Synergistic Activity of the Aurora Kinase Inhibitor MK-0457 (VX-680) with Idarubicin, Ara-C, and Inhibitors of BCR-ABL.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1384-1384 ◽  
Author(s):  
Russell R. Hoover ◽  
Matthew W. Harding
Keyword(s):  
Clinical Trials ◽  
Cell Viability ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Single Agent ◽  
Leukemia Cell ◽  
Parp Cleavage ◽  
Synergistic Activity ◽  
Dependent Manner ◽  
Wild Type

Abstract MK-0457 (VX-680) is a reversible small molecule kinase inhibitor that targets Aurora A, B, and C with Ki values of 0.7, 18, and 4.6 nM, respectively. MK-0457 also inhibits Flt3 (Ki = 30 nM), and both the wild type and the T315I mutant of BCR-ABL (Ki = 30 and 40 nM, respectively). Clinical trials are ongoing in patients with solid tumors and hematological malignancies. Recent data show that MK-0457 is active in patients against subtypes of AML, BCR-ABL T315I mutant CML, and Philadelphia positive (Ph+) ALL. To support multi-agent clinical trials, the activity of MK-0457 in combination with idarubicin, Ara-C, and BCR-ABL inhibitors was investigated. The viability of a panel of AML, ALL, and CML cell lines was assessed following single agent and either simultaneous or sequential combinations of agents. Combination effects were evaluated using the Bliss Independence Model. MK-0457 as a single agent markedly inhibited leukemia cell viability (at 72 hrs) with an IC50 range of 20–300 nM for MV4-11, Molt-4, Molm-13, K562, LAMA-84, MEG-01, and KU812F cells. Additionally, MK-0457 inhibited the viability of BaF3 cells transformed by wild type, T315I, or Y253F mutants of BCR-ABL with similar IC50s (approximately 300 nM). The sequential combination of MK-0457 followed by either idarubicin or Ara-C showed greater synergy than simultaneous combinations in a cell line dependent manner. MK-0457 displayed strong synergy in simultaneous combination with Gleevec (imatinib mesylate) in a panel of human CML-derived cell lines and BaF3 cells expressing wild type BCR-ABL. MK-0457 enhanced the Gleevec-mediated cell death of K562 leukemia cells as evidenced by increased caspase activity, PARP cleavage, and induction of the sub-G1 population. At concentrations where synergy was observed by cell viability analysis, the MK-0457/Gleevec combination resulted predominantly in aneuploidy and G2/M arrest, consistent with inhibition of Aurora kinases by MK-0457. These results support the clinical evaluation of MK-0457 combined with idarubicin and Ara-C in AML and with BCR-ABL inhibitors in CML and Ph+ ALL.

Mitochondrial-Mediated Apoptosis by the Sesquiterpene Oil α-Bisabolol and Its Synergism with Imatinib and Nilotinib In BCR-ABL+ Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4456-4456
Author(s):  
Massimiliano Bonifacio ◽  
Antonella Rigo ◽  
Elisabetta Cavalieri ◽  
Emanuele Guardalben ◽  
Christian Bergamini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Single Agent ◽  
Leukemic Cells ◽  
Parp Cleavage ◽  
Potential Candidate ◽  
Dependent Manner ◽  
Ros Production ◽  
Dna Laddering

Abstract Abstract 4456 Background. The plant-derived agent α-bisabolol is a small oily sesquiterpene alcohol that has been demonstrated to be cytotoxic against human malignant non-hematological and leukemic cells (Bonifacio M et al, Blood, 2009 ASH annual meeting abstracts;114:4800). Here we tested its activity against BCR-ABL+ cell lines and primary cells from patients, alone or in combination with the Tyrosine-Kinase Inhibitors (TKIs) Imatinib and Nilotinib. Also, the mechanism of α-bisabolol cytotoxicity in BCR-ABL+ cells was assessed. Methods. We used the BCR-ABL+ K562, LAMA-84 and CML-T1 cell lines and primary leukemic cells from 14 patients with BCR-ABL+ Acute Lymphoblastic Leukemia at diagnosis. First, the citotoxicity of single-agent α-bisabolol was determined by MTT. Then, mitochondrial membrane potential of treated cells was evaluated by the JC-1 dye in flow cytometry and fluorescence microscopy. Permeabilized leukemic cells were assayed for oxygen consumption by measuring mitochondrial state 3 and uncoupled respiration. Reactive oxygen species (ROS) production in α-bisabolol treated cells were quantified in flow cytometry by oxidation of CM-H2DCFDA, measuring the fluorescence intensity of the DCF products. Apoptosis was studied by the poly(ADP-ribose) polymerase (PARP) cleavage and internucleosomal DNA laddering analysis. Finally, the combination effects between α-bisabolol and Imatinib or Nilotinib (kindly provided by Novartis) were analyzed according to the median-effect method of Chou and Talalay using the CalcuSyn software. Results. α-bisabolol reduced the viability of BCR-ABL+ cells in a dose-dependent manner. The mean IC50 values of α-bisabolol were 46±11 μ M for primary leukemic cells and ranged from 62 to 115 μ M in the cell lines. JC-1 staining of BCR-ABL+ primary leukemic cells treated with 40 μ M α-bisabolol for 3 to 5 hours demonstrated a dissipation of the mitochondrial transmembrane potential (ΔΨm), thus indicating the start of the apoptotic process. Moreover, NADH-supported state 3 respiration in α-bisabolol treated leukemic cells was significantly decreased in comparison with untreated leukemic controls (140.0±70.5 vs 280.7±11.9 pmol O2/min/106 cells; p<.05). Finally, PARP cleavage and DNA laddering followed α-bisabolol exposure of leukemic BCR-ABL+ blasts. The apoptosis induction was accompanied by ROS production. When tested in combination at constant ratio with Imatinib or Nilotinib, α-bisabolol showed overall slight to strong synergistic effects, without evidence for antagonism across a range of doses (Table 1). In 3 patients with mutation of BCR-ABL (T315I, E255V and Y253H, respectively) we observed full activity of α-bisabolol as single agent and confirmed the synergism between α-bisabolol and Imatinib. Conclusion. This study indicates that α-bisabolol is an effective pro-apoptotic agent for human acute BCR-ABL+ leukemia cells via induction of mitochondrial membrane damage. The combination of α-bisabolol with Imatinib or Nilotinib allows a dose reduction up to 90% of each drug to obtain the same cytotoxic effect, so indicating a clear synergism. α-bisabolol may be a potential candidate for the treatment of BCR-ABL+ leukemias and the effective dose of TKIs could be reduced in a combined treatment with α-bisabolol. Disclosures: No relevant conflicts of interest to declare.

