Vikas Sehdev

The Aurora Kinase A Inhibitor MLN8237 Enhances Cisplatin-Induced Cell Death in Esophageal Adenocarcinoma Cells

Esophageal adenocarcinomas are poorly responsive to chemotherapeutics. This study aimed to determine the levels of Aurora kinase A (AURKA) and the therapeutic potential of MLN8237, an investigational AURKA inhibitor, alone and in combination with cisplatin. Using quantitative real-time PCR, we detected frequent AURKA gene amplification (15 of 34, 44%) and mRNA overexpression (37 of 44, 84%) in esophageal adenocarcinomas (P < 0.01). Immunohistochemical analysis showed overexpression of AURKA in more than two-thirds of esophageal adenocarcinoma tissue samples (92 of 132, 70%; P < 0.001). Using FLO-1, OE19, and OE33 esophageal adenocarinoma cell lines, with constitutive AURKA overexpression and mutant p53, we observed inhibition of colony formation with a single treatment of 0.5 μmol/L MLN8237 (P < 0.05). This effect was further enhanced in combination with 2.5 μmol/L cisplatin (P < 0.001). Twenty-four hours after treatment with the MLN8237 or MLN8237 and cisplatin, cell-cycle analyses showed a sharp increase in the percentage of polyploid cells (P < 0.001). This was followed by an increase in the percentage of cells in the sub-G1 phase at 72 hours, concordant with the occurrence of cell death (P < 0.001). Western blot analysis showed higher induction of TAp73β, PUMA, NOXA, cleaved caspase-3, and cleaved PARP with the combined treatment, as compared with a single-agent treatment. Using xenograft models, we showed an enhanced antitumor role for the MLN8237 and cisplatin combination, as compared with single-agent treatments (P < 0.001). In conclusion, this study shows frequent overexpression of AURKA and suggests that MLN8237 could be an effective antitumor agent, which can be combined with cisplatin for a better therapeutic outcome in esophageal adenocarcinomas. Mol Cancer Ther; 11(3); 763–74. ©2012 AACR.

Aurora kinase A promotes inflammation and tumorigenesis in mice and human gastric neoplasia.

BACKGROUND & AIMS Chronic inflammation contributes to the pathogenesis of gastric tumorigenesis. The aurora kinase A (AURKA) gene is frequently amplified and overexpressed in gastrointestinal cancers. We investigated the roles of AURKA in inflammation and gastric tumorigenesis. METHODS We used quantitative real-time reverse transcription polymerase chain reaction, immunofluorescence, immunohistochemistry, luciferase reporter, immunoblot, co-immunoprecipitation, and in vitro kinase assays to analyze AGS and MKN28 gastric cancer cells. We also analyzed Tff1(-/-) mice, growth of tumor xenografts, and human tissues. RESULTS We correlated increased expression of AURKA with increased levels of tumor necrosis factor-α and inflammation in the gastric mucosa of Tff1(-/-) mice (r = 0.62; P = .0001). MLN8237, an investigational small-molecule selective inhibitor of AURKA, reduced nuclear staining of nuclear factor-κB (NF-κB) p65 in human gastric cancer samples and mouse epithelial cells, suppressed NF-κB reporter activity, and reduced expression of NF-κB target genes that regulate inflammation and cell survival. Inhibition of AURKA also reduced growth of xenograft tumors from human gastric cancer cells in mice and reversed the development of gastric tumors in Tff1(-/-) mice. AURKA was found to regulate NF-κB activity by binding directly and phosphorylating IκBα in cells. Premalignant and malignant lesions from the gastric mucosa of patients had increased levels of AURKA protein and nuclear NF-κB, compared with healthy gastric tissue. CONCLUSIONS In analyses of gastric cancer cell lines, human tissue samples, and mouse models, we found AURKA to be up-regulated during chronic inflammation to promote activation of NF-κB and tumorigenesis. AURKA inhibitors might be developed as therapeutic agents for gastric cancer.

