scholarly journals IL-4 Mediated Upregulation of AMPK/SIRT1/PGC-1α Signalling Pathway Is Bypassed By Mitochondrial Complex I Downregulation By NAMPT Inhibition in Venetoclax and Ibrutinib Treated CLL Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2616-2616
Author(s):  
Subir Roy Chowdhury ◽  
Cheryl Peltier ◽  
Eileen M. McMillan-Ward ◽  
Ryan Saleh ◽  
Tricia Choquette ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Viability ◽  
B Cell ◽  
Combination Treatment ◽  
Mitochondrial Respiration ◽  
Drug Combination ◽  
Complex I ◽  
Single Agent ◽  
Respiration Rates

Abstract Introduction: Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults. Despite significant improvement in the treatment of CLL, drug resistance is emerging when using the single agents ibrutinib or venetoclax. To achieve greater depth of response, combination treatments are being used to eradicate disease. Altered mitochondrial metabolism is a key factor in CLL survival. In order to gain insights into the underlying biology of a promising drug combination treatment, we investigated the combination of venetoclax and ibrutinib on mitochondrial function as well as the B-cell receptor (BCR), apoptotic and adenosine monophosphate activated protein kinase /silent information regulator 1 / peroxisome proliferator-activated receptor-coactivator-1α (AMPK/SIRT1/PGC-1α) signaling pathways in CLL cells. We also evaluated a proposed mechanism of resistance using interleukin-4 (IL-4) to demonstrate the role of a nicotinamide phosphoribosyltransferase (NAMPT) specific inhibitor, FK866, in order to overcome resistance in vitro. Methods: Freshly isolated primary B-cells from CLL patients were treated with venetoclax, ibrutinib or their combination in a dose- and -time responsive fashion. CLL cells were also treated with IL-4 and FK866 in the presence or the absence of the combination treatment. Flow cytometry (Novocyte) was used to assess cell viability, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS). Mitochondrial respiration rates and specific substrate-dependent respiration of individual complexes of the respiratory chain were measured by respirometry (Orobooros O2k oxygraph) and ATP levels by luminometry (Lmax Luminometer, Molecular Devices). Cellular, mitochondrial, and lysosomal morphology was evaluated by Philips CM10 electron microscope and Olympus BX51 fluorescent microscope. Changes in protein levels of signaling pathways were detected by immunoblotting. Results: Each single agent venetoclax or ibrutinib reduced mitochondrial respiration profiles in CLL cells in vitro. The combined effect of these drugs on the respiration profiles, ATP, MMP, ROS and cell viability was more profound than with each agent alone. Proteins involved in 1. BCR [Bruton's tyrosine kinase (BTK); serine/threonine-specific protein kinase (AKT); phospholipase Cɣ2 (PLCɣ2) and extracellular signal-regulated kinase (ERK)], 2. Apoptotic B-cell lymphoma 2 (BCL-2); myeloid cell leukemia-1 (MCL-1) and 3. AMPK/SIRT1/PGC-1α signaling in the venetoclax and ibrutinib combination treated samples were significantly reduced when compared to DMSO and each single agent. AMPK/SIRT1/PGC-1α regulated transcription factors responsible for mitochondrial biogenesis [nuclear respiratory factor (NRF1 and NRF2)] and mitochondrial dynamics related proteins [mitofusin 2 (MFN2) and dynamin-related protein 1 (DRP1)] were preferentially downregulated by the combination treatment. These effects are seen in the morphological changes, as visualized by transmission electron microscopy demonstrating swelling of mitochondria (venetoclax) and vacuole formation (ibrutinib) in addition to the formation of multi-vesicular bodies in the combination. We also validated the impact of the mitochondria and lysosomes using immunofluorescence. In the presence of IL-4 (a secreted cytokine used to activate the BCR), the effects of the combination were negated by the addition of the NAMPT inhibitor, FK866. FK866 also preferentially decreased mitochondrial respiration rates in the presence of Complex I specific substrates and sustained this inhibition in all FK866 containing conditions regardless of IL-4. Conclusions: The combined effect of venetoclax and ibrutinib to target mitochondrial metabolism via the AMPK/SIRT1/PGC-1α signaling pathway provides a rationale for this drug combination treatment. The use of IL-4 identifies a potential path of resistance that can be overcome by NAMPT inhibition by directly targeting Complex I of the electron transport chain of the mitochondria. Disclosures No relevant conflicts of interest to declare.

