The Natural Product Borrelidin Induces UPR and Apoptosis in AML Cell Lines

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4260-4260
Author(s):  
Leah Jackson ◽  
Shelby Bechler ◽  
Justin Miller ◽  
Amy Brownell ◽  
Danielle Garshott ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Natural Product ◽  
Cell Lines ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Xenograft Model ◽  
Myelogenous Leukemia ◽  
Ic50 Values

Abstract Abstract 4260 Acute Myelogenous Leukemia (AML) is the most common form of leukemia. Current therapies are intense and even those fortunate enough to achieve remission often relapse extending extremely poor prognoses to these patient. The most commonly used therapeutics, namely cytarabine aribinoside, the anthracyclines and etoposide, are decades old and target ubiquitous cellular processes. We have previously reported that small molecules and natural products that activate and exacerbate the unfolded protein response (UPR) can effectively and selectively induce cell death in a wide variety of solid tumor cells. We hypothesized that the UPR might be a viable new therapeutic target in AML and sought to determine whether or not the novel UPR-inducing natural product borrelidin might be used as such an agent. A luminescent proliferation assay performed with panel of four AML cell lines treated with the ER stress-inducing antibiotic tunicamycin (Tm) revealed that three of the cell lines displayed IC50 values between 0.47–2.5μ M, doses of Tm which are known to induce a low to moderate level of ER stress. We then repeated the experiment with the more general UPR-inducing natural product borrelidin, which has been shown to have potent anti-inflammatory properties in several murine assays in vivo. All four cell lines were sensitive to borrelidin, displaying IC50 values between 0.032–0.29 μ M. Time course assays performed with borrelidin revealed 4–20 fold increases in active caspase 3 and 7 indicating borrelidin-induced AML decreases in cell proliferation might be the result of apoptosis. Quantitative reverse-transcription real time PCR performed with mRNA isolated from two AML cell lines revealed an increase in the UPR-related transcripts CHOP, ATF4, and GADD34 and the cell death genes Noxa, Puma, DR5 and Bim confirming that borrelidin could induce the UPR and apoptosis in AML cells. Studies currently underway in our laboratory will determine the ability of borrelidin and other UPR-inducing agents to reduce leukemic burden in an in vivo xenograft model. Disclosures: No relevant conflicts of interest to declare.

Azacitidine/Decitabine Synergism with the NEDD8-Activating Enzyme Inhibitor MLN4924 in Pre-Clinical AML Models

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 578-578 ◽  
Author(s):  
Peter G Smith ◽  
Tary Traore ◽  
Steve Grossman ◽  
Usha Narayanan ◽  
Jennifer S Carew ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Death ◽  
Synthetic Lethality ◽  
Single Agent ◽  
Xenograft Model ◽  
S Phase ◽  
Myelogenous Leukemia ◽  
Phase Accumulation

Abstract Abstract 578 MLN4924 is an investigational small molecule inhibitor of NEDD8-activating enzyme that has shown clinical activity in a Phase I clinical trial in Acute Myelogenous Leukemia (AML). To identify potential combination partners of MLN4924 we performed a high-throughput viability screen in AML cells with 40 approved and investigational agents. In vitro characterization of AML cell lines revealed two distinct cell cycle phenotypes suggesting alternate mechanism of action following MLN4924 inhibition of NAE. One group demonstrated moderate S-phase accumulation with greater than 4N DNA content consistent with DNA-rereplication as a result of CDT1 dysregulation. The second group demonstrated distinct and rapid accumulation of subG1 cells without S-phase accumulation or DNA re-replication suggesting induction of apoptosis and cell death. These observations led us to choose two cells lines representative of each mechanism to understand potential for synergy in AML cells. Two hypomethylating agents were included in the screen (decitabine and azacitidine) and were found to be synergistic with MLN4924 by Combination Index and Blending Synergy Analysis. These data were confirmed with a second NAE inhibitor that is structurally dissimilar to MLN4924. The combination of azacitidine and MLN4924 were shown to result in significantly increased DNA-damage and cell death compared to single agent alone as measured by Western Blotting and FACS analysis of cell cycle distributions. In vivo studies were performed in HL-60 and THP-1 xenografts using MLN4924 on a clinically relevant dosing schedule twice weekly. Single agent azacitidine at its Maximum Tolerated Dose (MTD) had minimal activity in the HL-60 model and was combined with a sub-optimal dose of MLN4924 that when combined induced complete and sustained tumor regressions. The mechanism for the apparent synthetic lethality in this in vivo model is currently under evaluation; however it is supported by a dramatic elevation in DNA damage and cleaved caspase-3 in vivo in the combination arm. A second xenograft model (THP-1) that was also insensitive to single agent azacitidine treatment underwent complete and sustained tumor regressions when combined with MLN4924. Thus MLN4924 and azacitidine can combine to produce synergistic antitumor activity in pre-clinical models of AML. Coupled with their non-overlapping clinical toxicities these data suggest the potential for future combination studies in clinical trials. Disclosures: Smith: Millennium Pharmaceuticals: Employment. Traore:Millennium Pharmaceuticals: Employment. Grossman:Millennium Pharmaceuticals: Employment. Narayanan:Millennium Pharmaceuticals: Employment. Carew:Millennium Pharmaceuticals: Research Funding. Lublinksky:Millennium Pharmaceuticals: Employment. Kuranda:Millennium Pharmaceuticals: Employment. Milhollen:Millennium Pharmaceuticals: Employment.

