Lapatinib sensitivity in nasopharyngeal carcinoma is modulated by SIRT2-mediated FOXO3 deacetylation

Abstract Background Chemoresistance is an obstacle to the successful treatment of nasopharyngeal carcinoma (NPC). Lapatinib is a targeted tyrosine kinase inhibitor therapeutic drug also used to treat NPC, but high doses are often required to achieve a result. To investigate the mechanism for the development of Lapatinib resistance, we characterised a number of NPC cell lines to determine the role of FOXO3 and sirtuins in regulating NPC resistance. Methods Sulforhodamine B (SRB) assays, Clonogenic assays, Protein extraction, quantification and western blotting, RT qPCR, Co-immunoprecipitation assay. Results To explore novel treatment strategies, we first characterized the Lapatinib-sensitivity of a panel of NPC cell lines by SRB and clonogenic cytotoxic assays and found that the metastatic NPC (C666–1 and 5-8F) cells are highly resistant whereas the poorly metastatic lines (6-10B, TW01 and HK-1) are sensitive to Lapatinib. Western blot analysis of the Lapatinib-sensitive 6-10B and resistant 5-8F NPC cells showed that the expression of phosphorylated/inactive FOXO3 (P-FOXO3;T32), its target FOXM1 and its regulator SIRT2 correlate negatively with Lapatinib response and sensitivity, suggesting that SIRT2 mediates FOXO3 deacetylation to promote Lapatinib resistance. In agreement, clonogenic cytotoxic assays using wild-type and foxo1/3/4−/− mouse embryonic fibroblasts (MEFs) showed that FOXO1/3/4-deletion significantly attenuates Lapatinib-induced cytotoxicity, confirming that FOXO proteins are essential for mediating Lapatinib response. SRB cell viability assays using chemical SIRT inhibitors (i.e. sirtinol, Ex527, AGK2 and AK1) revealed that all SIRT inhibitors can reduce NPC cell viability, but only the SIRT2-specific inhibitors AK1 and AGK2 further enhance the Lapatinib cytotoxicity. Consistently, clonogenic assays demonstrated that the SIRT2 inhibitors AK1 and AGK2 as well as SIRT2-knockdown increase Lapatinib cytotoxicity further in both the sensitive and resistant NPC cells. Co-immunoprecipitation studies showed that besides Lapatinib treatment, SIRT2-pharmaceutical inhibition and silencing also led to an increase in FOXO3 acetylation. Importantly, SIRT2 inhibition and depletion further enhanced Lapatinib-mediated FOXO3-acetylation in NPC cells. Conclusion Collectively, our results suggest the involvement of SIRT2-mediated FOXO3 deacetylation in Lapatinib response and sensitivity, and that SIRT2 can specifically antagonise the cytotoxicity of Lapatinib through mediating FOXO3 deacetylation in both sensitive and resistant NPC cells. The present findings also propose that SIRT2 can be an important biomarker for metastatic and Lapatinib resistant NPC and that targeting the SIRT2-FOXO3 axis may provide novel strategies for treating NPC and for overcoming chemoresistance.

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Lapatinib sensitivity in nasopharyngeal carcinoma is modulated by SIRT2-mediated FOXO3 deacetylation

10.21203/rs.2.11556/v3 ◽

2019 ◽

Author(s):

Sathid Aimjongjun ◽

Zimam Mahmud ◽

Yannasittha Jiramongkol ◽

Glowi Alasiri ◽

Shang Yao ◽

...

Keyword(s):

