Anti-CD74-doxorubicin immunoconjugate (IMMU-110) is cytotoxic in non-Hodgkin’s lymphoma (NHL) models and overcomes MDR

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 7598-7598 ◽  
Author(s):  
R. Blumenthal ◽  
R. Stein ◽  
R. Michell ◽  
D. M. Goldenberg
Keyword(s):  
Cell Lines ◽  
Day Treatment ◽  
Annexin V ◽  
In Vitro Cytotoxicity ◽  
Kaplan Meier ◽  
Free Doxorubicin ◽  
Daudi Cells ◽  
Cell Implantation

7598 Background: The internalizing LL1 anti-CD74 antibody is an optimal agent for delivering drugs, toxins, or radionuclides to CD74+ cancer cells. Here, we investigated the efficacy of IMMU-110 (Immunomedics, Inc.) in common follicular and aggressive types of NHL cells and in two disseminated non-Burkitt NHL models. Methods: CD74, MDR and MRP expression on NHL cell lines was determined by flow cytometry. In vitro cytotoxicity was assessed by cell cycle analysis of propidium iodide (PI)-stained cells and by measuring apoptotic cells using FITC-Annexin V and PI. In vivo therapy of a single 350-μg dose of IMMU-110 was evaluated in disseminated SUDHL4 and FSCCL. Results: Raji and Daudi Burkitt lines express similar amounts of CD74 (>93% positive cells and a MCF=35), yet a 3-day treatment with 0.8 μg/ml of IMMU-110 results is 18.4% of Raji and 67.9% of Daudi cells in Sub-Go. Aside from Daudi cells that respond with cells shifting into Sub-Go, most other NHL cell lines experience a G2/M block (44%-82% of cells) in response to a 3-day exposure to IMMU-110. Both MDR- and MDR+ NHL cells responded to IMMU-110. Kaplan Meier analysis showed a significant increase in survival of both SUDHL4 (MDR-/MRP-)- and FSCCL (MDR+/MRP+)-bearing SCID mice (P < 0.025) with 100% survival of treated mice vs. 38% survival of untreated mice at 70–77 days post cell implantation. Conclusions: IMMU-110 is cytotoxic in non-Burkitt and in Burkitt NHL cell lines. The magnitude of the cytotoxic response is not related to the amount of CD74 expressed on the cell surface. IMMU-110 is therapeutic in drug-sensitive (SUDHL4) and drug-resistant (FSCCL) NHL models, suggesting that antibody targeting can bypass the MDR drug efflux system that prevents free doxorubicin from being therapeutic. [Table: see text] [Table: see text]

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

NPI-2358, a Novel Vascular Disrupting Agent Blocks Growth and Angiogenesis in Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3842-3842
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Madhavi Bandi ◽  
Noopur Raje ◽  
Robert L Schlossman ◽  
...  
Keyword(s):  
Cell Lines ◽  
Annexin V ◽  
Vascular Endothelial ◽  
Caspase 9 ◽  
Migration Assays ◽  
Induced Apoptosis ◽  
Rpmi 8226 ◽  
Anti Tumor Activity

