Lumiliximab Triggers Apoptosis Mechanisms in CLL Cells through the Inhibition of PI3-K/Akt Pathway.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2371-2371
Author(s):  
Medhat Shehata ◽  
Susanne Schnabl ◽  
Elena Ponath ◽  
Stefanie Tauber ◽  
Dita Demirtas ◽  
...  
Keyword(s):  
Cell Viability ◽  
Research Funding ◽  
Repeated Exposure ◽  
Akt Pathway ◽  
Cross Linking ◽  
Serine Residue ◽  
Single Exposure ◽  
Previous Therapy ◽  
Apoptotic Effect

Abstract Abstract 2371 Poster Board II-348 Chronic lymphocytic leukemia (CLL) is a clonal expansion of B cells which is characterized by a defect in apoptosis and is associated with the co-expression of CD19, CD5 and CD23. Lumiliximab is a monoclonal antibody against CD23 which has been shown to exert a promising therapeutic effect in CLL in vivo and to induce apoptosis in CD23+ lymphoma cells in vitro. The aim of this study was to investigate the direct effect of lumiliximab on cell viability and to explore its molecular mechanism of action in primary CLL cells. PBMC from twenty CLL patients were used in this study. Nine patients had previous therapy and 11 were untreated. Nine patients had unmutated and 11 had mutated IgVH genes. The mean percentage of CD19+/CD5+/CD23+ cells was 80% (range 53-98%). PBMC were exposed to various concentrations of lumiliximab (1-50 μg/mL) for various durations (1-15 days). The long term incubation with lumiliximab was performed in a microenvironment co-culture model using primary human stromal cells which prevent spontaneous apoptosis of CLL cells. Cell viability was assessed by flow cytometric analysis using annexin V/PI staining and by MTT assays. The results showed that single exposure to lumiliximab had a minimal effect on cell viability (< 1 fold increase in apoptosis rate compared to untreated cells and to the isotype control antibody). Cross-linking with goat anti-human IgG was more effective in inducing cell death. Interestingly, repeated exposure to lumiliximab without cross-linking had a significant pro-apoptotic effect selectively in the CD19+/CD5+ cells. Western blotting analysis demonstrated a significant biological response to the single and repeated exposure to lumiliximab in spite of the moderate pro-apoptotic effect. Lumiliximab induced a significant decrease in Bcl-2, Mcl-1 and Hsp70 protein expression. In addition, it resulted in a significant decrease in the phosphorylation of Akt1 at serine residue 473 and dephosphorylation (activation) of the tumor suppressor PTEN at serine residue 380 suggesting the involvement of the PI3-K/Akt/PTEN cascade in priming CLL cells to undergo apoptosis by lumiliximab. Pre-incubation of CLL cells with lumiliximab enhanced the pro-apoptotic effect of PI3-K inhibitor LY292004 and the CK2 inhibitor apigenin. Consistent with previous observations, pre-exposure of CLL cells to lumiliximab augmented the cytotoxic effect of Fludarabine. The in vitro response to lumiliximab did not appear to be influenced by IgVH mutation status, cytogenetics or by previous therapy. To gain further insight into the downstream targets of lumiliximab in CLL, microarray analysis and pathway exploration was performed. The data revealed that lumiliximab regulates several sets of genes which are involved in chemokine signaling, cytokine/cytokine receptor interaction, oxidative stress, PI3-K and integrin signaling, complement cascade and Toll-like receptor signaling. Single exposure to lumiliximab led to down-regulation of several genes including LY9, GPR183, RGS1, HSPA6, and INPP5F and up-regulation of FN1, FAIM3 and LOX. Repeated exposure to Lumiliximab led to a significant down-regulation of TXNIP, CTSB, SODS, IFI44, IRF7, TNFSF13B, MS4A7, CCL4, CCL7, CCL8, cathepsin B, MARKS, ADAMS and FCER1G and up-regulation of SPP1, C10orf10, ANGPTL6, RBBP4 and GSTA4. In conclusion, these data demonstrate that repeated exposure to Lumiliximab is effective in priming CLL cells to undergo apoptosis through inactivation of the PI3-K/Akt pathway and render the cells more sensitive to cytotoxic compounds. The data also provide further evidence of a promising therapeutic role for lumiliximab in CLL and a rationale for lumiliximab-based drug combinations to improve treatment of this incurable disease. Disclosures: Shehata: Biogen Idec: Research Funding. Hughes:Biogen Idec: Employment. Maclaren:Biogen Idec: Employment. Jaeger:Biogen Idec: Research Funding.

scholarly journals β-elemene alleviates airway stenosis via the ILK/Akt pathway modulated by MIR143HG sponging miR-1275

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Guoying Zhang ◽  
Cheng Xue ◽  
Yiming Zeng
Keyword(s):  
Cell Cycle ◽  
Cell Viability ◽  
Cell Model ◽  
Protective Efficacy ◽  
Rabbit Model ◽  
Akt Pathway ◽  
Loss Of Function ◽  
Airway Stenosis

Abstract Background We have previously found that β-elemene could inhibit the viability of airway granulation fibroblasts and prevent airway hyperplastic stenosis. This study aimed to elucidate the underlying mechanism and protective efficacy of β-elemene in vitro and in vivo. Methods Microarray and bioinformatic analysis were used to identify altered pathways related to cell viability in a β-elemene-treated primary cell model and to construct a β-elemene-altered ceRNA network modulating the target pathway. Loss of function and gain of function approaches were performed to examine the role of the ceRNA axis in β-elemene's regulation of the target pathway and cell viability. Additionally, in a β-elemene-treated rabbit model of airway stenosis, endoscopic and histological examinations were used to evaluate its therapeutic efficacy and further verify its mechanism of action. Results The hyperactive ILK/Akt pathway and dysregulated LncRNA-MIR143HG, which acted as a miR-1275 ceRNA to modulate ILK expression, were suppressed in β-elemene-treated airway granulation fibroblasts; β-elemene suppressed the ILK/Akt pathway via the MIR143HG/miR-1275/ILK axis. Additionally, the cell cycle and apoptotic phenotypes of granulation fibroblasts were altered, consistent with ILK/Akt pathway activity. In vivo application of β-elemene attenuated airway granulation hyperplasia and alleviated scar stricture, and histological detections suggested that β-elemene's effects on the MIR143HG/miR-1275/ILK axis and ILK/Akt pathway were in line with in vitro findings. Conclusions MIR143HG and ILK may act as ceRNA to sponge miR-1275. The MIR143HG/miR-1275/ILK axis mediates β-elemene-induced cell cycle arrest and apoptosis of airway granulation fibroblasts by modulating the ILK/Akt pathway, thereby inhibiting airway granulation proliferation and ultimately alleviating airway stenosis.

scholarly journals In Vitro Evaluation of the Neuroprotective Effect of Panax notoginseng Saponins by Activating the EGFR/PI3K/AKT Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Shuang Wu ◽  
Tiantian Yang ◽  
Kai Cen ◽  
Yihuai Zou ◽  
Xiaowei Shi ◽  
...  
Keyword(s):  
Cell Viability ◽  
Expression Profiles ◽  
Neuroprotective Effect ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Panax Notoginseng ◽  
Akt Pathway ◽  
Neuroprotective Effects ◽  
Traditional Chinese Herbal Medicine

Context. About 15 million people worldwide suffer strokes each year and 5 million people are left with permanent disabilities which is due to the loss of local blood supply to the brain, resulting in a neurologic deficit. Panax notoginseng (Bruk.) F. H. Chen (Araliaceae) is a traditional Chinese herbal medicine widely used in the treatment of cardio-cerebrovascular diseases. Objective. This study investigated whether Panax notoginseng saponins (PNS) extracted from Panax notoginseng (Bruk.) F. H. Chen played a neuroprotective role by affecting the EGFR/PI3K/AKT pathway in oxygen-glucose deprived (OGD) SH-SY5Y cells. Materials and Methods. Different groups of OGD SH-SY5Y cells were treated with varying doses of PNS, PNS + AG1478 (a specific inhibitor of EGFR), or AG1478 for 16 hours. CCK8, Annexin V-FITC/PI apoptosis analysis, and LDH release analysis were used to determine cell viability, apoptosis rate, and amounts of LDH. Quantitative real-time PCR (q-RT-PCR) and western blotting were used to measure mRNA and proteins levels of p-EGFR/EGFR, p-PI3K/PI3K, and p-AKT/AKT in SH-SY5Y cells subjected to OGD. Results. PNS significantly enhanced cell viability, reduced apoptosis, and weakened cytotoxicity by inhibiting the release of LDH. The mRNA expression profiles of EGFR, PI3K, and AKT showed no difference between model and other groups. Additionally, ratios of p-EGFR, p-PI3K, and p-AKT to EGFR, PI3K, and AKT proteins expression, respectively, all increased significantly. Conclusions. These findings indicate that PNS enhanced neuroprotective effects by activating the EGFR/PI3K/AKT pathway and elevating phosphorylation levels in OGD SH-SY5Y cells.

scholarly journals The Development of a Precision Medicine Platform to Overcome Ibrutinib Resistance in Mantle Cell Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1487-1487
Author(s):  
Yang Liu ◽  
Shuangtao Zhao ◽  
Changying Jiang ◽  
Yixin Yao ◽  
Kelley Paige Murfin ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Akt Pathway ◽  
Therapeutic Resistance ◽  
Patient Sample ◽  
Mantle Cell ◽  
Btk Inhibitor ◽  
Ibrutinib Resistance

Background: While mantle cell lymphoma (MCL) initially responds to frontline therapies, this aggressive B-cell malignancy typically relapses or becomes resistant to treatment. Despite high overall response rates to the oral, covalent, first-in-class Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, most patients with relapsed/refractory MCL ultimately experience disease progression. To address the critical issue of BTK inhibitor resistance, novel therapies must be developed. Moreover, diversity in genetic alterations and the possibility that multiple pathway aberrations may contribute to disease progression and resistance makes overcoming this phenomenon with uniform treatment regimens extremely difficult, indicating that a personalized approach should be developed to overcome therapeutic resistance. In this study, molecular profiling of ibrutinib-resistant MCL patient samples has been conducted to identify targetable dysregulated signaling pathways and gene mutations associated with ibrutinib resistance. Clinical drug candidates targeting these potential pathways and their combinations with ibrutinib were analyzed in vitro to identify novel treatments that may potentially overcome ibrutinib resistance. Methods: Twenty-three agents targeting multiple pathways associated with ibrutinib resistance were deliberately chosen based on known targetable pathways in MCL to assess via high throughput drug screening. MCL tumor cells were isolated and purified from clinical apheresis and tumor excisional biopsies under established IRB-approved protocols. The same patient primary cells were subjected to gene expression analysis using a nanoString nCounter panel and whole exome sequencing (WES) to identify targetable dysregulated pathways and somatic mutations within each tumor. In vitro cell viability assays of single agents and drug combinations were tested per patient sample using the CellTiter-Glo luminescent assay (Promega), interrogating dysregulated pathways identified in each tumor. Subcutaneous, intravenous, and subrenal injections of the purified patient tumor cells were performed on NSG mice to create corresponding PDX mouse models for validation experiments. Results: nanoString nCounter analysis identified differentially expressed targetable pathways per patient sample such as BCR signaling, the PI3K/AKT pathway, NOTCH signaling, the cell cycle, and the NF-κB pathway. Correlations were identified between the WES and the nanoString nCounter analysis. For example, PTEN loss was observed in an MCL patient sample with high PI3K/AKT expression, demonstrating the potential underlying mechanism for the observed PI3K/AKT enrichment. Patients were divided into subgroups based on the identified responsive pathways in the in vitro screening. For example, PI3K/AKT pathway inhibitors were shown to be more potent against MCL samples in which the PI3K/AKT pathway was enriched. To further validate this finding, we created an MCL PDX model using a sample with enriched PI3K/AKT expression and successfully recapitulated the splenomegaly and hepatomegaly observed in the MCL patient. The MCL PDX mice were treated with the pan-PI3K inhibitor copanlisib (IP, 10 mg/kg) using a 5 on/2 off dosing schedule, which resulted in significantly reduced spleen (P &lt; 0.001) and liver size (P &lt; 0.01), as well as bone marrow involvement (P &lt; 0.05), compared with the vehicle control (Figure 1). Conclusions: Molecular matching with in vitro drug screening were utilized to develop a precision medicine platform for MCL to combat therapeutic resistance. This platform can be translated into a clinical setting to directly benefit the MCL patient population through treatment with therapies directly tailored to each patient. Disclosures Wang: Pulse Biosciences: Consultancy; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Acerta Pharma: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Dava Oncology: Honoraria; Kite Pharma: Consultancy, Research Funding; Juno Therapeutics: Research Funding; Celgene: Consultancy, Research Funding; MoreHealth: Consultancy, Equity Ownership; BioInvent: Consultancy, Research Funding; Aviara: Research Funding; BeiGene: Research Funding; Loxo Oncology: Research Funding; VelosBio: Research Funding.

Interaction between B-CLL Cells and Lymphoid Microenvironment Leads to Activation of PI3-K/Akt Pathway and Inhibition of Apoptosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2102-2102
Author(s):  
Medhat Shehata ◽  
Susanne Schnabl ◽  
Dita Demirtas ◽  
Josef D. Schwarzmeier ◽  
Martin Hilgarth ◽  
...  
Keyword(s):  
Cell Interaction ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Akt Pathway ◽  
Nf Kappa B ◽  
Stromal Fibroblasts ◽  
Apoptotic Effect ◽  
Induced Apoptosis ◽  
Novel Therapeutic Strategies

Abstract Inhibition of apoptosis and long survival leads to accumulation of the leukemic cells in B cell chronic lymphocytic leukemia (B-CLL). This could be due to activation of anti-apoptotic cascades in CLL cells through interaction with their lymphoid microenvironment. Therefore, we investigated the role of tumor microenvironment in prolongation of survival of B-CLL cells and activation of the potent anti-apoptotic PI3-K/Akt pathway. Stromal fibroblasts of bone marrow (BMFs), spleen (SF) and lymph gland (LGF) were used as an in vitro model for lymphoid microenvironment and we tested their ability to inhibit spontaneous apoptosis of B-CLL cells. Co-culture of B-CLL cells with human BMFs, LGF, and SF significantly inhibited apoptosis and prolonged survival of the leukemic cells in comparison to suspension cultures and to co-cultures with fibroblasts obtained from non-lymphoid organs. Trans-well culture experiments indicated that cell-cell interaction and soluble mediators are essential for this supportive effect. To explore the involvement of PI3-K/Akt pathway in the anti-apoptotic effect of stromal fibroblasts, co-cultures were performed in presence of PI3-K inhibitors (wortmannin or Ly294002) or siRNAs against PI3-K (p110ß subunit) and Akt1. These inhibitors significantly reduced the supportive effect of stromal fibroblasts and induced apoptosis in B-CLL cells. Interestingly, the leukemic cells were far more sensitive to PI3-K inhibition than T cells, monocytes and fibroblasts. Induction of apoptosis was associated with a significant decrease in the intracellular PIP3, PI3-K, PDK1 and Akt1, NF-kappa B, IKK and de-phosphorylation/activation of tumor suppressor protein PTEN. Studies using phosphospecific anti-PTEN antibody demonstrated that PBMC of CLL patients (n=40) highly express a phosphorylated form of PTEN. The results demonstrate that the PI3-K/Akt pathway is involved in inhibition of apoptosis of B-CLL cells and suggest that interaction of the leukemic cells with lymphoid microenvironment maintains the activation of this pathway. The data also suggest that targeting this pathway represents a new option for designing novel therapeutic strategies in B-CLL.

Lymphoid Microenvironment Inhibits Apoptosis in B-CLL Cells: Involvement of PI3-K/AKT Pathway and PTEN.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1443-1443
Author(s):  
Medhat Shehata ◽  
Susanne Schnabl ◽  
Dita Demirtas ◽  
Josef D. Schwarzmeier ◽  
Martin Hilgarth ◽  
...  
Keyword(s):  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Leukemic Cells ◽  
Prolonged Survival ◽  
Akt Pathway ◽  
Tail Domain ◽  
Stromal Fibroblasts ◽  
Apoptotic Effect ◽  
Induced Apoptosis

Abstract The activation of anti-apoptotic mechanisms in the leukemic B cells in chronic lymphocytic leukemia (B-CLL) through the interaction with their microenvironment may lead to prolonged survival and the accumulation of the malignant clone. The aim of this study is to elucidate the influence of the lymphoid microenvironment in the activation of the potent anti-apoptotic PI3-K/Akt pathway and to investigate the pattern of expression of the tumor suppressor PTEN in B-CLL. Stromal fibroblasts of bone marrow (BMF), spleen (SF) and lymph gland (LGF) were used as an in vitro model for lymphoid microenvironment. Pharmacological inhibitors and siRNAs against PI3-K and Akt were applied to explore the anti-apoptotic effect of this pathway in B-CLL. The results showed that co-cultivation of B-CLL cells with human BMF, LGF, and SF significantly inhibited apoptosis and prolonged survival of the leukemic cells in comparison to suspension cultures. To explore the involvement of PI3-K/Akt pathway in the anti-apoptotic effect of stromal fibroblasts, co-cultures were performed in presence of PI3-K inhibitors (wortmannin or LY294002) or siRNAs against PI3-K and Akt1. These inhibitors significantly reduced the supportive effect of stromal fibroblasts and induced apoptosis in B-CLL cells. The leukemic cells were more sensitive to PI3-K inhibition than T cells, monocytes and stromal fibroblasts. Induction of apoptosis was associated with a significant decrease in the intracellular levels of PIP3, PI3-K, PDK1, Akt1, NF-kappa B, IKK, and dephosphorylation (activation) of PTEN. Since PTEN activity, as a negative regulator for PI3-K signalling, is controlled by its phosphorylation at the tail domain, we studied the pattern of PTEN protein expression in B-CLL. Western blotting demonstrated that the total PTEN in PBMC of B-CLL patients (n=40) is comparable to healthy individuals (n=8). However, using phosphospecific anti-PTEN antibody demonstrated that samples of B-CLL patients highly express phosphorylated (inactive) forms of PTEN in comparison to healthy persons. In conclusion, the results demonstrate that PI3-K/Akt pathway is involved in inhibition of apoptosis of B-CLL cells and suggest that interaction of the leukemic cells with lymphoid microenvironment may lead to the activation of this pathway. The data also suggest that targeting this pathway represents a feasible therapeutic approach in B-CLL.

Effective Targeting of the PI3-K Pathway in CLL with NVP-BEZ235, a Novel Orally Available Dual PI3K/mTOR Inhibitor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3166-3166 ◽  
Author(s):  
Medhat Shehata ◽  
Susanne Schnabl ◽  
Dita Demirtas ◽  
Stefanie Tauber ◽  
Martin Hilgarth ◽  
...  
Keyword(s):  
Cell Viability ◽  
Suspension Cultures ◽  
Maximum Effect ◽  
Fish Analysis ◽  
Therapeutic Concept ◽  
Mutational Status ◽  
Apoptotic Effect ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis

Abstract There is growing evidence that the anti-apoptotic PI3-K/Akt pathway is involved in pathogenesis and progression of different types of cancer. We have evidence that PI3-K inhibitors such as LY294002 and wortmannin selectively induce apoptosis in CLL cells (Shehata et al Ab. Blood 2006). Recently, a new orally available PI3-K inhibitor, NVP-BEZ235 has been developed. This competitive ATP binding imidazo-quinoline derivative is already in phase I trials against solid tumors. Here we show, for the first time, the effects of NVP-BEZ235 on the viability of CLL cells in vitro. Primary CLL cells from 37 patients were investigated. Sixteen patients were in Binet stage C, 14 in B and 7 in stage A. Seventeen patients had mutated IgVH genes, 15 had unmutated IgVH and mutation status from 5 patients was not available. Fluorescence in situ hybridization (FISH) analysis showed that 23 patients had del(13q), 9 had del(17p), 8 had del(11q) and 4 patients had trisomy 12. Nineteen patients were untreated and 18 patients were previously treated. To overcome the experimental artifact due to the spontaneous apoptosis of CLL cells in vitro, which may mask the actual effect of the tested drugs, we applied a co-culture model using human bone marrow stromal fibroblasts which supports survival of CLL cells ex vivo. CLL cells were exposed to NVP-BEZ235 at different concentrations (1 nM-10 μM) and incubation times (1, 3, 7 days). Cell viability was assessed by annexin-V/propidium iodide staining, flow cytometry and MTT assays. The results showed that cell viability was significantly higher in co-cultures compared to suspension cultures (the percentage of apoptotic cells after 3 days in co-culture was 5±4 compared to 23±12 in suspension cultures, p&lt; 0,01). NVP-BEZ235 induced apoptosis in the majority of CLL samples under both experimental conditions. However, this effect tends to be more remarkable in co-culture than in suspension: 4-10 fold versus 3-fold increase in apoptosis rate respectively. The pro-apoptotic effect was dose and time dependent and could be observed within 16 hours after incubation at 10 nM. A maximum effect was obtained at a concentration of 5–10 μM. The IC50 values varied between patients and were in a range of 250–750 nM. At these concentrations, NVP-BEZ235 was significantly more effective in induction of apoptosis than LY294002. NVP-BEZ235 inhibited the adhesion of CLL cells to stromal cells suggesting that it may interfere with the survival signal provided by the lymphoid microenvironment in addition to its direct effect on the leukemic cells. FACS analysis demonstrated that NVB-BEZ235 specifically targets the leukemic CD19+ cells while a minimal effect on the viability of T cells and monocytes could be observed. The pro-apoptotic effect of NVP-BEZ235 was independent from the mutational status and cytogenetics. In addition, it induced apoptosis in vitro in CLL cells from patients resistant to fludarabine treatment. In parallel to induction of apoptosis in CLL cells, western blotting demonstrated that NVP-BEZ235 significantly inhibited Akt phosphorylation at Ser-473. This effect was also associated with dephosphorylation (activation) of the tumor suppressor PTEN at Ser-380. DNA microarray analysis using Affymetrix U133A Plus 2.0 GeneChips revealed more than 200 genes which were at least 2 fold up- or down-modulated by NVP-BEZ235 in vitro. These genes include LY9, DUSP10, CCR6, RGS2, IRS2, PI4K2A, ISG20, TFRC, EGR1, HSP90, LCK, TNFRSF17, LYZ, TGFBI and TLR10. In conclusion, the results demonstrate a significant and selective pro-apoptotic effect of NVP-BEZ235 in CLL cells. The data point also to the validity of PI3K-pathway inhibition as a novel therapeutic concept for CLL which should be evaluated in clinical trials.

PI3K Inhibition with GDC-0941 Has Greater Efficacy Compared to p110δ-Selective Inhibition with CAL-101 in Mantle Cell Lymphoma and May Be Particularly Advantageous in Multiply Relapsed Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1654-1654 ◽  
Author(s):  
Sunil Iyengar ◽  
Andrew J. Clear ◽  
Andrew Owen ◽  
Lenushka Maharaj ◽  
Janet Matthews ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Western Blotting ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Pi3k Inhibition ◽  
Mantle Cell ◽  
Morphological Variants ◽  
Gsk 3Β

Abstract Abstract 1654 Background: Mantle cell lymphoma (MCL) is an incurable, aggressive subtype of non-Hodgkin lymphoma in which there is a need for novel targeted therapies. Activation of the PI3K-Akt pathway and its role in the pathogenesis of MCL has been highlighted in a number of studies. Constitutive activation of the PI3K pathway inactivates GSK-3β, a downstream target of Akt, that can phosphorylate cyclin D1 resulting in its nuclear export. There is also evidence that cyclin D1 mRNA stability and translation is enhanced by this pathway. The class Ia PI3K p110 catalytic subunit isoforms α, β and δ are primarily implicated in oncogenesis. While the PI3K p110δ isoform is known to be enriched in lymphocytes, a gain of PIK3CA (the gene encoding PI3K p110α) copy number has been shown to be a frequent alteration in MCL. The expression and relative importance of the individual Class Ia PI3K isoforms has not been documented in this disease. With the development of isoform selective inhibitors, this is an important issue that needs to be addressed. Aims: We studied the expression of class Ia PI3K isoforms in primary MCL with relation to morphological variants and disease status. We also compared the efficacy of PI3K inhibition in MCL cell lines and primary samples using two novel inhibitors, GDC-0941(predominantly p110α/δ-selective) and CAL-101 (δ-selective), both of which are in early phase clinical trials. Methods: Tissue microarrays were constructed from triplicate 1mm cores from 144 MCL biopsies and 16 tonsil controls. The levels of p110α, p110β and p110δ isoforms were then determined by immunohistochemistry using isoform-specific antibodies. The in vitro effect of PI3K inhibitors on cell viability and apoptosis was studied in 4 MCL cell lines, (Jeko-1, Granta519, REC-1 and JVM-2), and 15 primary MCL samples. Expression of the class Ia PI3K isoforms and changes in downstream targets of PI3K were determined by western blotting. Results: P110δ was expressed at a consistently higher level in MCL samples and normal tonsil controls compared to the α and β isoforms, while p110β expression was weak and significantly lower than p110α expression. On comparing expression of isoforms at diagnosis and relapse, p110α expression was significantly increased beyond 1st relapse compared to diagnostic biopsies (p=0.04) and tonsil controls (p=0.02), an observation that was even more apparent in 6 paired samples [p=0.008, median IHC score 19.6 (5.0−53.2) at diagnosis vs. 91.5 (38.6 − 129) beyond 1st relapse]. No significant change was found in the expression of p110β or p110δ between diagnostic and relapse samples. There was no significant difference in expression levels of the 3 isoforms between blastoid and non-blastoid morphological variants. Expression of both the p110α and δ isoforms was detected by western blotting in 4 MCL cell lines, but only Jeko-1 cells were sensitive to inhibition with GDC-0941. CAL-101 produced little or no apoptosis in all 4 cell lines. In primary MCL samples, GDC-0941 was consistently more potent than CAL-101, with decrease in cell viability of 32 vs. 20% at 1μM (p=0.15), 51 vs. 25% at 5μM (p=0.02) and 67 vs. 35% at 10μM (p<0.0001) GDC-0941 and CAL-101 respectively. GDC-0941 was also able to partially overcome the stimulatory effect of sCD40L and IL4 on primary MCL samples. Western blotting showed a consistent reduction in the phosphorylation of Akt and GSK-3β in sensitive MCL cells. Conclusion: Our studies demonstrate that although p110δ is the most consistently expressed isoform, the expression of the p110α subunit increases significantly in multiply relapsed MCL. This observation, in combination with significantly greater in vitro sensitivity of MCL primary samples to GDC-0941, compared to the p110δ-selective inhibitor CAL-101, provides strong evidence for further evaluation of GDC-0941 in this disease. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria. Joel:Astra Zeneca: Research Funding; Intellikine: Research Funding.

scholarly journals Novel Bruton's Tyrosine Kinase Inhibitor Bgb-3111 Demonstrates Potent Activity in Mantle Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5374-5374 ◽  
Author(s):  
Carrie J Li ◽  
Yang Liu ◽  
Taylor Bell ◽  
Jack Wang ◽  
Hui Guo ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cell Viability ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Mantle Cell ◽  
Btk Inhibitor ◽  
Dose Dependent

Abstract Background: Aberrant B-cell receptor signaling is an important contributor to lymphomagenesis in mantle cell lymphoma (MCL). Bruton's Tyrosine Kinase (BTK), a component of the BCR signaling axis, has been validated as a clinically relevant target, and BTK inhibitor ibrutinib received FDA approval for treatment of MCL in 2013. Growing concerns that single agent ibrutinib exerts off-target effects that interfere with other treatments such as rituximab-induced antibody-dependent cell cytotoxicity limit its utility in combination treatments. In this study, we assessed the in vitro and in vivo effects of BGB-3111in MCL models. Methods: We performed cell viability assays with BGB-3111 treated MCL cell lines to determine inhibition of cellular proliferation. The same assays were conducted on primary human MCL cells and patient-derived xenograft (PDX) tumor samples. Dose-dependent inhibition of BTK auto-phosphorylation and inhibition of downstream targets such as PLC-γ were determined by phospho-protein immunoblotting and immunoprecipitation. A reverse-phase protein assay (RPPA) was conducted on BGB-3111-treated Mino cells to evaluate changes in MCL oncogenic signaling. Induction of apoptosis in MCL cells treated with increasing doses of BGB-3111 was quantified using flow cytometry. For in vivo experiments, an ibrutinib-sensitive MCL PDX mouse model was treated with 50 mg/kg/day BGB-3111 and monitored for mean tumor burden and survival. Results: BGB-3111 potently inhibited cell viability in a panel of MCL cell lines, with an activity range of 1-10 uM, and induced apoptosis in a dose-dependent manner in several MCL cell lines.BGB-3111 treatment of MCL cells demonstrated a dose-dependent decrease in p-BTK (Y223) and inhibition of downstream effectors without impacting total protein levels, while RPPA revealed upregulation of the PI3K-Akt signaling axes. In addition, BGB-3111 treatment did not impact phosphorylation of off-target kinases affected by ibrutinib treatment. In vivo, BGB-3111 suppressed tumor growth and prolonged tumor survival in BGB-3111 treated mice. Conclusion: The second generation BTK inhibitor BGB-3111 demonstrates selectivity for BTK in vitro and BTK inhibition in vivo. BGB-3111-treated PDX mouse models examining survival, tumor growth, and other factors point to BGB-3111 as an effective single agent BGB-3111 is being investigated in Phase I clinical trials. Disclosures Wang: Beigene: Employment. Wang:Asana BioSciences: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Honoraria; Acerta: Consultancy, Research Funding; Kite Pharma: Research Funding; BeiGene: Research Funding; Asana biosciences, Beigene, Celgene, Juno, Kite, Onyx, Pharmacyclics: Research Funding; Juno Therapeutics: Research Funding.

Phosphoinositide 3’-Kinase (PI3K) Delta Inhibition with CAL-101 Blocks B-Cell Receptor (BCR) Signaling and the Prosurvival Actions of Nurslike Cells (NLC), In Chronic Lymphocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 48-48
Author(s):  
Julia Hoellenriegel ◽  
Sarah A Meadows ◽  
William G. Wierda ◽  
Michael J Keating ◽  
Brian Lannutti ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Viability ◽  
B Cell ◽  
Cell Activation ◽  
Lymphocytic Leukemia ◽  
Cytotoxic Agents ◽  
Cross Linking ◽  
Clinical Activity ◽  
Bcr Signaling

Abstract Abstract 48 BCR signaling is increasingly recognized as a central mechanism for disease progression in CLL. PI3K transmits various external, microenvironment-derived signals that influence survival, drug resistance, and cell migration of CLL cells. PI3K isoforms have distinct functions and tissue distributions. P110δ mutant mice display defective BCR signaling, along with impaired B cell development and differentiation. Because of this unique role of p110δ in BCR signaling, p110δ inhibitors have been developed for treatment of B cell malignancies and autoimmune disorders. CAL-101, a potent and selective p110δ showing an IC50 of 2.5 nM in vitro and >200-fold selectivity relative to other PI3K isoforms, displays pre-clinical and clinical activity in CLL. Prior in vitro CLL studies revealed that CAL-101 induces caspase-dependent apoptosis, and inhibits CD40L-, BAFF-, TNF-alpha- and fibronectin-derived survival signals. Because of the importance of p110δ in BCR signaling, we tested the effects of CAL-101 on BCR-derived CLL cell activation, and confirmed our findings in correlative studies related to an ongoing CAL-101 phase I clinical trial. We found that BCR cross-linking with anti-IgM significantly increased CLL cell viability to 121 ± 5 % of controls (mean ± SEM, n=15, *P< 0.05).This pro-survival effect was abrogated by CAL-101, which reduced CLL cell viability to 85± 3 % of controls at 48 hours (mean ± SEM, n=15, *P< 0.05). CLL cell viability in co-culture with NLC was also significantly reduced by CAL-101 to 64± 6% of untreated controls at 48 hours (mean ± SEM, n=10, *P< 0.05). BCR cross-linking induces secretion of the chemokines CCL3 and CCL4 by CLL cells, which was quantified in CLL supernatants by ELISA. CAL-101 significantly reduced supernatant CCL3 concentrations from 4060 ± 1392 pg/mL to 2901 ± 1220 pg/mL, and CCL4 levels from 5721 ± 1789 pg/mL to 3223 ± 1311 pg/mL (mean ± SEM, n=6, *P<0.05). In CLL-NLC co-cultures, CAL-101 also inhibited CCL3/4 secretion by CLL cells.Here, the CCL3 concentrations were reduced by CAL-101 from 943 ± 535 pg/mL to 156 ± 8 pg/mL, and the CCL4 concentrations from 7433 ± 4463 pg/mL to 316 ± 53 pg/mL (mean ± SEM, n=5, *P<0.05).CAL-101 also decreased CXCL13 levels in CLL-NLC co-cultures from 151 ± 35 to 70 ± 27 pg/mL (mean ± SEM, n=4, *P=0.05), indicating that CAL-101 has pharmacological actions on both, CLL cells and the CLL microenvironment as represented by the NLC. Given the importance of cytotoxic agents in the therapy of patients with CLL, we evaluated whether a combination of CAL-101 with bendamustine, could overcome stroma-mediated drug resistance in CLL cells co-cultures with marrow stromal cells (MSC). Fig A shows contour plots of a representative case that depict CLL cell viability after treatment with CAL-101, bendamustine, or the two drugs combined. Fig. B displays a bar diagram that depicts the mean relative viabilities of CLL cells treated with CAL-101 (5 mM),bendamustine(10 mM),or the drug combination (mean ± SEM, n=4). The consistently higher CLL cell death of CLL cells treated with the combination of both drugs indicates an additive effect. Next, we used phospho-flow to demonstrate that CAL-101 inhibits constitutive and BCR-induced PI3K pathway activation in samples obtained from CLL patients undergoing treatment with CAL-101. CAL-101 treatment down regulated pAkt(T308) in peripheral CLL cells by >90% (n=12). Plasma samples from 14 CLL patients obtained before and after 28 days of daily treatment with CAL-101 were analyzed for concentrations of various cytokines. Interestingly, these analyses revealed substantial decreases from baseline to Day28+ of CAL-101 treatment in mean plasma levels of CCL3 (from 186 pg/mL to 29 pg/mL), CCL4 (from 303 to 70 pg/mL),CCL22 (1067 to 533 pg/mL), CXCL13 (316 pg/mL to 40 pg/mL), and TNFα(104 to 29 pg/mL), confirming our in vitro data related to CCL3/4 and CXCL13. Collectively, our data demonstrate that CAL-101 effectively inhibits BCR- and NLC-mediated CLL cell survival and activation in vitro. Also, CAL-101 enhances the activity of cytotoxic agents such as bendamustine against CLL cells. Our in vivo data indicate that CAL-101 decreases elevated pre-treatment CCL3 and CCL4 levels. These findings are consistent with the concept that inhibition of BCR-derived signals could be a key mechanism of action of CAL-101 in CLL. The results also support clinical evaluation of CAL-101 in combination with bendamustine for the treatment of CLL. Disclosures: Meadows: Calistoga Pharmaceuticals: Employment. Lannutti:Calistoga Pharmaceutical Inc.: Employment.

Activity of Rituximab and Ofatumumab Against Mantle Cell Lymphoma (MCL) in Vitro in MCL Cell Lines and Ex Vivo in Tumor Cells Derived From Patient Tumor Samples

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4972-4972
Author(s):  
Matthew J. Barth ◽  
Gopichand Pendurty ◽  
Cory Mavis ◽  
Natalie M Czuczman ◽  
John Gibbs ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Tumor Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Clinical Investigations ◽  
Mantle Cell ◽  
Anti Cd20

Abstract Abstract 4972 Mantle cell lymphoma (MCL) is an aggressive form of non-Hodgkin lymphoma (NHL) that frequently presents with advanced stage disease. The addition of rituximab, a monoclonal anti-CD20 antibody, to high dose chemotherapy regimens often followed by stem cell transplant has improved outcomes, but survival still remains low at 3–5 years. Novel agents are needed to improve outcomes in MCL. Ofatumumab is a fully human anti-CD20 monoclonal antibody directed against a novel epitope on the CD20 antigen. Ofatumumab has been shown to be more potent than rituximab against B-NHL cells in pre-clinical investigations. Ofatumumab is FDA approved for the treatment of CLL that is fludarabine and alemtuzumab refractory or with bulky disease resistant to fludarabine and is being investigated in clinical trials in NHL. In order to characterize the activity of ofatumumab against MCL, we performed pre-clinical investigations into the activity of ofatumumab against MCL cell lines and primary MCL tumor cells derived from patient tumor samples (n=2). Antibody-dependant cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) assays were performed in the MCL cell lines Mino, Jeko, Rec-1 and Z-138 to demonstrate sensitivity to rituximab and ofatumumab. Lymphoma cells were labeled with 51Cr prior to incubation with rituximab or ofatumumab at 10ug/mL plus human serum or effector cells (efector:target ratio of 20:1). 51Cr-release was measured and the percentage of lysis was calculated. Patient tumor cells were isolated from tumor biopsy samples by MACS sorting (negative selection). Patient tumor cells were incubated with ofatumumab or rituximab at 10ug/mL in the presence of human serum as a complement source. Cell viability was determined at 48 hours by CellTiterGlo assay. Means were compared using a t-test. Expression of CD20 and the complement inhibitory proteins (CIPs) CD55 and CD59 in MCL cell lines were determined by flow cytometry and compared to the rituximab-sensitive cell line Raji and the rituximab-resistant cell line Raji 4RH. Surface density of CD20, CD55 and CD59 were determined by Imagestream analysis. Western blot was performed to measure total CD20 protein expression. Ofatumumab induced significantly higher levels of cell lysis compared to rituximab in CDC assays of all MCL cell lines tested (Mino: 65.9% vs 0.5%; JeKo 43.9% vs 13.3%; REC-1 25.4% vs 4.7%; Z-138: 56.4% vs 0.65%; all p-values <0.05). The ADCC assays showed a similar degree of lysis with ofatumumab when compared to rituximab in all cell lines tested. In primary tumor cells, ofatumumab and rituximab demonstrated similar levels of decreased cell viability following 48 hours of antibody exposure. MCL cell lines demonstrated similar expression of surface and total CD20 when compared to the rituximab-sensitive B-NHL Raji cell line. CIP expression was increased in all MCL cell lines compared to Raji cells and was similar to the rituximab-resistant Raji 4RH cell line. Our data suggest ofatumumab is more potent than rituximab against MCL cells in vitro and retains CDC activity despite high expression levels of CIPs. This increased activity was not seen in patient tumor samples; however we were limited by the number of available patient samples. In vivo experiments investigating the activity of ofatumumab in a SCID mouse MCL xenograft model and investigations into the activity of ofatumumab in MCL cells in combination with cytotoxic agents and novel small molecule inhibitors are ongoing. Disclosures: Czuczman: Genmab: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Research Funding. Hernandez-Ilizaliturri:Genmab: Research Funding.

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