Investigation of a Novel Cyclin-Dependent-Kinase (CDK) Inhibitor Cdki-73 As an Effective Treatment Option for MLL-AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1365-1365 ◽  
Author(s):  
Ka Leung Li ◽  
Sarah C Bray ◽  
Diana Iarossi ◽  
Julian Adams ◽  
Longjin Zhong ◽  
...  
Keyword(s):  
Cell Death ◽  
Conflicts Of Interest ◽  
Xenograft Model ◽  
Lymphocytic Leukemia ◽  
Cyclin Dependent Kinase ◽  
T Complex ◽  
Mouse Xenograft Model ◽  
Orally Bioavailable ◽  
High Level

Abstract The Acute Myeloid Leukemia (AML) subtype characterised by translocations of the Mixed-Lineage Leukemia gene, MLL (t11q23; MLL-AML), is a particularly devastating disease with a median overall survival of only 9 months with current standard therapy. Cyclin dependent kinase (CDK) 9 inhibitors (CDK9i) directly target the CDK9/cyclin T complex (pTEFb) that is essential for activity of the MLL-fusion proteins and for transcriptional elongation, and therefore leads to reduction of transcript levels for multiple key leukemic oncogenes e.g. HOXA9, MYC and MCL1. Several observations suggest that utilising CDK9i to simultaneously target these oncogenes will be an effective strategy for AML, and MLL-AML in particular: (i) Leukemic stem cell (LSC) fractions of AML cells express a high level of MCL1, (ii) Targeting MCL1 has been demonstrated to reduce leukaemia cell survival in a murine model of MLL-ENL, (iii) MCL1 is consistently elevated in AML patients at relapse, (iv) HOXA9 is critical for leukemogenesis in many AMLs, in particular MLL-AML, (v) MYC has been shown to be a critical oncogene in MLL-AML, and (vi) CDK9 function has been shown to be important for MYC-driven tumorigenesis. Our in vitro and in vivo data support the clinical potential of a novel orally bioavailable inhibitor of CDK9, CDKI-73, as an effective therapy for MLL-AML patients. CDKI-73 is a potent inhibitor of CDK9 (Ki 3.5nM)1 and has been shown to induce down-regulation of MCL1, and cell death of Chronic Lymphocytic Leukemia (CLL) B-cells2 and Ovarian Cancer (OvCa) cells3 with nanomolar potency. At doses that are highly toxic for tumour cells, CDKI-73 shows limited toxicity for normal T- and B- Lymphocytes, Bone Marrow Mononuclear cells (BMMNC) and normal colony forming cells (CFC) from the BMMNC fraction. CDKI-73 has many favorable properties also making it an excellent clinical candidate for AML when compared to other CDK9i; in particular, CDKI-73 is (i) unique in its spectrum of inhibition, including targeting CDK6 (IC50 = 0.038 µM; a critical kinase for MLL-AML4), and is (ii) orally bioavailable (F = 56%)2, facilitating sustained in vivo target inhibition. Here we present data showing that in MLL-AML cell lines, CDKI-73 induces growth suppression and apoptosis associated with rapid loss of Myc and MCL1, and activation of PARP. In primary AML patient samples treated with 200nM CDKI-73, we have observed a similar decrease in MCL1 protein levels, with increased 7AAD uptake and Annexin-V staining, consistent with apoptotic cell death. Using a subcutaneous MV4;11 nude mouse xenograft model, we have shown that oral dosing of CDKI-73 (100 mg/kg once every 3 days for 18 days) resulted in a high level of anti-tumour efficacy (p<0.0001 compared to vehicle-treated mice), with minimal toxicity. Moreover, for an established MLL-AML patient-derived xenograft (PDX) generated in NOD/SCID-IL2RG-/- (NSG) mice we also observed significant inhibition of human AML in peripheral blood (p<0.0001), BM (p<0.05) and spleen (p<0.001) with administration of CDKI-73 at 75 mg/kg every 3 days for 15 days. In both models CDKI-73 was well-tolerated at these doses, consistent with our published and preliminary data showing differential effects of CDKI-73 on tumour versus normal cell populations. Given this data, our priority now is to establish the effectiveness of CDKI-73 across a larger panel of primary MLL-AML samples, in further patient derived AML xenografts, and as a combination treatment with AML chemotherapy. REFERENCES: 1. Shao H, Shi S, et al. (2013). J Med Chem. 56(3):640-59. 2. Walsby E, Pratt G, et al. (2014). Oncotarget. 5(2):375-85. 3. Lam F, Abbas AY, et al. (2014). Oncotarget. 5(17):7691-704. 4. Placke T, Faber K, et al. (2014). Blood. 124(1):13-23. Disclosures No relevant conflicts of interest to declare.