Functional RNAi Screen Identifies Novel Cytokine and Growth Factor Receptors Critical for Leukemia Cell Growth

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1308-1308
Author(s):  
Anupriya Agarwal ◽  
Ryan MacKenzie ◽  
Rita M. Braziel ◽  
Jeffrey W Tyner ◽  
Brian J. Druker
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Cell Viability ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Growth Factor Receptors ◽  
Rnai Screen ◽  
Wild Type ◽  
Downstream Signaling ◽  
Cancer Pathogenesis

Abstract Abstract 1308 Background: Despite the great strides that have been made in the treatment of acute myeloid leukemia (AML), one-third of patients are resistant to treatment. In many cases, disease-causing genetic targets still need to be elucidated. Cytokine and growth factor receptors contribute to cancer pathogenesis by regulating various downstream signaling cascades, including tyrosine kinase driven pathways, which represent amenable therapeutic targets. However, additional investigation is needed to understand the functional and therapeutic implications of cytokine and growth factor receptors in cancer pathogenesis. We have developed a novel RNAi-based screen to uncover the functional and prognostic relevance of growth factor/cytokine receptors for leukemia pathogenesis. This assay enables us to discover novel mechanisms of leukemogenesis involving various non-kinase receptors. Importantly, we show proof-of-principle data where IL2Rγ is found to be essential for JAK3 mutant mediated leukemogenesis. Methods and Results: To identify non-kinase cytokine and growth factor receptors that are critical for leukemia cell viability, we have designed a novel RNAi based functional screen targeting 188 growth factor receptors that were found to be highly expressed by gene microarray analysis of primary AML patient specimens. We have tested 40 AML patient samples and 5 AML cell lines for dependence on these receptors by electroporating cells with 188 individual siRNAs. After culturing cells for four days, effects on cell viability were measured using an MTS assay. Candidate targets found in primary patient samples include CD24, NCOA4, IL2Rα, IL15Rα and IL2Rγ. In many cases, we found that these targets have genetic abnormalities ranging from splice variation (IL2Rα) to intron retention (IL15Rα). In a few cases, the receptor was found to serve as a scaffold for regulating downstream oncogenic signaling and thus contributes to oncogenesis. For instance, we identified IL2Rγ as a potential target in the JAK3 A572V mutation positive AML cell line (CMK). Validation experiments demonstrated that knockdown of IL2Rγ significantly reduces the viability of CMK cells (90% decrease) and abrogates phosphorylation of JAK3 and downstream signaling molecules, STAT5, MAPK and the pS6 ribosomal protein. In a converse experiment, overexpression of IL2Rγ enhanced JAK3 A572V mediated signaling and increased its transformation potential in a ligand-independent manner. Similarly, IL2Rγ overexpression also increased the oncogenic potential of other JAK3 mutants such as M511I, which have previously been identified in AML patients. Intriguingly, the absence of IL2Rγ in murine bone marrow completely abrogated the clonogenic potential of JAK3 A572V as compared to IL2Rγ-wild type marrow. These effects can be rescued by co-expressing IL2Rγ with JAK3 A572V but not by co-expressing IL2Rγ with JAK3 Y100C, a FERM domain inactivating mutation. Additionally, overexpression of IL2Rγ made JAK3 A572V cells less sensitive to the JAK family inhibitor, JAK Inhibitor I (IC50is increased from 150 nM to 625 nM). Mechanistically, IL2Rγ contributes to constitutive JAK3 mutant signaling by increasing JAK3 protein levels and phosphorylation. In fact, mutant but not wild type JAK3 increased the expression of IL2Rγ, indicating IL2Rγ contributes to constitutive JAK3 signaling through a feedback mechanism. Overall these results demonstrate an oncogenic potentiating role of IL2Rγ. Conclusions: RNAi-based functional screening for AML cell dependence on non-kinase cytokine and growth factor receptors led to the identification of novel oncogenic etiologies for AML. These findings underscore the importance of cytokine and growth factor receptors in leukemia pathogenesis. This assay can identify genes that are crucial for malignant cell growth, regardless of the mutational status both in cell lines and in primary samples. Future studies integrating this RNAi screen with deep sequencing will lend additional power to this assay by accelerating our understanding of the genetic mechanisms underlying these functional gene targets such that these findings can be rapidly translated into novel therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.

