Heat Shock Protein 90 Inhibitor NVP-AUY922 Has Potent Anti-Tumor Activity With Adult T-Cell Leukemia-Lymphoma Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1829-1829
Author(s):  
Hiroaki Taniguchi ◽  
Hiroo Hasegawa ◽  
Daisuke Sasaki ◽  
Koji Ando ◽  
Yasushi Sawayama ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Survival ◽  
Cell Lines ◽  
Heat Shock Protein 90 ◽  
Hsp90 Inhibitor ◽  
Adult T Cell Leukemia ◽  
Pim Kinases ◽  
Client Proteins

Abstract Background Adult T-cell leukemia-lymphoma (ATL) is a chemo-resistant malignancy. Heat shock protein 90 (HSP90) is involved in folding and functions as a chaperone for multiple client proteins, many of which are important in tumorigenesis. The HSP90 inhibitor 17-AAG, derived from geldanamycin, has potent antitumor activity against ATL. However, geldanamycin derivatives have several limitations, including poor solubility, formulation difficulties, and severe hepatotoxicity in clinical settings, which have prompted development of second generation synthetic HSP90 inhibitors including NVP-AUY922 (AUY922), a second generation isoxazole-based non-geldanamycin HSP90 inhibitor that inhibits the ATPase activity of HSP90. AUY922 has shown nanomolar efficacy against a wide range of human cancer cells in vitro and also inhibits progression of a variety of tumors in vivo. Phase I/II studies of AUY922 with advanced solid tumors and hematological malignancies are presently underway. Here, we studied the effects of AUY922 on ATL in vitro and in vivo. Results We initially analyzed the effects of AUY922 (Novartis Pharmaceuticals) on survival of ATL-derived cell lines (KK1, SO4, LM-Y1, KOB, ST1) and HTLV-I-infected T-cell lines (MT2, HuT102). Cells cultured with various concentrations of AUY922 for 72 hours showed survival suppression in a dose-dependent manner in MTS assay findings. The concentrations of AUY922 required to inhibit cell survival by 50% (IC50) varied from 12.5 to 25.0 nM. We also found that the inhibitory effect of AUY was superior to that of 17-AAG. We further assessed AUY922-induced cell survival inhibition with peripheral blood mononuclear cells (PBMCs) obtained from patients with ATL and healthy donors. AUY922 induced apparent cell survival suppression in primary ATL cells, but not in normal PBMCs, while FACS analysis revealed that AUY922 induced cell-cycle arrest and apoptosis in these cell lines. Interestingly, AUY922 induced down-regulation of PIM kinases, which was confirmed by DNA microarray, qRT-PCR, and WB analysis results. Furthermore, SGI-1776, a PIM kinase inhibitor, successfully induced cell survival suppression in ATL and HTLV-1 infected cell lines in both dose- and cell-dependent manners. To elucidate the molecular mechanisms of cytotoxicity, we also examined the expressions of several client proteins using WB analysis. AUY922 treatment led to strong up-regulation of HSP70, a surrogate marker of HSP90 inhibition, and a dose-dependent decrease of HSP90 client proteins associated with cell survival, proliferation, and cell cycle in the G1 phase, including p-Akt, Akt, IκBα, IKKα, IKKβ, IKKγ, Cdk4, Cdk6, and survivin. In a xenograft model created with C.B-17/Icr-SCID mice, intraperitoneal administration of the vehicle or AUY922 was given after injection of HuT102 cells. In the control mice, bulky tumors grew within 4 weeks, whereas daily administrations of AUY922 significantly impaired tumor growth. Conclusion Together, our findings suggest that AUY922 may be an effective therapeutic agent for ATL and PIM kinases are a novel therapeutic target. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The HSP90 inhibitor Onalespib exerts synergistic anti-cancer effects when combined with radiotherapy: an in vitro and in vivo approach