Interfering with Arginine Metabolism As a New Treatment Strategy for Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3005-3005
Author(s):  
Bjoern Jacobi ◽  
Lea Stroeher ◽  
Nadine Leuchtner ◽  
Hakim Echchannaoui ◽  
Alexander Desuki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Caspase 3 ◽  
Xenograft Model ◽  
Myeloma Cell ◽  
Intracellular Protein ◽  
Myeloma Cells ◽  
Induction Of Apoptosis

Abstract Introduction Starvation of tumor cells from the amino acid arginine has recently gained particular interest because of the downregulation of the rate-limiting enzyme argininosuccinate synthethase 1 (ASS1) in various cancer entities. ASS1-deficient cells cannot resynthesize arginine from citrulline and are therefore considered arginine auxotrophic. The arginine depleting enzyme arginine deiminase (ADI-PEG20, Polaris Pharmaceuticals) is currently tested in phase I-III clinical trials for different arginine auxotrophic cancers. The natural arginine analogue canavanine can compete with arginine for arginyl-tRNA-binding sites and consequently be incorporated into nascent proteins instead of arginine. Canavanine could therefore potentially further disturb intracellular protein homeostasis, especially under arginine deprivation. The sensitivity of myeloma cells towards arginine depletion strategies has not been analyzed so far. Methods Human myeloma cell lines and CD138-sorted primary human myeloma cells from patient bone marrow were screened for ASS1 expression by western blotting (WB). The cells were cultured in arginine free medium and assessed for proliferation and metabolic activity (CFSE/MTT assays), apoptosis (caspase-3 cleavage) and cell death (annexinV/propidium iodide). Canavanine was supplied in both arginine-sufficient and -deficient conditions. The level of intracellular protein stress was determined by WB and/or flow cytometry analysis for ubiquitinated proteins, phosphorylated eukaryotic initiation factor 2α (peIF2α) and the spliced isoform of the X-Box binding protein 1 (Xbp1s). Repetitive ADI-PEG20 ± canavanine application i.p. were tested in vivo in an U266 myeloma xenograft model in NOD/SCID/IL2Rcg-/- (NSG) mice. Arginine and canavanine levels in plasma were determined by HPLC. Tumor growth was measured, mice were assessed for survival, weight and side effects. Tumor tissues were analyzed for caspase-3 cleavage and Ki67 expression by immunohistochemistry. Results 5 of 6 myeloma cell lines were negative for ASS1. Also, ASS1 was either not or only weakly expressed in the majority of primary CD138+ myeloma patient samples. Arginine starvation induced an arrest of cell proliferation and/or metabolic activity of primary myeloma cells and myeloma cell lines after 18-24 h. Addition of citrulline could only rescue ASS1 positive myeloma cells due to the intracellular resynthesis of arginine. Arginine starvation alone led to delayed induction of apoptosis (e.g. 35% cell death of NCI-H929 cells after 72 h of treatment). Addition of 100 mM canavanine strongly increased cell death specifically in the context of arginine deficiency (e.g. cell death in NCI-H929 cells: 87% after 24 h, 100 % after 48h) while it was non-toxic and had no effect on cell viability under physiological arginine conditions. Co-application of canavanine induced ubiquitination of cellular proteins and led to the prolongation of a fatal unfolded protein response (UPR) as measured by markedly elevated Xbp1s levels. Prolonged UPR ultimately led to the induction of apoptosis as reflected by annexin V binding and caspase-3 cleavage. In an U266 myeloma NSG xenograft model, systemic arginine depletion by ADI-PEG20 suppressed tumor growth in vivo and significantly prolonged median survival of mice when compared with the control group (22±3 vs. 15±3 days). Canavanine treatment alone had no influence on viability (13±0 days). However, the combination of ADI-PEG20 and canavanine demonstrated the longest median survival (27±7 days). Histological examination of explanted tumors showed the highest rates of caspase-3 cleavage in the ADI-PEG20/canavanine group. Conclusion Myeloma cells are mostly arginine auxotrophic and can be selectively targeted by arginine starvation. Combination of arginine depletion with the arginine analogue canavanine leads to a highly efficient and specific tumor cell eradication and should be further optimized in multiple myeloma preclinical models. Disclosures Bomalaski: Polaris Pharmaceuticals Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

In Vitro and In Vivo Activity of the Src/Abl Kinase Inhibitor AP23464 and Analogs Against Activation Loop Mutants of KIT.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 793-793 ◽  
Author(s):  
Amie S. Corbin ◽  
Shadmehr Demehri ◽  
Ian J. Griswold ◽  
Chester A. Metcalf ◽  
William C. Shakespeare ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Myelogenous Leukemia ◽  
Activation Loop ◽  
Wild Type ◽  
Abl Kinase ◽  
Ic50 Values

