Seongyeong Kim

AZD6738, A Novel Oral Inhibitor of ATR, Induces Synthetic Lethality with ATM Deficiency in Gastric Cancer Cells

Ataxia telangiectasia and Rad3-related (ATR) can be considered an attractive target for cancer treatment due to its deleterious effect on cancer cells harboring a homologous recombination defect. The aim of this study was to investigate the potential use of the ATR inhibitor, AZD6738, to treat gastric cancer. In SNU-601 cells with dysfunctional ATM, AZD6738 treatment led to an accumulation of DNA damage due to dysfunctional RAD51 foci formation, S phase arrest, and caspase 3–dependent apoptosis. In contrast, SNU-484 cells with functional ATM were not sensitive to AZD6738. Inhibition of ATM in SNU-484 cells enhanced AZD6738 sensitivity to a level comparable with that observed in SNU-601 cells, showing that activation of the ATM-Chk2 signaling pathway attenuates AZD6738 sensitivity. In addition, decreased HDAC1 expression was found to be associated with ATM inactivation in SNU-601 cells, demonstrating the interaction between HDAC1 and ATM can affect sensitivity to AZD6738. Furthermore, in an in vivo tumor xenograft mouse model, AZD6738 significantly suppressed tumor growth and increased apoptosis. These findings suggest synthetic lethality between ATR inhibition and ATM deficiency in gastric cancer cells. Further clinical studies on the interaction between AZD 6738 and ATM deficiency are warranted to develop novel treatment strategies for gastric cancer. Mol Cancer Ther; 16(4); 566–77. ©2017 AACR.

Anti‐tumor activity of the ATR inhibitor AZD6738 in HER2 positive breast cancer cells

Ataxia telangiectasia and Rad3‐related (ATR) proteins are sensors of DNA damage, which induces homologous recombination (HR)‐dependent repair. ATR is a master regulator of DNA damage repair (DDR), signaling to control DNA replication, DNA repair and apoptosis. Therefore, the ATR pathway might be an attractive target for developing new drugs. This study was designed to investigate the antitumor effects of the ATR inhibitor, AZD6738 and its underlying mechanism in human breast cancer cells. Growth inhibitory effects of AZD6738 against human breast cancer cell lines were studied using a 3‐(4,5‐dimethylthiazol‐2‐yl)−2,5‐diphenyltetrazolium bromide (methyl thiazolyl tetrazolium, MTT) assay. Cell cycle analysis, Western blotting, immunofluorescence and comet assays were also performed to elucidate underlying mechanisms of AZD6738 action. Anti‐proliferative and DDR inhibitory effects of AZD6738 were demonstrated in human breast cancer cell lines. Among 13 cell lines, the IC50 values of nine cell lines were less than 1 μmol/L using MTT assay. Two cell lines, SK‐BR‐3 and BT‐474, were chosen for further evaluation focused on human epidermal growth factor receptor 2 (HER2)‐positive breast cancer cells. Sensitive SK‐BR‐3 but not the less sensitive BT‐474 breast cancer cells showed increased level of apoptosis and S phase arrest and reduced expression levels of phosphorylated check‐point kinase 1 (CHK1) and other repair markers. Decreased functional CHK1 expression induced DNA damage accumulation due to HR inactivation. AZD6738 showed synergistic activity with cisplatin. Understanding the antitumor activity and mechanisms of AZD6738 in HER2‐positive breast cancer cells creates the possibility for future clinical trials targeting DDR in HER2‐positive breast cancer treatment.

