scholarly journals Targeting Polo-like Kinase 4 Triggers Polyploidy and Apoptotic Cell Death in TP53-Mutant Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1167-1167
Author(s):  
Edward Ayoub ◽  
Rafael Heinz Montoya ◽  
Vakul Mohanty ◽  
Wencke Walter ◽  
Tallie Patsilevas ◽  
...  
Keyword(s):  
Cell Death ◽  
Board Of Directors ◽  
Cell Lines ◽  
Intellectual Property Rights ◽  
Research Funding ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Advisory Committees ◽  
Grant Support ◽  
Support Research

Abstract Background: TP53 mutations in acute myeloid leukemia (AML) are associated with complex karyotype, high incidence of minimal residual disease (MRD), and high risk of relapse (Döhner et al., 2017; Giacomelli et al., 2018). While numerous novel treatment regimens, including the combination of the BCL2 inhibitor venetoclax (VEN) and hypomethylating agents (HMA), have emerged as partially effective treatments and resulted in higher remission rates in patients with TP53-mutant AML, full clearance of the mutant TP53 clone is rarely achieved and the majority of patients relapse (Short et al., 2021; Takahashi et al., 2016). The mechanisms responsible for response and relapse in TP53-mutant AML remain unclear and investigating novel mechanisms is critical to develop more effective therapies. Results: In order to shed light on the defective p53 signaling pathways underlying TP53 mutant AML, and to better understand mechanisms of resistance, we performed RNA-sequencing (RNA-seq) on FACS-sorted subpopulations using samples collected from TP53-mutant or TP53-wt high-risk AML patients. Samples were collected at diagnosis (DX) and post-treatment (POSTTX) (total number of samples n= 67, TP53-mutant=36, TP53-wt=31). Diagnostic samples include bulk AML, leukemic stem cells (LSCs), and post-treatment samples including bulk mononuclear cells (MNCS) and patient specific MRD (total n= 67, DX_Bulk=15, DX_LSCs=15, POSTTX_MNCs=14, POSTTX_MRD=23). Differential gene expression analysis of TP53-mutant samples indicates a positive enrichment of the following pathways: G2/M checkpoint, MYC targets, and mitotic spindle, among others. We focused here on genes associated with TP53-mutant AML enriched pathways, and identified a key regulator of centriole biogenesis, one of E2F targets: Polo-like kinase 4 (PLK4) as a potential target highly expressed in TP53-mutant AML samples . Previous publications showed that PLK4 is transcriptionally repressed by p53 and induces apoptosis upon RNAi silencing (Fischer et al., 2014; Li et al., 2005). Here we show that TP53-mutant AML samples lack the p53-dependent PLK4 repression and have higher levels of PLK4 compared to TP53-wt AML. To test the rigor of this finding, we interrogated the Munich Leukemia Laboratory (MLL) data base and analyzed their clinically annotated (e.g. karyotype, survival, complete blood counts, previous treatments ... etc) RNA-seq dataset of 726 AML samples (TP53-mutant=72, TP53-wt=654). TP53-mutant AML samples consistently showed significant PLK4 upregulation (p= 0.0003). We analyzed PLK4 expression and its correlation with TP53 mutations in The Cancer Dependency Map project dataset (1375 cell lines in 35 different types of cancers) (p= 0.004). Furthermore, we found significantly higher PLK4 protein levels in TP53-mutant AML MOLM13 cell lines when compared with syngeneic TP53-wt AML MOLM13 cells. Experimentally, we found that PLK4 inhibition using 25nM CFI-400945 in TP53-mutant AML MOLM13 cell lines triggers polyploidy > 2-fold higher than in TP53-wt AML MOLM13 cell lines 72 hours post treatment (Fig.1A p< 0.0001). Finally, we show that polyploidy is not reversible after drug removal and results in significantly increased levels of apoptotic cell death in TP53-mutant AML MOLM13 cells (Fig.1B). Conclusion: Our data suggest that TP53-mutant AML expresses higher levels of PLK4 in comparison to TP53-wt AML, and targeting PLK4 triggers polyploidy and apoptotic cell death in TP53-mutant AML. A clinical trial is ongoing testing the efficacy of PLK4 inhibition (CFI-400945) in AML (Clinical Trial ID: NCT04730258, TWT-202). Figure 1 Figure 1. Disclosures Issa: Kura Oncology: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding; Novartis: Consultancy, Research Funding. Borthakur: Takeda: Membership on an entity's Board of Directors or advisory committees; ArgenX: Membership on an entity's Board of Directors or advisory committees; Ryvu: Research Funding; Astex: Research Funding; University of Texas MD Anderson Cancer Center: Current Employment; Protagonist: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy. Konopleva: Ascentage: Other: grant support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Stemline Therapeutics: Research Funding; KisoJi: Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Sanofi: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Rafael Pharmaceuticals: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Cellectis: Other: grant support; Calithera: Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Andreeff: Senti-Bio: Consultancy; ONO Pharmaceuticals: Research Funding; Glycomimetics: Consultancy; Aptose: Consultancy; Breast Cancer Research Foundation: Research Funding; Oxford Biomedica UK: Research Funding; Karyopharm: Research Funding; Medicxi: Consultancy; Amgen: Research Funding; AstraZeneca: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Syndax: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company.