Abstract Oncogenic mutations of the KIT receptor tyrosine kinase have been identified in several malignancies including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), seminomas/dysgerminomas and acute myelogenous leukemia (AML). Mutations in the regulatory juxtamembrane domain are common in GIST, while mutations in the activation loop of the kinase (most commonly D816V) occur predominantly in SM and at low frequency in AML. Several ATP-competitive kinase inhibitors, including imatinib, are effective against juxtamembrane KIT mutants, however, the D816V mutant is largely resistant to inhibition. We analyzed the sensitivities of cell lines expressing wild type KIT, juxtamembrane mutant KIT (V560G) and activation loop mutant KIT (D816V,F,Y and murine D814Y) to a potent Src/Abl kinase inhibitor, AP23464, and analogs. IC50 values for inhibition of cellular KIT phosphorylation by AP23464 were 5–11 nM for activation loop mutants, 70 nM for the juxtamembrane mutant and 85 nM for wild type KIT. Consistent with this, IC50 values in cell proliferation assays were 3–20 nM for activation loop mutants and 100 nM for wild type KIT and the juxtmembrane mutant. In activation loop mutant-expressing cell lines, AP23464, at concentrations ≤50 nM, induced apoptosis, arrested the cell cycle in G0/G1 and down-regulated phosphorylation of Akt and STAT3, signaling pathways critical for the transforming capacity of mutant KIT. In contrast, 500 nM AP23464 was required to induce equivalent effects in wild-type KIT and juxtamembrane mutant-expressing cell lines. These data demonstrate that activation loop KIT mutants are considerably more sensitive to inhibition by AP23464 than wild type or juxtamembrane mutant KIT. Non-specific toxicity in parental cells occurred only at concentrations above 2 μM. Additionally, at concentrations below 100 nM, AP23464 did not inhibit formation of granulocyte/macrophage and erythrocyte colonies from normal bone marrow, suggesting that therapeutic drug levels would not impact normal hematopoiesis. We also examined in vivo target inhibition in a mouse model. Mice were subcutaneously injected with D814Y-expressing (D816V homologous) murine mastocytoma cells. Once tumors were established, compound was administered three-times daily by oral gavage. One hour post treatment we observed >90% inhibition of KIT phosphorylation in tumor tissue. Following a three-day treatment regimen, there was a statistically significant difference in tumor size compared to controls. Thus, AP23464 analogs effectively target D816-mutant KIT both in vitro and in vivo and inhibit activation loop KIT mutants more potently than the wild type protein. These data provide evidence that this class of kinase inhibitors may have therapeutic potential for D816V-expressing malignancies such as SM or AML.

Glucocorticoids Inhibit Cell Death Induced by Oncogenic Stress After Imatinib Withdrawal In TKI Resistant p190Bcr/Abl Overexpressing Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3150-3150
Author(s):  
Michael A. Dengler ◽  
Annette M. Staiger ◽  
Ute Hofmann ◽  
Matthias Gutekunst ◽  
Heiko van der Kuip ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Glycogen Synthase ◽  
Apoptotic Cell ◽  
Metabolic Stress ◽  
Nucleic Acid Content ◽  
Imatinib Resistance ◽  
Oncogenic Stress

Abstract Abstract 3150 Imatinib resistance is a major problem in treatment of Bcr-Abl positive leukemias, particularly in patients with ALL and advanced CML. One mechanism of this resistance is overexpression of Bcr-Abl. We investigated the effects of Imatinib deprivation in Bcr-Abl overexpressing Imatinib-resistant ALL cell lines. Removal of Imatinib from culture medium led to induction of a non-apoptotic cell death starting approximately 40 hours after Imatinib withdrawal. This cell death was preceded by a rapid and robust induction of Bcr-Abl autophosphorylation and concomitant overstimulation of PI3K-, MAPK-, and JAK/STAT signalling. Over-activation of Bcr-Abl downstream signalling was accompanied by a significant enhanced glucose and amino acid metabolism and an elevated intracellular protein and nucleic acid content. As a result of this enhanced metabolisms we observed a massive increase in cell size, multinucleation, cytoplasmic vacuolization, and reduced intracellular ATP level. The phase-lucent vacuoles did not incorporate the endosome/lysosome tracker, Lyso Tracker Red indicating that the vacuoles were formed by dilation of ER cisternae. To determine if Imatinib deprivation induces an ER stress response concurrent with vacuolization we investigated typical ER stress markers. Upon removal of Imatinib a massive induction of CHOP expression and eIF2 alpha phosphorylation, as well as alternative splicing of XPB could be detected. It has been demonstrated that severe ER stress promotes cell death either by induction of a BIM-dependent apoptitic process or by a TRAF2-RIP1 dependent pathway. Despite of a robust induction of BIM and posttranslational modification of RIP1, siRNA-mediated suppression of BIM and RIP1 had no effect on Imatinib deprivation-induced cell death whereas downmodulation of CHOP partially rescued those cells. Using different small molecule inhibitor libraries, we also identified inhibitors of glycogen synthase kinase-3 (GSK3) and p38-MAPK, as well as glucocorticoids as potent compounds which not only completely prevented metabolic stress and induction of cell death upon removal of Imatinib but also partially repressed induction of CHOP upon Imatinib withdrawal. Importantly, in the presence of glucocorticoids, we found a significant enhanced number of cell clones with Bcr-Abl overexpression in lympoid cell lines upon de novo transfection with Bcr-Abl. In conclusion, acute over-activation of the oncogene Bcr-Abl led to metabolic stress and ER stress followed by a delayed induction of a necrosis-like cell death. These cellular responses to Bcr-Abl-mediated oncogenic stress could be completely blocked by glucocorticoids. Therefore, treatment of glucocorticoid resistant ALL may generate selective pressure towards Bcr-Abl overexpession. Disclosures: No relevant conflicts of interest to declare.