Nasopharyngeal Carcinoma ◽

Cell Viability ◽

Cell Lines ◽

Kinase Inhibitor ◽

Protein Extraction ◽

Treatment Strategies ◽

Therapeutic Drug ◽

Clonogenic Assays ◽

High Doses ◽

Novel Treatment Strategies

Abstract Background Chemoresistance is an obstacle to the successful treatment of nasopharyngeal carcinoma (NPC). Lapatinib is a targeted tyrosine kinase inhibitor therapeutic drug also used to treat NPC, but high doses are often required to achieve a result. To investigate the mechanism for the development of Lapatinib resistance, we characterised a number of NPC cell lines to determine the role of FOXO3 and sirtuins in regulating NPC resistance. Methods Sulforhodamine B (SRB) assays, Clonogenic assays, Protein extraction, quantification and western blotting, RT qPCR, Co-immunoprecipitation assay Results To explore novel treatment strategies, we first characterized the Lapatinib-sensitivity of a panel of NPC cell lines by SRB and clonogenic cytotoxic assays and found that the metastatic NPC (C666-1 and 5-8F) cells are highly resistant whereas the poorly metastatic lines (6-10B, TW01 and HK-1)are sensitive to Lapatinib. Western blot analysis of the Lapatinib-sensitive 6-10Band resistant 5-8FNPC cells showed that the expression of phosphorylated/inactive FOXO3 (P-FOXO3;T32), its target FOXM1 and its regulator SIRT2 correlate negatively with Lapatinib response and sensitivity, suggesting that SIRT2 mediates FOXO3 deacetylation to promote Lapatinib resistance. In agreement, clonogenic cytotoxic assays using wild-type and foxo1/3/4 −/− mouse embryonic fibroblasts (MEFs) showed that FOXO1/3/4-deletion significantly attenuates Lapatinib-induced cytotoxicity, confirming that FOXO proteins are essential for mediating Lapatinib response. SRB cell viability assays using chemical SIRT inhibitors (i.e. sirtinol, Ex527, AGK2 and AK1) revealed that all SIRT inhibitors can reduce NPC cell viability, but only the SIRT2-specific inhibitors AK1 and AGK2 further enhance the Lapatinib cytotoxicity. Consistently, clonogenic assays demonstrated that the SIRT2 inhibitors AK1 and AGK2 as well as SIRT2-knockdown increase Lapatinib cytotoxicity further in both the sensitive and resistant NPC cells. Co-immunoprecipitation studies showed that besides Lapatinib treatment, SIRT2-pharmaceutical inhibition and silencing also led to an increase in FOXO3 acetylation. Importantly, SIRT2 inhibition and depletion further enhanced Lapatinib-mediated FOXO3-acetylation in NPC cells. Conclusion Collectively, our results suggest the involvement of SIRT2-mediated FOXO3 deacetylation in Lapatinib response and sensitivity, and that SIRT2 can specifically antagonise the cytotoxicity of Lapatinib through mediating FOXO3 deacetylation in both sensitive and resistant NPC cells.

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Lapatinib sensitivity in nasopharyngeal carcinoma is modulated by SIRT2-mediated FOXO3 deacetylation

10.21203/rs.2.11556/v2 ◽

2019 ◽

Author(s):

Sathid Aimjongjun ◽

Zimam Mahmud ◽

Yannasittha Jiramongkol ◽

Glowi Alasiri ◽

Shang Yao ◽

...

Keyword(s):