scholarly journals Anticancer effects of the combined Thai noni juice ethanolic extracts and 5-fluorouracil against cholangiocarcinoma cells in vitro and in vivo

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jeerati Prompipak ◽  
Thanaset Senawong ◽  
Banchob Sripa ◽  
Albert J. Ketterman ◽  
Suppawit Utaiwat ◽  
...  
Keyword(s):  
Nude Mice ◽  
Combination Treatment ◽  
Synergistic Effects ◽  
Single Agent ◽  
Xenograft Model ◽  
Ethanolic Extract ◽  
Model Combination ◽  
Mouse Xenograft Model ◽  
Ethanolic Extracts

AbstractApplication of 5-fluorouracil (5-FU) in cholangiocarcinoma (CCA) is limited by adverse side effects and chemoresistance. Therefore, the combination therapy of 5-FU with other substances, especially natural products may provide a new strategy for CCA treatment. The aim of this study was to evaluate the combination effects of 5-FU and two ethanolic extracts of Thai noni juice (TNJ) products on CCA cell lines and nude mice xenografts. The results of antiproliferative assay showed the combination treatment of 5-FU and each TNJ ethanolic extract exerted more cytotoxicity on CCA cells than either single agent treatment. Synergistic effects of drug combinations can enable the dose reduction of 5-FU. The mechanism underlying a combination treatment was apoptosis induction through an activation of p53 and Bax proteins. In the nude mouse xenograft model, combination treatments of 5-FU with each TNJ ethanolic extract suppressed the growth of CCA cells implanted mice more than single agent treatments with no effects on mouse body weight, kidney, and spleen. Moreover, low doses of TNJ ethanolic extracts reduced the hepatotoxicity of 5-FU in nude mice. Taken together, these data suggested that the ethanolic extracts of TNJ products can enhance the anti-CCA effect and reduce toxicity of 5-FU.

scholarly journals Targeted inhibition of PI3Kα/δ is synergistic with BCL-2 blockade in genetically defined subtypes of DLBCL

Blood ◽  
2019 ◽  
Vol 133 (1) ◽  
pp. 70-80 ◽  
Author(s):  
Kamil Bojarczuk ◽  
Kirsty Wienand ◽  
Jeremy A. Ryan ◽  
Linfeng Chen ◽  
Mariana Villalobos-Ortiz ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Xenograft Model ◽  
Myeloid Cell ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Synergistic Activity ◽  
Genetic Bases

Abstract Inhibition of the B-cell receptor (BCR) signaling pathway is a promising treatment strategy in multiple B-cell malignancies. However, the role of BCR blockade in diffuse large B-cell lymphoma (DLBCL) remains undefined. We recently characterized primary DLBCL subsets with distinct genetic bases for perturbed BCR/phosphoinositide 3-kinase (PI3K) signaling and dysregulated B-cell lymphoma 2 (BCL-2) expression. Herein, we explore the activity of PI3K inhibitors and BCL-2 blockade in a panel of functionally and genetically characterized DLBCL cell line models. A PI3K inhibitor with predominant α/δ activity, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs. The proapoptotic effect of copanlisib was associated with DLBCL subtype-specific dysregulated expression of BCL-2 family members including harakiri (HRK) and its antiapoptotic partner BCL extra large (BCL-xL), BCL2 related protein A1, myeloid cell leukemia 1 (MCL-1), and BCL2 interacting mediator of cell death. Using functional BH3 profiling, we found that the cytotoxic activity of copanlisib was primarily mediated through BCL-xL and MCL-1–dependent mechanisms that might complement BCL-2 blockade. For these reasons, we evaluated single-agent activity of venetoclax in the DLBCLs and identified a subset with limited sensitivity to BCL-2 blockade despite having genetic bases of BCL-2 dysregulation. As these were largely BCR-dependent DLBCLs, we hypothesized that combined inhibition of PI3Kα/δ and BCL-2 would perturb BCR-dependent and BCL-2–mediated survival pathways. Indeed, we observed synergistic activity of copanlisib/venetoclax in BCR-dependent DLBCLs with genetic bases for BCL-2 dysregulation in vitro and confirmed these findings in a xenograft model. These results provide preclinical evidence for the rational combination of PI3Kα/δ and BCL-2 blockade in genetically defined DLBCLs.

