scholarly journals Generation of continuous large granular lymphocyte lines by interleukin 2 from the spleen cells of mice infected with Moloney leukemia virus. Involvement of interleukin 3.

1987 ◽  
Vol 166 (4) ◽  
pp. 833-849 ◽  
Author(s):  
M Hattori ◽  
T Sudo ◽  
M Iizuka ◽  
S Kobayashi ◽  
S Nishio ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Cdna Probe ◽  
Interleukin 2 ◽  
Cell Lineage ◽  
Constitutive Expression ◽  
Morphological Characteristics ◽  
Spleen Cells

Continuous cell lines could be reproducibly established by culturing spleen cells from adult mice injected with MLV-producer cells or directly infected with Mo-MLV with rIL-2, whereas the culture of normal splenic cells with rIL-2 induced only transient and limited proliferation resulting in no such lines. All of the lines showed morphological characteristics as LGL with Thy-1+,Lyt-1-,L3T4-,Lyt-2-,AsGM1+,FcR gamma+ phenotype without exception, and most of them exhibited typical NK-patterned cytotoxicity. Analysis of reverse transcriptase activity of the culture supernatants as well as Southern hybridization of the DNA from the lines using an Mo-MLV-specific cDNA probe indicated no evidence of retroviral replication or proviral integration, suggesting that the generation of cell lines reflected a reactive process and viral infection was not directly responsible. It was subsequently revealed that Thy-1+,Lyt-1+,Lyt-2- spleen cells from mice infected with Mo-MLV in vivo spontaneously produced surprising amounts of IL-3 in vitro, leading to the possibility that IL-3 was responsible for the generation of lines. The possibility was directly supported by the observation that continuous lines with identical characteristics could be generated completely in vitro by sequential stimulation with rIL-3 and rIL-2 from normal spleen cells without any involvement of Mo-MLV. The C beta gene of TCR was shown to be rearranged in all the lines examined, indicating the LGL lines were all genetically committed to T cell lineage. Unlike the situation in normal splenic populations expanded by rIL-2, where the expression of IL-2-R was progressively lost, constitutive expression of high-affinity-IL-2-R was observed in all the lines and thus, this was considered to explain the unlimited proliferation of them in response to rIL-2 alone. These results suggested the probable role of IL-3 in the regulation of growth and differentiation of a set of LGL committed to T cell lineage. The possible implications of the phenomenon in the regulation of hematopoiesis as well as in the control of Mo-MLV-induced leukemogenesis were discussed.

Trial of zoledronic acid and interleukin-2 to expand tumoricidal γδ T cells in vivo in patients with refractory neuroblastoma.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. TPS10073-TPS10073
Author(s):  
Joseph Gerald Pressey ◽  
Julia M Adams ◽  
David R Kelly ◽  
Lawrence S Lamb
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Zoledronic Acid ◽  
Cell Lines ◽  
Interleukin 2 ◽  
Γδ T Cells ◽  
Effector Memory