ABL Regulation by AXL Promotes Cisplatin Resistance in Esophageal Cancer

Esophageal adenocarcinoma (EAC) is characterized by resistance to chemotherapy and poor outcome. Although cisplatin (CDDP) has been used as a first-line therapy in patients with EAC, resistance remains a major clinical problem. The AXL receptor tyrosine kinase, originally isolated as a transforming gene from leukemia, is overexpressed in several solid tumors. Herein, we assessed AXL protein expression in human EACs and examined its role in CDDP resistance in human EAC cells. AXL overexpression was detected in more than 50% of tumors examined. Elevating AXL in nonoverexpressing cells doubled the CDDP IC(50) and increased cell survival three-fold, while attenuating AXL in overexpressing cells reduced survival two-fold. The effects of AXL modulation on cell survival were associated with changes in cellular and molecular markers of apoptosis. Mechanistic investigations revealed that AXL blocked CDDP-induced activation of endogenous p73β (TP73), reducing its protein half-life, and inhibited CDDP-induced levels of p-c-ABL(Y412) and p-p73β(Y99). These changes were associated with a disruption of c-ABL/p73β protein interactions due to association with c-ABL in the cytoplasm, thereby blocking nuclear accumulation of c-ABL and phosphorylation of p73β in response to DNA damage. Together, our results establish that AXL promotes CDDP resistance in esophageal adenocarcinoma and argue that therapeutic targeting of AXL may sensitize these cancers to DNA-damaging drugs.

The combination of alisertib, an investigational Aurora kinase A inhibitor, and docetaxel promotes cell death and reduces tumor growth in preclinical cell models of upper gastrointestinal adenocarcinomas

Upper gastrointestinal adenocarcinomas (UGCs) respond poorly to current chemotherapeutic regimes. The authors and others have previously reported frequent Aurora kinase A (AURKA) gene amplification and mRNA and protein overexpression in UGCs. The objective of the current study was to determine the therapeutic potential of alisertib (MLN8237) alone and in combination with docetaxel in UGCs.

HDM2 Regulation by AURKA Promotes Cell Survival in Gastric Cancer

Purpose: Suppression of P53 (tumor protein 53) transcriptional function mediates poor therapeutic response in patients with cancer. Aurora kinase A (AURKA) and human double minute 2 (HDM2) are negative regulators of P53. Herein, we examined the role of AURKA in regulating HDM2 and its subsequent effects on P53 apoptotic function in gastric cancer. Experimental Design: Primary tumors and in vitro gastric cancer cell models with overexpression or knockdown of AURKA were used. The role of AURKA in regulating HDM2 and cell survival coupled with P53 expression and activity were investigated. Results: Overexpression of AURKA enhanced the HDM2 protein level; conversely, knockdown of endogenous AURKA decreased expression of HDM2 in AGS and SNU-1 cells. Dual co-immunoprecipitation assay data indicated that AURKA was associated with HDM2 in a protein complex. The in vitro kinase assay using recombinant AURKA and HDM2 proteins followed by co-immunoprecipitation revealed that AURKA directly interacts and phosphorylates HDM2 protein in vitro. The activation of HDM2 by AURKA led to induction of P53 ubiquitination and attenuation of cisplatin-induced activation of P53 in gastric cancer cells. Inhibition of AURKA using an investigational small-molecule specific inhibitor, alisertib, decreased the HDM2 protein level and induced P53 transcriptional activity. These effects markedly decreased cell survival in vitro and xenograft tumor growth in vivo. Notably, analysis of immunohistochemistry on tissue microarrays revealed significant overexpression of AURKA and HDM2 in human gastric cancer samples (P < 0.05). Conclusion: Collectively, our novel findings indicate that AURKA promotes tumor growth and cell survival through regulation of HDM2-induced ubiquitination and inhibition of P53. Clin Cancer Res; 20(1); 76–86. ©2013 AACR.

Biochanin A reduces pancreatic cancer survival and progression.

