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.

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.

scholarly journals Role of MiR-155 Signal Pathway in Regulating Podocyte Injury Induced by TGF-β1

2017 ◽  
Vol 42 (4) ◽  
pp. 1469-1480 ◽  
Author(s):  
Xu Lin ◽  
Xintng Zhen ◽  
Haiting Huang ◽  
Haohao Wu ◽  
Yanwu You ◽  
...  
Keyword(s):  
Protein Expression ◽  
Antisense Oligonucleotides ◽  
Negative Control ◽  
Signal Pathway ◽  
Caspase 9 ◽  
Podocyte Injury ◽  
Apoptosis Rate ◽  
Mrna And Protein Expression ◽  
Tgf Β1

Background/Aims: Transforming growth factor beta 1 (TGF-β1) plays a critical role in the pathogenesis of glomerulosclerosis. The purpose of this study was to examine the effects of inhibition of miR-155 on podocyte injury induced by TGF-β1 and to determine further molecular mediators involved in the effects of miR-155. Methods: Conditionally immortalized podocytes were cultured in vitro and they were divided into four groups: control; TGF-β1 treatment; TGF-β1 with miR-155 knockdown [using antisense oligonucleotides against miR-155 (ASO-miR-155)] and TGF-β1 with negative control antisense oligonucleotides (ASO-NC). Real time RT-PCR and Western blot analysis were employed to determine the mRNA and protein expression of nephrin, desmin and caspase-9, respectively. Flow cytometry was used to examine the apoptotic rate of podocytes and DAPI fluorescent staining was used to determine apoptotic morphology. In addition, we examined the levels of miR-155, TGF-β1, nephrin, desmin and caspase-9 in glomerular tissues of nephropathy induced by intravenous injections of adriamycin in rats. Results: mRNA and protein expression of desmin and caspase-9 was increased in cultured TGF-β1-treated podocytes, whereas nephrin was decreased as compared with the control group. Importantly, miR-155 knockdown significantly attenuated upregulation of desmin and caspase-9, and alleviated impairment of nephrin induced by TGF-β1. Moreover, the number of apoptotic podocytes was increased after exposure to TGF-β1 and this was alleviated after miR-155 knockdown. Knocking down miR-155 also decreased an apoptosis rate of TGF-β1-treated podocytes. Note that negative control antisense oligonucleotides failed to alter an increase of the apoptosis rate in TGF-β1-treated podocytes. Consistent with in vitro results, expression of miR-155, TGF-β1, desmin and caspase-9 was increased and nephrin was decreased in glomerular tissues with nephropathy in vivo experiments. Conclusions: TGF-β1 impairs the protein expression of nephrin and amplifies the protein expression of desmin and caspase -9 via miR-155 signal pathway. Inhibition of miR-155 alleviates these changes in podocytes-treated with TGF-β1 and attenuated apoptosis of podocytes. Our data suggest that miR-155 plays a role in mediating TGF-β1-induced podocyte injury via nephrin, desmin and caspase-9. Results of the current study also indicate that blocking miR-155 signal has a protective effect on podocyte injury. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of podocyte injury observed in glomerulosclerosis.

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.

INFORM2 exploratory multinational phase I/II combination study of nivolumab and entinostat in children and adolescents with refractory high-risk malignancies: INFORM2 NivEnt.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. TPS10065-TPS10065 ◽  
Author(s):  
Cornelis Martinus van Tilburg ◽  
Ruth Witt ◽  
Kristian W. Pajtler ◽  
Plass Christoph ◽  
Isabel Poschke ◽  
...  
Keyword(s):  
High Risk ◽  
Phase I ◽  
Children And Adolescents ◽  
Mrna Expression ◽  
Single Agent ◽  
Mutational Load ◽  
Duration Of Treatment ◽  
Open Label