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Diana Spiegelberg ◽  
Andris Abramenkovs ◽  
Anja Charlotte Lundgren Mortensen ◽  
Sara Lundsten ◽  
Marika Nestor ◽  
...  
Keyword(s):  
Tumor Growth ◽  
External Beam Radiotherapy ◽  
Heat Shock Protein 90 ◽  
Hsp90 Inhibitor ◽  
Control Group ◽  
A431 Cells ◽  
Anti Cancer ◽  
Client Proteins

AbstractOncogenic client-proteins of the chaperone Heat shock protein 90 (HSP90) insure unlimited tumor growth and are involved in resistance to chemo- and radiotherapy. The HSP90 inhibitor Onalespib initiates the degradation of oncoproteins, and might also act as a radiosensitizer. The aim of this study was therefore to evaluate the efficacy of Onalespib in combination with external beam radiotherapy in an in vitro and in vivo approach. Onalespib downregulated client proteins, lead to increased apoptosis and caused DNA-double-strands. Monotherapy and combination with radiotherapy reduced colony formation, proliferation and migration assessed in radiosensitive HCT116 and radioresistant A431 cells. In vivo, a minimal treatment regimen for 3 consecutive days of Onalespib (3 × 10 mg/kg) doubled survival, whereas Onalespib with radiotherapy (3 × 2 Gy) caused a substantial delay in tumor growth and prolonged the survival by a factor of 3 compared to the HCT116 xenografted control group. Our results demonstrate that Onalespib exerts synergistic anti-cancer effects when combined with radiotherapy, most prominent in the radiosensitive cell models. We speculate that the depletion and downregulation of client proteins involved in signalling, migration and DNA repair mechanisms is the cause. Thus, individually, or in combination with radiotherapy Onalespib inhibits tumor growth and has the potential to improve radiotherapy outcomes, prolonging the overall survival of cancer patients.

A Novel Heat Shock Protein (HSP) 90 Inhibitor KW-2478 shows Activity in B-Cell Malignancies in Vitro and in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1625-1625
Author(s):  
Simone Juliger ◽  
Takayuki Nakashima ◽  
Lenushka Maharaj ◽  
Toshihiko Ishii ◽  
Hiroshi Nakagawa ◽  
...  
Keyword(s):  
Cell Growth ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Hsp90 Inhibitor ◽  
Anti Tumour Activity ◽  
Client Proteins ◽  
Tumour Activity