Abstract Abstract 3842 Poster Board III-778 Background and Rationale Vascular disrupting agents (VDAs) act via selectively disrupting established tumor vasculature and have shown remarkable clinical success as anti-cancer therapies. NPI-2358 is a novel VDA with a distinct structure and mechanism of action from other available VDAs. NPI-2358 binds to the colchicine-binding site of beta-tubulin preventing polymerization and disrupting the cytoplasmic microtubule network, thereby causing loss of vascular endothelial cytoskeletal function, and inducing cytotoxicity in cancer cells. Here, we examined the anti-angiogenic and anti-tumor activity of NPI-2358 in multiple myeloma (MM) cells using both in vitro and in vivo model systems. Material and Methods We utilized MM.1S, MM.1R, RPMI-8226, U266, and INA-6 human MM cell lines, as well as purified tumor cells from MM patients relapsing after prior anti-MM therapies. Cell viability/apoptosis assays were performed using MTT, trypan blue exclusion, and Annexin V/PI staining. Angiogenesis was measured in vitro using Matrigel capillary-like tube structure formation assays: Since human vascular endothelial cells (HUVECs) plated onto Matrigel differentiate and form capillary-like tube structures similar to in vivo neovascularization, this assay measures anti-angiogenic effects of drugs/agents. Migration assays were performed using transwell insert assays. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, Bax, pJNK and GAPDH. Statistical significance was determined using a Student t test. Results Treatment of MM.1S, RPMI-8226, MM.1R, INA-6, and KMS-12BM with NPI-2358 for 24h induces a dose-dependent significant (P < 0.005) decrease in viability of all cell lines (IC50 range: 5-8 nM; n=3). To determine whether NPI-2358-induced decrease in viability is due to apoptosis, MM cell lines were treated with NPI-2358 for 24h; harvested, and analyzed for apoptosis using Annexin V/PI staining. A significant increase in NPI-2358-induced apoptosis was observed in all MM cell lines (% Annexin V+/PI- apoptotic cells: MM.1S, 48 ± 2.3%; MM.1R, 46.6 ± 3.1%; RPMI-8226, 61.7 ± 4.5%; and INA-6, 59.9 ± 3.2%; P < 0.05; n=3). Importantly, NPI-2358 decreased viability of freshly isolated MM cells from patients (IC50 range: 3-7 nM; P < 0.005), without affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for NPI-2358. Examination of in vitro angiogenesis using capillary-like tube structure formation assay showed that even low doses of NPI-2358 (7 nM treatment for 12h; IC50: 20 nM at 24h) significantly decreased tubule formation in HUVECs (70-80% decrease; P < 0.05). Transwell insert assays showed a marked reduction in serum-dependent migration of NPI-2358-treated MM cells (42 ± 2.1% inhibition in NPI-2358-treated vs. control; P < 0.05). NPI-2358 at the concentrations tested (5 nM for 12h) in the migration assays did not affect survival of MM cells (> 95% viable cells). A similar anti-migration activity of NPI-2358 was noted against HUVEC cells (48 ± 1.7% decrease in migration; P < 0.05). Mechanistic studies showed that NPI-2358-induced apoptosis was associated with activation of caspase-8, caspase-9, caspase-3 and PARP. Importantly, treatment of MM.1S cells with NPI-2358 (5 nM) triggered phosphorylation of c-Jun amino-terminal kinase (JNK), a classical stress response protein, without affecting Bcl-2 family members Bax and Bcl-2. Blockade of JNK using dominant negative strategy markedly abrogated NPI-2358-induced apoptosis. Conclusion Our preclinical data provide evidence for remarkable anti-angiogenic and anti-tumor activity of NPI-2358 against MM cells, without significant toxicity in normal cells. Ongoing studies are examining in vivo anti-MM activity of NPI-2358 in animal models. Importantly, a Phase-1 study of NPI-2358 as a single agent in patients with advanced malignancies (lung, prostrate and colon cancer) has already established a favorable pharmacokinetic, pharmacodynamic and safety profile; and, a Phase-2 study of the combination of NPI-2358 and docetaxel in non-small cell lung cancer showed encouraging safety, pharmacokinetic and activity data. These findings, coupled with our preclinical studies, provide the framework for the development of NPI-2358-based novel therapies to improve patient outcome in MM. Disclosures: Chauhan: Nereus Pharmaceuticals, Inc: Consultancy. Lloyd:Nereus Pharmaceuticals, In: Employment. Palladino:Nereus Pharmaceuticals, Inc: Employment. Anderson:Nereus Pharmaceuticals, Inc: Consultancy.

Effect of cabazitaxel on the growth of human castration-refractory prostate cancer cells in vitro compared to docetaxel and on the anti-tumor properties of the angio-inhibitor PEDF in vivo.

2015 ◽  
Vol 33 (7_suppl) ◽  
pp. 205-205
Author(s):  
Thomas Nelius ◽  
Courtney Jarvis ◽  
Dalia Martinez-Marin ◽  
Stephanie Filleur
Keyword(s):  
Prostate Cancer ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Cell Lines ◽  
In Vitro Cytotoxicity ◽  
In Vivo Studies ◽  
Raw264.7 Macrophages