scholarly journals PL3.6 Targeting GSK-3 activity promotes mitotic catastrophe via centrosome destabilisation and enhances the effect of radiotherapy in glioma models

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii4-iii4
Author(s):  
A Bruning-Richardson ◽  
H Sanganee ◽  
S Barry ◽  
D Tams ◽  
T Brend ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Progression ◽  
Combination Treatment ◽  
Single Agent ◽  
Drug Penetration ◽  
In Vivo Models ◽  
Human Astrocytes ◽  
Normal Human

Abstract BACKGROUND Targeting kinases as regulators of cellular processes that drive cancer progression is a promising approach to improve patient outcome in GBM management. The glycogen synthase kinase 3 (GSK-3) plays a role in cancer progression and is known for its pro-proliferative activity in gliomas. The anti-proliferative and cytotoxic effects of the GSK-3 inhibitor AZD2858 were assessed in relevant in vitro and in vivo glioma models to confirm GSK-3 as a suitable target for improved single agent or combination treatments. MATERIAL AND METHODS The immortalised cell line U251 and the patient derived cell lines GBM1 and GBM4 were used in in vitro studies including MTT, clonogenic survival, live cell imaging, immunofluorescence microscopy and flow cytometry to assess the cytotoxic and anti-proliferative effects of AZD2858. Observed anti-proliferative effects were investigated by microarray technology for the identification of target genes with known roles in cell proliferation. Clinical relevance of targeting GSK-3 with the inhibitor either for single agent or combination treatment strategies was determined by subcutaneous and orthotopic in vivo modelling. Whole mount mass spectroscopy was used to confirm drug penetration in orthotopic tumour models. RESULTS AZD2858 was cytotoxic at low micromolar concentrations and at sub-micromolar concentrations (0.01 - 1.0 μM) induced mitotic defects in all cell lines examined. Prolonged mitosis, centrosome disruption/duplication and cytokinetic failure leading to cell death featured prominently among the cell lines concomitant with an observed S-phase arrest. No cytotoxic or anti-proliferative effect was observed in normal human astrocytes. Analysis of the RNA microarray screen of AZD2858 treated glioma cells revealed the dysregulation of mitosis-associated genes including ASPM and PRC1, encoding proteins with known roles in cytokinesis. The anti-proliferative and cytotoxic effect of AZD2858 was also confirmed in both subcutaneous and orthotopic in vivo models. In addition, combination treatment with AZD2858 enhanced clinically relevant radiation doses leading to reduced tumour volume and improved survival in orthotopic in vivo models. CONCLUSION GSK-3 inhibition with the small molecule inhibitor AZD2858 led to cell death in glioma stem cells preventing normal centrosome function and promoting mitotic failure. Normal human astrocytes were not affected by treatment with the inhibitor at submicromolar concentrations. Drug penetration was observed alongside an enhanced effect of clinical radiotherapy doses in vivo. The reported aberrant centrosomal duplication may be a direct consequence of failed cytokinesis suggesting a role of GSK-3 in regulation of mitosis in glioma. GSK-3 is a promising target for combination treatment with radiation in GBM management and plays a role in mitosis-associated events in glioma biology.