Nasopharyngeal Carcinoma ◽

Cell Viability ◽

Cell Lines ◽

Kinase Inhibitor ◽

Protein Extraction ◽

Treatment Strategies ◽

Therapeutic Drug ◽

Clonogenic Assays ◽

High Doses ◽

Novel Treatment Strategies

Abstract Background Chemoresistance is an obstacle to the successful treatment of nasopharyngeal carcinoma (NPC). Lapatinib is a targeted tyrosine kinase inhibitor therapeutic drug also used to treat NPC, but high doses are often required to achieve a result. To investigate the mechanism for the development of Lapatinib resistance, we characterised a number of NPC cell lines to determine the role of FOXO3 and sirtuins in regulating NPC resistance. Methods Sulforhodamine B (SRB) assays, Clonogenic assays, Protein extraction, quantification and western blotting, RT qPCR, Co-immunoprecipitation assay Results To explore novel treatment strategies, we first characterized the Lapatinib-sensitivity of a panel of NPC cell lines by SRB and clonogenic cytotoxic assays and found that the metastatic NPC (C666-1 and 5-8F) cells are highly resistant whereas the poorly metastatic lines (6-10B, TW01 and HK-1)are sensitive to Lapatinib. Western blot analysis of the Lapatinib-sensitive 6-10Band resistant 5-8FNPC cells showed that the expression of phosphorylated/inactive FOXO3 (P-FOXO3;T32), its target FOXM1 and its regulator SIRT2 correlate negatively with Lapatinib response and sensitivity, suggesting that SIRT2 mediates FOXO3 deacetylation to promote Lapatinib resistance. In agreement, clonogenic cytotoxic assays using wild-type and foxo1/3/4 −/− mouse embryonic fibroblasts (MEFs) showed that FOXO1/3/4-deletion significantly attenuates Lapatinib-induced cytotoxicity, confirming that FOXO proteins are essential for mediating Lapatinib response. SRB cell viability assays using chemical SIRT inhibitors (i.e. sirtinol, Ex527, AGK2 and AK1) revealed that all SIRT inhibitors can reduce NPC cell viability, but only the SIRT2-specific inhibitors AK1 and AGK2 further enhance the Lapatinib cytotoxicity. Consistently, clonogenic assays demonstrated that the SIRT2 inhibitors AK1 and AGK2 as well as SIRT2-knockdown increase Lapatinib cytotoxicity further in both the sensitive and resistant NPC cells. Co-immunoprecipitation studies showed that besides Lapatinib treatment, SIRT2-pharmaceutical inhibition and silencing also led to an increase in FOXO3 acetylation. Importantly, SIRT2 inhibition and depletion further enhanced Lapatinib-mediated FOXO3-acetylation in NPC cells. Conclusion Collectively, our results suggest the involvement of SIRT2-mediated FOXO3 deacetylation in Lapatinib response and sensitivity, and that SIRT2 can specifically antagonise the cytotoxicity of Lapatinib through mediating FOXO3 deacetylation in both sensitive and resistant NPC cells.

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Lapatinib sensitivity in nasopharyngeal carcinoma is modulated by SIRT2-mediated FOXO3 deacetylation

10.21203/rs.2.11556/v1 ◽

2019 ◽

Author(s):

Eric W.-F. Lam ◽

Zimam Mahmud ◽

Sathid Aimjongjun ◽

Yannasittha Jiramongkol ◽

Glowi Alasiri ◽

...

Keyword(s):

Nasopharyngeal Carcinoma ◽

Western Blot ◽

Cell Viability ◽

Blot Analysis ◽

Treatment Strategies ◽

Wild Type ◽

Novel Treatment ◽

Novel Treatment Strategies ◽

Lapatinib Treatment ◽

Specific Inhibitors

Abstract Chemoresistance is an obstacle to the successful treatment of nasopharyngeal carcinoma (NPC). To explore novel treatment strategies, we first characterized the lapatinib-sensitivity of a panel of NPC cell lines by SRB and clonogenic cytotoxic assays and found that the highly metastatic NPC (C666-1 and 5-8F) cells are significantly more resistant and the poorly metastatic lines (6-10B, TW01 and HK-1) more sensitive to lapatinib. Western blot analysis of the lapatinib-sensitive 6-10B and resistant 5-8F NPC cells showed that the expression of phosphorylated/inactive FOXO3(P-FOXO3;T32), its target FOXM1 and its regulator SIRT2 correlates negatively with lapatinib response and sensitivity, suggesting that SIRT2 mediates FOXO3 deacetylation to promote lapatinib resistance. In agreement, clonogenic cytotoxic assays using wild-type and foxo1/3/4−/− MEFs showed that FOXO1/3/4-deletion significantly attenuates lapatinib-induced cytotoxicity, confirming that FOXO proteins are essential for mediating lapatinib response. SRB cell viability assays using chemical SIRT inhibitors (i.e. sirtinol, Ex527, AGK2 and AK1) revealed that all SIRT inhibitors can reduce NPC cell viability, but only the SIRT2-specific inhibitors AK1 and AGK2 further enhance the lapatinib cytotoxicity. Consistently, clonogenic assays demonstrated that the SIRT2 inhibitors AK1 and AGK2 as well as SIRT2-knockdown increase lapatinib cytotoxicity further in both the sensitive and resistant NPC cells. Co-immunoprecipitation studies showed that besides lapatinib treatment, SIRT2-pharmaceutical inhibition and silencing also led to an increase in FOXO3 acetylation. Importantly, SIRT2 inhibition and depletion further enhanced lapatinib-mediated FOXO3-acetylation in NPC cells. Collectively, our results suggest the involvement of SIRT2-mediated FOXO3 deacetylation in lapatinib response and sensitivity, and that SIRT2 can specifically antagonise the cytotoxicity of lapatinib through mediating FOXO3 deacetylation in both sensitive and resistant NPC cells. The present findings also propose that SIRT2 can be an important biomarker for metastatic and lapatinib resistant NPC and that targeting the SIRT2-FOXO3 axis may provide novel strategies for treating NPC and for overcoming chemoresistance.