Alvocidib Potentiates the Activity of Venetoclax in Preclinical Models of Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1652-1652
Author(s):  
Clifford J. Whatcott ◽  
James M Bogenberger ◽  
Wontak Kim ◽  
Hillary Haws ◽  
Nanna Hansen ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cell Viability ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Employment Equity ◽  
Fold Reduction ◽  
Remission Rates ◽  
Equity Ownership

Abstract Introduction Venetoclax (ABT-199) is an approved BCL-2 inhibitor for the treatment of patients with chronic lymphocytic leukemia (CLL). Multiple clinical trials are underway to explore its efficacy in additional indications. While venetoclax demonstrated high remission rates in combination with azacitidine in early stage clinical trials, the question of durability of responses and primary and acquired resistance remain, especially given the modest activity and rapid development of resistance as a single agent. One reported mechanism of intrinsic resistance is high expression of other BCL-2 family proteins, including MCL-1. We and others have demonstrated that the CDK9 inhibitor, alvocidib, can mediate transcriptional repression of anti-apoptotic MCL-1. It has also been shown that alvocidib can increase pro-apoptotic BIM, a dual activator and sensitizer BH3-only protein that can directly induce apoptosis and simultaneously inactivate anti-apoptotic BCL-2 family proteins such as MCL-1 and BCL-2, thus having the same effect on mitochondria-associated apoptosis as MCL-1 down-regulation, with the potential to directly induce apoptosis. An alvocidib-containing cytotoxic chemotherapy regimen demonstrated favorable remission rates in high-risk AML patients over standard therapy in a randomized Phase 2 trial indicating its potential role and safety in AML. We hypothesized that alvocidib and venetoclax would synergize against AML cells by shifting the overall balance of pro- and anti-apoptotic BCL-2 proteins in favor of apoptosis and thus represent a novel active treatment regimen in AML. Aims This study seeks to examine the efficacy of a treatment regimen containing alvocidib and venetoclax in multiple preclinical studies, including in vivo models of AML. Methods Cell viability assays interrogating alvocidib and venetoclax activity in cell lines were performed using CellTiter-Glo according to manufacturer's protocol. mRNA/miRNA expression changes were assessed using standard RT-PCR technique. Protein expression changes were assessed using standard western immunoblotting technique. To assess the efficacy of an alvocidib and venetoclax combination on tumor growth in an in vivo model, the OCI-AML3 xenograft mouse model and ex vivo studies with AML patient samples were performed. Results Herein we demonstrate that alvocidib inhibits both mRNA and protein expression of MCL-1 in a time and concentration-dependent fashion in 3 out of 4 AML cell lines analyzed, while in cells where alvocidib did not reduce MCL-1 protein levels (i.e. MOLM-13) a dose-dependent decrease in miR17-92, and concomitant increase in BIM protein was observed after 24 hours of alvocidib treatment. The alvocidib-venetoclax combination resulted in very strong synergistic reductions of cell viability (with combination indices [CI] of 0.4 to 0.7), both in venetoclax-sensitive and resistant cells. The venetoclax-sensitive lines, MV4-11 and MOLM-13, exhibited 5- to 10-fold reduction of venetoclax EC50 values in the low nM range when combined with only 80 nM alvocidib. Importantly, venetoclax-resistant lines, OCI-AML3 and THP-1, exhibited at least 20-fold reduction of venetoclax EC50 values from near 1 µM to 10-50 nM, when combined with 80 nM alvocidib.In the venetoclax-resistant OCI-AML3 xenograft model, single agent alvocidib and venetoclax achieved tumor growth inhibition (TGI) of 9.7 and 31.5%, respectively, while the combination achieved 87.9% TGI at the same dose levels of individual drugs. Conclusions Taken together, our data suggest that the combination of alvocidib with venetoclax is highly synergistic in vitro and in vivo, in both venetoclax-sensitive and -resistant AML across a heterogeneous genomic background. The particularly high level of synergy achieved in venetoclax-resistant cell lines highlights the central importance of both BCL-2 and MCL-1-mediated cell survival in AML. Importantly, the addition of alvocidib to venetoclax treatment reduced IC50s to clinically achievable concentrations. Therefore, we conclude that an alvocidib/venetoclax combination may be a novel approach for the treatment of AML and warrants further pre-clinical and clinical validation. Disclosures Whatcott: Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Haws:Tolero Pharmaceuticals: Employment. Mesa:Celgene: Research Funding; Galena: Consultancy; Promedior: Research Funding; Ariad: Consultancy; Novartis: Consultancy; CTI: Research Funding; Incyte: Research Funding; Gilead: Research Funding. Peterson:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Weitman:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Small Molecule ONC201/TIC10 Induces Caspase-Dependent Apoptosis in Acute Lymphoblastic Leukemia Cells Via Modulation of Bcl-2 and IAP Family Proteins