Anti-tumor Effect of KX-01 Through Inhibiting Src Family Kinases and Mitosis

Purpose KX-01 is a novel dual inhibitor of Src and tubulin. Unlike previous Src inhibitors that failed to show clinical benefit during treatment of breast cancer, KX-01 can potentially overcome the therapeutic limitations of current Src inhibitors through inhibition of both Src and tubulin. The present study further evaluates the activity and mechanism of KX-01 in vitro and in vivo. Materials and Methods The antitumor effect of KX-01 in triple negative breast cancer (TNBC) cell lines was determined by MTT assay. Wound healing and immunofluorescence assays were performed to evaluate the action mechanisms of KX-01. Changes in the cell cycle and molecular changes induced by KX-01 were also evaluated. A MDA-MB-231 mouse xenograft model was used to demonstrate the in vivo effects. Results KX-01 effectively inhibited the growth of breast cancer cell lines. The expression of phospho-Src and proliferative-signaling molecules were down-regulated in KX-01-sensitive TNBC cell lines. In addition, migration inhibition was observed by wound healing assay. KX-01-induced G2/M cell cycle arrest and increased the aneuploid cell population in KX-01-sensitive cell lines. Multi-nucleated cells were significantly increased after KX-01 treatment. Furthermore, KX-01 effectively delayed tumor growth in a MDA-MB-231 mouse xenograft model. Conclusion KX-01 effectively inhibited cell growth and migration of TNBC cells. Moreover, this study demonstrated that KX-01 showed antitumor effects through the inhibition of Src signaling and the induction of mitotic catastrophe. The antitumor effects of KX-01 were also demonstrated in vivo using a mouse xenograft model.

Pan-Pim Kinase Inhibitor AZD1208 Suppresses Tumor Growth and Synergistically Interacts with Akt Inhibition in Gastric Cancer Cells

Purpose Pim kinases are highly conserved serine/threonine kinases, and different expression patterns of each isoform (Pim-1, Pim-2, and Pim-3) have been observed in various types of human cancers, including gastric cancer. AZD1208 is a potent and selective inhibitor that affects all three isoforms of Pim. We investigated the effects of AZD1208 as a single agent and in combination with an Akt inhibitor in gastric cancer cells. Materials and Methods The antitumor activity of AZD1208 with/without an Akt inhibitor was evaluated in a large panel of gastric cancer cell lines through growth inhibition assays. The underlying mechanism was also examined by western blotting, immunofluorescence assay, and cell cycle analysis. Results AZD1208 treatment decreased gastric cancer cell proliferation rates and induced autophagy only in long-term culture systems. Light chain 3B (LC3B), a marker of autophagy, was increased in sensitive cells in a dose-dependent manner with AZD1208 treatment, which suggested that the growth inhibition effect of AZD1208 was achieved through autophagy, not apoptosis. Moreover, we found that cells damaged by Pim inhibition were repaired by activation of the DNA damage repair pathway, which promoted cell survival and led the cells to become resistant to AZD1208. We also confirmed that the combination of an Akt inhibitor with AZD1208 produced a highly synergistic effect in gastric cancer cell lines. Conclusion Treatment with AZD1208 alone induced considerable cell death through autophagy in gastric cancer cells. Moreover, the combination of AZD1208 with an Akt inhibitor showed synergistic antitumor effects through regulation of the DNA damage repair pathway.

A large and distinct skin impression on the cast of a sauropod dinosaur footprint from Early Cretaceous floodplain deposits, Korea

The occurrence and features of skin impressions in a sauropod footprint, the largest (>50 cm in diameter) reported to date for this taxon, from the Lower Cretaceous Haman Formation (Albian) in Korea are described, and its preservation and paleoenvironmental implications are interpreted. The skin impression-bearing deposits are floodplain sediments formed by sheetflood processes. The large impression is preserved in silty mudstone with microbial lenses and wisps overlying a planar- to cross-laminated and fine-grained sandstone to siltstone bed. The paleoenvironment of the skin impression-bearing deposits is interpreted as a saline sandflat to mudflat where microbial mats can form around lakes or ponds under semi-arid paleoclimatic conditions with alternating wetting and drying intervals. These paleoenvironmental conditions would have permitted the distinct preservation of skin impressions in a dinosaur footprint. The observations here suggest that some sauropod dinosaurs in the Cretaceous had a well-developed polygonal skin texture covering nearly the whole of their foot pads, as seen in modern elephants, which would increase stability when walking on muddy and wet ground.