TPS10073 Background: CD3+ γδ+ T cells comprise 2 to 5% of the circulating T cells. Vγ9Vδ2+ cells are the dominant circulating γδ T cell and recognize non-peptide phosphoantigens and stress-associated NKG2D ligands expressed on malignant cells. Strategies that incorporate the tumoricidal properties of γδ T cells represent a promising means of harnessing the innate immune system to treat malignancies including neuroblastoma (NB). Indeed, γδ T cells from both healthy volunteers and NB patients exert a potent cytotoxic effect on NB cell lines and autologous NB in vitro following expansion and activation in culture. Vγ9Vδ2+ cells can also be induced to proliferate in vivo. By blocking farnesyl pyrophosphate synthase in the mevalonate pathway of isoprenoid synthesis in monocytes, the aminobisphosphonate zoledronic acid (ZOL) promotes the accumulation of isopentenyl pyrophosphate which is sensed by γδ T cells. IL-2 is also required for robust expansion γδ T cells. Methods: The trial is a prospective, non-randomized trial that assesses two dose levels of recombinant IL-2 (aldesleukin) in combination with ZOL. To be eligible for the study patients must be 2 to 21 years of age with refractory neuroblastoma with no known curative therapeutic options. Patients must also have adequate organ function and performance status. ZOL is given intravenously on day 1, and aldesleukin is given subcutaneously on days 1 to 5 and 15 to 19 of every 28 day cycle. The single-institution study is being conducted at the University of Alabama at Birmingham. Correlative studies include evaluating the absolute count, phenotype, activation, and effector/memory progression of γδ T cells by flow cytometry and the biological function of autologous expanded/activated γδ T cells by in vitro cytotoxicity assays employing established human NB cell lines. In order to determine the ability of in vivo expanded/activated γδ T cells to infiltrate NB tissue, bone marrow core biopsies obtained before and after the first course of therapy from patients with bone marrow metastasis are assayed for T cell infiltration by immunohistochemistry. The study has currently enrolled 1 of the planned 6 patients. Clinical trial information: NCT01404702.

scholarly journals Alterations of p53 and c-myc in the clonal evolution of malignant lymphoma

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 452-459 ◽  
Author(s):  
H Chang ◽  
S Benchimol ◽  
MD Minden ◽  
HA Messner
Keyword(s):  
Growth Factors ◽  
T Cell ◽  
Cell Lines ◽  
P53 Protein ◽  
Clonal Evolution ◽  
Cdna Probe ◽  
Cell Phenotype ◽  
Coding Region ◽  
P53 Genes

Abstract We derived the lymphoma cell lines OCI-Ly 13.1 and OCI-Ly 13.2 from a patient with non-Hodgkin's lymphoma at the time of presentation and during chemotherapy-resistant relapse. These lines were of T-cell phenotype and contained the identical T-cell receptor beta-chain rearrangement, indicating that both lines were members of the same malignant clone. The lines differed in their growth characteristics; OCI-Ly 13.1 grew slowly and required growth factors for colony formation, whereas OCI-Ly 13.2 grew rapidly and formed colonies without addition of growth factors. To test whether or not these biologic differences were associated with specific genetic changes, we evaluated the status of the c-myc and p53 genes of both cell lines. The p53 and c- myc genes of OCI-Ly 13.1 were in germline configuration and produced normal-sized transcripts. The p53 protein expressed in OCI-Ly 13.1 was recognized by the anti-p53 monoclonal antibody, PAb240, indicating a conformation typical of p53 proteins expressed by p53 alleles containing a missense mutation. However, sequencing studies of the entire p53 coding region did not reveal any point mutations. In contrast, the cell line OCI-Ly 13.2 contained structural abnormalities of both the c-myc and p53 genes. In addition, one of the p53 alleles was lost as determined by a cDNA probe for the p53 gene (17p 13.1) and the YNZ22.1 probe (17p 13.3). These changes resulted in the absence of p53 protein and mRNA in OCI-Ly 13.2 as detected by immunoprecipitation and Northern blot analysis, respectively. They may be a reflection of disease progression and may be associated with the altered behavior of the malignant cell population within the patient and in vitro.

scholarly journals MATURATION OF THE HUMORAL IMMUNE RESPONSE IN MICE

1974 ◽  
Vol 139 (2) ◽  
pp. 249-263 ◽  
Author(s):  
Patricia G. Spear ◽  
Gerald M. Edelman
Keyword(s):  
T Cell ◽  
B Cells ◽  
Cell Function ◽  
Spleen Cells ◽  
Con A ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Humoral Antibodies