Galectin-9 Overcomes Various Types of Treatment Resistance through ATF-3/Noxa Pathway-Mediated Apoptosis In Chronic Myelogenous Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4930-4930
Author(s):  
Junya Kuroda ◽  
Mio Yamamoto ◽  
Hisao Nagoshi ◽  
Tsutomu Kobayashi ◽  
Nasa Sasaki ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Cell Lines ◽  
Chronic Myelogenous Leukemia ◽  
Inhibitory Effect ◽  
Myelogenous Leukemia ◽  
Growth Inhibitory Effect ◽  
Growth Inhibitory ◽  
Anti Cancer ◽  
New Treatment

Abstract Abstract 4930 Tyrosine kinase inhibitors (TKIs) against Bcr-Abl fusion oncoprotein, such as imatinib mesylate (IM), nilotinib, or dasatinib, are the first-line molecular targeted therapeutics for chronic myelogenous leukemia (CML). However, the resistance to Bcr-Abl TKIs is induced in leukemic cells not only by loss of sensitivity to TKIs through Bcr-Abl-related molecular mechanisms, such as the acquisition of Abl mutation or the overexpression of Bcr-Abl, but also by loss of addiction to Bcr-Abl TK activity by acquiring Bcr-Abl-unrelated additional oncogenic mutations. Therefore, a new treatment approach that induces an anti-leukemic effect via Bcr-Abl-unrelated molecular pathways is urgently needed for achievement of a complete cure and to overcome TKI resistance. Galectins are a family of animal lectins that show specific affinity for beta-galactosides. Among fourteen mammalian galectins, galectin-9 (Gal9) has been shown to possess the anti-cancer properties by regulating various cellular functions, such as cell adhesion, cell proliferation, or apoptosis. These prompted us to investigate whether Gal9 can have an anti-CML effect via signaling cascades distinct from the pathway utilized by Bcr-Abl TKIs or by other commonly utilized anti-cancer agents. Modified human Galectin-9 (hGal9) inhibits the proliferation of six CML-derived cell lines, BV173, KT-1, KCL22, K562, KBM5 and MYL, by inducing apoptosis at their IC50s from 17.5 to 164.9 nM, with the activation of caspases-3, -4, - 8 and -9. The addition of 25 mM lactose prevented the growth inhibitory effect by hGal9 in K562, indicating the essential role of beta-galactoside binding activity in the anti-CML activity of hGal9. Because hGal9 treatment caused upregulation of Noxa, a pro-apoptotic BH3-only protein of Bcl-2 family proteins, and Mcl-1, a member of anti-apoptotic Bcl-2 proteins, in CML cell lines, we next investigated the involvement of Bcl-2-regulated apoptosis pathway in cell death by hGal9. K562 sublines overexpressing Bcl-2, Bcl-XL, or Mcl-1, showed resistance to cell death induced by IM, but were as sensitive to hGal9-induced cell death as the parental cells, suggesting the involvement of a pathway which is independent of Bcl-2 family proteins. These results also indicate that the accumulation of Mcl-1 following hGal9 treatment does not hamper apoptotic induction by hGal9. Besides, the expression of dominant-negative FADD protein did not hamper the effect of hGal9, also indicating that the death receptor pathway was not responsible for apoptosis induced by hGal9. In contrast, our study revealed that hGal9 caused the upregulation of activating transcription factor 3 (ATF3), a member of the ATF/CREB family transcription factors, within 3 hour treatment, and the gene knockdown experiments using RNA interference (RNAi) technique revealed that ATF3 is the critical mediator for cell killing by hGal9. Moreover, RNAi experiments indicated that Noxa is one of the downstream effector molecules of ATF3, and that Noxa partly mediates cell death induction by hGal9. Bim, on the other hand, the BH3-only protein essential for apoptosis by Bcr-Abl TKIs, was not associated with hGal9-induced cell death. Considering that the activation of caspase-4 and caspase-8 is involved in ER stress-induced apoptosis, and that Noxa induction by ATF3 has been shown to be crucial in the cell death induced by inhibitors for ER-associated protein degradation, we suggest that hGal9-induced cell death may at least partly involve ER stress. ATF3-mediated cell death by hGal9 was not hampered by the absence of p53, the presence of mutant AblT315I, or by P-glycoprotein overexpression. In addition, hGal9 showed the additive growth inhibitory effect with IM on CML cell lines. Collectively, hGal9 is a candidate agent that may overcome various kinds of resistance to treatment for CML, and suggest that ATF3 may be a new target molecule for the development of new treatment modalities that can overcome resistance to currently available chemotherapeutics. Disclosures: No relevant conflicts of interest to declare.