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Advantages of Tyrosine Kinase Anti-Angiogenic Cediranib over Bevacizumab: Cell Cycle Abrogation and Synergy with Chemotherapy

Pharmaceuticals ◽

10.3390/ph14070682 ◽

2021 ◽

Vol 14 (7) ◽

pp. 682

Author(s):

Jianling Bi ◽

Garima Dixit ◽

Yuping Zhang ◽

Eric J. Devor ◽

Haley A. Losh ◽

...

Keyword(s):

Cell Cycle ◽

Endometrial Cancer ◽

Tyrosine Kinase ◽

Cell Viability ◽

Cell Lines ◽

Cell Cycle Progression ◽

Kinase Inhibitor ◽

Cell Cycle Checkpoint ◽

Mitotic Catastrophe ◽

M Cell

Angiogenesis plays a crucial role in tumor development and metastasis. Both bevacizumab and cediranib have demonstrated activity as single anti-angiogenic agents in endometrial cancer, though subsequent studies of bevacizumab combined with chemotherapy failed to improve outcomes compared to chemotherapy alone. Our objective was to compare the efficacy of cediranib and bevacizumab in endometrial cancer models. The cellular effects of bevacizumab and cediranib were examined in endometrial cancer cell lines using extracellular signal-related kinase (ERK) phosphorylation, ligand shedding, cell viability, and cell cycle progression as readouts. Cellular viability was also tested in eight patient-derived organoid models of endometrial cancer. Finally, we performed a phosphoproteomic array of 875 phosphoproteins to define the signaling changes related to bevacizumab versus cediranib. Cediranib but not bevacizumab blocked ligand-mediated ERK activation in endometrial cancer cells. In both cell lines and patient-derived organoids, neither bevacizumab nor cediranib alone had a notable effect on cell viability. Cediranib but not bevacizumab promoted marked cell death when combined with chemotherapy. Cell cycle analysis demonstrated an accumulation in mitosis after treatment with cediranib + chemotherapy, consistent with the abrogation of the G2/M checkpoint and subsequent mitotic catastrophe. Molecular analysis of key controllers of the G2/M cell cycle checkpoint confirmed its abrogation. Phosphoproteomic analysis revealed that bevacizumab and cediranib had both similar and unique effects on cell signaling that underlie their shared versus individual actions as anti-angiogenic agents. An anti-angiogenic tyrosine kinase inhibitor such as cediranib has the potential to be superior to bevacizumab in combination with chemotherapy.

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Compensatory Signaling From ROR1 and the Pre-B Cell Receptor Promote Survival of t(1;19) Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.2466.2466 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2466-2466

Author(s):

Vincent T Bicocca ◽

Bill H Chang ◽

Markus Muschen ◽

Brian J. Druker ◽

Jeffrey W Tyner

Keyword(s):