Pancreatic cancer has dismally low mean survival rates worldwide. Only a few chemotherapeutic agents including gemcitabine have been shown to improve the survival of pancreatic cancer patients. Biochanin A, an isoflavone, is known to exert an anticancer effect on various cancer types. In this study, we examined the anticancer properties of biochanin A on pancreatic cancer cells. The effect of biochanin A on cellular survival, apoptosis, and proliferation was analyzed using MTT, flow cytometry, and colony formation assay. The effect of biochanin A on pancreatic cancer’s mitogenic signaling was determined using western blot analysis. Migration assay and zymography were used to determine biochanin A’s effect on pancreatic cancer progression. Biochanin A induced dose-dependent toxicity on pancreatic cancer cells (Panc1 and AsPC-1). It reduced colony formation ability of Panc1 cells and induced dose-dependent apoptosis. Activation of Akt and MAPK was inhibited. Furthermore, the migratory and invasive potential of the cancer cells was also reduced. The results suggest that biochanin A is effective in reducing pancreatic cancer cell survival by inhibiting their proliferation and inducing apoptosis. It affects mitogenic, migratory, and invasive processes involved in cancer progression. These findings may lead to novel approaches to treat pancreatic cancer using isoflavones in combination with other therapeutic drugs.

Abstract 4180: RS-41, a multi-targeted kinase inhibitor, induces cell cycle arrest and apoptosis in p53 mutant and wild type models of upper gastrointestinal cancers

Introduction: Upper Gastrointestinal Cancers (UGCs) are a leading cause of cancer-related mortality and account for approximately 1.1 million deaths worldwide. UGCs respond poorly to conventional chemotherapy due to constitutive over activity of multiple oncogenic signaling mechanisms, including the epidermal growth factor receptor (EGFR), ERBB2/HER-2, Aurora kinases, and JAK-STAT pathways. In addition, presence of mutant P53 further imparts resistance to conventional chemotherapeutic agents. Therefore, novel small molecule inhibitors that target multiple kinases associated with oncogenic progression could pave the way for improved chemotherapy and better therapeutic outcomes. In this study, we characterized the anticancer activity of RS-41, an investigational 4-phenylbenzamidopyrrolo[2,3-d]-pyrimidin-4-amine multi-kinase inhibitor, in P53 mutant and wild type models of UGC. Methods: Target kinase inhibition and selectivity screening assays were performed to determine potency and selectivity of kinase inhibition for RS-41. In addition, MTT-cell viability assay, clonogenic cell survival assay, cell cycle analyses, and western blot analyses were done to evaluated the effect of RS-41 treatment on cell viability, survival, cell cycle progression, and expression of apoptotic markers in P53 mutant (FLO-1) and P53 wild type (AGS) UGC cells, respectively. Results: The kinase selectivity screening assay characterized RS-41 for its selectivity against a panel of 90 human kinases. The kinase screening analyses showed that RS-41 selectively inhibits AURKA (IC50-0.96±0.03µM), JAK2 (IC50-1.21±0.17µM), and EGFR (IC50-5.92±0.75µM) kinases, respectively. The cell viability data indicate that treatment with RS-41 mediates significant (P≤0.05) inhibition of FLO-1 and AGS UGC cell viability. The clonogenic cell survival data showed that treatment with RS-41 for 24 hrs. suppresses subsequent formation of colonies in both FLO-1 and AGS UGC cells. The cell cycle data exhibited a marked increase (P≤0.05) in the percentage of FLO-1 and AGS cells in the sub-G1-phase (cell death) after treatment with RS-41 for 24 and 72 hrs., respectively. The western blotting data further confirmed induction of apoptosis in FLO-1 and AGS cells as evidenced by an increase in expression of various markers of apoptosis (P73/P53, cleaved PARP, and/or cleaved caspase 3) following treatment with RS-41 for 24 and 72 hrs. Conclusions: Our in vitro data indicate that inhibition of various oncogenic kinases with RS-41 is an effective therapeutic strategy for inducing apoptosis in both P53 mutant and wild type UGC cells. Our study suggests that RS-41 is a promising multi-kinase inhibitor with a potential to further enhance chemotherapeutic options for treatment of UGC. Citation Format: Tanvi Visal, Shraddha Patel, Priya Pancholi, Samhita Bapat, Amruta Samant, Dhvanir Kansara, Sonali Kurup, Vikas Sehdev. RS-41, a multi-targeted kinase inhibitor, induces cell cycle arrest and apoptosis in p53 mutant and wild type models of upper gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4180. doi:10.1158/1538-7445.AM2017-4180