Abstract Background : HSP90 plays an important role in chaperoning key proteins implicated in malignant disease and is a promising therapeutic target. We now report the in vitro and in vivo activity of a novel HSP90 inhibitor of non-ansamycin, non-purine analogue class, KW-2478, (Kyowa Hakko Kirin) in B-cell malignancies including multiple myeloma (MM), B-cell lymphoma (BCL) and mantle cell lymphoma (MCL) cells, and in primary tumour cells from MM and BCL patients. Procedures: The binding affinity of KW-2478 to HSP90 was examined using immobilised human HSP90a and a biotinylated HSP90 binding agent, radicicol (bRD). The effect of KW-2478 on cell viability, cell growth and apoptosis induction were evaluated in cell lines, with KW-2478 induced changes in major HSP90 client proteins studied by Western blotting analysis. The in vivo anti-tumour activity of KW-2478 was evaluated in a human MM xenograft mouse model,. Primary MM cells were studied using a co-culture system with the HS-5 bone marrow stromal cell line (BMSCs), while primary BCL samples were cultured on CHO cells stably transfected to produce CD40L. Results: KW-2478 inhibited the binding of bRD to HSP90α in concentration-dependent manner with an IC50 value of 3.8 nM. KW-2478 clearly inhibited cancer cell growth in all cell lines, with EC50 values from 101–252 nM in BCL, 81.4–91.4 nM in MCL and 120–622 nM in MM. The drug also exhibited potent growth inhibitory activity in primary CLL (n=3) and NHL (n=2) cells with EC50 values of 40–170 nM and 200–400 nM, respectively. In 2 of 4 human primary myeloma cells, KW-2478 at 2 μM inhibited cell growth by at least 50%. The presence of BMSCs did not affect drug activity against primary MM cells and importantly there was little or no effect on cell number or viability of normal BMSCs at up to 20 μM KW-2478. Exposure of MM and BCL cell lines to KW-2478 for 24 hours resulted in the degradation of HSP90 client proteins (IGF-1Rβ and Raf-1) and the induction of HSP70. KW-2478 also induced PARP cleavage and dephosphorylated Erk1/2 in NCI-H929 cells. Further studies in selected cell lines showed that exposure to 1 μM KW-2478 or lower resulted in the depletion of p53 and Akt proteins, a reduction in nuclear NFKB, and the cleavage of caspase-3. In the NCI-H929 xenograft model, KW-2478 (qd×5, i.v.) showed a statistically significant suppression of tumour growth at the doses of 25, 50, 100 and 200 mg/kg. Moreover, tumour regressions were observed at doses of 100 and 200 mg/kg, with a significant decrease in serum M protein concentration at doses of 50, 100 and 200 mg/kg. No severe KW-2478 toxicity was observed as assessed by treatment-related mortality and body weight change. Conclusions: The novel HSP90 inhibitor KW-2478 showed a potent anti-tumour activity both in vitro and in vivo, including activity in primary patient samples. The agent retained its activity in primary myeloma cells in the presence of BMSCs, suggesting that KW-2478 can overcome the protective effect of the bone marrow microenvironment. Additional pharmacokinetic and safety data support the further development of KW-2478 and the drug is currently undergoing clinical evaluation in a phase I trial.

scholarly journals The Pim kinases control rapamycin-resistant T cell survival and activation

2005 ◽  
Vol 201 (2) ◽  
pp. 259-266 ◽  
Author(s):  
Casey J. Fox ◽  
Peter S. Hammerman ◽  
Craig B. Thompson
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Cell Survival ◽  
Growth And Survival ◽  
Pim Kinases ◽  
T Cell Survival

Although Pim-1 or Pim-2 can contribute to lymphoid transformation when overexpressed, the physiologic role of these kinases in the immune response is uncertain. We now report that T cells from Pim-1−/−Pim-2−/− animals display an unexpected sensitivity to the immunosuppressant rapamycin. Cytokine-induced Pim-1 and Pim-2 promote the rapamycin-resistant survival of lymphocytes. The endogenous function of the Pim kinases was not restricted to the regulation of cell survival. Like the rapamycin target TOR, the Pim kinases also contribute to the regulation of lymphocyte growth and proliferation. Although rapamycin has a minimal effect on wild-type T cell expansion in vitro and in vivo, it completely suppresses the response of Pim-1−/−Pim-2−/− cells. Thus, endogenous levels of the Pim kinases are required for T cells to mount an immune response in the presence of rapamycin. The existence of a rapamycin-insensitive pathway that regulates T cell growth and survival has important implications for understanding how rapamycin functions as an immunomodulatory drug and for the development of complementary immunotherapeutics.

scholarly journals A Brain Penetrating Heat-Shock Protein 90 Inhibitor NXD30001 Inhibits Glioblastoma as a Monotherapy or in Combination With Radiation

Neurosurgery ◽  
2019 ◽  
Vol 66 (Supplement_1) ◽  
Author(s):  
Hao Chen ◽  
Jialiang Wang ◽  
Hengli Tian
Keyword(s):  
Stem Cells ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Median Survival ◽  
Therapeutic Potential ◽  
Heat Shock Protein 90 ◽  
Tumor Bearing ◽  
Client Proteins