Methyljasmonate, An Inhibitor of Glycolytic Energy Production, Displays Pre-Clinical Activity against Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1741-1741
Author(s):  
Steffen Klippel ◽  
Jana Jakubikova ◽  
Jake Delmore ◽  
Melissa G. Ooi ◽  
Douglas McMillin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cancer Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Energy Production ◽  
Advisory Committees ◽  
Normal Cells

Abstract Abstract 1741 Poster Board I-767 Background In contrast to most normal cells, cancer cells typically produce energy predominantly by glycolysis as demonstrated by O. Warburg more than 50 years ago. Methyljasmonate (MJ), a hormone produced by plants in response to biotic & abiotic stresses such as herbivory and wounding, has been shown to prevent the interaction of hexokinase (Hxk) and voltage dependent anion channels (VDACs), thereby significantly impacting the onset of glycolytic energy production. This may explain promising preclinical results observed with MJ against a variety of cancer cells, including myeloid leukemia and B-cell lymphoma cell lines. Methods and Results We tested the potential of MJ against Multiple Myeloma (MM) cells. We first evaluated the response of 16 different MM cell lines to 24 h of exposure to MJ concentrations of 0.5 – 3.5 mM using MTT assays. 15/16 of the MM cell lines tested displayed an IC50 of < 1.5 mM. In contrast, HS-5 stroma cells and peripheral blood mononuclear cells (PBMCs) did not respond to that MJ concentration, and even at a concentration of 2.5 mM MJ showed a maximal reduction of cell viability of 40%. Similarly to MM cell lines, purified CD138+ primary tumor cells of 3 MM patients displayed an IC50 of < 1.5 mM, suggesting that the differential sensitivity of MM vs. normal cells to MJ is not restricted to cell lines, but is also observed with primary tumor cells. Importantly, neither co-culture with HS-5 stroma nor IL-6 protected MM cells against MJ. Cell death commitment assays revealed that 1h exposure of 1.5 mM MJ induced cell death. Annexin V/PI FACS analysis of MJ-exposed MM cells showed that the cell death is mainly driven by apoptosis, evidenced by cleavage of caspases 3, 8 and 9 as well as of PARP. However, pre-incubation of MM cells with specific caspase inhibitors such as 10 mM of AC-DEVD-CHO, Z-IETD-fmk, Z-LEHD-fmk or 50 mM of Z-VAD only minimally protects the cancer cells from MJ exposure. Therefore, the impact of the MJ is not solely due to caspase triggered proteolytic cascades. Measurements of cellular ATP content by cell titer glow (CTG; Promega, Madison, WI) assay showed rapid depletion of ATP triggered by MJ action in sensitive MM cell lines. Additionally, we observed that 1 h exposure to 2 mM MJ modulated signaling pathways including IRS1/PI3K/AKT, MEK1/2, as well as Stat3 and JNK. FACS-based cell cycle analysis after propidium iodide staining did not show cell cycle arrest, but rather a rapid transition of cells to G0/G1 No correlation of sensitivity of MM cell lines and the number of mitochondria per cancer cell, as determined by Mitotracker Green (Invitrogen, Carlsbad, CA) -based flow analysis, was observed. We next examined if MJ exhibits either significant antagonism or synergy with established or novel anti-MM agents, including Bortezomib, Lenalidomide, Doxorubicin, Rapamycin or Dexamethasone, but discovered neither. However, MJ displayed synergy when combined with 2-Deoxyglucose. Finally, MJ was tested in vivo in scid/nod mice irradiated with 150 rads, injected with 1× 106 MM1S cells, and then, treated at 500 mg/kg by IP administration on a 5 days on / 2 days off schedule starting two weeks after tumor cell injection, There was an overall survival advantage of MJ-treated animals over the respective controls, with all treated mice (n=10) still alive but 6/10 control mice dead after 27 d. Conclusions Based on its rapidity of anti-MM action, favorable safety profile in preclinical models, distinct pattern of molecular sequelae, and compatibility with established anti-MM agents, MJ represents a promising investigational anti-MM agent. Disclosures Laubach: Novartis: Consultancy, Honoraria. Richardson:Millennium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Novartis Pharmaceuticals: Consultancy, Honoraria; Milllennium: Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.

Role Of Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) and Its Ligands In The Regulation Of Functional OCT-1 Activity In CML Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1470-1470
Author(s):  
Jueqiong Wang ◽  
Chung Hoow Kok ◽  
Richard J. D'Andrea ◽  
Timothy P. Hughes ◽  
Deborah L. White
Keyword(s):  
Cell Death ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
K562 Cells ◽  
Vehicle Control ◽  
Peroxisome Proliferator ◽  
Advisory Committees ◽  
Peroxisome Proliferator Activated Receptor