In spite of the prenatal appearance of immunoglobulin-bearing lymphocytes and θ-positive lymphocytes in the spleens of Swiss-L mice, these mice are not able to produce detectable levels of humoral antibodies in response to antigen until after 1 wk of age. Adult levels of response are not achieved until 4–8 wk of age. In the presence of bacterial lipopolysaccharides, which can substitute for or enhance T-cell function, the B cells from young Swiss-L mice were found to be indistinguishable in function from adult B cells, both with respect to the numbers of plaque-forming cells (PFC) produced in vitro in response to antigen and with respect to the kinetics of PFC induction. The spleen cells from young Swiss-L mice are significantly less sensitive than adult spleen cells, however, to stimulation by the T cell mitogens, concanavalin A (Con A) and phytohemagglutinin (PHA). Very few Con A-responsive cells could be detected at birth but the numbers increased sharply with age until 3 wk after birth. On the other hand, PHA-responsive cells could not be detected in the spleen until about 3 wk of age. The latter cells were found to respond also to Con A, but at a lower dose (1 µg/ml) than that required for the bulk of the Con A-responsive cells (3 µg/ml). The cells that respond both to PHA and to Con A appear in the spleen at about the time that Swiss-L mice acquire the ability to produce humoral antibodies, and these cells can be depleted from the spleen by the in vivo administration of antithymocyte serum. The development of humoral immune responses in these mice therefore appears to be correlated with the appearance of recirculating T lymphocytes that are responsive both to PHA and to Con A.

scholarly journals Stable Transduction of the Interleukin-2 Gene Into Human Natural Killer Cell Lines and Their Phenotypic and Functional Characterization In Vitro and In Vivo

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3850-3861 ◽  
Author(s):  
Shigeki Nagashima ◽  
Robbie Mailliard ◽  
Yoshiro Kashii ◽  
Torsten E. Reichert ◽  
Ronald B. Herberman ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Lines ◽  
Killer Cell ◽  
Interleukin 2 ◽  
Human Natural Killer Cell ◽  
Antitumor Effects ◽  
Packaging Cell Line

Abstract A variety of strategies have been attempted in the past to stably transduce natural killer (NK) cells with cytokine or other cellular genes. Here, we demonstrate the successful delivery of the interleukin-2 (IL-2) gene into two human NK cell lines, IL-2–dependent NK-92 and IL-2–independent YT, by retroviral transduction. An MuLV-based retroviral vector expressing human IL-2 andneor markers from a polycistronic message was constructed and transduced into a CRIP packaging cell line. By coincubation of NK cells with monolayers of CRIP cells or by using retrovirus-containing supernatants in a flow-through method, 10% to 20% of NK cells were stably transduced. Upon selection in the presence of increasing G418 concentrations, transduced NK cells were able to proliferate independently of IL-2 for more than 5 months and to secrete up to 5.5 ng/106 cells/24 h of IL-2. IL-2 gene-transduced NK-92 cells had an in vitro cytotoxicity against tumor targets that was significantly higher than that of parental cells and secreted interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) in addition to IL-2. Moreover, the in vivo antitumor activity of IL-2 gene-transduced NK-92 cells against established 3-day liver metastases in mice was greater than that of parental nontransduced NK cells. Stable expression of the IL-2 transgene in NK cells improved their therapeutic potential in tumor-bearing hosts. Thus, transduced NK cells secreted sufficient quantities of bioactive IL-2 to proliferate in vitro and mediated the antitumor effects both in vitro and in vivo in the absence of exogenous IL-2. These results suggest that genetic modification of NK cells ex vivo could be useful for clinical cancer therapy in the future.