Cell Viability ◽

B Cell ◽

Signaling Pathways ◽

Cell Lines ◽

Tyrosine Kinases ◽

Kinase Inhibitor ◽

Lymphoblastic Leukemia ◽

Immunoblot Analysis ◽

Cell Receptor ◽

Cell Surface Receptor

Abstract Abstract 2466 BACKGROUND: Aberrant tyrosine kinase activity is commonly implicated in the pathogenesis of leukemia and other cancers. Identification of these leukemogenic tyrosine kinases has proven invaluable for diagnostic and prognostic stratification of patients as well as for the development of novel strategies for therapeutic intervention. We previously demonstrated that siRNA screening of mononuclear cells from leukemia patients can determine sensitivity to individual tyrosine kinases. With the goal of uncovering novel viability-dependent tyrosine kinases in leukemia patients, we have employed an RNAi-assisted protein target identification (RAPID) assay to screen cytogenetic subtypes of acute lymphoblastic leukemia (ALL). ALL is the most common pediatric cancer, accounting for one-quarter of all childhood malignancies. Childhood ALL has a primarily B cell precursor phenotype and is characterized by chromosomal abnormalities, primarily translocations and duplications. One of the most common recurring translocations associated with pediatric ALL, t(1;19)(q23;p13.3), generates the E2A-PBX1 fusion product. Here we show unique viability-dependent expression of a receptor tyrosine kinase, ROR1, in the t(1;19) ALL background. In addition, we identify a kinase inhibitor, dasatinib, with significant activity against t(1;19) ALL cells due to its capacity to inhibit tyrosine kinases necessary for transduction of pre-B cell receptor (preBCR) signaling. Finally, we show that ROR1 and the preBCR activate mutually compensatory signaling pathways, suggesting that optimal therapeutic regimens would include agents targeting both pathways. METHODS: To identify targets required for viability of leukemic cells, we screened cell lines as well as primary cells from ALL patients with siRNAs and determined cell viability using an MTS assay. ROR1 expression levels were determined by RT-PCR, immunoblot analysis and flow cytometry. Kinase inhibitor screening was performed on both cells lines and primary ALL cells by treating samples with a library of small-molecule inhibitors and cell viability was assessed by MTS. Signaling pathways disrupted by inhibitor treatment or ROR1 knockdown were interrogated by phospho-protein arrays and confirmed by immunoblot analysis. RESULTS: The RAPID assay identified a unique sensitivity to the cell surface receptor ROR1 in a subject identified with t(1;19) pediatric ALL. Similar sensitivity was not observed in ALL patients of alternative cytogenetic subtypes. Examination of additional ALL patient samples revealed conserved overexpression of the ROR1 transcript in t(1;19)-positive specimens with absence of ROR1 expression in t(1;19)-negative samples. Cell lines and early passage xenograft cells confirmed overexpression and functional dependence of t(1;19)-positive cells on ROR1. A subsequent kinase inhibitor screen of t(1;19) ALL cell lines and patient samples revealed universal sensitivity to the FDA-approved drug dasatinib. Further examination revealed the dasatinib targets, BTK and LYN, which signal downstream of the preBCR as the viability dependent targets of dasatinib in t(1;19) ALL. Inhibition of the preBCR results in transient loss of AKT activity and, surprisingly, upregulation of ROR1. Analysis of signaling pathways after silencing of ROR1 or dasatinib treatment revealed compensatory signaling pathways emanating from ROR1 and the preBCR that both serve to regulate AKT activity. Consequently, combination of ROR1 knockdown and dasatinib treatment resulted in additive impairment of cell viability compared with either treatment alone. CONCLUSION: The cell surface receptor ROR1 is consistently overexpressed in t(1;19) ALL. RNAi mediated downregulation of ROR1 impairs the viability of these cells. Similarly, t(1;19) cells are sensitive to the kinase inhibitor dasatinib due to activity against the preBCR. Combined targeting of ROR1 and the preBCR with dasatinib yields an additive viability effect due to compensatory signaling pathways aimed at regulating AKT. These results demonstrate a novel mechanism of AKT regulation in t(1;19) ALL as well as a therapeutic strategy for treatment of t(1;19) ALL. Disclosures: Druker: MolecularMD: Equity Ownership, OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee and t; Ariad Pharmaceuticals: Consultancy; OHSU patent #843: Mutated ABL Kinase Domains: Patents & Royalties; Bristol-Myers Squibb: OHSU has clinical trial contracts with Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Novartis: OHSU has clinical trial contracts with Novartis to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Cylene: Consultancy.