TPS10065 Background: Immune checkpoint inhibition in children has shown limited success rates until now. This is most likely due to the fact that the vast majority of pediatric cancers are so-called immunologic cold tumors, and that patients have been enrolled in an unselected manner in single agent trials. Recently, it has been shown that the class I selective HDAC inhibitor entinostat has significant immune enhancing activity in vitro and in vivo. This is mediated through multiple mechanisms including depletion of myeloid-derived suppressor cells, activation of neoantigen transcription and increase of MHC expression. Methods: INFORM2 NivEnt is an exploratory nonrandomized, open-label, multinational and multicenter seamless phase I/II basket trial of nivolumab and entinostat in children and adolescents with relapsed, refractory or progressive high-risk solid and CNS tumors. Patients aged 6-21 will be allocated to the following biomarker-defined groups: high mutational load ( > 100 somatic SNVs/exome; group A), high PD-L1 mRNA expression (RPKM > 3; group B), focal MYC(N) amplification (group C), low mutational load and low PD-L1 mRNA expression and no MYC(N) amplification (Biomarker low group D). Phase I determines the recommended phase 2 dose for the combination for the age groups 6-11 and 12-21 years. Patients will receive nivolumab 3mg/kg every 2 weeks. Entinostat has 2 dose levels: 2mg/m2 and 4mg/m2 once per week. Patients can seamlessly enter the phase II which investigates activity (defined as best response during the first 6 cycles) in the 4 biomarker groups A-D. The duration of treatment is 12 cycles, preceded by 1 entinostat priming week. Interim analyses for futility will be performed after every 10 patients in each group. The study will enroll a maximum of 128 patients in Germany, The Netherlands, Sweden, France, Australia and additional countries under discussion. A comprehensive accompanying biomarker program will investigate a series of immune and epigenetic pharmacodynamic biomarkers. Clinical trial information: NCT03838042.

scholarly journals YKL-40 Aggravates Early-Stage Atherosclerosis by Inhibiting Macrophage Apoptosis in an Aven-dependent Way

2021 ◽  
Vol 9 ◽  
Author(s):  
Wei Huan ◽  
Liu Yandong ◽  
Wang Chao ◽  
Zou Sili ◽  
Bai Jun ◽  
...  
Keyword(s):  
Early Stage ◽  
Expression Level ◽  
Atherosclerotic Plaques ◽  
Caspase 9 ◽  
Apoptosis Rate ◽  
Macrophage Apoptosis ◽  
Apoptosis Inhibitor ◽  
Human Carotid

Objective: programmed cell removal in atherosclerotic plaques plays a crucial role in retarding lesion progression. Macrophage apoptosis has a critical role in PrCR, especially in early-stage lesions. YKL-40 has been shown to be elevated as lesions develop and is closely related to macrophages. This study aimed to determine the effect of YKL-40 on regulating macrophage apoptosis and early-stage atherosclerosis progression.Research design and Methods: The correlations among the expression level of YKL-40, the area of early-stage plaque, and the macrophage apoptosis rate in plaques have been shown in human carotid atherosclerotic plaques through pathological and molecular biological detection. These results were successively confirmed in vivo (Ldlr−/- mice treated by YKL-40 recombinant protein/neutralizing antibody) and in vitro (macrophages that Ykl40 up-/down-expressed) experiments. The downstream targets were predicted by iTRAQ analysis.Results: In early-stage human carotid plaques and murine plaques, the YKL-40 expression level had a significant positive correlation with the area of the lesion and a significant negative correlation with the macrophage apoptosis rate. In vivo, the plaque area of aortic roots was significantly larger in the recomb-YKL-40 group than that in IgG group (p = 0.0247) and was significantly smaller in the anti-YKL-40 group than in the IgG group (p = 0.0067); the macrophage apoptosis rate of the plaque in aortic roots was significantly lower in the recomb-YKL-40 group than that in IgG group (p = 0.0018) and was higher in anti-YKL-40 group than that in VC group. In vitro, the activation level of caspase-9 was significantly lower in RAW264.7 with Ykl40 overexpressed than that in controls (p = 0.0054), while the expression level of Aven was significantly higher than that in controls (p = 0.0031). The apoptosis rate of RAW264.7 treated by recomb-YKL40 was significantly higher in the Aven down-regulated group than that in the control group (p &lt; 0.001). The apoptosis inhibitor Aven was confirmed as the target molecule of YKL-40. Mechanistically, YKL-40 could inhibit macrophage apoptosis by upregulating Aven to suppress the activation of caspase-9.Conclusion: YKL-40 inhibits macrophage apoptosis by upregulating the apoptosis inhibitor Aven to suppress the activation of caspase-9, which may impede normal PrCR and promote substantial accumulation in early-stage plaques, thereby leading to the progression of atherosclerosis.

scholarly journals Apoptosis of Platelets Inhibited By Herba Sarcandrae Extract through the Mitochondria Pathway

2018 ◽  
Vol 2018 ◽  
pp. 1-12
Author(s):  
Xiaoqin Zhu ◽  
Yiling Jiang ◽  
Qin Zheng ◽  
Aiping Zhang ◽  
Ling Shi ◽  
...  
Keyword(s):  
Bone Marrow Failure ◽  
Underlying Mechanism ◽  
Mitochondrial Pathway ◽  
Total Flavonoids ◽  
Suspension Cells ◽  
Caspase 9 ◽  
Immune Mediated ◽  
Mitochondria Pathway

The purpose of the present study is to decode the underlying mechanism of Herba Sarcandrae that indicated antipurpuric effect and to unveil one of its primary components, flavonoids, which play an important role. An immune mediated bone marrow failure (BMF) model in mouse was established by infusion thymus suspension cells after radiation in vivo. Platelets isolated in vitro were prepared from normal mice and BMF mice, respectively. The expressions of PS, P-selectin, PAC-1, Bax, Bad, Bid, and caspase-9 were examined by flow cytometry, and alteration of morphology of platelets under different conditions was observed. Our results indicated that the number of platelets was increased by addition of total flavonoids, and some of apoptotic markers such as Bax, Bad, Bid, and Caspase-9 were downregulated. In addition, the phosphatidylserine (PS) exposure on platelets was inhibited by total flavonoids, and the expressions of PAC-1 and P-selectin were decreased. In conclusion, it is suggested that the total flavonoids of Herba Sarcandrae may inhibit the excessive platelet apoptosis through mitochondrial pathway. In addition, activation of platelets may be also involved in mediating apoptosis of platelets.

An Inducible Caspase 9 Suicide Gene to Improve the Safety of Mesenchymal Stromal Cell Therapies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1444-1444
Author(s):  
Carlos A. Ramos ◽  
Zahra Asgari ◽  
Eric Yvon ◽  
Cliona M. Rooney ◽  
Helen E. Heslop ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Suicide Gene ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Caspase 9 ◽  
Growth Modulation ◽  
Ectopic Ossification ◽  
Specific Chemical ◽  
Immunodeficient Mice

Abstract Abstract 1444 Poster Board I-467 Given their immunomodulatory properties and potential for tissue differentiation, mesenchymal stromal cells (MSCs) are attractive vehicles for the treatment of graft-versus-host disease and autoimmune disorders and for regenerative cellular therapies. While MSCs have been infused in hundreds of patients to date with minimal reported side effects, follow-up is limited and little is known of their longer term potential for autonomous growth or unwanted activity or differentiation. Several in vitro and animal models have recently raised safety concerns, including reports of spontaneous osteosarcoma formation in culture, and ectopic ossification and calcification foci in the myocardium and lung. In light of those concerns, we sought to develop a system that could control the growth and survival of MSCs used therapeutically. We previously described a suicide gene system for T lymphocytes which was based on an inducible caspase-9 (iCasp9) protein that can be activated using a specific chemical inducer of dimerization (CID), which has been safely tested in a phase I study. Because caspase 9 should induce apoptosis even in differentiated and non-dividing cells, we tested this approach in MSC before and after induction of differentiation. MSCs isolated from healthy donors were transduced with a retroviral vector encoding iCasp9 together with a truncated CD19 (ΔCD19), to allow selection of transduced cells. After a single transduction, 47% ± 6% of the cells were iCasp9/CD19-positive, a percentage that was stable over more than two weeks in culture, suggesting no growth modulation of MSC by the construct. Transduced cells were readily selected by an immunomagnetic column to &gt;97% purity. The phenotype of the iCasp9/CD19-positive cells was identical to that of untransduced cells, with &gt;98% cells positive for CD73, CD90 and CD105, and negative for hematopoietic markers. Non-transduced MSCs had a spontaneous rate of apoptosis in culture of approximately 18% (±7%), and this was not increased following iCasp9-ΔCD19 transduction and selection (apoptotic rate 15% ± 6%, P = 0.47). Addition of CID to MSC cultures after transduction and selection with iCasp9-ΔCD19 resulted in the apoptotic death of 93% ± 1% of iCasp9-positive cells within 24 hrs (P &lt; 0.0001 compared to control), while iCasp9-negative cells retained an apoptosis index similar to that of non-transduced controls (20% ± 7%, P = 0.99 and P = 0.69 vs. non-transduced controls with or without CID, respectively). Furthermore, iCasp9-positive MSCs injected subcutaneously in immunodeficient mice were selectively eliminated after administration of CID to the animals, as assessed by in vivo imaging, without systemic toxicity. Hence, iCasp9-positive MSCs can be selectively killed by exposure to this drug in vitro or in vivo. Moreover, addition of CID to cultures of MSCs differentiated to adipocytes, osteoblasts or chondroblasts also resulted in &gt;90% apoptosis as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. In summary, we have demonstrated that MSCs can express an inducible caspase gene without affecting their phenotype, survival or capacity to differentiate, that the transduced cells can be selected with clinical grade procedures and maintain their basic physiology, and that both the MSC and their differentiated progeny can be selectively eliminated in vitro and in vivo by exposure to a small dimerizer molecule. This approach may provide an added margin of safety to the increasing clinical applications of MSCs and their progeny. Disclosures No relevant conflicts of interest to declare.

CXCL12 Enhances Survival of Chronic Lymphocytic Leukemia Cells Via a RAF-Dependent Pathway That Can Be Inhibited by Sorafenib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2445-2445
Author(s):  
Davorka Messmer ◽  
Jessie-F Fecteau ◽  
Morgan O'Hayre ◽  
Tracy Handel ◽  
Thomas J. Kipps
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Survival ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Growth Factor Receptor ◽  
Lymphocytic Leukemia ◽  
Increased Risk ◽  
Dependent Pathway

Abstract Abstract 2445 The cellular microenvironment is critical for the survival of Chronic Lymphocytic Leukemia (CLL) cells. CLL cells die rapidly in vitro unless they receive survival signals from stromal cells or “nurse-like” cells (NLCs). CLL cell survival is in part mediated by the stromal cell-derived factor-1 (SDF-1alpha, designated as CXCL12), which is expressed by NLCs. CXCL12 is a highly conserved chemokine that can promote CLL-cell survival through its receptor CXCR4. Prior studies showed that treatment of CLL cells with CXCL12 induced activation of Extracellular Signal-Regulated Kinase (ERK). In this study, we examined CXCL12 signaling in CLL cells to characterize the mechanism (s) accounting for its ability to enhance CLL-cell survival. For this we examined CLL cells with high- or low- level expression of the zeta-associated protein of 70 kD (ZAP-70), a tyrosine kinase that is expressed by CLL cells of patients who have an increased risk for early disease progression and short survival. We found that CXCL12 induced a robust intracellular Ca2+ flux in ZAP-70+ CLL cells but only modest-to-poor Ca2+ flux in ZAP-70-negative CLL cells. Furthermore, ZAP-70+ CLL cells (n=10) responded to CXCL12 stimulation with increased and prolonged phosphorylation of ERK and MEK compared to ZAP-70-negative CLL cells (n=9). To investigate the underlying mechanism for MEK activation in ZAP-70+ CLL, we used small molecule inhibitors and found that CXCL12-induced phosphorylation of ERK and MEK could be blocked by sorafenib, a small molecule inhibitor of RAF. The role of RAF was further supported using KG5, a kinase inhibitor of RAF signaling through B-RAF and C-RAF in addition to platelet-derived-growth-factor-receptor (PDGFR) alpha and beta, Flt3, and Kit. As a control, we used a kinase inhibitor that targets all of these kinases except B- and C-RAF (KG1) and found it could not inhibit MEK activation. The involvement of Raf was further substantiated using GW5074, an inhibitor of B-RAF and C-RAF. Both KG5 and GW5074 could inhibit CXCL12-induced MEK activation in ZAP-70+ CLL samples. CXCL12-induced activation of MEK/ERK was not affected by sunitinib, an inhibitor of non-RAF kinases that also are inhibited by sorafenib, including VEGFR, PDGFR, Flt3, and c-Kit. Sorafenib not only inhibited MEK/ERK activation but also caused apoptosis of CLL cells whereby ZAP-70+ CLL cells showed incresed sensitivity to lower doses of sorafenib. Consistent with these results we found that ZAP-70+ CLL cells had a greater responsiveness to CXCL12 for survival in vitro than did ZAP-70-negative CLL cells. We conclude that CXCL12 can enhance survival particularly of ZAP-70+ CLL cells via a RAF dependent pathway, which can be targeted by the kinase inhibitor sorafenib. As such, sorafenib might be effective in blocking the protective influence of the microenvironment on CLL cells, suggesting that this drug could have activity in the treatment of patients with this disease. Disclosures: No relevant conflicts of interest to declare.