Differential Effects of Milatuzumab On Human Antigen-Presenting Cells in Comparison to Malignant B Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2744-2744
Author(s):  
Xiaochuan Chen ◽  
Rhona Stein ◽  
Chien-Hsing Chang ◽  
David M. Goldenberg
Keyword(s):  
T Cell ◽  
B Cells ◽  
Cell Lines ◽  
Hairy Cell ◽  
Employment Equity ◽  
Cross Presentation ◽  
Equity Ownership ◽  
Antigen Presenting

Abstract Abstract 2744 Poster Board II-720 Introduction: The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab, is in clinical evaluation as a therapeutic mAb for non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and multiple myeloma after preclinical evidence of activity in these tumor types. In addition to its expression in malignant cells, CD74 is also expressed in normal B cells, monocytes, macrophages, Langerhans cells, follicular and blood dendritic cells. A question therefore arises whether milatuzumab is toxic to or affects the function of these immune cells. This has important implications, not only for safe therapeutic use of this mAb, but also for its potential application as a novel delivery modality for in-vivo targeted vaccination. Methods: We assessed the binding profiles and functional effects of milatuzumab on human antigen-presenting cell (APC) subsets. Studies on the effect of milatuzumab on antigen presentation and cross-presentation are included. In addition, binding and cytotoxicity on a panel of leukemia/lymphoma cell lines and CLL patient cells were tested to demonstrate the range of malignancies that can be treated with this mAb. Results: Milatuzumab bound efficiently to different subsets of blood dendritic cells, including BDCA-1+ myeloid DCs (MDC1), BDCA-2+ plasmacytoid DCs (PDC), BDCA-3+ myeloid DCs (MDC2), B lymphocytes, monocytes, and immature DCs derived from human monocytes in vitro, but not LPS-matured DCs, which correlated well with their CD74 expression levels. In the malignant B-cells tested, milatuzumab bound to the surface of 2/3 AML, 2/2 mantle cell (MCL), 4/4 ALL, 1/1 hairy cell leukemia, 2/2 CLL, 7/7 NHL, and 5/6 multiple myeloma cell lines, and cells of 4/6 CLL patient specimens. Significant cytotoxicity (P<0.05) was observed in 2/2 MCL, 2/2 CLL, 3/4 ALL, 1/1 hairy cell, 2/2 NHL, and 2/2 MM cell lines, and 3/4 CD74-positive CLL patient cells, but not in the AML cell lines following incubation with milatuzumab. In contrast, milatuzumab had minimal effects on the viability of DCs or B cells that normally express CD74. The DC maturation and DC-mediated T-cell functions were not altered by milatuzumab treatment, which include DC-induced T-cell proliferation, CD4+CD25+FoxP3+ Treg expansion, and CD4+ naïve T-cell polarization. Moreover, milatuzumab had little effect on CMV-specific CD8- and CD8+ T cell interferon-g responses of peripheral blood mononuclear cells stimulated in vitro with CMV pp65 peptides or protein, suggesting that milatuzumab does not influence antigen presentation or cross-presentation. Conclusion: These results demonstrate that milatuzumab is a highly specific therapeutic mAb against B-cell malignancies with potentially minimal side effects. It also suggests that milatuzumab may be a promising novel delivery mAb for in vivo targeted vaccinations, given its efficient binding, but lack of cytotoxicity and functional disruption on CD74-expressing normal APCs. (Supported in part by NIH grant PO1-CA103985.) Disclosures: Chang: Immunomedics Inc.: Employment, Equity Ownership, Patents & Royalties. Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

The Combination of Histone Deacetylase Inhibitors and Hypomethylating Agents Exhibits Marked Synergy In Preclinical Models of T-Cell Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3937-3937 ◽  
Author(s):  
Enrica Marchi ◽  
Danielle C Bongero ◽  
Matko Kalac ◽  
Luigi Scotto ◽  
Owen A. O'Connor
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Histone Deacetylase Inhibitors ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Hypomethylating Agents ◽  
Cytotoxicity Assays ◽  
Nanomolar Range