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Preclinical Evaluation of Oncolytic Reovirus in Targeted Therapeutics for High-Risk Pediatric Leukemia

Blood ◽

10.1182/blood.v120.21.3571.3571 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3571-3571

Author(s):

Matthew F. Clarkson ◽

Aru Narendran ◽

Randal N. Johnston

Keyword(s):

Cell Death ◽

High Risk ◽

Cell Viability ◽

Cell Lines ◽

Leukemia Cell ◽

Treatment Strategies ◽

Leukemia Cells ◽

Western Blots ◽

Pediatric Leukemia ◽

Leukemia Cell Lines

Abstract Abstract 3571 Purpose: Leukemia is the most common malignancy in children. Improved treatment strategies in recent decades have yielded substantially enhanced outcomes for children with leukemia, reaching survival rates >80%. However, there remain significant issues with current treatment. Certain subgroups of patients who are resistant to or relapse from current treatments have a dismal prognosis. Furthermore, there are significant late effects of intensive treatments, including secondary cancers, neurocognitive defects, cardiotoxicity, obesity and infertility. For these reasons, novel treatment strategies are urgently needed for high-risk leukemia in children. Reovirus type 3 Dearing is a wild-type double-stranded RNA virus that has shown great promise as a selective oncolytic agent by its ability to replicate in transformed cells but not in normal cells. Although a number of early phase clinical studies have been completed in patients with advanced, refractory solid tumors in adults, systematic evaluation of this agent in the treatment of refractory pediatric leukemia has not been reported. As an initial step towards developing an oncolytics based treatment approach, we report preclinical data with respect to the activity, target validation, target modulation and drug combinability of reovirus in childhood leukemia cells. Experimental Design: A panel of pediatric leukemia cell lines representing high-risk molecular features such as Bcr-Abl, MLL rearranged and mixed lineage was used (n =6). Expression of JAM-A, the cell surface receptor for reovirus, was assessed by flow cytometry. The Ras Activation Assay Kit (EMD Millipore) was used to assess activity of the RAS protein. Western Blots were used to assess the activation (phosphorylation) of the signaling partners downstream of RAS. Cells treated with reovirus, chemotherapy drugs, or both for distinct treatment schedules were assessed for cell viability by the CellTiter-Glo© Luminescent Cell Viability Assay (Promega), and cell death by apoptosis was confirmed by cleavage of PARP. Productive viral infection was assessed by measuring reoviral protein synthesis by Western Blots, and reoviral replication was assessed by virus plaque titration assay. Drug synergies were calculated according to the method of Chou and Talalay. Results: Target validation assays showed the expression of JAM-A, which facilitates effective viral entry into malignant cells, in five of six cell lines. These cell lines also demonstrated differential activation of RAS and downstream kinases, suggesting targeted susceptibility of these cells to reovirus oncolysis. To further test this, we infected cells with reovirus for 1–4 days and assessed cytopathic effects. Using phase contrast microscopy, we observed the virus treated cell lines to demonstrate morphological changes characteristic of cell death following infection. Cell viability assays were used to quantify this effect, and the mechanism of cell death was determined to be apoptotic as evidenced by caspase-dependent cleavage of PARP. Reovirus-induced cell death was correlated with viral protein production and replication. Next, we screened for the ability of reovirus to induce synergistic activity in a panel of conventional and novel targeted therapeutic agents. Our studies showed that, in contrast to the current antileukemic agents, the Bcl-2 inhibitor BH3 mimetic ABT-737 was able to significantly synergize with reovirus in all cell lines tested. Conclusions: In our in vitro studies, oncolytic reovirus as a single agent showed potent oncolytic activity against all pediatric leukemia cell lines tested that express the receptor for reovirus, regardless of the status of the RAS signaling pathway. Further, we found reovirus-induced oncolysis can be enhanced by combination with Bcl-2 inhibition but was unaltered or antagonized by the other drugs indicating a key relationship between the two pathways. As such, our data for the first time, show that pediatric leukemia cells carry the potential to be targeted by reovirus induced oncolysis and the identification of drug synergy and the biomarkers of target modulation provide the basis for further studies to develop this novel therapeutic approach for clinical studies in the near future. Disclosures: No relevant conflicts of interest to declare.

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RDNA-14. RADIATION-INDUCED miR-4516 CONTRIBUTES TO RADIO-RESISTANCE AND PROMOTES AGGRESSIVE PHENOTYPE IN GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noz175.873 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi209-vi210

Author(s):

Ebin Sebastian ◽

Tiantian Cui ◽

Erica Hlavin Bell ◽

Joseph McElroy ◽

Benjamin Johnson ◽

...