Abstract INTRODUCTION It has been increasingly recognized that glioblastoma multiforme (GBM) is a highly heterogeneous disease, which is initiated and sustained by molecular alterations in an array of signal transduction pathways. Heat-shock protein 90 (Hsp90) is a molecular chaperone to be critically implicated in folding and activation of a diverse group of client proteins, many of which are key regulators of important glioblastoma biology. METHODS To determine the therapeutic potential of targeting Hsp90 in glioblastoma, we assessed the anti-neoplastic efficacy of NXD30001, a brain-penetrating Hsp90 inhibitor as a monotherapy or in combination with radiation, both in Vitro and in Vivo. RESULTS Our results demonstrated that NXD30001 potently inhibited neurosphere formation, growth and survival of CD133 + glioblastoma stem cells (GSCs) with the half maximal inhibitory concentrations (IC50) at low nanomolar concentrations. At suboptimal concentrations, inhibition of Hsp90 did not exert cytotoxic activity but rather increased radiosensitivity in GSCs. CD133- GBM cells were less sensitive and not radiosensitized by NXD30001. In lines with its cytotoxic and radiosensitizing effects, NXD30001 dose-dependently decreased phosphorylation protein levels of multiple Hsp90 client proteins, including those playing key roles in GSCs, such as EGFR, Akt, c-Myc, and Notch1. In addition, combining NXD30001 with radiation could impair DNA damage response and ER stress response to induce apoptosis of GSCs. Treatment of orthotopic glioblastoma tumors with NXD30001 extended median survival of tumor-bearing mice by approximately 20% (treated 37 days vs vehicle 31 d, P = .0026). Radiation alone increased median survival of tumor-bearing mice from 31 to 38 d, combination with NXD30001 further extended survival to 43 d (P = .0089). CONCLUSION Our results suggest that GBM stem cells (CD133+) are more sensitive to NXD30001 than non-stem GBM cells (CD133-). Furthermore, combination NXD30001 with radiation significantly inhibits GBM progression than use it as a monotherapy by targeting GSCs.

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.

Preclinical Evaluation of A Potent 2nd Generation Small-Molecule Hsp90 Inhibitor STA-9090 In Hematological Cell Lines

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2899-2899
Author(s):  
Weiwen Ying ◽  
David Proia ◽  
Suqin He ◽  
Jim Sang ◽  
Kevin Foley ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Small Molecule ◽  
First Generation ◽  
Cell Lymphoma ◽  
Hsp90 Inhibitor ◽  
Phase 2 ◽  
Hsp90 Inhibitors

Abstract Abstract 2899 STA-9090 is a potent, second generation, small-molecule Hsp90 inhibitor, with a chemical structure unrelated to the first-generation, ansamycin family of Hsp90 inhibitors. In preclinical in vitro and in vivo studies, STA-9090 has shown potency up to 100 times greater than the first-generation Hsp90 inhibitors against a wide range of solid and hematological cancer types including those resistant to imatinib, sunitinib, erlotinib, and dasatinib. STA-9090 is currently being evaluated two Phase 1 and four Phase 2 trials (non-small cell lung, GIST, colon, and gastric) in solid tumor cancers; and two trials in hematologic cancers. Additional Phase 2 trials in several other indications are planned for 2H 2010. Inhibition of Hsp90 by STA-9090 results in the destabilization of a broad range of oncogenic kinases often overexpressed or mutated in hematological cancers. For example, the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressed in the anaplastic large cell lymphoma (ALCL) cell line Karpas 299, is degraded rapidly in the presence of STA-9090 in vitro, resulting in the loss of viability. Similar results were shown in other NPM-ALK driven ALCL cells including SU-DHL-1 and SR-786 with IC50 less than 20 nM. Stability of other kinases common to hematological malignancies, such as Bcr-Abl, FLT3 and c-Kit, were also shown to be highly sensitive to STA-9090, resulting in potent cell death of cell lines addicted to signaling by these kinases. In vivo, STA-9090 was highly effective in a subcutaneous xenograft model of diffuse large B-cell lymphoma SU-DHL-4 with resulting %T/C values of 26, 4, -90 and -93 when dosed at 25, 50, 75 and 100 mg twice per week, respectively. Importantly, 75 and 100 mg/kg STA-9090 dosed 2 times per week for a total of 3 weeks (150 and 200 mg/kg weekly) resulted in 25% and 50% of the animals in each group being free of tumors by the end of the study, respectively. MV4-11, an AML (FLT3ITD) cell line, turned out to be one of the most sensitive xenograft models to STA-9090 treatment. STA-9090 at 100 mg/kg or125mg/kg once weekly was highly efficacious with 37.5% of mice achieving tumor free with acceptable toxicity at the end of the 3-week treatment period. In conclusion, STA-9090 exhibits preferable biological profiles both in vitro and in vivo in treating hematological malignances. Clinical studies for using STA-9090 both once weekly and twice weekly are ongoing. Disclosures: Ying: Synta Pharmaceuticals: Employment. Proia:Synta Pharmaceuticals: Employment. He:Synta Pharmaceur: Employment. Sang:Synta Pharmaceuticals: Employment. Foley:Synta Pharmaceuticals: former employee. Du:Synta Pharmaceuticals: former employee. Blackman:Synta Pharmaceuticals: Employment. Wada:Synta Pharmaceuticals: Employment. Sun:Synta Pharmaceuticals: Employment. Koya:Synta Pharmaceuticals: Employment.

