Mudong Kim, Ahrum Min, Sohyeon Kim, Seongyeong Kim, Yu-jin Kim, Sujin Ham, Miso Lee, Eunice Yoojin Lee, Jinyong Kim, Dae-Won Lee, Kyung-Hun Lee, Seock-Ah Im
Received November 19, 2024 Accepted April 1, 2025 Published online April 7, 2025
Purpose
Sustained cell proliferation and cell cycle acceleration in cancer cells inherently increase DNA damage, which interferes with homeostatic replication and transcription. Ataxia telangiectasia and Rad3-related (ATR) is crucial for initiation of the DNA damage response, and ATR inhibitors, such as elimusertib, induce increased replication stress and DNA damage. We investigated the anti-tumor effects of elimusertib and its mechanism of action in relation to replication stress.
Materials and Methods
Anti-tumor effects were evaluated by MTT assay and colony formation assay in breast cancer cell lines in vitro, in breast cancer cell xenografts in vivo, and in patient-derived xenograft models. Cell cycle was assessed by flow cytometry and BrdU assay was used to measure replicating cells and S-phase progression. Alkaline and neutral comet assay was used to measure single and double stranded DNA damages, respectively.
Results
Elimusertib delayed S-phase progression in MDA-MB-453 and MDA-MB-231 cells and induced caspase-7-dependent apoptosis. Furthermore, the increase in sub-G1 population in the FACS analysis and Annexin V assay also confirmed apoptotic cell death. In the BrdU assay, single stranded DNA (ssDNA) increased in sensitive cells and aberrant ssDNA induced DNA damage in S-phase and eventually caused replication catastrophe. Finally, these anti-tumor effects were proven in in vivo xenograft and patient-derived xenograft models.
Conclusion
Elimusertib had anti-tumor effects and induced replication catastrophe in breast cancer cells with a high replication rate. Moreover, cells under high DNA replication stress were sensitive to elimusertib. Further studies and treatment strategies with elimusertib are warranted for cancers with a high replication rate.
Miso Lee, Kyung-Hun Lee, Ahrum Min, Jeongeun Kim, Seongyeong Kim, Hyemin Jang, Jee Min Lim, So Hyeon Kim, Dong-Hyeon Ha, Won Jae Jeong, Koung Jin Suh, Yae-Won Yang, Tae Yong Kim, Do-Youn Oh, Yung-Jue Bang, Seock-Ah Im
Cancer Res Treat. 2019;51(2):451-463. Published online June 6, 2018
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.
Citations
Citations to this article as recorded by
Prognostic Model Construction of Disulfidptosis-Related Genes and Targeted Anticancer Drug Research in Pancreatic Cancer Hongtao Duan, Li Gao, Aiminuer Asikaer, Lingzhi Liu, Kuilong Huang, Yan Shen Molecular Biotechnology.2025; 67(4): 1463. CrossRef
A literature review of recent advances in gastric cancer treatment: exploring the cross-talk between targeted therapies Reza Panahizadeh, Padideh Panahi, Vahid Asghariazar, Shima Makaremi, Ghasem Noorkhajavi, Elham Safarzadeh Cancer Cell International.2025;[Epub] CrossRef
PIM1 attenuates cisplatin-induced AKI by inhibiting Drp1 activation Yuzhen Li, Lang Shi, Fan Zhao, Yanwen Luo, Mingjiao Zhang, Xiongfei Wu, Jiefu Zhu Cellular Signalling.2024; 113: 110969. CrossRef
PIM1 kinase and its diverse substrate in solid tumors Rituparna Choudhury, Chandan Kumar Bahadi, Ipsa Pratibimbita Ray, Pragyanshree Dash, Isha Pattanaik, Suman Mishra, Soumya R. Mohapatra, Srinivas Patnaik, Kumar Nikhil Cell Communication and Signaling.2024;[Epub] CrossRef
The evaluation of six genes combined value in glioma diagnosis and prognosis Ping Lin, Lingyan He, Nan Tian, Xuchen Qi Journal of Cancer Research and Clinical Oncology.2023; 149(13): 12413. CrossRef
Toxic effects of AZD1208 on mouse oocytes and its possible mechanisms Feng‐Ze Yan, Ying‐Chun Ouyang, Tie‐Gang Meng, Hong‐Yong Zhang, Wei Yue, Xin‐Ran Zhang, Yue Xue, Zhen‐Bo Wang, Qing‐Yuan Sun Journal of Cellular Physiology.2022; 237(9): 3661. CrossRef
Therapeutic targeting of PIM KINASE signaling in cancer therapy: Structural and clinical prospects Aanchal Rathi, Dhiraj Kumar, Gulam Mustafa Hasan, Mohammad Mahfuzul Haque, Md Imtaiyaz Hassan Biochimica et Biophysica Acta (BBA) - General Subjects.2021; 1865(11): 129995. CrossRef