Combined Targeting of BCR-ABL and JAK2 with ABL and JAK2 Inhibitors Is Effective Against CML Patients' Leukemic Stem/Progenitor Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3404-3404
Author(s):  
Donna DeGeer ◽  
Paolo Gallipoli ◽  
Min Chen ◽  
Ivan Sloma ◽  
Heather Jorgensen ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Integration Site ◽  
Single Agent ◽  
K562 Cells ◽  
High Rate ◽  
Jak2 Inhibitors

Abstract Abstract 3404 Imatinib mesylate (IM) is a tyrosine kinase inhibitor (TKI) that induces clinical responses in most chronic myeloid leukemia (CML) patients. Nevertheless, early relapses and later emergence of IM-resistant disease pose serious concerns for many. The inadequacies of IM therapy are due, at least in part, to the unique properties of CML stem/progenitor cells that make them generally less responsive to IM and, indeed, other TKIs, and also confer on them a genetic instability that leads to a high rate of formation of BCR-ABL mutants. Improved treatment approaches to prevent the development of resistant subclones by targeting other key molecular elements active in CML stem/progenitor cells are thus clearly needed. One candidate is a complex that forms in CML stem/progenitor cells between the oncoproteins encoded by AHI-1 (Abelson helper integration site 1), BCR-ABL and the JAK2 kinase. This complex contributes to the transforming activity of BCR-ABL both in vitro and in vivo and also plays a role in the IM response/resistance of primary CML stem/progenitor cells. We now describe the results of experiments designed to test the ability of ABL and JAK2 inhibitors to block the activity of this protein complex in CML cells. K562 cells engineered to stably overexpress AHI-1 showed a significantly reduced sensitivity to both IM (at 1 and 5 μM) and TG101209, a JAK2 inhibitor, (at 0.5 and 1 μM), as determined by assays for cell viability, apopotosis, and colony-forming activity. K562 cells engineered to suppression AHI-1 showed an opposite effect, with a heightened sensitivity to IM at concentrations as low as 1 μM. In addition, IM together with TG101209 was more effective at killing AHI-1-overexpressing K562 cells, IM-resistant K562 cells and IM-resistant T315I-mutant cells than either treatment alone. Western blot and co-IP experiments demonstrated a significant reduction of p-BCR-ABL, p-JAK2 and p-STAT5 in cells treated with IM plus TG101209 compared to cells treated with IM or TG101209 alone. Importantly, treatment with 5 μM IM, 150 nM dasatinib (DA) or 5 μM nilotinib (NL) in combination with 100 nM TG101209 caused a significantly greater reduction in the viability of primary CD34+CD38− and CD34+CD38+ CML cells when these responses were compared to any of the TKIs or TG101209 alone (~2-4 fold, n=3). Apoptotic cells at 72 hours were also significantly increased for all drug combinations compared to single agent treatments (40%-52% for the combinations vs 15%-18% for the single agents). CFSE tracking analysis of cell division in these cells further demonstrated additive anti-proliferative activity from the TKI plus TG101219 combinations, although some rare undivided cells were not eliminated. Nevertheless, exposure of CD34+ CML cells from IM-nonresponders (n=4) to TG101209 plus IM or DA did cause a greater inhibition (81% and 85%) of patients' colony-forming cells as compared to the same cells treated with the combination of IM plus DA only, or IM or DA only (60%, 41% and 50% inhibition, p<0.05). Long-term culture-initiating cell assays were undertaken to compare the effect of these combination treatments versus the effects of TKIs or TG101209 alone on very primitive CML cells. The results again showed a more significant reduction of these cells treated with the combination (n=3). Intracellular staining revealed a greater reduction in the levels of p-CrKL and p-STAT5 in CD34+ CML cells treated for 24 hours with the combination of TKIs plus TG101219 as compared to single TKI-treated cells (~44% vs 65% for p-CrKL and 36% vs 57% for p-STAT5, n=3). Strikingly, the combination treatment produced an even greater inhibition of both p-CrKL and p-STAT5 after 72 hours while p-CrKL was almost fully reactivated with TKIs alone (~29% vs 89% for p-CrKL and 23% vs 50% for p-STAT5). These results point to the possibility of achieving improved therapeutic outcomes in CML patients by simultaneously targeting both BCR-ABL and JAK2 activities in the critical TKI-insensitive CML stem/progenitor reservoir. Disclosures: No relevant conflicts of interest to declare.