Abstract LB-107: Androgen receptor inhibitor enhances the antitumor effect of PARP inhibitor in breast cancer cells via modulation of DNA damage response

Introduction: Olaparib, a PARP inhibitor has produced promising antitumor efficacy in patients with BRCA mutation. However, cancers with BRCAness represent only a small proportion of breast cancer cases. To make olaparib useful to the broader cancer population, the development of new combinational strategies are required. The androgen receptor (AR) is expressed in 60∼70% of breast cancers regardless of ER status and has been proposed as a therapeutic target in breast cancers that retain AR. AR signaling was recently demonstrated to be a key regulator of the DNA damage response (DDR) in prostate cancer cells. AR inhibition increased cell death induced by DNA damage from cytotoxic insults. Based on these evidence, we hypothesized that AR inhibition using AZD3514, a novel AR inhibitor, would enhance the anti-tumor effect of PARP inhibitor in breast cancer cells by blocking DNA repair pathway. Materials and Methods: The cytotoxic assay, cell cycle analysis and western blotting were conducted to determine whether AZD3514, an AR inhibitor could enhance the anti-tumor effects of olaparib on breast cancer cells. The regulation of DDR activity by AZD3514 was accessed by the comet and IFA analysis. The molecular mechanism of DDR regulation by AZD3514 was also determined through gene knockdown experiments. These in vitro data were validated in vivo model as well. Results: AR inhibition had a minimal anti-proliferative effect on most of the breast cancer cell lines irrespective of the degree of AR expression levels as a monotherapy. However, AZD3514 downregulated the expressions of DDR molecules, including ATM and chk2 in some breast cancer cell lines. Breast cancer cell lines exhibited a various level of response of the combination treatment irrespective of their subtype as well as their AR expression levels. Co-targeting AR and PARP suppressed the proliferation and induced G2/M cell cycle arrest and apoptosis in MDA-MB-468 cells, but not that of MDA-MB-453 cells. Furthermore, AZD3514 treatment decreased the activity of ATM-chk2 axis resulting in the accumulation of DNA damage via compromising DDR activity in MDA-MB-468 cells. The results indicated that the mechanism underlying that AZD3514 enhances cellular sensitivity to olaparib would be through abrogation of the DNA DSB repair pathway in MDA-MB-468 cells. We also found that down-regulation of NKX3.1 expression correlated with suppressed activation of ATM-chk2 axis in MDA-MB-468 cells. In addition, the suppressed levels of NKX3.1 following AZD3514 treatment were a result of that AZD3514 induced TOPORS expression resulting in acceleration of NKX3.1 degradation. Finally, these in vitro findings were validated in a MDA-MB-468 xenograft model. Conclusions: Our data revealed that post-translational regulation of NKX3.1 by modulation of TOPORS expression following AZD3514 treatment induced ATM inactivation which can increase olaparib sensitivity in AR positive and TOPORS expressed breast cancer cells. This study firstly demonstrated the anti-tumor effect of AZD3514 in a combination with olaparib via compromising DDR activity in breast cancer cell lines as well as in a xenograft model. Our results provide a rationale for the future clinical trials of olaparib combined with AR inhibition in the treatment of breast cancers. Citation Format: Ahrum Min, Seock-Ah Im, Hyemin Jang, Seongyeong Kim, So Hyeon Kim, Yu Jin Kim, Debora Keunyoung Kim, Yaewon Yang, Koung Jin Suh, Kyung-Hun Lee, Tae-Yong Kim, Do-Youn Oh, Yung-Jue Bang. Androgen receptor inhibitor enhances the antitumor effect of PARP inhibitor in breast cancer cells via modulation of DNA damage response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-107.

Abstract 2422: Pan-pim kinases inhibitor, AZD1208 suppresses tumor growth and synergistically interacts with an AKT inhibitor in gastric cancer cells