Abstract Abstract 3937 CHOP and CHOP-like chemotherapy programs remain the most commonly used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite often sub-optimal results. Histone deacetylase inhibitors (HDACIs) are epigenetic agents known to be active in T-cell lymphoma. Recently romidepsin (R) was approved for patients with relapsed or refractory CTCL. Both R and belinostat (B) are being investigated in patients with relapsed or refractory PTCL. We have previously shown that hypomethylating agents as decitabine (D) produce synergistic interactions with HDACIs in B-cell lymphomas. We investigated the in vitro and in vivo activity of D, R and B alone or in combination in different T-cell lymphoma and leukemia cell lines including CTCL (H9, HH), and T- acute lymphoblastic leukemia (T-ALL) lines resistant to gamma-secretase inhibitors (GSI) (P12, PF-382). For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-Glo™ followed by acquisition on a Biotek Synergy HT. The IC50s for D, B and R were calculated using the Calcusyn software (Biosoft). Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR) based on the GraphPad software (RRR<1 are defining synergism). Apoptosis was assessed by staining with Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition. Whole cell lysate proteins were extracted and quantified according to Bradford assay. After electrophoresis on a gradient 4–20% SDS-PAGE gels the proteins were transferred to nitrocellulose membrane. After blocking and incubation with the primary and the secondary antibodies, the chemiluminescent agent was added and the x-ray films were exposed to the membranes. The IC50s for belinostat alone at 24, 48 and 72 hours were generally in the nanomolar range: H9: 108.1nM – 35.7nM – 29.1nM; HH: 240.1nM - 67.6nM – 39.01nM; P12: 386.9nM – 99.9nM – 99.8nM; PF 382: 267.1nM – 135nM – 118.3nM. The IC50s for romidepsin alone at 24, 48 and 72 hours were generally in the low nanomolar range: H9: 5nM – 2.1nM – 2.2nM; HH: 14nM – 2.6nM - 2.5nM; P12: 6.2nM – 2.4nM – 2.1nM; PF382: 6.1nM – 1.7nM – 1.5nM. The IC50s for D alone at 72 and 96 hours were in the micromolar range: H9: 7.4uM – 3.7uM; HH: > 20 uM. In the cytotoxicity assays, the combination of D and B or R at 72 hours showed synergism in all the cell lines studied. The most representative RRRs are showed in table 1. Table 1 D 0.5 uM 1uM B (nM) RRR H9 50 0.7 0.7 70 0.6 0.6 100 0.4 0.5 PF 382 150 0.8 0.7 0.5 uM 1 uM R (nM) RRR H9 0.5 0.9 0.9 1 0.8 0.8 2 0.3 0.3 PF 382 1 0.8 0.7 1.5 0.4 0.4 2 0.1 0.1 When H9, HH, P12 and PF382 cell lines were treated with D and B or R for 72 hours, all the combination groups showed significantly more apoptosis than the single drug exposures and controls. Table 2 displays the range of apoptosis induction for B, R and D or for them used in combination and the RRR value after the analysis for the most significant data. Table 2 B D B + D RRR (% Apoptotic + Dead Cells) H9 100nM (22.9%) 500nM (17.9%) 51.5% 0.7 HH 100nM (42.9%) 1uM (46.9%) 61.3% 0.8 P 12 150nM (16%) 1uM (42.7%) 80.1% 0.4 PF 382 100nM (8.3%) 1uM (27.9%) 40.1% 0.8 R D R + D H9 2nM (22.2%) 500nM (17.9%) 63.6% 0.5 HH 2nM (80%) 1uM (46.9%) 89.7% 0.6 P 12 2nM (9.9%) 10uM (58.7%) 98% 0.03 PF 382 2nM (54.5%) 500nM (17.9%) 88.7% 0.2 Increased acetylation of H3 was observed when H9 cells were treated with R alone and synergistically increased after exposing cells to the combination of D + B and D + R. The expression of phosphorylated Stat3 was decreased after exposure of H9 cells to the combination of D and R. Additional interrogation of the effects of this epigenetic therapy on the JAK-STAT signaling pathway are now underway. An in vivo xenograft study in six to eight weeks old female SCID beige mice injected subcutaneously with 2 × 107 HH cells has also begun and will be reported. Mice were separated into different cohorts and treated with intraperitoneal injections of D or B or their combination according to the following schedules: D alone at 1.5 mg/kg on days 1, 5; B alone at 35 mg/Kg/day for 7 days. Collectively, the data suggest that the combination of a hypomethylating agent like D and a HDACI (B and R) are synergistic in in vitro models of human T-cell lymphoma, and may lead to a new platform for the treatment of these diseases. Disclosures: O'Connor: Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

MHC Class II and T Cell Receptor Signals Are Dispensable for IL-2-Induced Regulatory T Cell Proliferation In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2181-2181
Author(s):  
Tao Zou ◽  
Atsushi Satake ◽  
Jonathan Maltzman ◽  
Taku Kambayashi
Keyword(s):  
T Cell ◽  
Conflicts Of Interest ◽  
Interleukin 2 ◽  
Adaptor Protein ◽  
Class Ii ◽  
Sh2 Domain ◽  
Tcr Signaling ◽  
Mhc Ii

Abstract Abstract 2181 Regulatory T cells (Tregs) protect the host from autoimmunity and inappropriate immune activation. Thus, to ensure immune tolerance in the steady state, an adequate number of peripheral Tregs must be constantly maintained. Prior work has suggested that major histocompatibility class II (MHC II) and interleukin-2 (IL-2) are both necessary to maintain peripheral Treg homeostasis and proliferation in vivo. However, we have recently reported that Treg proliferation may not strictly depend on MHC II, as the provision of IL-2 was sufficient to drive proliferation of Tregs in an MHC II-independent manner in vitro, as long as the Tregs interacted with dendritic cells (DC)s. Here, extending our previous in vitro observations, we tested the dependence of Treg proliferation on IL-2, DCs, and TCR signaling in vivo. Proliferation of adoptively transferred Tregs was detected in wildtype (WT) mice. This proliferation was markedly enhanced when the mice were injected with IL-2 immune complexes (IC)s but not when the IL-2 IC-injected mice lacked DCs, suggesting that IL-2-induced Treg proliferation was dependent on DCs in vivo. As previously reported, adoptively transferred Tregs did not proliferate in MHC II-deficient hosts. However, the injection of IL-2 ICs into these mice induced Treg proliferation comparable to those transferred into IL-2 IC-injected WT mice, suggesting that IL-2 signaling by Tregs obviated the need of MHC II for their proliferation. Furthermore, while the ablation of TCR signaling by conditional deletion of the adaptor protein SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) rendered Tregs unable to proliferate by themselves, IL-2 IC treatment partially rescued this deficiency. We next examined the signaling pathways involved in Treg proliferation downstream of the IL-2 receptor. Despite the importance of the Stat5 pathway in IL-2 receptor signaling during Treg development in the thymus, activation of Stat5b alone was insufficient to rescue proliferation of SLP-76-deficient Tregs, indicating that alternative pathways must also be activated for Treg proliferation. Additional studies investigating the role of other signaling molecules downstream of the IL-2 receptor are currently underway. In summary, we have demonstrated for the first time that Tregs do not require TCR signaling through interaction with MHC II for their proliferation in vivo. We propose that this MHC II-independent mode of Treg proliferation allows Tregs with multiple antigen specificities to proliferate, which ensures that a diverse TCR repertoire is continuously maintained in the Treg pool. Furthermore, we believe that exploitation of these pathways may be therapeutically beneficial in autoimmunity and in transplantation. Disclosures: No relevant conflicts of interest to declare.