Keyword(s):

Brain Tumor ◽

Cell Viability ◽

Cell Lines ◽

Poor Prognosis ◽

Radiation Treatment ◽

Critical Role ◽

Luciferase Reporter ◽

Glioblastoma Cell ◽

Clonogenic Assays ◽

Glioblastoma Cell Lines

Abstract BACKGROUND Glioblastoma is the most aggressive brain tumor with poor prognosis despite the best available treatment. MicroRNAs (miRNAs) are emerging as promising, novel prognostic biomarkers and therapeutic targets in glioblastoma. In a previous study, we demonstrated that miR-4516 predicts poor prognosis and functions as an oncogene in glioblastoma. Aim of the current study is to examine the role miR-4516 in radiation resistance and identify downstream targets contributing to this phenotype METHODS Radiosensitization was evaluated by cell viability and clonogenic assays. Cell apoptosis was evaluated using flow cytometry and immunoblotting. Potential targets of miR-4516 were identified using bioinformatic analysis (Targetscan and miRDB) and confirmed by luciferase reporter assays. Results were validated using immunoblotting. miR-4516 expression in glioblastoma cell lines after radiation treatment was quantified by qRT-PCR. RESULTS Expression of miR-4516 was increased up to 15 fold following radiation treatment, peaking at around 15min-60 min in primary and established glioblastoma cell lines including GBM 08-387, GBM 30 and U87-MG. Furthermore, inhibition of miR-4516 sensitized GBM 08-387, GBM30 and U87-MG cells to radiation in comparison to control groups as determined by cell viability and clonogenic assays. Further, miR-4516 inhibition induced apoptosis in these cell lines following radiation treatment. While conducting mechanistic studies, we found that the tumor-promoting function of miR-4516 was, in part, mediated by inhibition of p21 and PTPN14, two direct targets of miR-4516 CONCLUSION Our data suggest that radiation induces the expression of miR-4516 in glioblastoma cell lines. This miRNA plays a critical role in radio-resistance and promotes aggressive phenotypes in glioblastoma and therefore, functional analyses of its target pathways may uncover novel therapeutically vulnerable target(s) in glioblastoma. FUNDING: R01CA108633, R01CA169368, RC2CA148190, U10CA180850-01(NCI), Brain Tumor Funders Collaborative Grant, and OSU-CCC (all to AC). The Ton and Patricia Bohnenn Fund for Neuro_Oncology Research (to PR).

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

Blood ◽

10.1182/blood.v108.11.1384.1384 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1384-1384 ◽

Cited By ~ 3

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.

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Combination of the IGF-1 Receptor Inhibitor Picropodophylin and the BH3 Mimetic ABT-737 Has Synergistic Anti-Myeloma Activity

Blood ◽

10.1182/blood.v120.21.4010.4010 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4010-4010

Author(s):

Liesbeth Bieghs ◽

Ken Maes ◽

Els Van Valckenborgh ◽

Eline Menu ◽

Hans Erik Johnsen ◽

...

Keyword(s):

Growth Factors ◽

Cell Viability ◽

Cell Lines ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Differential Sensitivity ◽

