Agents; Ionomycin; Lymphoma, B-Cell; Mice; Polyenes; Receptors, Antigen, B-Cell; Signal Transduction; Sirolimus; Tacrolimus; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured