scholarly journals Venetoclax Is Synergistic with Idelalisib or MK2206 Against Primary CLL Cells in an in Vitro Model of the Microenvironment

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5443-5443
Author(s):  
Yandong Shen ◽  
Kyle R Crassini ◽  
Narjis Fatima ◽  
Richard Christopherson ◽  
Stephen P. Mulligan ◽  
...  
Keyword(s):  
Clinical Trials ◽  
High Risk ◽  
Signaling Pathway ◽  
Conflicts Of Interest ◽  
Single Agent ◽  
Pi3 Kinase ◽  
Lymphocytic Leukemia ◽  
Pi3 Kinase Pathway ◽  
Kinase Pathway

Background The PI3-kinase signaling pathway and the Bcl-2-family of proteins play crucial roles in regulating the survival and proliferation of chronic lymphocytic leukemia (CLL) cells in the bone marrow and lymph nodes. Trials of ibrutinib, idelalisib and venetoclax illustrate the potential of targeting the B-cell receptor (BCR) signaling pathway and Bcl-2, however disease relapse is still common. Several pre-clinical studies and on-going clinical trials [Rogers et al., 2018, Jain et al., 2019] suggest that combinations of BCR inhibitors with venetoclax may be an effective treatment strategy for CLL patients with high risk disease. We sought to investigate the effects of combining idelalisib or the AKT inhibitor MK2206 with venetoclax against CLL cells under in vitro conditions that mimic the tumor microenvironment. Methods Primary CLL cells were co-cultured with CD40L-expressing mouse L-fibroblasts. Cell viability was assessed using the mitochondrial membrane potential dye DilC1(5), propidium iodide and flow cytometry (n = 6). Synergy between idelalisib or MK2206 and venetoclax was evaluated by calculating combination indices (CI) using the Compusyn software. The mechanisms of action of the drugs and synergies between the drugs were investigated by immunoblotting (n = 6). Results Venetoclax was highly synergistic in combination with idelalisib or MK2206 against CLL cells co-cultured with CD40L-fibroblasts, with CI values of 0.2 and 0.5 at a fractional effect of 0.9, respectively (Figure 1). This synergy was consistent with a significant (P < 0.05) reduction in the IC50 for venetoclax, idelalisib and MK2206. Immunoblotting suggests that MK2206, as a single agent or in combination with venetoclax, was more effective than idelalisib in inhibiting the phosphorylation of AKT and NF-κB. Both MK2206 and idelalisib as single agents and in combination with venetoclax significantly reduced expression of Mcl-1 and Bfl-1, two pro-survival members of the Bcl-2 family of proteins in primary CLL cells co-cultured with CD40L-fibroblasts. Conclusions The synergy observed, which was associated with a significant decrease in the IC50s for idelalisib and MK2206, may mitigate some of the toxicities associated with PI3-kinase pathway inhibitors. Comparison of the two PI3-kinase-pathway inhibitors suggests that MK2206 may be more effective than idelalisib at blocking BCR-mediated signaling as a single agent and in combination with venetoclax. The mechanisms underlying the synergy include down-regulation of expression of Bcl-2 family proteins that are not targeted by venetoclax as a single agent. The data presented support the rationale for on-going and future clinical trials of combination therapies incorporating a PI3-kinase inhibitor with venetoclax for the treatment of high risk CLL. Figure 1 Disclosures No relevant conflicts of interest to declare.

IL-21 Promotes Apoptosis through Induction of the BH3 Only Protein Bim and Enhacnes Direct and Innate Immune-Promoted Death of Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3118-3118
Author(s):  
Julie M. Roda ◽  
Aruna Gowda ◽  
Rehan Hussain ◽  
Asha Ramanunni ◽  
Amy Lehman ◽  
...  
Keyword(s):  
Clinical Trials ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Nk Cell ◽  
Single Agent ◽  
Innate Immune ◽  
Lymphocytic Leukemia ◽  
Clinical Activity ◽  
Specific Lysis

Abstract Chemoimmunotherapy with fludarabine and the anti-CD20 monoclonal antibody rituximab has demonstrated promising clinical activity in chronic lymphocytic leukemia (CLL). We hypothesized that the gamma chain receptor cytokine IL-21, which is currently in clinical trials for lymphoma, might enhance the efficacy of this regimen by augmenting both immune-mediated clearance of CLL cells and a direct apoptotic mechanism involved in the homeostasis of normal B cells. CLL cells expressed variable levels of the IL-21 receptor alpha subunit (<10–80% positive cells, n = 16). IL-21 induced direct apoptosis in CLL cells from a subset of patients (40% ± 15.9 apoptotic cells; n = 9; p <0.0001), which directly correlated with increased expression of surface IL-21R (p = 0.001). The in vitro apoptosis occurred at physiologically attainable concentrations (25 ng/ml) and was time- and dose-dependent. As a single agent, IL-21 did not activate CLL cells, as evidenced by lack of surface expression of CD86, HLADR, CD95, or CD40. However, IL-21 induced phosphorylation of STAT-1Tyr-701 and STAT-3 Tyr-705 in CLL cells exhibiting > 20% apoptosis at 72 hours, whereas phosphorylation of these proteins was not seen in CLL cells failing to undergo apoptosis. Similar to normal murine B cells, IL-21-mediated death was associated with up-regulation of the pro-apoptotic BH3 only domain protein Bim, whereas Bim was not up-regulated in CLL cells insensitive to IL-21-induced apoptosis. Furthermore, silencing of Bim in primary CLL cells with siRNA antagonized IL-21-mediated death. Preliminary studies examining the mechanism of Bim up-regulation demonstrated that both total levels and phosphorylation of FOXO3AThr32 increases following IL-21 treatment. FOXO3a is involved in transcriptional regulation of Bim, and further mechanistic studies are ongoing and will be presented. Given the favorable modulation of Bim, we examined the ability of IL-21 to enhance apoptosis in response to rituximab, alemtuzumab, or fludarabine. Our studies confirm that CLL cells pre-treated with IL-21 are sensitized to rituximab and fludarabine, whereas IL-21 had no effect on fludarabine-mediated apoptosis of normal T cells. IL-21 also enhanced NK cell ADCC against rituximab-coated autologous CLL cells (42 ± 4.4% rituximab-specific lysis vs. 28 ± 3.1% at an E:T ratio of 25:1; p < 0.0001). These data provide evidence that IL-21 promotes direct apoptosis through induction of Bim and also enhances fludarabine- and rituximab-mediated apoptosis. Additionally, IL-21 enhances autologous innate immune activation of NK cells toward primary CLL cells coated with rituximab. Overall, these findings provide justification for combination studies of IL-21 with fludarabine and rituximab chemoimmunotherapy in CLL and point to Bim induction as a pharmacodynamic endpoint to predicting surrogate biologic activity of IL-21 in vivo as part of planned clinical trials with this agent.

Anergy in CLL: Moving Towards the Clinic

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4677-4677
Author(s):  
Benedetta Apollonio ◽  
Tania Veliz Rodriguez ◽  
Cristina Scielzo ◽  
Maria Teresa Sabrina Bertilaccio ◽  
Lydia Scarfò ◽  
...  
Keyword(s):  
Mouse Model ◽  
B Cell ◽  
Nuclear Translocation ◽  
Conflicts Of Interest ◽  
Single Agent ◽  
Volume Growth ◽  
Lymphocytic Leukemia ◽  
Functional Features

Abstract B-Cell Receptor (BCR) triggering and responsiveness play a crucial role in the survival and expansion of Chronic Lymphocytic Leukemia (CLL) clones. In the recent past, several groups including ours have investigated the activation status of the signaling pathways originating from the leukemic BCR. Specifically we found that around 50% of CLL patients display a biochemical signature characterized by constitutive phosphorylation of ERK1/2 (pERK(+)) and constitutive nuclear translocation of NF-ATc1. These cases are unable to respond in vitro to BcR stimulation and are resistant to spontaneous apoptosis, thus resembling B lymphocytes previously anergized in vivo. Similar biochemical and functional features have been recently demonstrated in B leukemic cells persisting in the blood in patients treated with the BTK inhibitor, Ibrutinib, thereby making anergy an attractive target on the way to obtain eradication of the disease. CLL-associated B cell anergy can be specifically targeted by using different MAPK-inhibitors that have been shown to induce apoptosis selectively in the group of pERK(+) CLL. These data suggested that MAPK signalling can be efficiently inhibited in CLL for therapeutic purpose and that the phosphorylation status of ERK1/2 may represent a reliable biomarker to predict and monitor treatment response. However, even if the tested compounds were shown to be extremely efficient in inhibiting ERK1/2 phosphorylation in vitro, a lack of clinical activity was reported for many of them when tested in patients, mostly with solid tumors. In the present work, we used Trametinib, a specific MEK1/2 inhibitor, recently approved as a single-agent for the treatment of V600E mutated metastatic melanoma, and we investigated, at preclinical level, its activity in both primary CLL samples and a xenograft leukemic mouse model. Trametinib treatment completely inhibited constitutive ERK1/2 phosphorylation in 10 pERK1/2(+) samples at 3uM after 30 minutes treatment. Additionally, in 23 patients Trametinib treatment for 48 hours reduced cell viability in the cells from all 12 pERK1/2(+) patients (28,2% ± 3,5 mean survival) tested as compared to those from the pERK(-) group (11 cases, 58,1% ± 3,8 mean survival, p< 0,0001). To strengthen our in vitro data, we evaluated the effect of Trametinib administration in the xenograft Rag2-/-gc-/- mouse model subcutaneously transplanted with the CLL cell line MEC1, characterized by specific features of anergy. Mice were subcutaneously injected with 10x106 cells and then challenged with Trametinib (oral gavage with 1mg/kg or with vehicle alone) starting from day 21 after tumour injection for 14 days. The effect of the inhibitor was monitored by tumour volume growth. Trametinb administration delayed tumour growth (p<0.05 starting at days 27) and inhibited leukemic cell dissemination in the peripheral blood, peritoneal cavity and bone marrow. In summary, our data further support the idea that blocking anergic pathways may be highly effective not only in vitro but also in vivo with potential clinical implications at least in the subset of patients whose cells are characterized by anergic features, including those with persistent lymphocytosis when treated with Ibrutinib. The preclinical efficacy shown by Trametinib, a drug already approved for clinical use, warrants the implementation of controlled studies in CLL patients. Disclosures No relevant conflicts of interest to declare.

Artesunate Enhanced Apoptosis of Human High Risk MDS Cells Induced By the DNMT Inhibitor Decitabine

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5212-5212
Author(s):  
Wang Ying ◽  
Niu Zhiyun ◽  
Wen Shupeng ◽  
Wang Fuxu ◽  
Zhang Xuejun
Keyword(s):  
High Risk ◽  
Conflicts Of Interest ◽  
Single Agent ◽  
Mitochondrial Pathway ◽  
Caspase 9 ◽  
Apoptosis Rate ◽  
Single Agent Therapy ◽  
Laser Confocal Microscope ◽  
Dependent Pathway

Abstract Abstract: The present study evaluated whether the artesunate (ART) could enhance the apoptosis induced by decitabine (DAC) in SKM-1 cells, and to explore its potential mechanisms. Cytotoxicity of the combination of ART and DAC on SKM-1 cells was detected by CCK-8 assay and the apoptosis rate of high-risk myelodysplastic syndrome (MDS) cell line, SKM-1 cells was measured by flow cytometry. Protein expression levels of activated caspase-3, caspase-9, caspase-8, cleaved PARP and AIF in SKM-1 cells were measured by Western blot. Laser confocal microscope analysis showed AIF transfer to the nucleus. The growth inhibition rate and the apoptosis rate of SKM-1cells treated with the combination of ART and DAC was significantly increased compared with that of SKM-1 cells treated with the single agent (P<0.05). Both ART and DAC could induce caspase-dependent apoptosis, while ART, but not DAC, also induced caspase-independent apoptosis via AIF transfer from mitochondria to the nucleus. Additionally, the combination of the two agents induced cell death was not attenuated by caspase3,7 inhibitor Ac-DEVD-CHO. Our result suggested that the ART–DAC combination was more effective than single-agent therapy in vitro. Both compounds not only activated caspase-dependent pathway but also activated a caspase-independent mitochondrial pathway. Disclosures No relevant conflicts of interest to declare.

Integration of stimulatory and inhibitory signals in pituitary tumor cells through the PI3 kinase pathway

2006 ◽  
Vol 114 (08) ◽  
Author(s):  
T Colaco ◽  
C Onofri ◽  
M Theodoropoulou ◽  
M Kowarik ◽  
GK Stalla ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Pituitary Tumor ◽  
Pi3 Kinase ◽  
Pi3 Kinase Pathway ◽  
Kinase Pathway

scholarly journals Loss of Phosphatase and Tensin Homolog or Phosphoinositol-3 Kinase Activation and Response to Trastuzumab or Lapatinib in Human Epidermal Growth Factor Receptor 2–Overexpressing Locally Advanced Breast Cancers

2011 ◽  
Vol 29 (2) ◽  
pp. 166-173 ◽  
Author(s):  
Bhuvanesh Dave ◽  
Ilenia Migliaccio ◽  
M. Carolina Gutierrez ◽  
Meng-Fen Wu ◽  
Gary C. Chamness ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Single Agent ◽  
Growth Factor Receptor ◽  
Breast Cancers ◽  
Trastuzumab Resistance ◽  
Epidermal Growth

Purpose Phosphatase and tensin homolog (PTEN) loss or activating mutations of phosphoinositol-3 (PI3) kinase (PIK3CA) may be associated with trastuzumab resistance. Trastuzumab, the humanized human epidermal growth factor receptor 2 (HER2) monoclonal antibody, and lapatinib, an epidermal growth factor receptor/HER2 tyrosine kinase inhibitor, are both established treatments for HER2-overexpressing breast cancers. Understanding of the cellular response to HER2-targeted therapies is needed to tailor treatments and to identify patients less likely to benefit. Methods We evaluated the effect of trastuzumab or lapatinib in three HER2-overexpressing cell lines. We confirmed the in vitro observations in two neoadjuvant clinical trials in patients with HER2 overexpression; 35 patients received trastuzumab as a single agent for the first 3 weeks, then docetaxel every 3 weeks for 12 weeks (trastuzumab regimen), whereas 49 patients received lapatinib as a single agent for 6 weeks, followed by trastuzumab/docetaxel for 12 weeks before primary surgery (lapatinib regimen). Apoptosis, Ki67, p-MAPK, p-AKT, and PTEN were assessed by immunohistochemistry. Genomic DNA was sequenced for PIK3CA mutations. Results Under low PTEN conditions, in vitro data indicate that lapatinib alone and in combination with trastuzumab was effective in decreasing p-MAPK and p-AKT levels, whereas trastuzumab was ineffective. In the clinical trials, we confirmed that low PTEN or activating mutation in PIK3CA conferred resistance to the trastuzumab regimen (P = .015), whereas low PTEN tumors were associated with a high pathologic complete response rate (P = .007). Conclusion Activation of PI3 kinase pathway is associated with trastuzumab resistance, whereas low PTEN predicted for response to lapatinib. These observations support clinical trials with the combination of both agents.

scholarly journals APTO-253 Induces KLF4 to Promote Potent in Vitro Pro-Apoptotic Activity in Hematologic Cancer Cell Lines and Antitumor Efficacy As a Single Agent and in Combination with Azacitidine in Animal Models of Acute Myelogenous Leukemia (AML)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4813-4813 ◽  
Author(s):  
William G Rice ◽  
Avanish Vellanki ◽  
Yoon Lee ◽  
Jeff Lightfoot ◽  
Robert Peralta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Animal Models ◽  
Antitumor Activity ◽  
Single Agent ◽  
Xenograft Model ◽  
Myelogenous Leukemia ◽  
Acute Myelogenous ◽  
Xenograft Models