Expression Ratio ◽

Therapy Strategy ◽

Induced Apoptosis ◽

Bh3 Mimetic

Abstract Abstract 4010 Despite intensive research multiple myeloma (MM) is still an incurable disease. MM cells are strongly dependent on the BM micro-environment where growth factors are secreted. IGF-1 is one of the most important growth factors in MM and thus forms an attractive target for anti-cancer therapy. Previously, we demonstrated that picropodophylin (PPP), an IGF-1R kinase inhibitor, indeed has potent anti-MM effects both on human cells and in the 5T2MM and 5T33MM murine models. However, mice eventually relapsed and showed signs of morbidity. Therefore it would be an attractive approach to combine PPP with other cytotoxic drugs. ABT-737 is a BH3 mimetic that binds with high affinity to Bcl-xL, Bcl-2 and Bcl-w, but not Mcl-1. In MM, elevated expression of the Bcl-2 pro-survival family of proteins, especially Mcl-1 and to a lesser extent Bcl-2, has extensively been shown to cause resistance to drug induced apoptosis in MM cells. Consequently, ABT-737 was shown to have potent anti-MM activity but only on a subset of human cell lines. Only very recently, it was shown that the ABT-737 sensitivity appears to be determined both by the Bcl-2/Mcl-1 expression ratio and the interaction of these anti-apoptotic proteins with Bim. Interestingly, we demonstrated that IGF-1R inhibition reduces the expression of Mcl-1 and Bcl-xL and that IGF-1 down-regulates the expression of Bim. In addition, a protective effect of IL-6 and BMSC against ABT-737 has been reported. Together, all the above suggests that it would be beneficial to combine ABT-737 with agents that target growth factors, like PPP. Here, we investigated the potential synergistic anti-MM effects of PPP and ABT-737 and studied the underlying mechanisms using two human myeloma cell lines (OPM-2 and RPMI-8226) and the murine 5T33MM model. Both PPP and ABT-737 (kindly provided by Abbott Laboratories) alone were found to significantly decrease cell viability and induce apoptosis dose and time dependently as evidenced by a decrease in ATP levels and an increase in the number of AnnexV/7'AAD positive cells. However, in agreement with previous reported data, we observed differential sensitivity to ABT-737 between the cell lines used. Nevertheless, treatment with PPP/ABT-737 synergistically decreased cell viability and induced apoptosis in all cell lines. In addition, by western blot analysis we could observe increased cleavage of caspase- 3,- 9 and PARP. Mechanistically, PPP was found to circumvent the adverse effect of ABT-737 by blocking the ABT-737 induced Mcl-1 expression and increasing the expression of Noxa. Interestingly, while CD138+ 5T33MM cells were more sensitive to PPP and the CD138- cells more sensitive to ABT-737, treatment with PPP/ABT-737 targeted both MM cell subpopulations to an equal extent. Finally, we tested the combination of PPP and ABT-737 in the 5T33MM model in a prophylactic setting. Whereas, vehicle and ABT-737 treated mice exhibited progressive MM growth, PPP, and to a significant greater extent, PPP/ABT-737 reduced the tumor burden and prolonged overall survival (p≤0.001). In conclusion, PPP combined with ABT-737 appears to have synergistic anti-MM activity and might thus be a novel and promising therapy strategy for MM. Disclosures: No relevant conflicts of interest to declare.

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PNU-74654 Suppresses TNFR1/IKB Alpha/p65 Signaling and Induces Cell Death in Testicular Cancer

Current Issues in Molecular Biology ◽

10.3390/cimb44010016 ◽

2022 ◽

Vol 44 (1) ◽

pp. 222-232

Author(s):

Wen-Jung Chen ◽

Wen-Wei Sung ◽

Chia-Ying Yu ◽

Yu-Ze Luan ◽

Ya-Chuan Chang ◽

...

Keyword(s):

Cell Death ◽

Testicular Cancer ◽

Cell Viability ◽

Cell Lines ◽

Cancer Progression ◽

Treatment Strategies ◽

Annexin V ◽

Western Blots ◽

Execution Phase ◽

Induced Apoptosis

Testicular cancer (TC) is a rare malignancy worldwide and is the most common malignancy in males aged 15–44 years. The Wnt/β-catenin signaling pathway mediates numerous essential cellular functions and has potentially important effects on tumorigenesis and cancer progression. The search for drugs to inhibit this pathway has identified a small molecule, PNU-74654, as an inhibitor of the β-catenin/TCF4 interaction. We evaluated the therapeutic role of PNU-74654 in two TC cell lines, NCCIT and NTERA2, by measuring cell viability, cell cycle transition and cell death. Potential pathways were evaluated by protein arrays and Western blots. PNU-74654 decreased cell viability and induced apoptosis of TC cells, with significant increases in the sub G1, Hoechst-stained, Annexin V-PI-positive rates. PNU-74654 treatment of both TC cell lines inhibited the TNFR1/IKB alpha/p65 pathway and the execution phase of apoptosis. Our findings demonstrate that PNU-74654 can induce apoptosis in TC cells through mechanisms involving the execution phase of apoptosis and inhibition of TNFR1/IKB alpha/p65 signaling. Therefore, small molecules such as PNU-74654 may identify potential new treatment strategies for TC.

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