The 2nd Generation Proteasome Inhibitor, MLN2238: Potent Induction of Cell Death in T-Cell Lymphoma (TCL) and Hodgkin Lymphoma (HL) Cell Lines and in Two Human Lymphoma Xenograft Models.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2767-2767
Author(s):  
Ravi Dashnamoorthy ◽  
Irawati Kandela ◽  
Savita Bhalla ◽  
Jasmine Galloway ◽  
Irina Zaretsky ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Molecular Mechanisms ◽  
Annexin V ◽  
2Nd Generation ◽  
Ic50 Values ◽  
Xenograft Models ◽  
Dose Dependent Increase

Abstract Abstract 2767 Background: MLN2238 is a novel 2nd generation proteasome inhibitor with significant anti-neoplastic activity. We investigated the preclinical therapeutic efficacy of MLN2238 in TCL and HL cells through in vitro and in vivo tumor models and examined the related molecular mechanisms of action. Methods: TCL cell lines (Jurkat, Hut78 and HH) and HL cell lines (L428, L540, L1236) were treated with increasing concentrations of MLN2238 from 24 to 48 hours. Inhibitor concentrations at 50% cell viability (IC50) values were determined using Calcusyn software. Biological function of MAPK, AKT/PI3K, NFκB, and proteasome activity were analyzed using Western blot. We also interrogated pertinent signaling pathways with shRNA stable knock outs (KO) in the presence and absence of MLN2238. Cell viability was assessed by MTT and apoptosis in was measured with Annexin-V/propidium iodide (PI) flow cytometric analysis; this was confirmed by Western blot analysis for caspase activation and PARP cleavage. In vivo tumor growth inhibition and survival of tumor bearing SCID mice was determined using xenografts derived from Jurkat (TCL) or L540 (HL) cell lines. Cells were inoculated at density of 5×106 subcutaneously (SQ). The in vivo study started with 7–8 mice for each control group and 7–8 mice for each treatment group. Once the tumor volume average reaches 100–250 mm3, the treatment and control groups were injected (5 days per week) with similar volumes of MLN2238 SQ daily or 5% cyclodextrin, respectively, for a total of 3 weeks. Results: Treatment with 50–100 nanomolar (nM) of MLN2238 resulted in time- and dose-dependent increase in cytotoxicity in all TCL and HL cell lines. The IC50 values for 72-hour treatment with MLN2238 were 38nM, 52nM, and 41nM for Jurkat, Hut78, and HH respectively, and 117nM and 39nM for L428 and L540, respectively. Further, MLN2238 resulted in dose-dependent increase in apoptosis as detected by Annexin-V/PI (p<0.001) and cleavage of PARP and caspases 3, 8, and 9 in all TCL and HL cell lines. In vivo experiments with tumor xenografts derived from Jurkat (TCL) and L540 (HL) showed significant inhibition of tumor growth (P<0.001) (see Figure) as well as improved survival (P<0.001) in MLN2238-treated mice with low concentrations of MLN2238 compared with untreated control. We next examined proteasome activity; we detected significant intracellular accumulation of ubiquitnylated proteins in all TCL and HL cell lines following 25–50nM of MLN2238. We also observed decreased levels of total NFkB-p65 (anti-apoptotic) in all TCL and HL cell lines, except L540. In addition, examination of relevant signaling pathways after MLN2238 treatment (25–50nM) showed activation of the MAPK pathway as detected by increased phosphorylation of pERK in TCL (Jurkat, HH, and Hut78) and in HL (L428 and L1236); pERK was undetectable in L540. Next, we tested cytotoxicity of MLN2238 in MEK and ERK KO cell lines using stably transfected shRNA in L540, Hut78, and Jurkat lines. There was minimal effect of these KOs in the TCL lines, while there was increased cytotoxic effect in L540 with ERK shRNA. Treatment with MLN2238 also resulted in decreased levels of total AKT in all TCL and HL cell lines. The role of the JNK signaling pathway in apoptosis is complex where its activation could either lead to induction or suppression of apoptosis. MLN2238 treatment in TCL resulted in decreased levels of pJNK in Jurkat, HH, and Hut78. In contrast, treatment of HL cell lines with MLN2238 resulted in increased levels of pJNK in L428, L540, and L1236. Conclusions: Altogether, we found that the novel 2nd generation proteasome inhibitor, MLN2238, induced potent cell death at nanomolar and clinically achievable concentrations in multiple TCL and HL cells lines and in associated TCL and HL in vivo xenograft models. In HL cells, the cytotoxic effect of MLN2238 appeared to be mediated in part through ERK. Further investigation is required to continue to elucidate the molecular mechanisms of cell death induced by MLN2238 and to identify potential rational novel therapeutic combinations. Clinical investigation of this novel agent in TCL and HL is warranted. Disclosures: Off Label Use: MLN2238 for treatment of T cell lymphoma and Hodgkin's lymphoma.