Constitutive and Immunoproteasome Inhibitors Increase IκB and Decrease NF-κB Expression In Dendritic Cell

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3493-3493
Author(s):  
Ahmad-Samer Samer Al-Homsi ◽  
Zhongbin Lai ◽  
Tara Sabrina Roy ◽  
Niholas Kouttab
Keyword(s):  
Multiple Myeloma ◽  
T Cell ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Peripheral Blood ◽  
Research Funding ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell

Abstract Introduction Constitutive and immunoproteasome inhibitors (C&IPI) were thought to suppress nuclear factor-κB (NF-κB) pathway by preventing IκB degradation, which prevents NF-κB translocation into the nucleus. This mechanism of action has since been questioned by a number of studies. First, bortezomib promoted constitutive NF-κB activity in endothelial cell carcinoma. Second, NF-κB constitutive activity was resistant to bortezomib in multiple myeloma cell lines. Third, bortezomib increased IκB mRNA but post-transcriptionally downregulated IκB in normal cells and in multiple myeloma cell lines resulting in induced canonical NF-κB activation. Lastly, bortezomib increased nuclear levels of IκB as opposed to lowering cytoplasmic levels in cutaneous T cell lymphoma cell line suggesting that nuclear translocation of IκB was possibly responsible for NF-κB inhibition. The inhibitory activity of C&IPI on dendritic cells (DC) is of interest in the prevention of graft versus host disease (GvHD). It has been shown that different C&IPI impede DC maturation and T cell priming both in vitro and in vivo. Herein we sought to understand the mechanism of action of proteasome and immunoproteasome inhibitors on DC and to test their effect on IκB and NF-IκB expression. Materials and Methods We first performed RT PCR on lysates of DC obtained from the peripheral blood of 7 patients who received post-transplant cyclophosphamide and bortezomib as prevention of GvHD on a phase I clinical trial. Patients received allogeneic transplantation from matched-related or unrelated donors. Patients received no other immunosuppressive therapy except for rabbit anti-thymocyte globulin for those receiving graft from unrelated donor. Steroids were not allowed on the study. Samples were obtained on days +1, +4, and +7. The results were analyzed in comparison to samples obtained on day 0 before stem cell infusion. We then performed the same experiment on lysates of DC obtained from the peripheral blood of healthy volunteer donors. DC were untreated or incubated with bortezomib (10 nM for 4 h), carfilzomib (30 nM for 1 h), oprozomib (100 nM and 300 nM for 4 h), ONX 0914 (200 nM for 1 h), PR-825 (125 nM for 1 h), or PR-924 (1000 nM for 1 h). The drug concentration and duration of exposure were chosen based on the IC50 on proteasome activity and to reproduce in vivo conditions. We also performed IκB western blot on DC isolated from peripheral blood of healthy volunteers, untreated or incubated with bortezomib (10 nM for 4 h) or oprozomib (300 nM for 4 h). Each experiment was performed at least in triplicate. Results We found that the combination of cyclophosphamide and bortezomib significantly and progressively increased IκB mRNA while decreasing NF-κB mRNA in DC studied ex vivo. We also found that all studied C&IPI increased IκB mRNA to a variable degree while only oprozomib (300 nM) decreased NF-κB mRNA in DC in vitro. Finally, both bortezomib and oprozomib increased IκB protein level in DC in vitro (figure). Conclusion Our data suggest that C&IPI increase IκB expression in DC. As opposed to the previously reported data in other cell types, the effect is not associated with post-transcriptional downregulation. Cyclophosphamide and bortezomib also decrease NF-κB expression in DC in vivo while only oprozomib had the same effect in vitro. The effect of C&IPI on IκB and NF-κB expression may represent a new mechanism of action and suggests their effect may be cell-type dependent. Disclosures: Al-Homsi: Millennium Pharmaceuticals: Research Funding. Off Label Use: The use of cyclophosphamide and bortezomib for GvHD prevention. Lai:Millennium Pharmaceuticals: Research Funding.