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5237-5237 ◽  
Author(s):  
Varun Vijay Prabhu ◽  
Amriti R. Lulla ◽  
Jessica M Wagner ◽  
Liz J. Hernandez-Borrero ◽  
Mala K. Talekar ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Viability ◽  
Solid Tumors ◽  
Cell Lines ◽  
Small Molecule ◽  
Lymphoblastic Leukemia ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Equity Ownership ◽  
The Impact

Abstract ONC201/TIC10 is a potent small molecule anti-tumor agent in several types of solid tumors and lymphomas. ONC201/TIC10 is on track to enter clinical trials for patients with advanced cancer in 2014, with IND issued by the FDA in March, 2014. Early trials will evaluate the safety and efficacy of ONC201/TIC10 as a monoagent in hematological malignancies. In the current study, we evaluated the anti-cancer effects of the small molecule in Acute Lymphoblastic Leukemia (ALL). Analysis of cell viability by the CellTiter-Glo method revealed that ONC201/TIC10 treatment reduces the viability of three ALL cell lines (Reh, Jurkat, MOLT-4) in a dose- (2.5/5/10 μM) and time-dependent manner (24/48/72 h). We have previously reported that ONC201/TIC10-mediated reduction in cell viability and apoptosis in various types of solid tumors occurs at 60/72 h. Interestingly, ONC201/TIC10 reduces the viability of ALL cell lines within 24/48 h at the indicated doses. An inactive TIC10 isomer compound synthesized by Medkoo Biosciences with a structure related to the active ONC201/TIC10 compound does not reduce the viability of ALL cells. Sub-G1 analysis indicated that ONC201/TIC10 induces apoptosis in ALL cells and a pan-caspase inhibitor reduces ONC201/TIC10-mediated apoptosis. Western blot analysis was used to further investigate the mechanism of ONC201/TIC10-mediated apoptosis. ONC201/TIC10-mediated apoptosis involves PARP cleavage and caspase-9 activation. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl are downregulated while the pro-apoptotic Bcl-2 family member Bim is upregulated in response to ONC201/TIC10 treatment. ONC201/TIC10 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2. We have previously shown that the anti-tumor effect of ONC201/TIC10 involves inhibition Akt and ERK phosphorylation resulting in Foxo3a activation and TRAIL-gene transcription. We observed inhibition of Akt phosphorylation upon ONC201/TIC10 treatment of ALL cells. Thus, ONC201/TIC10 holds promise as a novel agent for the treatment of ALL based on its robust activity in preclinical models of the disease. Our ongoing studies are evaluating the impact of this novel therapy on ALL cells with different translocations, and are introducing combination therapy with ONC201/TIC10 for ALL. Figure 1 Figure 1. Disclosures Allen: Oncoceutics: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc.: Equity Ownership, Patents & Royalties.

scholarly journals Targeting XIAP Via Degradation of MDM-2 By MX69 in Aggressive Lymphomas

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5379-5379
Author(s):  
Sumera Khan ◽  
Kyle Runckel ◽  
Cory Mavis ◽  
Matthew J. Barth ◽  
Francisco J. Hernandez-Ilizaliturri
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Parp Cleavage ◽  
Synergistic Activity ◽  
In Vitro Exposure ◽  
Induction Of Apoptosis