Obinutuzumab (GA101) Significantly Enhances Cell Death and ADCC Compared to Rituximab Against CD20+ sensitive and Rituximab Resistant B-Cell Non-Hodgkin Lymphoma (NHL) and Lymphoblastic Leukemia (BLL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4865-4865 ◽  
Author(s):  
Aradhana Awasthi Tiwari ◽  
Janet Ayello ◽  
Carmella van de Ven ◽  
Danielle Glassman ◽  
Anthony Sabulski ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Dismal Prognosis

Abstract Abstract 4865 Background: Patients who relapse with CD20+ B-NHL and B cell lymphoblastic leukemia (B-LL) have a dismal prognosis, often associated with chemotherapy resistance (Cairo et al. JCO, 2012,Mils/Cairo et al. BJH,2012) and often require alternative therapeutic strategies. Rituximab (RTX) in combination with FAB 96 chemotherapy is a safe, well-tolerated treatment that is associated with > 90% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Cairo M.S. et al. ASCO, 2010). Resistance to RTX, however, may predispose patients with CD20+ NHL to an increase risk of relapse and or disease progression (Barth/Cairo et al. BJH, 2012; Tsai et al. Cl. Can. Res, 2012). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, mediates enhanced cell death vs RTX and has a glycoengineered Fc region that induces significantly enhanced ADCC (Mössner et al. Bld, 2010; Niederfellner G. et al. Bld, 2011; Bologna L et al. JI, 2012). Objective: To evaluate the in-vitro efficacy of GA101 compared to RTX against RTX sensitive and resistant CD20+ B-NHL and B-LL cell lines. Methods: Raji (CD20+,ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany), Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) and Raji-2R and Raji-4RH (generously supplied by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS and incubated with GA101 and/or RTX at 100 μg/ml for 24 hrs (n=6), 48 and 72 hrs (n=5). Cell death was evaluated by staining with AnnexinV/7AAD and flow-cytometry. Loucy cells (CD20−) were used as the negative control. The caspase 3/7 activity was measured by FAM caspase 3/7 assay kit by FLICA™ methodology. RSCL, RRCL, U698-M and Loucy were incubated with GA101 and RTX treatment for 24, 48 and 72 hrs, and caspase3/7 activity was detected by FACS using 488 nm excitation and emission filter (n=3). ADCC were performed with K562-IL-15–41BBL expanded NK cells (Ayello/Cairo et al. ASH, 2010) as well as IL-2 expanded NK cells, at 20:1 effector: target ratio (E: T, n=3) using europium release assay (Perkin-Elmer). Results: GA101 induced significantly more cell death compared to RTX in B-NHL and BLL cell lines. (Table-1) GA101 vs RTX shows a significantly increase in caspase 3/7 activity in Raji 16.92±0.84% vs 11.76±0.08% compared to Raji2R 6.7±0.62% vs 2.8±0.7%, Raji4RH 5.8±0.35% vs 2.0±0.3% and U698-M 12.54±0.44% vs 9.6±0.95% compared to Loucy 3.22±0.45% vs 2.59±0.05%, respectively, at 24 hrs of treatment (p<0.0001). GA101 vs RTX also elicited a significant increase a ADCC with K562-IL15–41BBL expanded NK cells, in Raji 73.8±8.1% vs 56.81±4.6% compared to Raji-2R 38.0±2.0% vs 21.6±1.2%, Raji-4RH 40.0±1.6% vs 0.5±1.1% and U698-M 70.0±1.6% vs 45.56±0.1%, compared to Loucy 21.67±0.48% vs 15.92±0.52%, respectively (p<0.001) at day 7.The IL-2 alone expanded Hu-NK cells demonstrated a reduction of 10–20% cytotoxicity compared to K562-IL15–41BBL Hu-NK cells at day 7 against BLL, RSCL and RRCL, in-vitro. Conclusion: Obinutuzumab compared to RTX significantly enhanced cell death, caspase3/7 activity and NK mediated ADCC in sensitive and RTX resistant B-NHL and B-LL. Obinutuzumab represents a promising candidate for treating RTX sensitive and resistant CD20+ B-Cell Lymphomas and lymphoblastic leukemia. Further studies will investigate the combination of activated NK cells or chemotherapy that may enhance or synergize with the efficacy of GA101 (Obinutuzumab) both in -vitro and in-vivo in xenografted NOD/SCID mice. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Role of SMAD7 Downstream of TEL-AML1 Signalling in t(12;21) Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4823-4823 ◽  
Author(s):  
Aishwarya Sundaresh ◽  
Maurizio Mangolini ◽  
Jasper de Boer ◽  
Mike Hubank ◽  
Nicholas Goulden ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Xenograft Model ◽  
Direct Role ◽  
Hematopoietic Stem ◽  
Childhood All