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.

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.

Effect of a combination of epigenetic agents on the malignant phenotype in models of T-cell lymphoma.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e13569-e13569
Author(s):  
Enrica Marchi ◽  
Matko Kalac ◽  
Danielle Bongero ◽  
Christine McIntosh ◽  
Laura K Fogli ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Cell Lines ◽  
Molecular Level ◽  
Cell Lymphoma ◽  
Single Agent ◽  
T Cell Lymphoma ◽  
Cytotoxicity Assays

e13569 Background: CHOP and CHOP-like chemotherapy are the most used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) have shown class activity in PTCLs. The interaction between the HDACIs (depsipeptide (R), belinostat (B), vorinostat (V) and panobinostat (P)) and a DNMT inhibitor (decitabine (D) was investigated in vitro, in vivo and at the molecular level in T-cell lymphoma and leukemia cell lines (H9, HH, P12, PF-382). Methods: For cytotoxicity assays, luminescence cell viability assay was used (CellTiter-Glo). Drug:drug interactions were analyzed with relative risk ratios (RRR) based on the GraphPad software (RRR<1 defining synergism). Apoptosis was assessed by Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition. Gene expression profiling was analyzed using Illumina Human HT-12 v4 Expression BeadChip microarrays and Gene Spring Software for the analysis. Results: The IC50s for B, R, V, P, D and 5-Azacytidine alone were assessed at 24, 48 and 72 hours. In cytotoxicity assays the combination of D plus B, R, V or P at 72 hours showed synergism in all the cell lines (RRRs 0.0007-0.9). All the cell lines were treated with D, B or R for 72 hours and all the combinations showed significantly more apoptosis than the single drug exposures and controls (RRR < 1). In vivo, HH SCID beige mice were treated i.p. for 3 cycles with the vehicle solution, D or B or their combination at increasing dose. The combination cohort showed statistically significant tumor growth inhibition compared to all the other cohorts. Gene expression analysis revealed differentially expressed genes and modulated pathways for each of the single agent treatment and the combination. The effects of the two drugs were largely different (only 39 genes modified in common). Most of the effects induced by the single agent were maintained in the combination group. Interestingly, 944 genes were modulated uniquely by the combination treatment. Conclusions: The combination of a DNMTI and HDACIs is strongly synergistic in vitro, in vivo and at the molecular level in model of T-cell lymphoma and these data will constitute the basis for a phase I-II clinical trials.

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