Abstract Background: The addition of Rituximab to front-line therapy has improved clinical outcomes in diffuse large B-cell lymphoma (DLBCL), but it has also altered the biology of relapsed/refractory disease. To better understand the mechanisms responsible for Rituximab associated chemotherapy cross-resistance our group developed and characterized several Rituximab resistance cell lines (RRCL). We previously demonstrated using SiRNA interference, that X-linked inhibitor of apoptosis (XIAP) is critical for chemotherapy sensitivity and survival in RRCL. MX69, a dual inhibitor of Mdm2 and XIAP that indirectly downregulates XIAP, is undergoing pre-clinical testing. MX69 affects XIAP levels by its effects on the ubiquitination and degradation of endogenous MDM-2, resulting in decrease XIAP translation and activation of caspase 3, 7 and 9 as well as PARP cleavage leading to apoptosis of cancer cells. In our current work, we pharmacologically inhibited XIAP in lymphoma pre-clinical models using MX69. Materials and Methods: A panel of Burkitt's Lymphoma (BL, including RRCL), germinal center B-cell (GCB)-DLBCL (including RRCL), activated B-cell (ABC)-DLBCL, Mantle cell Lymphoma (MCL) and Pre-B cell Leukemia cell lines were exposed to MX69 as a single agent (0-80uM) over 24, 48, 72 hrs and IC50 concentrations were calculated for each cell line. Changes in Mdm2, p53, XIAP and PARP expressions were determined following MX69 exposure (at IC50 doses) for 24 hrs. Induction of apoptosis was evaluated by Annexin V/propidium iodine staining. Subsequently, cell lines were exposed to MX69 (0-80 uM), in combination with Doxorubicin (0-1uM), Cytarabine(0-50uM), Vincristine (0-10nM), Etoposide(0-50uM), Carboplatin (0-20uM), Ixazomib (0-1.5uM), Ibrutinib (0-20uM) and Venetoclax (0-10uM) for 48 hours. Cell viability was determined by Cell Titerglo. Coefficient of synergy was calculated using CalcuSyn. Results: In vitro, MX69 single agent exposure induced cell death in a dose and time-dependent manner in all cell lines tested. Western blotting studies confirmed downregulation of Mdm2, XIAP and changes in P53 and PARP, following in vitro exposure to MX69. Induction of apoptosis was observed by flow cytometry in all cell lines tested. The combination of MX69 with Doxorubicin, Cytarabine, Vincristine, Ixazomib, Carboplatin, Etoposide, Ibrutinib, and Venetoclax resulted in significant synergistic activity. The strongest CI of synergy was observed when cell lines were exposed to MX69 and Venetoclax, Ixazomib, Etoposide or Ibrutinib. Conclusion: Our data suggests that in vitro exposure of a wide variety of B-cell lymphoma cell lines (including BL, DLBCL, MCL or RRCL) to MX69 resulted in anti-tumor activity. Perhaps related to its anti-tumor effects, MX69 inhibited XIAP levels. These findings are similar to prior SiRNA XIAP knockdown experiments. Strong synergistic activity was observed when XIAP was combined with various chemotherapy agents and small molecules inhibitors (such as Venetoclax, ixazomib or ibrutinib). Ex vivo experiments using primary tumor cells isolated from lymphoma patients and lymphoma mouse models are been planned. Targeting Mdm2 and XIAP can be an attractive therapeutic strategy in patients with Rituximab-sensitive or -resistant B-cell lymphoma. Disclosures No relevant conflicts of interest to declare.

Serum Dependency of t(8;21) AML Cell Line Is Associated with VEGF/VEGFR Pathway and Early Phosphorylation of Akt.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4571-4571
Author(s):  
Norikazu Imai ◽  
Masato Shikami ◽  
Hiroshi Miwa ◽  
Akiko Hattori ◽  
Akihito Hiramatsu ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Calf Serum ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Dependent Manner ◽  
Growth And Survival ◽  
Akt Phosphorylation ◽  
Leukemia Cell Lines

Abstract Most human leukemia cell lines are dependent on serum supplementation (usually fetal calf serum (FCS)), although the extent of serum dependency differs among each cell line. Kasumi-1, a t(8;21) AML cell line is one of the most serum-dependent cell lines. Since growth and survival of many leukemia cell lines are associated with phosphorylation of Akt, we examined the Akt phosphorylation by FCS treatment. In Kasumi-1, Akt was phosphorylated by culture with FCS in a dose-dependent manner, although no such Akt phosphorylation was observed in NB-4, a t(15;17) cell line. By FCS stimulation, Akt (Thr308, Ser473) was phosphorylated from 0.5 hr and the phosphorylation sustained until 48 hours in Kasumi-1. Then, we tested the effect of VEGF/VEGFR signaling in phosphorylation of Akt by FCS. The addition of VEGFR1/Fc and VEGFR2/Fc (which bind external VEGF and abrogate its function) inhibited the Akt phosphorylation from 2 hours until 10 hours, although the growth of Kasumi-1 was not inhibited. The addition of VEGFR2 kinase inhibitor (which inhibits internal VEGF signal) inhibited the Akt phosphorylation from 0.5 hr until 2 hours, and the growth of Kasumi-1 was greatly inhibited. Taken together, it is suggested that serum dependency of Kasumi-1 is at least in part attributed to VEGF/VEGFR pathway. Then, both external and internal VEGF/VEGFR pathways work in Kasumi-1, which in turn phosphorylate Akt. However, blockade of only internal VEGF signal (by VEGFR2 kinase inhibitor) inhibit the early Akt phosphorylation (0.5 hr), which resulted in growth inhibition, indicating the importance of early Akt phosphorylation.

scholarly journals Single Agent and Synergistic Activity of Maritoclax with ABT-263 in Nasopharyngeal Carcinoma (NPC) Cell Lines