Abstract Introduction The human organic cation transporter-1 (hOCT-1) is the primary active influx protein for imatinib in BCR-ABL positive cells. The functional activity of the OCT-1 protein (OCT-1 activity, OA) is predictive of molecular response in de-novo chronic phase chronic myeloid leukemia (CP-CML) patients. We have previously demonstrated that diclofenac, a competitive peroxisome proliferator-activated receptor-γ (PPARγ) antagonist, can significantly increase OA in CML cells 1. However, the role of PPARγ and its ligands in OA regulation remain unknown. Thus, the link between OA and PPARγ in CML cells has been investigated in this study. Methods OA was determined by intracellular uptake and retention assay (IUR) in the presence and absence of the OCT-1 inhibitor, prazosin 2. To assess the effect of PPARγ ligands on OA, BCR-ABL positive cell lines (KU812, K562) were incubated with PPARγ antagonist (GW9662, T0070907) or agonists (GW1929, rosiglitazone) respectively for 1 hour immediately prior to the IUR assays. The OA was also assessed in the mononuclear cells (MNCs) of 77 CP-CML patients enrolled to the TIDEL II trial. PPARγ activity in CML MNC nuclear extracts was determined through the use of a PPARγ Transcription Factor Assay Kits according to the manufacturer's instructions. To assess the effect of PPARγ ligands on cell death, KU812 or K562 cells were stained with AnnexinV and 7-AAD for detection of apoptosis after the co-administration of imatinib and PPARγ ligands for 72 hours. Results A significant increase in OA was observed in KU812 and K562 cells treated with PPARγ antagonists. In contrast, PPARγ agonists significantly decreased the OA in both cell lines (Table 1). A negative link between OA and PPARγ activity was observed in CML MNC samples (R=-0.585, p<0.001). PPARγ activity was significantly elevated in CML patients who had a low OA at diagnosis (less than 4 ng/200,000 cells) compared with those who had higher OA (p<0.001). After 72 hours co-administration with 0.1µM imatinib, KU812 cells treated with PPARγ antagonists (GW9662 and T0070907) showed a significantly lower cell viability (40% and 18% respectively) compared with vehicle control (70%, p<0.001). Similar results were also observed in K562 cells after co-administration with 1.0µM imatinib for 72 hours. K562 cells treated with PPARγ antagonists (GW9662: 51% and T0070907: 47%) showed a significantly lower cell viability (51% and 47% respectively) compared with vehicle control (61%, p<0.05). Conclusion Ligand-activation or inhibition of PPARγ is a regulator of OA in CML cell lines, and the low MNC OCT-1 activity in CML patients is consistent with the high level of PPARγ activity in these cells. Low PPARγ activity may be the key driver for low OA and poor imatinib response observed in a subset of CML patients. Importantly, the enhanced OA as a result of PPARγ antagonist treatment resulted in increased cell death following co-administration with imatinib. Ongoing studies relating to the upstream pathways involved in PPARγ activation aim to reveal the possible mechanism of OA modulation by PPARγ. Enhancement of OA by PPARg antagonists is likely to provide an important axis for clinical application to improve the clinical efficacy of imatinib. This would be particularly important in patients with low OA who currently have inferior outcomes with imatinib therapy. 1. Wang J, Hughes TP, Kok CH, et al. Contrasting effects of diclofenac and ibuprofen on active imatinib uptake into leukaemic cells. British Journal of Cancer. 2012;106(11):1772-1778. 2. White DL, Saunders VA, Dang P, et al. Most CML patients who have a suboptimal response to imatinib have low OCT-1 activity: Higher doses of imatinib may overcome the negative impact of low OCT-1 activity. Blood. 2007;110(12):4064-4072. Disclosures: Hughes: Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; CSL: Research Funding. White:Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding.

scholarly journals A New Therapeutic Hypothesis: Nonsense-Mediated Decay Is an Exploitable Target in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1937-1937
Author(s):  
Alexander Leeksma ◽  
Ingrid A.M. Derks ◽  
Brett Garrick ◽  
Torsten Trowe ◽  
Aldo Jongejan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Er Stress ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Protein Production ◽  
Splicing Factors ◽  
Advisory Committees ◽  
Nonsense Mediated Decay

Abstract Background Nonsense-mediated decay (NMD) is a cellular quality control system that degrades mRNAs containing premature termination codons (PTCs) as well as ~10% of normal mRNAs (Kurosaki and Maquat, 2016). NMD thus prevents translation of misfolded proteins, and potential activation of the unfolded protein response (UPR). Mutations in splicing factors such as SF3B1, SRSF2, U2AF1 and ZRSR2 found in hematological as well as solid tumors, can lead to generation of aberrant mRNAs that contain PTCs. Aberrant splicing patterns in cancer cells can possibly result in increased pressure on the NMD machinery. CC-115, a potent inhibitor of mTOR kinase (TORK) and of DNA-dependent protein kinase, (DNA-PK; Mortensen et al., 2015; Tsuji et al., 2017), is in clinical development for the treatment of solid and hematologic malignancies (Thijssen et al., 2016). Preclinical data revealed an additional target of CC-115 and its differential effect on NMD. Our hypothesis was that a subset of tumor cells, especially hematologic tumors with high protein production and/or splicing factor mutations, would be susceptible to NMD inhibition by CC-115. Methods In total, 141 cell lines were screened for sensitivity to CC-115-mediated inhibition of proliferation and induction of cell death, in comparison to specific inhibition of TORK (CC-223). Isogenic DNA-PK knockout cell lines HCT116/HCT116 DNA-PK-/- and M059K/M059J DNA-PK-/- were treated with CC-115 and CC-223. Activity on NMD in vivo was tested using HCT-116 xenograft tumors treated with Vehicle or CC-115. Dependence on CC-115 sensitivity was determined using CRISPR/Cas9 technology of apoptosis or UPR genes in various MM cell lines. RNA sequencing was used for identification of potential targets in sensitive and resistant cell lines. Results A subset of cancer cell lines underwent cell death at sub-micromolar concentrations of CC-115 due to inhibition of NMD, but this was independent of mutations in splicing factors such as SF3B1. We next focused on MM cells as these generally produce high levels of (immunoglobulin) proteins and are prone to ER stress, and therefore potentially susceptible to NMD inhibition. Indeed, treatment with CC-115 resulted in activation of the UPR independent of TORK and DNA-PK inhibition, and cell death in 11/12 MM cell lines. Activity of CC-115 correlated strongly with cell death by the known ER-stress inducer, thapsigargin. Cell death by CC-115 occurred by the mitochondrial pathway of apoptosis, as it depended on caspase activity and the presence of Bax-Bak. Analysis of RNA sequencing data is ongoing and has indicated potential targets dictating sensitivity to CC-115-mediated cell death. Conclusions We describe that hematologic tumors with high protein production are specifically sensitive to CC-115, a novel and clinically exploitable inhibitor of NMD. This might lead to application in malignancies that depend on NMD to avoid excessive protein stress, such as multiple myeloma. Disclosures Garrick: Celgene: Employment. Trowe:Celgene: Employment. Kater:Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Eldering:Celgene: Research Funding. Filvaroff:Celgene: Employment.