Rational Combinations Including a Novel Syk Inhibitor, Fostamatinib Disodium (FosD) in Diffuse Large B Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 283-283
Author(s):  
Randall M Rossi ◽  
Valerie Grose ◽  
Polly Pine ◽  
Richard I Fisher ◽  
Craig T. Jordan ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cell Viability ◽  
B Cell ◽  
Cell Line ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
In Vivo Studies

Abstract Abstract 283 Certain malignant B-cells rely upon B-cell receptor-mediated survival signals. Spleen tyrosine kinase (Syk) initiates and amplifies the B-cell receptor-mediated signal. We and others have demonstrated that fostamatinib disodium (FosD: a prodrug of R406, a potent and specific inhibitor of Syk) induces apoptosis in lymphoma cell lines and primary tumors. A recent clinical trial has demonstrated significant clinical activity of FosD in relapsed/refractory B-cell non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia, and minimal overlap in toxicities with conventional agents. Given this background, future development in B-cell NHL will include rational combinations of FosD and currently available therapies. Therefore, we conducted in vitro and in vivo studies of rational combinations including FosD, in anticipation of clinical trial development. First, using a human DLBCL cell line of GCB genotype, (OCI-Ly19), we analyzed in vitro the combination of R406 with the following agents: fludarabine, rapamycin, rituximab, bendamustine and bortezomib. Increased cytotoxicity was observed using in vitro culture assays with the addition of fludarabine, rapamycin, or rituximab to R406. Cell viability at 72 hours was 25% with R406 alone, 27% for fludarabine alone, and only 9% for the fludarabine/R406. At 48 hours, cell viability was 49% using R406 alone, 31% using rituximab alone, and 21% for rituximab/R406. At 120 hours using primary lymphoma cells (DLCL27), there were no viable cells treated with the rapamycin/FosD combination, compared with rapamycin alone (7%) or FosD alone (25%) The addition of bortezomib or bendamustine to FosD resulted in only a minimal additive increase in cytotoxicity. Results with all combinations were similar with the OCI-Ly10 human DLBCL line of ABC genotype. We then performed in vivo studies by subcutaneous transplantation of the DLBCL cell line OCI-Ly19, (engineered to express luciferase allowing for real time in vivo imaging) into immune deficient NOD/SCID mice which reproducibly formed tumors. Recipient animals were separated into uniform cohorts when the tumors were less than or equal to 500 mm3 in size. The animals were then simultaneously treated with FosD (n=7; 3 gm/kg ad. lib.; translates into 2-5 micromolar R406 systemically throughout the 24h period) and either bortezomib, (n=6; 0.4 mg/kg weekly IP), or rituximab, (n=13; 3 mg/kg, 2x weekly IP). Analysis of the OCI-Ly19 tumor volumes at day 46 showed a median of 2364 mm3 with bortezomib alone compared with 1823 mm3 with bortezomib and FosD. When FosD was combined with rituximab the most significant cytotoxicity was observed: (p=0.01; median tumor volume of 497 mm3 following the combination) in comparison to either FosD alone (3150 mm3) or rituximab alone (1764 mm3). We conclude that the addition of FosD appears to increase activity against NHL of several drugs, including fludarabine and rapamycin. These agents have significant activity in indolent and mantle cell NHL as well as CLL. Moreover, there is no evidence that FosD impedes rituximab responses in vitro or in vivo; in fact we have suggested possible synergy with the combination of rituximab and FosD. Based upon the documented single agent activity of FosD in humans, and this data, clinical trials are now indicated using these promising combinations in NHL and CLL. Disclosures: Pine: Rigel: Employment. Friedberg:Rigel: 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.

A phase II, multicenter study of bendamustine HCl plus rituximab in relapsed indolent B-cell and mantle-cell non-Hodgkin’s lymphoma (NHL)

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 7528-7528 ◽  
Author(s):  
M. E. Williams ◽  
P. Cohen ◽  
A. Tulpule ◽  
R. H. Van der Jagt ◽  
J. A. Herst ◽  
...  
Keyword(s):  
B Cell ◽  
Phase Ii ◽  
Multicenter Study ◽  
Single Agent ◽  
Objective Response ◽  
Progression Free Survival ◽  
Mitotic Catastrophe ◽  
Hematologic Toxicity ◽  
Mantle Cell

7528 Background: Bendamustine HCl, a novel alkylating hybrid agent, has single-agent activity in multiple hematologic and solid tumors. In vitro data have demonstrated the multifunctional mechanisms of bendamustine by which cell death occurs via both apoptosis and mitotic catastrophe. Bendamustine has shown activity in NHL cell lines that are refractory to conventional alkylator chemotherapies. The combination of bendamustine and rituximab has shown a synergistic effect on NHL cells. The efficacy and safety of bendamustine in combination with rituximab in patients with relapsed NHL were evaluated. Methods: This phase II, multicenter study enrolled adult patients with relapsed, indolent, rituximab-sensitive B-cell or mantle-cell NHL. Patients received rituximab 375 mg/m2 IV on day 1 and bendamustine 90 mg/m2 IV on days 2 and 3 of a 28-day cycle for 4 to 6 cycles. An additional dose of rituximab 375 mg/m2 IV was given 1 week prior to the first cycle of bendamustine and 4 weeks after the last cycle. Results: The intent-to-treat population included 67 patients (57% males; median age, 60 years) with indolent NHL (81%) or mantle-cell NHL (16%) (data not available [3%]). A total of 81% of patients had stage III/IV disease. Patients had relapsed from a median of 1 prior therapy; 37% had prior treatment with rituximab. In the 57 evaluable patients, the overall objective response rate (ORR) was 87% (complete response [CR], 33%). The ORR for the 9 evaluable mantle-cell NHL patients was 89% (CR, 33%). For all patients, the median duration of response and progression-free survival had not been reached after a median follow-up of 3.7 months (range, 0–14.2 months). This therapy was well tolerated. Most commonly reported nonhematologic toxicities were grade 1/2 gastrointestinal events, with nausea being the greatest (38%). The primary grade 3/4 hematologic toxicity was neutropenia (29%), with 1 event of sepsis. Conclusions: Bendamustine in combination with rituximab was well tolerated and produced high response rates in patients with relapsed indolent and mantle-cell NHL. These results suggest bendamustine in combination with rituximab provides a potential benefit over single-agent rituximab therapy. [Table: see text]

Mevalonate pathway inhibitors in the treatment of B-cell chronic lymphocytic leukemia.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 6561-6561
Author(s):  
Ravi Kiran Bobba ◽  
Indira Benakanakere ◽  
Smitha Bearelly ◽  
Monica Arya ◽  
Richard Sleigtholm ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Viability ◽  
B Cell ◽  
Combination Treatment ◽  
Mevalonate Pathway ◽  
Chemotherapeutic Agents ◽  
Lymphocytic Leukemia ◽  
Cell Chronic Lymphocytic Leukemia

6561 Background: B-cell chronic lymphocytic leukemia (CLL) cells are arrested in G0/G1 phase of the cell cycle and are resistant to programmed cell death, hypothesized to contribute to the resistance of CLL cells to standard chemotherapy with curative intent. Methods: Mec-2 cells and Wac-3 cells are CLL cells that have been shown to be resistant to fludarabine and rituximab. We tested a novel enzyme inhibitor’s ability to render CLL cells sensitive to fludarabine and rituximab. Results: BIBB515, a lanosterol synthase inhibitor, at a concentration of 10μM, was able to reduce the cell viability from 82% in controls to 65% after 72 hours. Fludarabine 10μM alone did not reduce the cell viability, 82 % in controls compared to 80%. BIBB515+ fludarabine treatment for 72 hours, at the dose of 10μM each decreased the cell viability to 37%. Cell proliferation by MTT assay was 0.66±0.010 in control compared to 0.37±0.01 in BIBB515+fludarbine and 0.21±0.01 in BIBB515+ fludarabine+ rituximab. There is a 68% down-regulation of cell proliferation using this treatment. There was a two fold induction of CD 20 with combination treatment, and BIBB515 treatment. The mechanism of cell death in the combination treatment of BIBB515 and fludarabine may be due to the up regulation of cell surface marker CD-20. WAC-3 is another CLL cell line that is sensitive to fludarabine, and resistant to rituximab. BIBB515 sensitizes WAC-3 cells to CD 20 antibody rituximab. There is a 68.7% decrease in cell proliferation with combination treatment of BIBB515 and rituximab. Proliferation of Mec-2 cells were inhibited by 60µM and 30µM terbinafine. Ro-48-8071, showed dose-dependent activity, alone or in combination to fludarabine was seen to induce cell death in Mec-2 cells. Fludarabine alone did not have any effect on these cells. Conclusions: Inhibitors of the mevalonate pathway make resistant CLL cells sensitive to current chemotherapeutic agents. Exploiting this mechanism could alter the current treatment regimens, leading to control of the disease in advanced stages by either inducing the leukemic cells to be static or to regress. This strategy may also limit the toxicities involved with chemotherapy.

Metal-induced hormesis requires cPKC dependent glucose transport and lowered respiration

2001 ◽  
Vol 20 (7) ◽  
pp. 347-358 ◽  
Author(s):  
L H Damelin ◽  
J J Alexander
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Mitochondrial Respiration ◽  
In Vitro Toxicity ◽  
Protein Kinase Activity ◽  
Energy Status ◽  
Independent Events ◽  
Cytotoxicity Studies

Previously, cytotoxicity studies using an 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT)based in vitro toxicity assay found that low concentrations of mercuric, cadmium and cupric chloride (0.7, 1 and 3 μM, respectively) induced hormesis in McCoy cells after 24 h exposure. An investigation of the biochemical events required for the induction of this phenomenon revealed that hormesis was dependent on two simultaneous but independent events, namely, an 11-15% conventional protein kinase C (cPKC)-dependent increase in glucose uptake and a protein synthesis-dependent 19-23% drop in mitochondrial respiration. The inhibition of either event was sufficient to abolish hormesis for all three metal toxicants. Furthermore, an investigation of the energy status of cells prior to and during hormesis revealed an oscillating level of ATP production found to be in phase with mitochondrial respiration, independent of cPKC activated glucose transport and found to coincide with a 16-20% drop in AMP-activated protein kinase activity. These findings suggest that hormesis is not a form of energy compensation but is most likely a reductive burst where an increase in glucose uptake together with a simultaneous reduction in oxygen consumption results in a significant increase in reduction equivalents, which may then be utilized by cells to counteract the effects of oxidative stress induced by heavy metal toxicants.

scholarly journals Targeted Inhibition of PI3K α/δ Is Synergistic with BCL-2 Blockade in Genetically Defined Subtypes of DLBCL

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 39-39
Author(s):  
Kamil Bojarczuk ◽  
Kirsty Wienand ◽  
Jeremy A. Ryan ◽  
Linfeng Chen ◽  
Mariana Villalobos-Ortiz ◽  
...  
Keyword(s):  
Tumor Growth ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Research Report ◽  
Genetic Bases