2020 ◽  
Vol 31 (3) ◽  
pp. 1-13
Author(s):  
Shi Xiang Benedict Lian ◽  
Kwok-Wai Lo ◽  
Soo-Beng Alan Khoo ◽  
Nethia Mohana-Kumaran
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Cell Lines ◽  
Single Agent ◽  
Treatment Strategies ◽  
Synergistic Activity ◽  
Dependent Manner ◽  
Apoptotic Protein ◽  
Apoptotic Proteins ◽  
Bh3 Mimetic ◽  
3D Spheroids

The BCL-2 anti-apoptotic proteins are over-expressed in many cancers and hence are attractive therapeutic targets. In this study, we tested the sensitivity of two Nasopharyngeal Carcinoma (NPC) cell lines HK1 and C666-1 to Maritoclax, which is reported to repress anti-apoptotic protein MCL-1 and BH3 mimetic ABT-263, which selectively inhibits anti-apoptotic proteins BCL-2, BCL-XL and BCL-w. We investigated the sensitisation of the NPC cell lines to these drugs using the SYBR Green I assay and 3D NPC spheroids. We report that Maritoclax repressed anti-apoptotic proteins MCL-1, BCL-2, and BCL-XL in a dose- and time-dependent manner and displayed a single agent activity in inhibiting cell proliferation of the NPC cell lines. Moreover, combination of Maritoclax and ABT-263 exhibited synergistic antiproliferative effect in the HK1 cells. Similar results were obtained in the 3D spheroids generated from the HK1 cells. More notably, 3D HK1 spheroids either treated with single agent Maritoclax or combination with ABT-263, over 10 days, did not develop resistance to the treatment rapidly. Collectively, the findings illustrate that Maritoclax as a single agent or combination with BH3 mimetics could be potentially useful as treatment strategies for the management of NPC.

Tyrosine Kinase Inhibitor Screen Identifies the Multikinase Inhibitor, Foretinib, As a Sensitisor of Treatment-resistant B-Precursor ALL to Core Chemotherapeutic Agents

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1509-1509
Author(s):  
Shaun R Wilson ◽  
Victoria J Weston ◽  
Tatjana Stankovic ◽  
Pamela R Kearns
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Philadelphia Chromosome ◽  
Chemotherapeutic Agents ◽  
Significant Heterogeneity ◽  
Synergistic Activity ◽  
Multikinase Inhibitor ◽  
Childhood All

Abstract Abstract 1509 Acute lymphoblastic leukaemia (ALL) is the most frequent malignancy in childhood with resistance or relapse occurring in up to 20% of patients. The precise mechanisms of resistance to conventional therapy leading to relapse have not been elucidated. Deregulation of tyrosine kinases (TKs) have been implicated in resistant solid tumours and the of aetiology haemopoietic tumours, Philadelphia – chromosome positive ALL (Ph+ ALL), FLT3 in MLL+ infant ALL and FLT3-ITD subset in acute myeloid leukaemia. The role of TK inhibitors (TKIs) has not been extensively investigated in non-Ph + ALL. We screened 5 B-cell precursor ALL cell lines and 20 primary samples with a library of 34 TKIs. Nalm 6 (t(5;12)), Nalm 17 (normal karyotype), REH (t(12;21)), SD1 and Sup15 (Ph+ ALL) and primary cells were tested at 1μM and 10μM and alterations in cell viability assessed with the Promega CellTiter-Glo assay. A drug was considered to be effective if it induced >50% reduction in cell viability at 1μM. While we demonstrated significant heterogeneity in response to many of the TKIs, we observed reduction in viability to lestaurtinib (FLT3/JAK2), dovitinib (FLT3/FGFR/PDGFR/VEGFR) and bosutinib (Abl/Src) in all cell lines. Compared with Nalm 6 and Nalm 17 which only exhibited sensitivity to these 3 TKIs, REH demonstrated additional sensitivity to crizotinib (ALK/Met) and the quinazoline pan-EGFR inhibitors, afatinib and canertinib. The Ph+ cell lines SupB15 and SD1 responded to the highest number of TKIs, 12 and 14 respectively. These included the expected Bcr/Abl and Aurora kinase inhibitors. Activity of the putative PDGFR/VEGFR TKIs axitinib, linifanib, vargatef and also foretinib (MET/VEGFR2/FLT3) appeared limited to Ph+ cell lines. The cell lines, REH and SD-1, which are resistant to ionizing radiation–induced apoptosis, were selectively inhibited by both the quinazolines. Baseline mRNA expression of the ErbB family was present in all cell lines and therefore did not correlate with response. TKIs inducing the greatest reduction in cell viability across the cell lines were those that target class III/IV/V RTKs. Although all cell lines expressed FLT3 mRNA, reduction in cell viability was not universally induced by the specific FLT3 inhibitor tandutinib at doses of up to 10μM. As observed in previous studies, the level of mRNA transcript did not predict or directly correlate with the response to TKI. A panel of 20 primary ALL samples, representative of common biological features, were screened. We found no correlation between cytogenetics, age, white cell count, post – induction MRD status and response to TKI groups or individual inhibitor. Only 5/20 did not respond to any of the tested TKIs. Lestaurtinib, dovitinib and foretinib reduced cell viability in 7/20 primary ALLs. In addition, canertinib reduced cell viabililty in 6/20 primary ALL samples, afatinib and TAE684 (ALK/MET) both in 5/20 ALL samples respectively and vargatef in 4/20 samples. Based on our preliminary screen, the multikinase inhibitor foretinib was selected as one of several promising candidates for further pre-clinical testing. Recent adult phase 1 solid tumor trials have shown limited toxicity and good bioavailability. Foretinib inhibited leukaemia proliferation with LD50 in nanomolar and low micromolar range; SupB15 (333nM ±49), SD-1 (381nM ±239), Nalm 17 (484nM ±124), REH (689nM ±92) and Nalm 6 (1.84μM ±0.25). Annexin/PI staining, DNA fragmentation and PARP protein cleavage confirmed that the mechanism of cell death was apoptosis. We next investigated whether foretinib could sensitise ALL cell lines to dexamethasone, cytarabine, methotrexate, doxorubicin or mitoxantrone. Drug interactions were modelled using the Biosoft Calcusyn software package. We found that the addition of foretinib resulted in predominantly synergistic interactions in all cell lines (CI<1). The most striking example of synergism was in the dexamethasone-resistant cell line, REH. Addition of a sub–LD50 dose of foretinib led to >50% reduction in cell viability when combined with 1nM dexamethasone compared with no response at 10μM dexamethasone alone. Overall these data support further exploration of TKIs as potential therapeutic agents in childhood ALL. Specifically, we are currently investigating the direct anti-leukemic activity of foretinib in childhood ALL and its synergistic activity with dexamethasone in vivo using our NOG mouse primograft model for ALL. Disclosures: No relevant conflicts of interest to declare.