scholarly journals The Unfolded Protein Response Mediates Resistance in AML and Its Therapeutic Targeting Enhances TKI Induced Cell Death in FLT3-ITD + AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2246-2246
Author(s):  
Timo Jaquet ◽  
Christian Preisinger ◽  
Marlena Bütow ◽  
Stefan Tillmann ◽  
Nicolas Chatain ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Death ◽  
Board Of Directors ◽  
Research Funding ◽  
Bone Marrow Niche ◽  
Advisory Committees ◽  
Clonogenic Potential ◽  
The Unfolded Protein Response ◽  
Support Research

Abstract Introduction: The unfolded protein response (UPR) is a stress sensing signaling network that is activated upon endoplasmic reticulum (ER) stress, a condition characterized by an accumulation of mis- and unfolded proteins in the ER. To retain a functional cell metabolism, UPR activation increases protein folding and degradation. Acute myeloid leukemia (AML) stem cells are prone to develop ER stress, due to their oncogene-driven metabolism and the bone marrow niche, where they face stressors like hypoxia or nutrient fluctuations. Our preliminary work showed enhanced UPR gene expression levels, especially of IRE1α and XBP1, in different AML subtypes. Patients with high XBP1 mRNA expression had an inferior overall survival rate compared to patients with low XBP1 mRNA expression. Aims: We studied the role of elevated UPR signaling in AML therapy resistance and assessed the therapeutic potential of IRE1α-XBP1 inhibitor STF-083010 (STF) as a new strategy in different AML subtypes, including FLT3-ITD + AML. Methods: Human MV4-11 (FLT3-ITD), RS4-11 (FLT3 wildtype; WT), NB-4 (PML-RARα), THP-1 (MLLr) cells, and murine 32D cells transduced with FLT3-ITD or FLT3 WT were analyzed via western blot and RT-PCR. Metabolic activity was assessed by MTT assay, cell death and apoptosis were measured with propidium iodide (PI) or Annexin V staining using flow cytometry. FLT3 cell surface expression was measured via flow cytometry. The clonogenic potential was determined in CFU assays, using patient-derived mononuclear and CD34 + cells. For hypoxic experiments, MV4-11 cells were cultivated under hypoxia (3 % O 2) and cells were subjected to phosphoproteomic analysis, which was performed by mass spectrometry. Conditional Mx1-Cre/XBP1 fl/fl knockout mice were generated and deletion of XBP1 was induced by IP injection of Polyinosinic-polycytidylic acid (Poly(I:C)). Bone marrow and spleen cells were analyzed via flow cytometry and RT-PCR. Results: Treatment with FLT3 TKI AC220 specifically enhanced IRE1α mRNA (9.3-fold, p&lt;0.05) and increased IRE1α protein in 32D FLT3-ITD cells. Likewise, the percentage of dead cells was significantly elevated in 32D FLT3-ITD upon IRE1α inhibition by STF compared to 32D FLT3 WT cells. Treatment with STF prevented XBP1 splicing and reduced the metabolic activity of human AML cell lines in a dose-dependent manner. Furthermore, IRE1α inhibition significantly induced apoptosis in human MV4-11 (6-fold, p&lt;0.05), NB-4 (8-fold, p&lt;0.01) and THP-1 (7-fold, p&lt;0.01) cells and reduced their clonogenic potential. The combination of STF and AC220 strongly enhanced the percentage of apoptotic cells in MV4-11 cells compared to single treatments (by 3-fold, p&lt;0.001). This strong induction of cell death was specific for FLT3-ITD + MV4-11 cells and not observed in FLT3 WT + RS4-11 cells. Similarly, the clonogenic potential of MV4-11 cells and FLT3-ITD + AML mononuclear patient cells was significantly decreased by the combinatorial treatment, while healthy donor cells were not affected. Likewise, conditional XBP1 knockout did not significantly alter normal hematopoiesis in mice. Hypoxia further enhanced IRE1α signaling in MV4-11 cells and strongly reduced the efficacy of AC220 (normoxia: 58.4-fold induction of dead cells, p&lt;0.01; hypoxia: 2.2-fold induction, p&gt;0.05). Analysis of phosphoproteomics revealed a less active FLT3 signaling under hypoxia. Intriguingly, the combination of IRE1α and FLT3 inhibition overcame the resistance towards AC220 under hypoxia and significantly induced cell death. Conclusion: IRE1α-XBP1 signaling is activated in different AML subtypes including FLT3-ITD + and is further enhanced by hypoxia present in the bone marrow niche. Targeting IRE1α in FLT3-ITD + cells effectively decreases clonogenic growth and induces apoptosis. Our data demonstrate that hypoxia-mediated resistance against AC220 can be overcome by simultaneous IRE1α inhibition. Genetic deletion of XBP1 does not harm steady-state murine hematopoiesis, rendering XBP1 an excellent therapeutic target. Disclosures Koschmieder: CTI: Membership on an entity's Board of Directors or advisory committees, Other; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Baxalta: Membership on an entity's Board of Directors or advisory committees, Other; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); AOP Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Shire: Honoraria, Other; Image Biosciences: Other: Travel support; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Geron: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Karthos: Other: Travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Alexion: Other: Travel support; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Abbvie: Other: Travel support; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Brümmendorf: Bristol Myers: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Repeat Diagnostics: Research Funding; Takepart Media: Honoraria.