Azacitidine (AZA) Combined with Idarubicin in Untreated Patients with High Risk MDS – Results of a Phase I/II Study of the Groupe Francophone Des Myelodysplasies

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1720-1720 ◽  
Author(s):  
Lionel Ades ◽  
Benoit de Renzis ◽  
Ramzi Jeddi ◽  
Jacques Delaunay ◽  
Thorsten Braun ◽  
...  
Keyword(s):  
High Risk ◽  
Phase I ◽  
Disease Progression ◽  
Stable Disease ◽  
Overall Response Rate ◽  
Response Rate ◽  
Conflicts Of Interest ◽  
Performance Status ◽  
Single Agent ◽  
Overall Response

Abstract Abstract 1720 Background: hypomethylating agents, especially AZA, have become the reference treatment of higher risk MDS, but the median survival of about 2 years obtained with AZA remains modest, and must be further improved. In addition, if it is able to increase overall survival in MDS, AZA yields only about 30% of marrow response (including CR+PR+ mCR), Idarubicin given at conventional dose (12 mg/m2/d during 3 days) is the anthracycline of choice in the intensive chemotherapy given with cytarabine in patients with high risk MDS and, given as a single agent, induces up to 30% of complete remission (CR) in elderly AML patients. Thus, we designed a phase I/II study evaluating the safety and efficacy of 2 doses of Idarubicin combined with Azacitidine in high risk MDS patients (clinical trial NCT01305135). Methods: For this trial Azacitidine was combined with increasing doses of Idarubicin. Main Inclusion criteria were: (1) IPSS int 2 or high MDS, or CMML with WBC < 13,000/mm3 and marrow blasts > 10% or AML with 20–30% marrow blasts (corresponding to EU label for AZA) (2) Age 3 18 years (3) Performance Status (PS) <=2 (4) no prior treatment except ESAs. Patients received Azacytidine 75 mg/m2/d SC during 7 days every 4 weeks combined on day 8 of each cycle to Idarubicin 5 mg/m2 (administered by 1 hour IV infusion) in the first cohort of 10 patients, escalated to Idarubicin 10 mg/m2 IV in the second cohort of 10 patients after review of toxicity (especially hematological) of the 1st cohort by the independent DSMB r. The primary endpoint of the study was response after 6 cycles according to IWG criteria. Data were analyzed at the reference date of June, 1St 2012. Results: The 20 study patients (from 8 centers) were enrolled between Dec 2010 and Feb 2012, including 7 women and 13 men with a median age of 75 years. At inclusion, WHO classification was RCMD in 1 pt, CMML in 1 pt, RAEB-1 in 6 pts, RAEB-2 in 7 pts, AML in 3 pts and unclassified in 2 pt. Median marrow blasts were 6.5% (0–26) Karyotype (IPSS) was favorable in 7 pts, int in 3 pts and unfav in 8 pts (2 pts had cytogenetic failure). IPSS was high in all patients. PS was 0 in 28% pts, 1 in 50% and 2 in 22%. A total of 92 cycles of treatment had been administrated with a median number of 5 cycles/patient and 10 pts had received 6 or more cycles. 14 patients had terminated the study due to side effects (severe febrile pancytopenia, n=2), disease progression (n=5, after 2–10 cycles), death (disease progression, severe septic shock after Cycle 2, and unrelated coma), stable disease after 6 cycles (n=3), and patient decision (n=1). Overall 7 pt had died. 18 SAEs were reported observed in 9 patients, including 10 episodes of febrile neutropenia, 3 episodes of bleeding and 5 unrelated SAE. Of the 20 patients enrolled in the study, 19 were evaluable for response after 3 cycles, including 10/10 in the First cohort and 9/10 in the second cohort. One patient achieved CR, 2 PR, 1 mCR and 2 additional patients achieved stable disease with HI, leading to an Overall response rate of 6/19 (32%). Two patients were still on study but did not reached cycle 6. Thus, after 6 cycles, 17 patients, only could be evaluated. Among them 9/17 (53%) patients were still on study, 2 pts had died, 3 progressed, 2 had experienced sides effects and had terminated the study and 1 pt had withdrawn consent. Two patients achieved CR (including 1 already in CR at cycle 3), 2 PR and 2 additional patients achieved stable disease with HI leading to an Overall response rate of 6/17 (35%). At the time of the present analysis, none of the responder had relapsed. Conclusion: The phase I/II results presented here show that Idarubicin can be combined to Azacitidine with acceptable toxicity. Whether the azacitidine- Idarubicin combination can improve the outcome of higher risk MDS patients will be evaluated in a phase II randomized trial comparing this combination (and other combinations of azacitidine with other drugs) to azacitdine alone alone. Data of the present phase I/II trial will be updated at the meeting. Disclosures: No relevant conflicts of interest to declare.

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