ONC201 Overcomes Chemotherapy Resistance By Upregulation of Bim in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4476-4476 ◽  
Author(s):  
Yong-sheng Tu ◽  
Jin He ◽  
Huan Liu ◽  
Richard Eric Davis ◽  
Robert Z. Orlowski ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Viability ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Parp Cleavage ◽  
Wild Type ◽  
Myeloma Cells ◽  
Induced Apoptosis ◽  
Tumor Types

Abstract In multiple myeloma, disease relapse and drug resistance occurs in the majority of myeloma patients after standard treatment despite recent improvements offered by new therapies. Therefore, there is an urgent need for new drugs that can overcome drug resistance and prolong patient survival after failure of standard therapies. ONC201, the founding member of a novel class of anti-tumor agents called impridones, has selective preclinical efficacy against a variety of tumor types. It is currently in phase I and phase II clinical trials for patients with advanced solid tumors and hematological malignancies. Given the pronounced sensitivity of B-cell lymphomas to ONC201, we assessed the efficacy of ONC201 in preclinical models of multiple myeloma. We treated human myeloma cell lines and primary myeloma cells isolated from bone marrow aspirates of myeloma patients with ONC201 for 72 hours. CellTiter-Glo Luminescent and annexin-V binding assays for assessing myeloma cell viability and apoptosis were performed, along with immunoblotting for cleavage of caspases, phosphorylation of signaling kinases, and expression of pro- or anti-apoptotic proteins. ONC201 treatment decreased myeloma cell viability, with IC50 values that were 1 μM to 1.5 μM, even in high risk myeloma cell line RPMI8226. The status of TP53 did not appear to affect the efficacy of ONC201, as MM.1S or NCI-H929 cells with wild-type TP53 and OPM-2 or RPMI8226 with mutated TP53 had a similar sensitivity towards ONC201. These results agree with prior reports in other tumor types that have demonstrated that the efficacy of ONC201 is independent of TP53. Western blot analysis showed increased apoptosis, cleavage of caspase-9, caspase-3, and PARP. We also found that ONC201 induced expression of the pro-apoptotic protein Bim in myeloma cells, which can occur downstream of ERK inactivation. Knockdown of Bim expression in myeloma cells by shRNAs abrogated ONC201-induced apoptosis. Phosphorylation of Bim at Ser69 by Erk1/2 has been shown to promote proteasomal degradation of Bim. In accordance with this mechanism, we observed that ONC201 treatment reduced levels of phosphorylated Erk1/2, an indicator of Erk1/2 kinase activity, and Bim pSer69. In addition, ONC201 induced apoptosis in dexamethasome-, bortezomib-, and carfilzomib-resistant myeloma cell lines with the same efficacy as in wild-type cells. As a rational strategy to increase the efficacy of ONC201 by enhancing its inhibition of proteasome-mediated Bim degradation, we tested combinations of ONC201 with proteasome inhibitors bortezomib or carfilzomib. These combinations were synergistic in reducing cell viability and enhancing Bim expression and PARP cleavage in myeloma cells. Overall, these findings demonstrate that ONC201 inhibits the Erk1/2 signaling pathway and induces Bim expression to induce apoptosis in multiple myeloma regardless of resistance to standard-of-care therapies. Our studies suggest that ONC201 should be evaluated clinically in relapsed/refractory multiple myeloma. Disclosures Allen: Oncoceutics: Employment, Equity Ownership.