205 Background: Docetaxel/DTX and cabazitaxel/CBZ have shown promise in the treatment of metastatic Castration-Refractory Prostate Cancer/mCPRC however, comparative studies are missing. Toxicities of these drugs are significant, urging the need to modify taxane regimens. Recently, low-dose metronomic/LDM treatments using conventional chemotherapeutic drugs have shown benefits in CPRC in improving the effect of anti-angiogenic agents. Previously, we have demonstrated that LDM-DTX in combination with PEDF curbs significantly CRPC growth, limits metastases formation and prolongs survival in vivo. In this study, we intended to compare the cytotoxic effect of CBZ and DTX on CRPC cells in vitro and CL1 tumors in vivo. Methods: PC3, DU145 cell lines were from ATCC.CL1 cells were obtained from androgen-deprived LNCaP cells. Cell proliferation was assessed by crystal violet staining and cell cycle analyses. In vitro cytotoxicity assays were performed on CL1 cells/RAW264.7 macrophages co-cultures treated with PEDF and increasing doses of taxanes. For the in vivo studies, CL1 cells were engineered to stably express the DsRed Express protein +/- PEDF. PEDF anti-tumor effects were assessed on s.c. xenografts treated with DTX (5mg/kg ip ev. 4 day) as reference, CBZ (5mg/kg ip ev. 4 days, 1mg/kg for 10 days, 0.5mg/kg q.a.d. and 0.1mg/kg daily) or placebo. Results: CBZ limits cell proliferation with a greater efficacy than DTX in all CRPC cell lines tested. DU145 presented the largest difference. High doses of taxane blocked tumor cells in mitosis, whereas LDM increased the SubG1 population. This effect was significantly higher in DU145 cells treated with CBZ. In vivo, 5mg/kg CBZ delayed tumor growth more efficiently than 5mg/kg DTX. PEDF/5mg/kg CBZ markedly delayed tumor growth compared to all treatments. Finally, engulfment of tumor cells by macrophages was higher in combined treatments suggesting an inflammation-related process. Conclusions: CBZ is more efficient than DTX both in vitro and in vivo.The data also reinforce PEDF as a promising anti-neoplasic agent in combination with LDM taxane chemotherapies.

Effect of inducing HR deficiency using AVB500, a receptor tyrosine kinase AXL inhibitor, on response to olaparib in uterine serous cancer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e18108-e18108
Author(s):  
Michael Driscoll Toboni ◽  
Barbara Blachut ◽  
Mary M Mullen ◽  
Jo'an Tankou ◽  
Hollie M Noia ◽  
...  
Keyword(s):  
Treatment Period ◽  
Cell Lines ◽  
Day Treatment ◽  
Parp Inhibitor ◽  
Tumor Burden ◽  
Scid Mice ◽  
In Vivo Studies ◽  
Treatment Groups

e18108 Background: Evidence suggests DNA repair is a therapeutic target in endometrial cancer (EC). Given this, we determined whether combination therapy with AVB500, an AXL inhibitor, could improve response in a uterine serous cancer (USC) model. Methods: Two USC cell lines (ARK1 & ARK4) were treated with AVB500 (Aravive Biologics, Houston, TX) in combination with the poly ADP ribose polymerase (PARP) inhibitor, olaparib. Colony forming assays were assessed after 4 days of treatment with either AVB500 alone, olaparib alone or combination treatment (olaparib + AVB500); colonies were stained and absorbance was obtained to calculate relative cell viability using Graph Pad Prism. Baseline homologous recombination (HR) status was determined after radiating cells with 10Gy and identifying RAD51 foci by immunofluorescence (IF). Cell lines were considered to be HR proficient if over 30% of the cells expressed RAD51 ( > 5 foci per cell). IF was conducted using a Leica confocal microscope and foci were quantified using FociCounter. In vivo studies were performed using NOD-SCID mice injected with 1 x 107 ARK1 cells intraperitoneally followed by treatment q3 days for a 14 and 21 day treatment period. Treatment groups were vehicle control, AVB500 alone, olaparib alone and olaparib with AVB500. Results: The absorbance for olaparib + AVB500 was significantly less than the olaparib only group in two assays involving ARK1s (0.417nm vs 0.756nm, p = 0.001; 0.320nm vs 0.620nm, p = 0.008) as well as in ARK4s (0.186nm vs 0.641nm, p = 0.003). The HR assay indicated both cell lines were HR proficient. After baseline HR proficiency was established, the cell lines were pretreated with AVB500 prior to radiation. When compared to cells without treatment with AVB500, IF showed a decrease in RAD51 foci per cell in ARK1 (2.7 vs 7.3, p = 0.0003) and ARK4 (6.3 vs 13.0, p = 0.0054). The proportion of ARK1 cells expressing RAD51 decreased to 21%, indicating HR deficiency. Lastly, NOD-SCID mice receiving olaparib + AVB500 had less tumor weight than those treated with olaparib alone (0.008g vs 0.138g, p = 0.002) and AVB500 alone (0.008g vs 0.145g, p = 0.0006) in a 14 day and a 21 day treatment period (0.212g vs 0.586g, p = 0.027 and 0.212 vs 0.494g, p = 0.005, respectively). Conclusions: HR proficient USC cell lines treated in vitro and in vivo with the combination of AVB500 and olaparib demonstrate an improved response to olaparib or AVB500 alone with a greater decrease in tumor burden. AVB500 appears to induce HR deficiency. Additional therapeutic and mechanistic experiments are ongoing.