Abstract The single most frequent chromosomal translocation associated with childhood ALL is the t(12;21) rearrangement that creates a fusion gene between TEL (ETV6) and AML1 (RUNX1). Although TEL-AML1+ patients have very good prognoses, relapses occur in up to 20% of patients and many patients face long-term side effects of chemotherapy. Recent data has shown that TEL-AML1 has a direct role in inducing signal transducer and activator of transcription 3 (STAT3) activation in human t(12;21) leukemia. This activation has been shown to transcriptionally induce MYC and is critical for survival of TEL-AML+ leukemia cells. Here, we demonstrate that STAT3 also regulates SMAD7 gene expression. SMAD7 is an antagonist of TGF-β signaling, functioning through a negative feedback mechanism, but is also known to function in other biological pathways. Interestingly, SMAD7 has also been shown to play a role in promoting self-renewal of hematopoietic stem cells. We show that both pharmacological and mechanistic inhibition of STAT3 results in down regulation of SMAD7 gene expression in TEL-AML1+ cell lines. This result was specific to TEL-AML1+ cells and not found in cells of other ALL subtypes. To understand the role played by SMAD7 in TEL-AML1+ cells, we used lentiviral vectors expressing shRNA targeting SMAD7. Interestingly, SMAD7 silencing was found to inhibit proliferation of TEL-AML1+ cell lines, eventually leading to growth arrest and apoptosis. Furthermore, we have established that this effect is not mediated through TGF-β signalling. This poster highlights the results of RNA-seq performed on TEL-AML1+ cells with SMAD7 knockdown and in vivo xenograft model of SMAD7 shRNA in TEL-AML+ ALL. Disclosures No relevant conflicts of interest to declare.

ENMD-981693 Is an Orally-Active Kinase Inhibitor with Activity towards Human Hematologic Cancers In Vitro and In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1377-1377
Author(s):  
Mark R. Bray ◽  
Graham C. Fletcher ◽  
Trisha A. Denny ◽  
John Xu ◽  
Xiaoru Chen ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Rapid Development ◽  
Xenograft Model ◽  
Myelogenous Leukemia ◽  
Aurora B ◽  
Ic50 Value ◽  
Ic50 Values ◽  
Orally Active

Abstract The clinical success of Gleevec (imatinib) for chronic myelogenous leukemia has demonstrated that small-molecule inhibitors of specific kinases can be developed into effective oncology therapies. However, the rapid development of resistance in leukemic cells to drugs such as Gleevec and the limited therapeutic indications addressed with these molecules suggests that considerable opportunity exists for new inhibitors with a distinctive spectrum of activities against multiple kinase targets. ENMD-981693 is a novel, orally-active molecule that was discovered through a screening effort directed towards Aurora kinases, a family of serine/threonine kinases that are essential for mitotic progression. ENMD-981693 is selective for the Aurora A isoform, with an IC50 value of 25 nM, compared to an IC50 value of ~700 nM for Aurora B. The activity of ENMD-981693 was evaluated against a panel of 100 recombinant kinases, and the compound was shown to inhibit a broad range of tyrosine kinase targets including Flt3, CSF1R, Lck, JAK2, and c-Kit. ENMD-981693 inhibited the in vitro growth of human hematopoietic cancer lines including MV4;11, K562, THP-1, Jurkat, TF-1, U937, and HL-60 with IC50 values ranging from 0.04 – 21 μM. ENMD-981693 was shown to induce G2/M cell cycle arrest followed by apoptosis in U937 cells, without induction of the endo-reduplication phenotype (≥4N DNA content) associated with Aurora B-acting inhibitors such as MK-0457 (VX-680) and AZD1152. Primary cells derived from AML patients were sensitive to treatment with ENMD-981693 in vitro, resulting in IC50 values from 0.2 – 6.0 μM. Sensitivity of primary CML samples was more variable in in vitro cytotoxicity assays, with IC50s in the range of 0.1 – 40 μM. ENMD-981693 shows significant antitumor activity and is well tolerated in xenograft studies, with no weight loss or morbidity observed in administration schedules of up to 100 mg/kg bid or 200 mg/kg qd, given continuously for more than 30 days. Results from in vivo efficacy studies with ENMD-981693 using the MV4;11 xenograft model will be described. In conclusion, ENMD-981693 is a novel kinase inhibitor with potent activity towards a number of targets important in hematologic cancers.

Sign in / Sign up

Export Citation Format

Share Document