scholarly journals BCL-2, a Therapeutic Target for High Risk Hypodiploid B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 280-280 ◽  
Author(s):  
Ernesto Diaz-Flores ◽  
Evan Q. Comeaux ◽  
Kailyn Kim ◽  
Kyle Beckman ◽  
Kara L. Davis ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Advisory Committees ◽  
Chromosomal Number ◽  
Patient Derived Xenograft

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood. Specific genetic subsets, including hypodiploid ALL, are associated with particularly high rates of relapse. Despite the poor outcomes of hypodiploid B-ALL with traditional therapeutic approaches, there have been no known effective alternative therapies or novel candidates tested to improve outcome. We hypothesized that new therapeutic targets could by identified by integrated biochemical and genomic profiling, combined with functional drug assays in order to determine which pathways play an essential role in transformation. For biochemical profiling, we analyzed multiple pathways commonly deregulated in leukemias using phosphoflowcytometry (including receptor tyrosine kinases, JAK/STAT, MAPK, PI3K, PTEN, Bcl-2 survival and pro-apoptotic family members and p53). We subjected hypodiploid cell lines (NALM-16, MHH-CALL2) and patient derived xenograft samples in vitro to inhibitors against each of these pathways (PP2:Src family;Ruxolitinib: JAK/STAT; PD235901/CI1040: MAPK; GDC-0941, PI-90, PI-103, p110 (a, b, g, d): PI3K isoform specific; PP-242:mTOR; ABT-263/ABT-737: Bcl-2/Bcl-xl, and ABT-199: Bcl-2 specific). We found that the Bcl-2 inhibitors (ABT-263, ABT-737 and ABT-199) and to a lesser extent PI3K pathway inhibitors GDC-0941 and PP-242, but not the MAPK or RTK inhibitors, efficiently reduced proliferation of hypodiploid cells. However, only ABT-263/ABT-199 induced high levels of apoptosis at nanomolar concentrations. Based on the consistent efficacy observed with ABT-199 against hypodiploid patient-derived cells and cell lines in culture, we selected eight cryopreserved, previously xenografted (F3 generation) hypodiploid patient samples (4 low hypodiploid, chromosomal number between 32 and 39; and 4 Near Haploid, chromosomal number between 24 and 31) and three non-hypodiploid patient samples (Ph-positive,Ph-Like and Erg+) for a preclinical trial in immunodeficient mice. Each patient sample was engrafted into six mice, which were randomized to receive vehicle or ABT-199 daily over 60 days (Figure 1). Treatment started when the peripheral blood (PB) human CD45 count reached 15%. A rapid decrease in PB blasts was noted at 7 days (Figure 1). Eighty-five percent of the hypodiploid xenografts survived 60 days with either undetectable or low levels of leukemia in the PB. In contrastPh+ andPh-Like xenografts died within 10-20 days regardless of treatment. Importantly, hypodiploid leukemic blasts gradually emerged after discontinuing ABT-199 after 60 days. Additionally, despite low or undetectable levels of leukemic blasts in PB and reduced levels in bone marrow and spleen, all mice had high percentages of leukemic cells in the liver (Figure 2). In conclusion we have identified the survival protein Bcl-2 as a promising molecular target in hypodiploid B-ALL. ABT-199 for dramatically reduced leukemia cells in vitro and in vivo in patient-derived xenograft models of hypodiploid B-ALL. However, the liver represented a protective niche for these leukemias. In addition, our biochemical characterization of the organ infiltrating blasts collected from mice on trial indicate that the sensitivity of hypodiploid ALL to ABT-199 relies not only on high levels of Bcl-2 and deficiency for other survival proteins such as Bcl-xl but also on high levels of proapoptotic proteins, providing two different signatures that correlate with response to ABT-199. Using genome editing (CRISPR/Cas9) we interrogated the necessity for individual proapoptotic genes, including PUMA, NOXA, and BAD, for ABT-199-induced cell death. This study provides encouraging preclinical data that Bcl-2 may be a promising target for the treatment of hypodiploid B-ALL. Our studies identify signature biomarkers that correlate with drug response and identify essential proteins mediating ABT-199-induced cell death. Importantly, this report also identifies the limitations of using ABT-199 as single drug, and provides the rationale for using combinatorial therapies in order to improve the efficacy of the drug. Disclosures Mullighan: Loxo Oncology: Research Funding; Amgen: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees. Loh:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.

scholarly journals Targeting Hypersumoylation in Mantle Cell Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4060-4060 ◽  
Author(s):  
Walter Hanel ◽  
Liudmyla Tsyba ◽  
Dennis Huszar ◽  
Alex Prouty ◽  
Xiaoli Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Committees ◽  
Human B Cells ◽  
Mantle Cell ◽  
Normal Human ◽  
Protein Sumoylation