Abstract Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease that is transcriptionally classified into germinal center B-cell (GCB) and activated B-cell (ABC) subtypes. A subset of both GCB- and ABC-DLBCLs are dependent on B-cell receptor (BCR) signaling. Previously, we defined distinct BCR/PI3K-mediated survival pathways and subtype-specific apoptotic mechanisms in BCR-dependent DLBCLs (Cancer Cell 2013 23:826). In BCR-dependent DLBCLs with low baseline NF-κB activity (GCB tumors), targeted inhibition or genetic depletion of BCR/PI3K pathway components induced expression of the pro-apoptotic HRK protein. In BCR-dependent DLBCLs with high NF-κB activity (ABC tumors), BCR/PI3K inhibition decreased expression of the anti-apoptotic NF-κB target gene, BFL1. Our recent analyses revealed genetic bases for perturbed BCR/PI3K signaling and defined poor prognosis DLBCL subsets with discrete BCR/PI3K/TLR pathway alterations (Nat Med 2018 24:679). Cluster 3 DLBCLs (largely GCB tumors) exhibited frequent PTEN deletions/mutations and GNA13 mutations. Cluster 5 DLBCLs (largely ABC tumors) had frequent MYD88L265P and CD79B mutations that often occurred together. These DLBCL subtypes also had different genetic mechanisms for deregulated BCL2 expression - BCL2 translocations in Cluster 3 and focal (18q21.33) or arm level (18q) BCL2 copy number gains in Cluster 5. These observations prompted us to explore the activity of PI3K inhibitors and BCL2 blockade in genetically defined DLBCLs. We utilized a panel of 10 well characterized DLBCL cell line models, a subset of which exhibited hallmark genetic features of Cluster 3 and Cluster 5. We first evaluated the cytotoxic activity of isoform-specific, dual PI3Kα/δ and pan-PI3K inhibitors. In in vitro assays, the PI3Kα/δ inhibitor, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs. We next assessed the transcriptional abundance of BCL2 family genes in the DLBCLs following copanlisib treatment. In BCR-dependent GCB-DLBCLs, there was highly significant induction of the pro-apoptotic HRK. In BCR-dependent ABC-DLBCLs, we observed significant down-regulation of the anti-apoptotic BFL1 protein and another NF-κB target gene, BCLxL (the anti-apoptotic partner of HRK). We then used BH3 profiling, to identify dependencies on certain BCL2 family members and to correlate these data with sensitivity to copanlisib. BCLxL dependency significantly correlated with sensitivity to copanlisib. Importantly, the BCLxL dependency was highest in DLBCL cell lines that exhibited either transcriptional up-regulation of HRK or down-regulation of BCLxL following copanlisib treatment. In all our DLBCL cell lines, PI3Kα/δ inhibition did not alter BCL2 expression. Given the genetic bases for BCL-2 deregulation in a subset of these DLBCLs, we next assessed the activity of the single-agent BCL2 inhibitor, venetoclax, in in vitro cytotoxicity assays. A subset of DLBCL cell lines was partially or completely resistant to venetoclax despite having genetic alterations of BCL2. We postulated that BCR-dependent DLBCLs with structural alterations of BCL2 might exhibit increased sensitivity to combined inhibition of PI3Kα/δ and BCL2 and assessed the cytotoxic activity of copanlisib (0-250 nM) and venetoclax (0-250 nM) in the DLBCL cell line panel. The copanlisib/venetoclax combination was highly synergistic (Chou-Talalay CI<1) in BCR-dependent DLBCL cell lines with genetic bases of BCL2 deregulation. We next assessed copanlisib and venetoclax activity in an in vivo xenograft model using a DLBCL cell line with PTENdel and BCL2 translocation (LY1). In this model, single-agent copanlisib did not delay tumor growth or improve survival. Single-agent venetoclax delayed tumor growth and improved median survival (27 vs 51 days, p<0.0001). Most notably, we found that the combination of copanlisib and venetoclax delayed tumor growth significantly longer than single-agent venetoclax (p<0.0001). Additionally, the combined therapy significantly increased survival in comparison with venetoclax alone (median survival 51 days vs not reached, p<0.0013). Taken together, these results provide in vitro and in vivo pre-clinical evidence for the rational combination of PI3Kα/δ and BCL2 blockade and set the stage for clinical evaluation of copanlisib/venetoclax therapy in patients with genetically defined relapsed/refractory DLBCL. Disclosures Letai: AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Novartis: Consultancy, Other: Lab research report; Vivid Biosciences: Equity Ownership; AstraZeneca: Consultancy, Other: Lab research report. Shipp:AstraZeneca: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.

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