Introduction: Pim kinases are highly conserved serine/threonine kinases that control proliferation and survival pathways in cancer cells. Different expression patterns of each isoforms (Pim-1, Pim-2, and Pim-3) have been observed in various types of human cancer including gastric cancer. AZD1208 is a potent and selective inhibitor that affects all three isoforms of Pim. We investigated the effect of AZD1208 as a single agent or in combination with an Akt inhibitor on the growth and cell death in gastric cancer cells. Materials and Methods: The antitumor activity of AZD1208 with/without an Akt inhibitor was evaluated in a large panel of gastric cancer cell lines through growth inhibition assay. The underlying mechanism was also examined by western blotting, immunofluorescence assay (IFA), and cell cycle analysis. Results: Direct substrates of Pim kinases were downregulated by the treatment with AZD1208, but we could not find any reduction of cell viability in short-term cell cultures. In contrast, cell viability decreased significantly in long-term cultures. Light chain 3B (LC3B), a marker for autophagy, increased in sensitive cells in dose dependent manner with AZD1208 treatment, which suggest that the growth inhibition effect of AZD1208 was achieved through autophagy, but not through apoptosis. Moreover, we found that ATM and Chk2, two critical components of the DNA repair pathway, were activated in an AZD1208-resistant cell line. We also confirmed that the combination of an Akt inhibitor with AZD1208 produced a highly synergistic effect in gastric cancer cell lines. Conclusion: AZD1208 induced cell death through autophagy in gastric cancer cells. Additionally, the combination of AZD1208 with an Akt inhibitor shows synergistic antitumor effects. Citation Format: Miso Lee, Kyung-Hun Lee, Ahrum Min, Jungeun Kim, Seock-Ah Im, Seon-Gyeong Kim, Hyemin Jang, Yae-Won Yang, Tae-Yong Kim, Sae-Won Han, Do-Youn Oh, Tae-You Kim, Yung-Jue Bang. Pan-pim kinases inhibitor, AZD1208 suppresses tumor growth and synergistically interacts with an AKT inhibitor in gastric cancer cells. [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 2422. doi:10.1158/1538-7445.AM2015-2422

Abstract 5479: Antitumor effect of KX-01, a novel Src and tubulin inhibitor, in triple negative breast cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Src kinases have been involved in cell proliferation, invasion, and metastasis and is one of the highly expressed oncogenes in breast cancer. Therefore, it could be a reasonable target for therapeutic strategy. Although several Src inhibitors, targeting the ATP binding site, were developed, none of them have shown remarkable responses as monotherapeutic agents in breast cancer clinical trials. KX-01 (KX2-391) is a novel peptidomimetic agent, and a non ATP-competitive Src inhibitor, which has potent efficacy for inhibiting both Src as well as tubulin polymerization. Its dual inhibition effects could potentially overcome the limitations of the Src inhibitors that have been evaluated previously. Materials & Methods: We determined the anti-tumor effects of KX-01 on breast cancer cell lines using a cytotoxicity assay, cell cycle analysis, a wound healing assay and western blotting following KX-01 treatment. Results: KX-01 effectively inhibited cell growth in most breast cancer cells including ER/PR/HER2 negative breast cancer cells. Short exposure of KX-01 down-regulated the expression level of Src phosphorylation. Phosphorylated FAK, ERK, AKT and STAT3 were also down-regulated by KX-01 treatment in MDA-MB-231, MDA-MB-468 and BT-549 cells. By wound healing assay, we confirmed migration inhibitory effect of KX-01 in BT-549 cells. Increase of G2/M cell cycle arrest was observed in dose dependent manner. In addition, mitotic catastrophes were also observed in KX-01 sensitive cell lines. Consistent with induction of mitotic catastrophe, the increased levels of aneuploidy in sensitive cell lines were examined following the treatment of KX-01. Conclusion: Inhibition of cell growth and migration, as well as an induction of mitotic catastrophy, were observed in triple negative breast cancer cell lines treated with KX-01. Our data strongly supports utilizing KX-01 as a new therapeutic agents for treating triple negative breast cancer. Citation Format: Seongyeong Kim, Seock-Ah Im, Ahrum Min, Miso Lee, Heymin Jang, Kyung-Hun Lee, Hee-Jun Kim, Tae-Yong Kim, Sae-Won Han, Do-Youn Oh, Tae-You Kim, Young-Kwang Yoon, David Hangauer, Johnson Lau, Yung-Jue Bang. Antitumor effect of KX-01, a novel Src and tubulin inhibitor, in triple negative breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5479. doi:10.1158/1538-7445.AM2014-5479