Mantle cell lymphoma (MCL) is an aggressive and incurable subtype of B-cell Non-Hodgkin's lymphoma (NHL) characterized by genetic dysregulation of CyclinD1. Despite the improvement in response rates with current therapies, MCL patients inevitably relapse and outcomes remain poor. This is particularly true for MCL patients progressing on novel targeted therapies such as ibrutinib, highlighting the continued need for new therapeutic approaches. SUMOylation is a post-translational modification regulated by SUMO Activating Enzymes 1 and 2 (SAE1/2) affecting function, stability, and subcellular localization of a multitude of proteins such as Cyclin D1 and regulating multiple cellular functions such as cell cycle control and DNA damage response. While not yet explored in MCL, it is known that hyper-SUMOylation is associated with augmented cell proliferation and tumor growth of a number of cancers including B-cell NHL. We evaluated the expression levels of SAE1/2, total SUMO1, and SUMO 2/3 in normal human B cells, primary MCL patient samples, and a panel of 8 MCL cell lines via immunoblotting. We found significantly increased levels of SAE1/2 and total protein SUMOylation in 4 out of 5 MCL patient samples and all MCL cell lines compared to normal human B-cells. To validate the SAE complex as a potential therapeutic target in MCL, we performed genetic knockdown of SAE1 and SAE2 using both shRNA and an inducible CRISPR/Cas9 system and found significant reduction in viability of MCL cells (p < 0.001) thus confirming that SUMOylation is essential for MCL survival. TAK-981 (Takeda Pharmaceuticals) is a potent and selective inhibitor of the SAE1/2 complex currently in a phase 1 clinical trial (NCT036483). We found that treatment of MCL cell lines with TAK-981 resulted in time- and dose-dependent cell death in 7 of 8 MCL cell lines (IC50 17 - 62.5 nM at 72 hr) which was associated with relevant decrease in protein sumoylation. MCL cells were sensitive to TAK regardless of ATM or p53 mutations. Finally, TAK-981 treatment prolonged the survival of SCID mice engrafted with a human MCL cell line (Jeko) compared with placebo control [median overall survival (OS): TAK-981, 34 days; placebo, 29 days, p = 0.008] and also extended the survival of a novel patient derived xenograft (PDX) mouse model of ibrutinib-resistant MCL (median OS: TAK-981, 60 days; placebo, 55 days, p = 0.001), thus establishing the in vivo efficacy of TAK-981 in models of aggressive MCL. Mechanistically, 24 hours of treatment with TAK-981 resulted in a profound G2M cell cycle arrest in 6 out of 7 TAK-981-sensitive MCL cell lines. Cell synchronization with palbociclib followed by release into TAK-981 showed significant apoptosis upon G2M re-entry. In addition, in p53-deficient MCL cell lines, we found rapid accumulation of polyploid and aneuploid cells followed by rapid cell death following 48 hours of drug exposure. These findings strongly support mitotic catastrophe as a significant mechanism of tumor cell death mediated by TAK-981. Upon fractionation of cells at distinct phases of the cell cycle, we found significantly increased levels of protein SUMOylation by both SUMO1 and SUMO2/3 at the G2M transition. Further mechanistic data will be presented at the meeting. Given the multiple immune dampening mechanisms of SUMOylation, we are currently studying the anti-MCL immune effects of TAK-981. To do this, we are employing a novel immunocompetent mouse model of MCL in which murine lymphoma cells from Eμ-SOX11/CCND1 double transgenic animals are adoptively transferred into syngeneic mice. These mice develop a systemic lymphoma with morphological, molecular, and phenotypic features characteristic of MCL resulting in death within 3-4 weeks. Preliminary results with this model show that treatment with TAK-981 leads to decrease in lymphoma burden and significant prolongation of survival. Studies into the immune mediated anti-lymphoma effects of TAK-981 using this model are ongoing and will be presented at the meeting. Together, our data strongly support further development of TAK-981 as a novel MCL therapeutic. Disclosures Huszar: Takeda Pharmaceuticals: Employment, Equity Ownership. Parekh:Karyopharm Inc.: Research Funding; Foundation Medicine Inc.: Consultancy; Celgene Corporation: Research Funding. Maddocks:BMS: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Merck: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees. Baiocchi:Prelude: Consultancy.

scholarly journals Preclinical Activity of Novel MCL1 Inhibitor AZD5991 in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 952-952 ◽  
Author(s):  
Shannon M Matulis ◽  
Vikas A. Gupta ◽  
Izabelle Brown ◽  
Jonathan J Keats ◽  
Paul Secrist ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Contact ◽  
Advisory Committees ◽  
Highly Sensitive ◽  
Ic50 Values ◽  
Cell Cell

Abstract We and others have previously demonstrated that MM is often dependent on MCL1 or co-dependent on MCL1 and BCLXL or BCL2 for survival. Therefore, drug development targeting MCL1 has been a top priority. Here we report on AZD5991, a specific small molecule inhibitor of MCL1. We treated 17 MM cell lines with increasing concentrations of AZD5991 for 24 h and measured Annexin V staining to determine the IC50s. Nine of the cell lines tested were highly sensitive to AZD5991 with IC50 values below 100 nM, 6 lines exhibited an intermediate sensitivity (IC50 100-1000 nM), and only 2 cell lines tested were resistant (IC50 >1000 nM). Six of the highly sensitive lines are t(11;14) and sensitive to venetoclax suggesting co-dependence on BCL2 and MCL1 for survival. We also determined the effect of the bone marrow microenvironment on the response of MM cell lines to AZD5991. We reported that IL-6 protects MM cell lines and patient samples from apoptosis by making the cells more MCL1 dependent. Based on this, we predicted IL-6 would have little to no effect on AZD5991-induced cell death. We treated 12 cell lines with AZD5991 in the presence of 1 ng/mL IL-6 or 10% Hs5 conditioned medium (CM) for 24 h and found that only 3/12 and 2/12 lines were protected from apoptosis in the presence of IL-6 and CM, respectively. Interestingly, when co-cultured with the stromal cell line Hs5, 7/11 lines tested were protected from AZD5991-induced cell death, suggesting cell-cell contact is influencing the response. This is in contrast to ABT-737 and venetoclax where cell-cell contact provided no additional protection than CM. Mechanistically apoptosis induced via MCL1 inhibition is not dependent on BIM expression as is the case with BCLXL and BCL2 inhibition. KMS26 and LP1 MM cell lines contain a bi-allelic deletion of BIM and we have reported their resistance to ABT-737. However, both cell lines respond to AZD5991 with IC50 values in an intermediate sensitivity range. Co-immunoprecipitation (CoIP) studies were employed to determine the protein bound to MCL1 that could be promoting apoptosis upon release. We found NOXA and BAK bound in KMS26 and LP1 and both were released from MCL1 in response to AZD5991. Additionally, CoIPs performed on cell lines expressing BIM showed NOXA, BIM, and BAK bound to MCL1 and released following treatment. To further investigate we used CRISPR-cas9 to generate MM cell lines lacking expression of NOXA, BAK, BAX, or BIM. In KMS26 and LP1, deletion of NOXA and BAX had little effect on AZD5991-induced cell death while the BAK deletion significantly inhibited apoptosis in both cell lines. Similar results were observed in the BIM expressing cell line OCI-My5, with no protection from AZD5991-induced apoptosis in the NOXA and BAX edited lines, significant protection in the BAK-deleted line, and an intermediate degree of protection in the BIM knockout line. In KMS18, BIM deficiency had a minimal effect on apoptosis following MCL1 inhibition, however both BAX and BAK were required for AZD-induced cell death. Additionally, we have tested 41 samples from 37 patients for sensitivity to AZD5991. Samples were treated with increasing concentrations to determine IC50 values in the same manner as the MM cell lines. The samples segregated into 4 groups based on IC50. The most sensitive group (N=3) had an IC50 below 10 nM. The largest group had an IC50 range of 50-114 nM (N=26). The last two cohorts were more resistant with a range of 500-916 nM (N=10) and 2 samples with an IC50 over 1300 nM. Since MCL1 is on 1q21, a frequently amplified region in MM, we determined if 1q21 gain was associated with sensitivity. For the 35 samples where FISH data were available, 18 had 1q21 gains by FISH while 17 were negative. There is a trend for the 1q21 gain cohort to be more sensitive (P=0.0573), with only 2/18 having an IC50 above 109 nM. In contrast for the 1q21 negative 7/17 were in the resistant groups. Thus 1q21 may be a marker of sensitivity to MCL1 inhibitors. The data reported here demonstrate that AZD5991 is effective at inducing apoptosis in MM and can overcome soluble microenvironment resistance factors that influence the response to venetoclax. This appears to be due to differential requirements for pro-apoptotic factors for BCL2 and MCL1 inhibition and suggests an underappreciated complexity in the role of BCL2 and MCL1 in cell survival. Finally these findings also suggest that 1q21 gain may be a marker for AZD5991 sensitivity. A clinical trial is currently ongoing in myeloma. Disclosures Secrist: AstraZeneca: Employment. Cidado:AstraZeneca: Employment, Equity Ownership. Tron:AstraZeneca: Employment. Neri:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Bahlis:Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Kaufman:Roche: Consultancy; Abbvie: Consultancy; Karyopharm: Other: data monitoring committee; Janssen: Consultancy; BMS: Consultancy. Heffner:Pharmacyclics: Research Funding; Genentech: Research Funding; ADC Therapeutics: Research Funding; Kite Pharma: Research Funding. Lonial:Amgen: Research Funding. Nooka:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive technologies: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Spectrum Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Boise:AstraZeneca: Honoraria; Abbvie: Consultancy.

scholarly journals Characterization of Mechanisms Underlying Acquired Venetoclax-Resistance in Mantle Cell Lymphoma: BDA-366 As a Potential Treatment Option

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1580-1580
Author(s):  
Daniela Steinbrecher ◽  
Felix Seyfried ◽  
Johannes Bloehdorn ◽  
Billy Michael Chelliah Jebaraj ◽  
Lüder Hinrich Meyer ◽  
...  
Keyword(s):  
Cell Death ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Resistant Cell ◽  
Advisory Committees ◽  
Apoptotic Protein ◽  
Mantle Cell ◽  
Resistant Cells