Activity of vincristine, L-ASP, and dexamethasone against acute lymphoblastic leukemia is enhanced by the BH3-mimetic ABT-737 in vitro and in vivo

Blood ◽  
2007 ◽  
Vol 110 (6) ◽  
pp. 2057-2066 ◽  
Author(s):  
Min H. Kang ◽  
Yun Hee Kang ◽  
Barbara Szymanska ◽  
Urszula Wilczynska-Kalak ◽  
Michael A. Sheard ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Clinical Investigation ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Cytochrome C Release ◽  
Synergistic Cytotoxicity ◽  
Bh3 Mimetic

Abstract Defects in apoptosis signaling contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL), and overexpression of antiapoptotic Bcl-2 (Bcl-2 and Bcl-XL) family proteins has been observed in ALL. ABT-737 is a small-molecule BH3-mimetic that inhibits the antiapoptotic Bcl-2 family proteins. We evaluated the cytotoxicity of ABT-737 in combination with vincristine, dexamethasone, and L-asparaginase (VXL) in 7 ALL cell lines. Multilog synergistic cytotoxicity was observed in all 7 cell lines with ABT-737 plus L-asparaginase or vincristine, and in 5 of 7 cell lines with ABT-737 plus dexamethasone or VXL. In leukemia cells, but not in normal lymphocytes, ABT-737 plus L-asparaginase induced greater mitochondrial depolarization (JC-1 staining); mitochondrial cytochrome c release; activation of Bax, Bid, and caspases (immunoblotting); and eventually apoptosis (annexin V staining) than did either drug alone. In mouse xenografts derived from patients with ALL at diagnosis (ALL-7) or at relapse (ALL-19), event-free survival (EFS) was significantly enhanced with ABT-737 plus VXL relative to VXL or ABT-737 alone (P ≤ .02). Thus, ABT-737 synergistically enhanced VXL cytotoxicity in ALL cell lines via a mitochondrial death pathway and enhanced EFS in VXL-treated mice bearing ALL xenografts. Combining VXL with a BH3-mimetic warrants clinical investigation in ALL at relapse and potentially in chemotherapy-resistant ALL subgroups.

scholarly journals Assessment of the toxicity and antiproliferative activity of hemocyanins from Helix lucorum, Helix aspersa and Rapana venosa

2021 ◽  
Vol Volume 53 (Special Issue A) ◽  
pp. 15-21
Author(s):  
I. Yankova ◽  
E Ivanova ◽  
K. Todorova ◽  
A. Georgieva ◽  
V. Dilcheva ◽  
...  
Keyword(s):  
Cell Lines ◽  
Antiproliferative Activity ◽  
Helix Aspersa ◽  
In Vitro Cytotoxicity ◽  
Histopathological Analysis ◽  
Rapana Venosa ◽  
Toxicological Assessment ◽  
Helix Lucorum