Targeting the PI3K/mTOR Pathway in Lymphoma with PQR309 and PQR620: Single Agent Activity and Synergism with the BCL2 Inhibitor Venetoclax

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3017-3017
Author(s):  
Chiara Tarantelli ◽  
Eugenio Gaudio ◽  
Petra Hillmann ◽  
Filippo Spriano ◽  
Ivo Kwee ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Mtor Inhibitor ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Apoptosis Induction ◽  
In Vivo Experiments

Abstract Background. The PI3K/AKT/mTOR pathway is an important therapeutic target in lymphomas. PQR309 is a dual PI3K/mTOR inhibitor that has shown in vitroanti-lymphoma activity (Tarantelli et al, ASH2015) and is in phase 2 trial (NCT02249429, , NCT02723877, NCT02669511). PQR620 is a novel mTORC1/2 inhibitor that has shown preclinical activity in solid tumor models (Beaufils et al, AACR 2016). Here, we present the in vitro and in vivo anti-lymphoma activity of PQR620 as single agent and also the in vivo results of PQR620 or PQR309 containing combinations with the BCL2 inhibitor venetoclax. Materials and Methods. The drug concentration causing 50% inhibition of cell proliferation (IC50) was obtained in lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), no.=26; mantle cell lymphoma (MCL), no.=8; anaplastic large T-cell lymphoma, no.=5; others, no=5] exposed to increasing doses of PQR620 for 72h using a Tecan D300e Digital Dispenser on 384well plates. For in vivo experiments, NOD-Scid (NOD.CB17-Prkdcscid/J) mice were subcutaneously inoculated with 10 x106 (RIVA) or with 5 x106(SU-DHL-6) cells. Results. PQR620 had a median IC50 of 250 nM (95%CI, 200-269 nM) when tested on 44 lymphoma cell lines. Activity was higher in B cell (no.=36) than in T cell tumors (no.=8) (median IC50s: 250 nM vs 450 nM; P=0.002). At 72h, anti-tumor activityof PQR620 was mostly cytostatic and apoptosis induction was seen only in 6/44 cell lines (13%), Sensitivity to PQR620 or apoptosis induction did not differ between DLBCL and MCL, and they were not affected by the DLBCL cell of origin, by TP53 status or by the presence of MYC or BCL2 translocations. The activity of PQR620 as single agent underwent in vivo evaluation in two DLBCL models, the germinal center B cell type DLBCL (GCB-DLBCL) SU-DHL-6 and the acivated B cell-like DLBCL (ABC-DLBCL) RIVA. Treatments with PQR620 (100mg/kg dose per day, Qdx7/w) started with 100-150 mm3 tumors and were carried for 14 (SU-DHL-6) or 21 days (RIVA). In both models, PQR620 determined a 2-fold decrease of the tumor volumes in comparison with control, with significant differences in both SU-DHL-6 (D7, D9, D11, D14; P < 0.005) and RIVA (D14, D16, D19, D21; P < 0.005). Based on the previously reported synergy between the dual PI3K/mTOR inhibitor PQR309 and venetoclax (Tarantelli et al, ASH 2015), we evaluated the combination of the PQR620 or PQR309 with the BCL2 inhibitor venetoclax (100 mg/kg, Qdx7/w) in the SU-DHL-6 model. Both the venetoclax combination with the dual PI3K/mTOR inhibitor and the venetoclax combination with mTORC1/2 inhibitor were superior to the compounds given as single agents, leading to the eradication of the xenografts. The combination of PQR620 with venetoclax showed highly significant differences either versus control or single agents during all days of the experiment (D4, D7, D9, D11, D14; P < 0.001). Similarly, the combination of PQR309 with venetoclax showed highly significant differences versus venetoclax (D7, D9, D11, D14; P < 0.001) and PQR309 (D7, D9, D11; P < 0.005) alone. Conclusions. The novel mTORC1/2 inhibitor PQR620 had in vitro and in vivo anti-lymphoma activity as single agent. In vivo experiments showed that both PQR620 and the dual PI3K/mTOR inhibitor PQR309 can strongly benefit from the combination with the BCL2 inhibitor venetoclax. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Fabbro:PIQUR Therapeutics AG: Employment. Cmiljanovic:PIQUR Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees.

Sign in / Sign up

Export Citation Format

Share Document