Abstract APTO-253, a small molecule that mediates anticancer activity through induction of the Krüppel-like factor 4 (KLF4) tumor suppressor, is being developed clinically for the treatment of acute myelogenous leukemia (AML) and high risk myelodysplastic syndromes (MDS). APTO-253 was well tolerated in a Phase I study in patients with solid tumors using a dosing schedule of days 1, 2, 15, 16 of a 28 day cycle (2T-12B-2T-12B), but recent scientific observations guided APTO-253 toward AML and high risk MDS. Indeed, suppression of KLF4 was reported as a key driver in the leukemogenesis of AML and subsets of other hematologic diseases. The vast majority (~90%) of patients with AML aberrantly express the transcription factor CDX2 in human bone marrow stem and progenitor cells (HSPC) (Scholl et al., J Clin Invest. 2007, 117(4):1037-48). The CDX2 protein binds to CDX2 consensus sequences within the KLF4 promoter, thereby suppressing KLF4 expression in HSPC (Faber et al., J Clin Invest. 2013, 123(1):299-314). Based on these observations, the anticancer activity of APTO-253 was examined in AML and other hematological cancers. APTO-253 showed potent antiproliferative activity in vitro against a panel of blood cancer cell lines, with ηM IC50values in AML (6.9 - 305 ηM), acute lymphoblastic leukemia and chronic myeloid leukemia (39 – 250 ηM), non-Hodgkin’s lymphoma (11 – 190 ηM) and multiple myeloma (72 – 180 ηM). To explore in vivo efficacy, dose scheduling studies were initially conducted in the H226 xenograft model in mice. In the H226 model, APTO-253 showed improved antitumor activity when administered for two consecutive days followed by a five day break from dosing (2T-5B) each week, i.e. on days 1,2, 8,9, 15,16, 22,23, compared to the 2T-12B-2T-12B schedule. The 2T-5B schedule was used to evaluate antitumor activity of APTO-253 in several AML xenograft models in mice. In Kasumi-1 AML and KG-1 AML xenograft models, APTO-253 showed significant antitumor activity (p = 0.028 and p=0.0004, respectively) as a single agent when administered using the 2T-5B schedule each week for four weeks compared to control animals. Mice treated with APTO-253 had no overt toxicity based on clinical observations and body weight measurements. Mice bearing HL-60 AML xenograft tumors were treated with APTO-253 for one day or two consecutive days per week for three weeks, either as a single agent or combined with azacitidine, or with azacitidine alone twice per week (on days 1,4, 8, 11, 15 and 18). APTO-253 as a single agent inhibited growth of HL-60 tumors to approximately the same extent as azacitidine. Furthermore, both once weekly and twice weekly dosing of APTO-253 in combination with azacitidine resulted in significantly enhanced antitumor activity relative to either single agent alone (p = 0.0002 and p = 0.0006 for 1X and 2X weekly APTO-253 treatment, respectively, compared to control). Likewise, using a THP-1 AML xenograft model, APTO-253 administered as a single agent using the 2T-5B per week schedule showed significant efficacy, similar to that of azacitidine, while the combination of APTO-253 and azacitidine demonstrated greatly improved antitumor effects relative to either drug alone. APTO-253 was effective and well tolerated as a single agent or in combination with azacitidine in multiple AML xenograft models, plus APTO-253 does not cause bone marrow suppression in animal models or humans. Taken together, our results indicate that APTO-253 may serve as a targeted agent for single agent use and may provide enhanced efficacy to standard of care chemotherapeutics for AML and other hematological malignancies. Disclosures Rice: Lorus Therapeutics Inc.: Employment. Vellanki:Lorus Therapeutics Inc.: Employment. Lee:Lorus Therapeutics Inc.: Employment. Lightfoot:Lorus Therapeutics Inc.: Employment. Peralta:Lorus Therapeutics Inc.: Employment. Jamerlan:Lorus Therapeutics Inc.: Employment. Jin:Lorus Therapeutics Inc.: Employment. Lum:Lorus Therapeutics Inc.: Employment. Cheng:Lorus Therapeutics Inc.: Employment.

scholarly journals c-Cbl Is Tyrosine-Phosphorylated by Interleukin-4 and Enhances Mitogenic and Survival Signals of Interleukin-4 Receptor by Linking With the Phosphatidylinositol 3′-Kinase Pathway

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 46-53 ◽  
Author(s):  
Hiroo Ueno ◽  
Ko Sasaki ◽  
Hiroaki Honda ◽  
Tetsuya Nakamoto ◽  
Tetsuya Yamagata ◽  
...  
Keyword(s):  
Specific Inhibitor ◽  
Pi3 Kinase ◽  
Interleukin 4 ◽  
Phosphatidylinositol 3 Kinase ◽  
Proliferation And Differentiation ◽  
Interleukin 4 Receptor ◽  
Pi3 Kinase Pathway ◽  
Kinase Pathway ◽  
Phosphatidylinositol 3 ◽  
Survival Signals

Interleukin-4 (IL-4) is a cytokine that induces both proliferation and differentiation and suppresses apoptosis of B cells. Although IL-4 has been shown to activate the phosphatidylinositol 3′ (PI3)-kinase pathway, the role of PI3 kinase in the IL-4 receptor (IL-4R) signaling remains unclear. In this study, we demonstrated that c-Cbl proto-oncogene product is inducibly phosphorylated on tyrosine residues and is associated with the p85 subunit of PI3-kinase by IL-4 stimulation. Overexpression of c-Cbl enhances the PI3-kinase activity and, at the same time, mitogenic activity and survival of cells in the presence of IL-4. However, these effects of c-Cbl were abolished by wortmannin, a specific inhibitor for the PI3 kinase pathway, or by a point mutation at tyrosine 731 of c-Cbl, which is a major binding site for p85. These results indicate that c-Cbl plays a role in linking IL-4R with the PI3 kinase pathway and thus enhancing the mitogenic and survival signals.

scholarly journals PI3 Kinase Pathway and MET Inhibition is Efficacious in Malignant Pleural Mesothelioma

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Rajani Kanteti ◽  
Jacob J. Riehm ◽  
Immanuel Dhanasingh ◽  
Frances E. Lennon ◽  
Tamara Mirzapoiazova ◽  
...  
Keyword(s):  
Malignant Pleural Mesothelioma ◽  
Pi3 Kinase ◽  
Pleural Mesothelioma ◽  
Pi3 Kinase Pathway ◽  
Kinase Pathway
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