Hemocyanins (Hcs) are respiratory, oxygen-carrying metalloproteins that are freely dissolved in the hemolymph of many molluscs and arthropods. The interest in hemocyanins has grown significantly since it was found that they can be successfully used in immunotherapy of neoplastic diseases as non-specific or active stimulators of the immune system. The present study aims to assess the cytotoxicity, in vivo toxicity and antiproliferative activity of hemocyanins isolated from marine snail Rapana venosa (RvH), garden snails Helix lucorum (HlH) and Helix aspersa (HaH). For in vitro safety testing, 3T3 Neutral Red Uptake (NRU) test was used. The experiments for antiproliferative activity of the hemocyanins were performed by MTT assay on a panel of cell lines - a model of breast cancer. The in vivo toxicological assessment was performed by regular clinical examinations of hemocyanin-treated laboratory mice and histopathological analysis of hematoxylin/eosin stained preparations of parenchymal organs. The evaluation of the in vitro cytotoxicity showed that the tested hemocyanins does not induce toxic effects in nontumorigenic epithelial cell lines. In contrast, significant reduction of the viability of human breast carcinoma cell lines was found after treatment with high concentrations of hemocyanins. The in vivo experiments showed no signs of organ and systemic toxicity in the hemocyanin-treated animals. The presented data indicate that Hcs show a potential for development of novel anticancer therapeutics due to their beneficial properties, biosafety and lack of toxicity or side effects. Key words: hemocyanins (Hcs); cytotoxicity; antitumor activity; in vivo biosafety testing.

Reversion of physiological drug resistance of weakly basic drugs: The discovery of a new mechanism of PEG-PCL nanoparticles

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e13528-e13528
Author(s):  
R. Li ◽  
L. Xie ◽  
X. Li ◽  
Q. Liu ◽  
X. Qian ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Lines ◽  
Cellular Uptake ◽  
Antitumor Effect ◽  
In Vitro Cytotoxicity ◽  
Extracellular Ph ◽  
Fluorescent Particle ◽  
Tumor Ph

e13528 Background: A number of studies have reported the superior antitumor effect of nanoparticles loading chemotherapeutics than the free agents, yet the underlying mechanism has not attract enough attention. The extracellular pH of cancer cells is lower than that of the intracellular pH. Due to this pH gradient, weakly basic drug will protonated extracellularly and display decreased intracellular concentration. In this study, we aimed to reveal a new mechanism of PEG-PCL nanoparticles, namely the reversion of physiological drug resistance. Methods: Tetradrine (Tet), an alkaloid isolated from traditional Chinese medicine, was incorporated into the diblock copolymer methoxy poly(ethylene glycol)-polycaprolactone (mPEG-PCL). In vitro cytotoxicity of free Tet and Tet-loaded nanoparticles at pH7.4 and pH6.8 was compared on four different cancer cell lines. Fluorescent particle cellular uptake study was also used. To evaluate the antitumor effect of the nanoparticles in a more complex model rather than monolayer cell culture, we used Histoculture Drug Resistance Assay (HDRA). The in vivo antitumor effect of the nanoparticles was also studied in ICR mice bearing H22 tumor with different in vivo pH values. Results: In vitro cytotoxicity study in four tumor cell lines showed that the cytotoxicity of free Tet decreased significantly (P<0.05) when the extracellular pH decreased from 7.4 to 6.8, while the cytotoxicity of Tet-loaded nanoparticles increased or didn’t change significantly. The possible mechanism may mainly be the endocytosis of nanoparticles, which was proven by fluorescent particle cellular uptake study. HDRA indicated better tissue penetration of nanoparticles over free Tet. As to in vivo study, the mice with in vivo tumor pH 6.8 and treated with Tet-loaded nanoparticles exhibited best tumor inhibit rate and mildest side effect, suggesting that the use of nanoparticles was more preferable than the manipulation of tumor pH by the use of basic water. Conclusions: Our study clearly demonstrated that the mPEG-PCL nanoparticles could overcome the drug resistance caused by low extracellular pH and enhance drug penetration in the tumor tissue, thus increasing the antitumor efficacy of weakly basic agents. No significant financial relationships to disclose.