Abstract 2172: ON 123300, an orally administered novel CDK4/6 + ARK5 inhibitor, exhibits potent antitumor activity in vivo: comparative studies with Palbociclib

Background: The overexpression of cyclin-dependent kinases 4/6 (CDK4/6) is known to cause cell cycle dysregulation in certain cancer types, making these cell cycle kinases attractive targets for pharmacological inhibition. The effectiveness of first-generation non-selective cyclin-dependent kinases, such as roscovitine and flavopiridol, was hampered by toxicities, leading to the development of second-generation compounds like IBRANCE®/Palbociclib that specifically inhibit CDK4 and 6. ON 123300 is a third-generation potent CDK4/6 inhibitor that also inhibits ARK5 with low nanomolar potency and has the potential to improve upon second-generation compounds. Previous studies have demonstrated the inhibitory effect of single-agent ON 123300 in various pre-clinical cancer models of MM and leukemia. In this study, we investigated the comparative therapeutic potential of ON 123300 as an oral anticancer agent and a second-generation inhibitor, Palbociclib, in xenografted Rb+ve mouse models. Methods: MDA-MB-435S xenografted mice were treated once a day for 21 days with ON 123300 (125mg/kg) or Palbociclib (125mg/kg). Tumor volumes were measured and peripheral blood was gathered to evaluate the effects on hematological parameters. Separately, Western blot analyses were performed to determine the effect of CDK4/6 inhibition on p-Rb following intra-tumoral treatment with ON 123300 (2.5µM) or Palbociclib (2.5µM). Results: ON 123300 and Palbociclib reduced tumor growth with an equivalent magnitude during the 21-day treatment period, suggesting that the two compounds were equally effective in this model. Both compounds decreased RBC and platelet counts, however Palbociclib had a more prominent and statistically significant (P≤0.05) inhibitory effect on neutrophil counts when compared to ON 123300 (30.70 ± 3.55 vs. 45.10 ± 2.04). Western blot analysis of tumor tissues demonstrated equivalent effects on p-Rb for both compounds. Conclusions: Xenograft data indicates that a third-generation CDK4/6 inhibitor, ON 123300, is as effective as Palbociclib in an Rb+ve xenograft model. Moreover, this study also suggests that ON 123300 may have the added advantage of reduced neutropenia compared to Palbociclib. Prior preclinical data suggest that ON 123300 may be efficacious in Rb-ve tumors, where second-generation compounds have diminished single-agent activity, and our ongoing studies are aimed at further characterizing the in vivo activity of ON 123300 in this setting. Citation Format: Shraddha Patel, Priya Pancholi, Tanvi Visal, Amruta Samant, Dhvanir Kansara, V J. Rajadhyaksha, Benjamin S. Hoffman, Manoj Maniar, Vikas Sehdev. ON 123300, an orally administered novel CDK4/6 + ARK5 inhibitor, exhibits potent antitumor activity in vivo: comparative studies with Palbociclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2172. doi:10.1158/1538-7445.AM2017-2172

Abstract 1649: Potent anticancer activity of an orally bioavailable small molecule, ON 013100, and its water soluble derivative, briciclib, a clinical-stage eIF4E-targeted agent