scholarly journals Development of an MDM2 Degrader for Treatment of Acute Leukemias

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1866-1866
Author(s):  
Bridget Marcellino ◽  
Xiaobao Yang ◽  
He Chen ◽  
Karie Chen ◽  
Claudia Brady ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ubiquitin Ligase ◽  
Research Funding ◽  
Leukemia Cell ◽  
Data Safety Monitoring Board ◽  
Feedback Mechanism ◽  
Dependent Manner ◽  
Wild Type ◽  
Leukemia Cell Lines ◽  
Direct Binding

Abstract Introduction Acute myeloid leukemias (AMLs) are characterized by suppressed cell death pathways which promote leukemic blast survival. TP53 acts as a tumor suppressor gene in AML and is found mutated or deleted in 10-15% of patients. In a majority of cases though, TP53 is wild-type. Other mechanisms including MDM2 over-expression lead to reduced TP53 activity. MDM2 acts as a negative regulator by direct binding of TP53 and mediating TP53 degradation through ubiquitination. MDM2 itself is a transcriptional target of TP53 as a negative feedback mechanism limiting the function of TP53. Small molecule inhibition of MDM2 , blocking its ability to bind TP53, can activate TP53 and trigger cell cycle arrest and apoptosis through increased transcription of TP53 target genes. Increased MDM2 expression has been observed in hematologic malignancies including AML, providing rationale for clinical trials with MDM2 inhibitors. These agents such as RG7388 and AMG232 have shown efficacy as monotherapy and in combination. However, these agents have also exhibited toxicity and have yet to demonstrate sufficient benefit for their approval. To create more effective agents against MDM2, we have developed an MDM2 degrader XY-27 that functions as a proteolysis-targeting chimera (PROTAC). Based on relatively higher expression in AML compared to other cancer types, we selected VHL as the E3 ubiquitin ligase target for XY-27 , as this may improve specificity and potency in AML. Results The PROTAC degrader XY-27 concurrently binds MDM2 and VHL, and by bringing these targets in proximity, VHL can then ubiquitinate MDM2, leading to its degradation by the proteasome. XY-27 can mediate degradation of MDM2 in a concentration dependent manner in the U937 leukemia cell line (Fig 1a). MDM2 degradation with XY-27 is blocked by proteasome inhibition and competitive binding of the VHL ligand. A control compound, which only differs in that it cannot bind to VHL, lacks degrader activity. Although MDM2 is itself an E3 ligase, VHL expression is not appreciably changed with XY-27 (Fig 1a). Treatment with XY-27 leads to apoptosis and decreased proliferation of leukemia cell lines in a TP53 dependent manner. Inhibition of MDM2 leads to up-regulation of TP53 and in TP53 wild-type cells, downstream targets CDKN1A (p21) and PUMA. MDM2 is also up-regulated through a feedback mechanism. XY-27 demonstrated greater potency than the MDM2-binding inhibitor AMG232 in the MOLM13 and MV4-11 leukemia cell lines (Fig 1b). Treatment with XY-27 led to higher levels of TP53 and p21 protein than with AMG232. CRISPR-mediated knock-out of VHL leads to reduced XY-27 potency. XY-27 also shows efficacy when combined with other chemotherapeutic agents such as azacytidine and cytarabine. In a long-term co-culture model with an OP9 feeder layer, XY-27 was capable of inducing apoptosis in primary patient AML samples (Fig 1c). Conclusion We describe a new MDM2 PROTAC, XY-27 that demonstrates TP53 dependent activity against leukemia cells. It also demonstrates increase potency compared to an MDM2 binding inhibitor. Utilization of the PROTAC system has potential advantages through selection of the VHL E3 ubiquitin ligase. Because of negative feedback mechanisms involving TP53 and MDM2, direct binding inhibitors of MDM2 may be limited in activity through continued accumulation of MDM2. PROTAC degraders have catalytic activity and may overcome this inhibition by continued degradation of the target MDM2, and thus achieve greater TP53 activity. Figure 1. Activity of the MDM2-PROTAC XY-27 in leukemia. (a) Western blot from treatment of U937 leukemia cells with XY-27 for 24 hrs, at various concentrations (5 nM to 1 μM), resulting in the degradation of MDM2. (b) Dose response curves from treatment of MOLM13 and MV4-11 cell lines with XY-27 (blue) and AMG232 (red) for 48 hrs, demonstrating greater potency of XY-27. (c) Induction of apoptosis in primary AML cells treated with XY-27 at 1μM using a co-culture system for 3 days. *p&lt;.05 Figure 1 Figure 1. Disclosures Hoffman: Protagonist Therapeutics, Inc.: Consultancy; AbbVie Inc.: Other: Data Safety Monitoring Board, Research Funding; Novartis: Other: Data Safety Monitoring Board, Research Funding; Kartos Therapeutics, Inc.: Research Funding.

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