Abstract In many cancers the equilibrium of pro- versus anti-apoptotic BCL-2 proteins is deregulated. BCL-2 inhibitors like Venetoclax (VEN) have been shown to be highly active drugs in BCL-2 dependent cancers like chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Despite being highly efficient in cell killing, resistance to VEN can be acquired over time. In addition to understanding the underlying mechanisms of resistance to VEN it is important to identify additional treatment options. BDA-366 is a BCL-2 inhibitor with a different mode of action than the BH3 mimetic VEN. BDA-366 acts by inhibiting the BH4 domain and thereby inducing a conversion of anti-apoptotic BCL-2 into a pro-apoptotic protein. BDA-366 showed high effectivity in inducing apoptosis in CLL cells, in primary as well as in cell lines, while all of the CLL cell lines (n=7) tested were resistant to VEN. Furthermore all of the MCL cell lines (n=5) tested were sensitive to the treatment with BDA-366 while only a subset (3 out of 5) responded to treatment with VEN. In order to investigate whether BDA-366 would be a treatment option for VEN-resistant patients, we generated VEN-resistant MCL cell lines (MINO and MAVER-1) by chronic exposure to the drug. In the resistant cell lines, BCL-2 protein levels were not deregulated. In variance to previous reports in diffuse large B cell lymphoma (DLBCL) (Choudhary et al, Cell Death Dis 2015), resistance in MCL cell lines was not mediated by MCL-1 upregulation. In VEN-resistant MINO cells, MCL-1 expression was similar to the parental cells, while MCL-1 was significantly downregulated in VEN-resistant MAVER-1 cells. In contrast, VEN-resistant MCL cell lines showed BCL-XL upregulation as compared to parental cells, which is in line with results obtained in DLBCL (Choudhary et al, Cell Death Dis 2015). Furthermore, dynamic BH3 profiling validated a dependency on BCL-XL in resistant cells and confirmed that resistance was not mediated by MCL-1. The significance of BCL-XL in mediating resistance to VEN was underlined by additional experiments using navitoclax. In contrast to VEN, navitoclax inhibits BCL-2, BCL-XL and BCL-W and was sufficient to induce apoptosis in both parental and resistant cells. In contrast to the BH3 domain inhibitor VEN, the BCL-2 inhibitor BDA-366 acts by converting BCL-2 into a pro-apoptotic molecule. BDA-366 efficiently induced dose dependent apoptosis in VEN-resistant cells. MINO as well as MINO VEN-resistant cells showed the same sensitivity to BDA-366 while VEN-resistant MAVER-1 cells showed reduced sensitivity to BDA-366 as compared to the parental cells. However, with increased BDA-366 concentrations efficient cell killing was achieved in the VEN-resistant cell lines Overall, these results suggest that VEN-resistance is mostly mediated by permanent upregulation of BCL-XL. BCL-2 levels are not deregulated upon development of resistance to VEN. The inhibition of the BH4 domain and thereby converting BCL-2 into a pro-apoptotic protein proved to be a promising therapeutic option even in cancers with acquired resistance to VEN. Disclosures Döhner: Pfizer: Research Funding; Amgen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria; Pfizer: Research Funding; AROG Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Stilgenbauer:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Inhibition of Replication Induces Non-Apoptotic Cell Death in Fibroblast Cell Lines Derived from LEC Rats

2003 ◽  
Vol 65 (2) ◽  
pp. 249-254 ◽  
Author(s):  
Masanobu HAYASHI ◽  
Taku HAMASU ◽  
Daiji ENDOH ◽  
Reiko SHIMOJIMA ◽  
Toyo OKUI
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Fibroblast Cell ◽  
Apoptotic Cell Death ◽  
Lec Rats ◽  
Fibroblast Cell Lines

scholarly journals ADP-Ribosylation of Actin by the Clostridium botulinum C2 Toxin in Mammalian Cells Results in Delayed Caspase-Dependent Apoptotic Cell Death

2008 ◽  
Vol 76 (10) ◽  
pp. 4600-4608 ◽  
Author(s):  
Karin Heine ◽  
Sascha Pust ◽  
Stefanie Enzenmüller ◽  
Holger Barth
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Clostridium Botulinum ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Adp Ribosylation ◽  
Mammalian Cell Lines ◽  
Adp Ribosyltransferase ◽  
C2 Toxin

ABSTRACT The binary C2 toxin from Clostridium botulinum mono-ADP-ribosylates G-actin in the cytosol of eukaryotic cells. This modification leads to depolymerization of actin filaments accompanied by cell rounding within 3 h of incubation but does not immediately induce cell death. Here we investigated the long-term responses of mammalian cell lines (HeLa and Vero) following C2 toxin treatment. Cells stayed round even though the toxin was removed from the medium after its internalization into the cells. No unmodified actin reappeared in the C2 toxin-treated cells within 48 h. Despite actin being completely ADP-ribosylated after about 7 h, no obvious decrease in the overall amount of actin was observed for at least 48 h. Therefore, ADP-ribosylation was not a signal for an accelerated degradation of actin in the tested cell lines. C2 toxin treatment resulted in delayed apoptotic cell death that became detectable about 15 to 24 h after toxin application in a portion of the cells. Poly(ADP)-ribosyltransferase 1 (PARP-1) was cleaved in C2 toxin-treated cells, an indication of caspase 3 activation and a hallmark of apoptosis. Furthermore, specific caspase inhibitors prevented C2 toxin-induced apoptosis, implying that caspases 8 and 9 were activated in C2 toxin-treated cells. C2I, the ADP-ribosyltransferase component of the C2 toxin, remained active in the cytosol for at least 48 h, and no extensive degradation of C2I was observed. From our data, we conclude that the long-lived nature of C2I in the host cell cytosol was essential for the nonreversible cytotoxic effect of C2 toxin, resulting in delayed apoptosis of the tested mammalian cells.

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