A Novel SIRT1 Activator SIRT1720 Triggers In Vitro and In Vivo Cytotoxicity In Multiple Myeloma Via ATM-Dependent Mechanism

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3007-3007
Author(s):  
Dharminder Chauhan ◽  
Madhavi Bandi ◽  
Ajita V Singh ◽  
Teru Hideshima ◽  
Nikhil C. Munshi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Cell Lines ◽  
Annexin V ◽  
Immunoblot Analysis ◽  
Advisory Committees ◽  
Rpmi 8226 ◽  
Sirt1 Activator

Abstract Abstract 3007 Background and Rationale: SIRT1 belongs to the silent information regulator 2 (Sir2) family of proteins and functions as NAD+-dependent deacetylase. Previous studies showed that resveratrol, a polyphenolic SIRT1 activator, inhibits tumorigenesis in various solid tumor and hematologic malignancies, including human multiple myeloma (MM) cells. This notion led to the discovery and development of more potent and selective pharmacological activators of SIRT1 as potential anti-cancer therapeutics. In this context, a recent medicinal chemistry research using high-throughput screening, and mass spectrometry identified SRT1720, a small molecule activator of SIRT1 that is structurally distinct from resveratrol. Here, we examined the anti-tumor activity of SRT1720 in MM cells using in vitro and in vivo model systems. Methods and Model: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12BM, H929, and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability, proliferation, and apoptosis assays were performed using trypan blue, MTT, thymidine incorporation, and Annexin V staining. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Results: We first confirmed the functional specificity of SRT1720 against SIRT1 using different experimental strategies. First, we utilized Fluor de Lys Deacetylase Assay to measure whether SRT1720 affects the SIRT1 deacetylase enzymatic activity. Treatment of MM.1R and RPMI-8226 MM cells with SRT1720 markedly increased the deacetylating activity; conversely, pre-treatment of cells with nicotinamide (NAM) - an inhibitor of SIRT1 – significantly blocked SRT1720-triggered deacetylating activity. Second, immunoblot analysis using antibodies specific against acetylated p53 (a known substrate of SIRT1) showed a marked decrease in acetylated state of p53 in SRT1720-treated MM cells. These findings in MM cells confirm SIRT1 as a selective target of SRT1720. We next examined the efficacyof SRT1720 in MM cells. Treatment of MM cell lines and primary patient cells for 24h significantly decreased their viability (IC50 range 3–7 uM) (P < 0.005; n=3) without markedly affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for SRT1720. SRT1720-triggered apoptosis was confirmed in MM.1R and RPMI-8226 cells, evidenced by a marked increase in Annexin V+ and PI- cell population (P < 0.001, n=3). Importantly, SRT1720 induced apoptosis in MM cells even in the presence of bone marrow stromal cells. Mechanistic studies showed that SRT1720-triggered apoptosis in MM cells is associated with 1) activation of caspase-8, caspase-9, caspase-3, and PARP; 2) activation of pATM, CHK2, endoplasmic reticulum stress molecules pEIF2, and BIP; as well as an increase in reactive oxygen species (ROS); 3) inhibition of MM cell growth and survival pathway via NF-kB; and 4) inhibition of VEGF-induced migration of MM cells and associated angiogenesis. Importantly, blockade of pATM using a biochemical inhibitor KU-5593 significantly attenuated SRT1720-induced MM cell death (P value < 0.002; n=2). These data suggest that SRT1720-induced MM cell apoptosis is predominantly mediated by an ATM-dependent apoptotic pathway. We next examined the in vivo efficacy of SRT1720 using a human plasmacytoma xenograft mouse model. Treatment of tumor-bearing mice with SRT1720 (200 mg/kg, 5 days a week for three weeks), but not vehicle alone, significantly (P < 0.008) inhibits MM tumor growth in these mice. Finally, the combination of SRT1720 with bortezomib or dexamethasone triggered synergistic anti-MM activity. Conclusions: These preclinical studies provide the rationale for novel therapeutics targeting SIRT1 to improve patient outcome in MM. Disclosures: Munshi: Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

scholarly journals Venetoclax Synergistically Enhances the Antileukemic Activity of Imipridone ONC213, a Novel Imipridone ONC201 Analog, in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3936-3936
Author(s):  
Yongwei Su ◽  
Xinyu Li ◽  
Holly Edwards ◽  
Lisa Polin ◽  
Juiwanna Kushner ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Annexin V ◽  
Vehicle Control ◽  
Antileukemic Activity ◽  
Hematopoietic Progenitor