Introduction: Eukaryotic translation initiation factor 4E (eIF4E) is a master regulator that controls translation of mRNA in mammalian cells. eIF4E is a proto-oncogene that promotes translation of several genes essential for cellular proliferation (cyclin D1, c-Myc, mTOR), survival (Akt, survivin), angiogenesis (VEGF), and metastasis (MMP9). Overexpression of eIF4E has been observed in almost all major groups of cancers and has been shown to induce increased expression of cyclin D1 and c-Myc. Briciclib is a small molecule, water soluble derivative of ON 013100 that binds to eIF4E. An intravenous formulation of briciclib is currently being investigated in a Phase 1 clinical trial. Recent advancements in formulation technology have made feasible a stable, orally bioavailable version of ON 013100, which may allow for more convenient administration. In this study we investigated and compared the anticancer activity of briciclib to ON 013100. We determined the susceptibility of various breast, mantle cell leukemia (MCL), gastric, and esophageal cancer cell lines to treatment with briciclib or ON 013100. In addition, we investigated the effect of briciclib and ON 013100 on expression of markers associated with eIF4E activity (cyclin D1 and c-Myc) and apoptosis (P53 and Cleaved Caspase 3). Methods: MTT cell viability assays, Western blot analysis, and ELISA assays were used to evaluate cellular viability, survival, and protein expression levels. Results: Briciclib and ON 013100 inhibited the proliferation of MCL (JEKO-1 and MINO), breast (MCF7 and MDA-MB-231), gastric (AGS), and esophageal (OE19, OE33, and FLO-1) cancer cell lines at nanomolar concentrations (Briciclib: GI50 = 9.8 - 12.2 nM; ON 013100 GI50 = 6.7 - 11.2 nM). By comparison, briciclib and ON 013100 were relatively non-toxic to normal endothelial cells. Western blot analysis indicated that treatment with briciclib or ON 013100 significantly reduced the expression of cyclin D1 and c-Myc in breast and MCL cancer cell lines within 8 hours and in a dose-dependent manner. These observations were supported by ELISA analysis of cyclin D1 and c-Myc protein levels. Furthermore, treatment with these agents enhanced the expression of P53 and Cleaved Caspase 3 pro-apoptotic proteins in breast and MCL cancer cell lines. Our ongoing tumor xenograft experiments are in agreement with the aforementioned in vitro observations. Conclusions: Our findings suggest that both an orally bioavailable ON 013100, and its water soluble derivative, briciclib, have the same novel mechanism of action involving translation. Our in vitro and in vivo data demonstrate the potential of briciclib in targeting eIF4E for hematopoietic and solid cancers and the possibility for developing an oral version of this promising clinical agent. Citation Format: Neel Jasani, Bina Desai, Justine M. Betzu, Tanmay Dichwalkar, Samhita Bapat, V. J. Rajadhayksha, Benjamin S. Hoffman, Manoj Maniar, Vikas Sehdev. Potent anticancer activity of an orally bioavailable small molecule, ON 013100, and its water soluble derivative, briciclib, a clinical-stage eIF4E-targeted agent. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1649. doi:10.1158/1538-7445.AM2015-1649

Regulation of HDM2 E3-ubiquitin ligase in esophageal adenocarcinoma cells by AURKA.

35 Background: Esophageal adenocarcinomas (EAC) exhibit intrinsic resistance against chemotherapy. AURKA regulates cell cycle progression and its overexpression is associated with oncogenic transformation. We have recently reported that AURKA is significantly overexpressed in about 70% of human EAC tissue samples and EAC cell lines. We have previously shown that AURKA inhibits p53- and p73-mediated apoptotic pathways in GI adenocarcinomas. HDM2 is an E3-ubiquitin ligase which is closely involved in regulating p53 and p73 protein stability and activity. In this study we demonstrate that AURKA directly interacts with HDM2 and regulates HDM2 protein expression and phosphorylation in both FLO-1 and OE33 EAC cells. Methods and Results: Western blot analyses were done following AURKA overexpression with adenovirus, knockdown with si-RNA or inhibition with MLN 8237 (0.5µM) in FLO-1 and OE33 EAC cell lines. The data indicated that overexpression of AURKA induced both total and phospho-HDM2-(Ser166) protein levels...