Abstract Although standard induction therapy initially elicits a promising response in the majority of acute myeloid leukemia (AML) patients, the majority relapse. Leukemia stem cells (LSCs) that survive chemotherapy are believed to be responsible for AML relapse. Therefore, new therapies that eliminate LSCs are desperately needed. ONC201 is a TRAIL inducer and the founding member of the imipridone family. It has been shown to induce apoptosis in LSCs (Ishizawa et al, Science Signaling. 2016; 9:ra17). ONC201 was chemically modified to increase the potency and selectivity against cancer cells, resulting in the new analog ONC213. In this study, we investigated the antileukemic activity and the underlying molecular mechanism of ONC213 in preclinical AML models. ONC213 activity in AML cell lines and primary AML patient samples was first tested in vitro. MTT assay results revealed that ONC213 IC50s ranged from 91.7 nM to 2.4 µM in AML cell lines and primary AML patient samples, which are achievable in vivo based on results from a PK study in mice (a single dose of 50 and 100 mg/kg ONC213 resulted in peak plasma concentrations of 3.7 μM and 8 μM, respectively). Annexin V/propidium iodide staining and flow cytometry analysis results showed variable responses for the AML cell lines tested. After 48 h treatment with 500 nM ONC213, striking induction of cell death in MOLM-13 and MV4-11 cells was detected (at least 72% Annexin V+ cells), while THP-1 and U937 cells showed little to no increase in Annexin V+ cells (6-11%). Similar results were obtained in primary AML patient samples. In contrast to the 48 h treatment of THP-1 and U937 cells, increasing the treatment duration to 120 h resulted in greater than 50% Annexin V+ cells, suggesting that a longer exposure time is necessary in some cell lines. In MV4-11 and MOLM-13 cells, initiation of cell death was detected 8 to 12 h post ONC213 treatment. Colony formation assays revealed that ONC213 treatment significantly reduced colony formation capacity of primary AML patient samples to less than 5% compared to vehicle control, while having no significant effect on normal hematopoietic progenitor cells. A primary AML patient sample was treated with or without ONC213 for 48 h, transplanted into NSG mice, and ten weeks later bone marrow was harvested and human CD45+ cells were measured. ONC213 treatment significantly reduced human AML engraftment compared to vehicle control (0.6% vs. 21.3%; p<0.05), demonstrating that ONC213 kills LSCs in vitro. Next, we examined in vivo efficacy of ONC213 against an AML cell line derived xenograft mouse model. MV4-11 cells were injected into NSGS mice through the tail vein. Three days post-injection, the mice were randomized into vehicle control or 125 mg/kg ONC213 cohorts (5 mice per cohort) and treated daily for 8 days. Modest weight loss was noted but was entirely manageable. ONC213 treatment extended the survival of mice by 88% (median survival 62 vs 33 days). Unlike ONC201, ONC213 treatment of AML cells did not increase the expression of TRAIL. Interestingly, RNAseq results showed that 500 nM ONC213 treatment for 48 h downregulated 33 mRNAs in the oxidative phosphorylation (OXPHOS) pathway, suggesting that ONC213 treatment decreases OXPHOS in AML cells. Thus far, six of the downregulated mRNAs (UQCRQ, SDHA, COX6C, NDUFS5, ATP5D, and NDUFB1) were verified by real-time RT-PCR after both 8 h and 48 h ONC213 treatment. LSCs have been shown to be highly reliant on OXPHOS, while normal hematopoietic stem cells and some bulk AML cells can switch to glycolysis for ATP production during times of OXPHOS inhibition. Thus, ONC213 may kill LSCs through inhibition of OXPHOS. In addition to downregulation of OXPHOS related genes, we found that ONC213 treatment downregulates Mcl-1. Since Mcl-1 mediates resistance to the promising Bcl-2-selective inhibitor ABT-199 (Venetoclax) and inhibition of Bcl-2 impairs OXPHOS, ONC213 would likely synergize with ABT-199 in AML cells. Indeed, combined treatment resulted in striking synergistic induction of apoptosis in both AML cell lines and primary patient samples. Enhanced cell death was detected 8 h post combination treatment in both MOLM-13 and MV4-11 cells. Results from colony formation assays revealed that the combination spares normal hematopoietic progenitor cells. Taken together, ONC213 is active as a single agent and in combination with ABT-199 in AML. Disclosures Allen: Oncoceutics: Employment. Stogniew:Oncoceutics: Employment. Prabhu:Oncoceutics: Employment. Ge:MEI Pharma: Research Funding.

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