TDP1 and TOP1 Modulation in Olaparib-Resistant Cancer Determines the Efficacy of Subsequent Chemotherapy Jin Won Kim, Ahrum Min, Seock-Ah Im, Hyemin Jang, Yu Jin Kim, Hee-Jun Kim, Kyung-Hun Lee, Tae-Yong Kim, Keun Wook Lee, Do-Youn Oh, Jee-Hyun Kim, Yung-Jue Bang Cancers.2020; 12(2): 334. CrossRef
Inhibition of PIM1 attenuates the stem cell–like traits of breast cancer cells by promoting RUNX3 nuclear retention Hui Liu, Cheng Chen, Dongshen Ma, Yubing Li, Qianqian Yin, Qing Li, Chenxi Xiang Journal of Cellular and Molecular Medicine.2020; 24(11): 6308. CrossRef
Antitumor effect of a WEE1 inhibitor and potentiation of olaparib sensitivity by DNA damage response modulation in triple-negative breast cancer Dong-Hyeon Ha, Ahrum Min, Seongyeong Kim, Hyemin Jang, So Hyeon Kim, Hee-Jun Kim, Han Suk Ryu, Ja-Lok Ku, Kyung-Hun Lee, Seock-Ah Im Scientific Reports.2020;[Epub] CrossRef
Pim kinase inhibitors in cancer: medicinal chemistry insights into their activity and selectivity Soraya Alnabulsi, Enas A. Al-Hurani Drug Discovery Today.2020; 25(11): 2062. CrossRef
New Mechanistic Insight on the PIM-1 Kinase Inhibitor AZD1208 Using Multidrug Resistant Human Erythroleukemia Cell Lines and Molecular Docking Simulations Maiara Bernardes Marques, Michael González-Durruthy, Bruna Félix da Silva Nornberg, Bruno Rodrigues Oliveira, Daniela Volcan Almeida, Ana Paula de Souza Votto, Luis Fernando Marins Current Topics in Medicinal Chemistry.2019; 19(11): 914. CrossRef
Recent Studies on Ponatinib in Cancers Other Than Chronic Myeloid Leukemia Francesca Musumeci, Chiara Greco, Giancarlo Grossi, Alessio Molinari, Silvia Schenone Cancers.2018; 10(11): 430. CrossRef
Seongyeong Kim, Ahrum Min, Kyung-Hun Lee, Yaewon Yang, Tae-Yong Kim, Jee Min Lim, So Jung Park, Hyun-Jin Nam, Jung Eun Kim, Sang-Hyun Song, Sae-Won Han, Do-Youn Oh, Jee Hyun Kim, Tae-You Kim, David Hangauer, Johnson Yiu-Nam Lau, Kyongok Im, Dong Soon Lee, Yung-Jue Bang, Seock-Ah Im
Cancer Res Treat. 2017;49(3):643-655. Published online October 6, 2016
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.
Citations
Citations to this article as recorded by
Clinical and experimental aspects of tirbanibulin treatments Annabel Shen, Rebecca A. Simonette, Peter L. Rady, Stephen K. Tyring Archives of Dermatological Research.2025;[Epub] CrossRef
The Potential Strategies for Overcoming Multidrug Resistance and
Reducing Side Effects of Monomer Tubulin Inhibitors for Cancer
Therapy Yingjie Cui, Jing Zhang, Guifang Zhang Current Medicinal Chemistry.2024; 31(14): 1874. CrossRef
Efficacy and tolerability of tirbanibulin 1% ointment in the treatment of cancerization field: a real‐life Italian multicenter observational study of 250 patients Gianluca Nazzaro, Andrea Carugno, Paolo Bortoluzzi, Stefano Buffon, Chiara Astrua, Elena Zappia, Emanuele Trovato, Stefano Caccavale, Vincenzo Pellegrino, Giovanni Paolino, Riccardo Balestri, Rossella Lacava, Giulia Ciccarese, Alice Verdelli, Stefania Bar International Journal of Dermatology.2024; 63(11): 1566. CrossRef
Dickkopf-1 (DKK1) drives growth and metastases in castration-resistant prostate cancer Letizia Rinella, Gloria Fiorentino, Mara Compagno, Cristina Grange, Massimo Cedrino, Francesca Marano, Ornella Bosco, Elena Vissio, Luisa Delsedime, Patrizia D’Amelio, Benedetta Bussolati, Emanuela Arvat, Maria Graziella Catalano Cancer Gene Therapy.2024; 31(8): 1266. CrossRef
Anti-tumor effects of tirbanibulin in squamous cell carcinoma cells are mediated via disruption of tubulin-polymerization Viola K. DeTemple, Antje Walter, Sabine Bredemeier, Ralf Gutzmer, Katrin Schaper-Gerhardt Archives of Dermatological Research.2024;[Epub] CrossRef
Tirbanibulin decreases cell proliferation and downregulates protein expression of oncogenic pathways in human papillomavirus containing HeLa cells Stephen Moore, Veda Kulkarni, Angela Moore, Jennifer R. Landes, Rebecca Simonette, Qin He, Peter L. Rady, Stephen K. Tyring Archives of Dermatological Research.2024;[Epub] CrossRef
Topical Pharmacological Treatment of Actinic Keratoses: Focus on Tirbanibulin 1% Ointment Mario Valenti, Matteo Bianco, Alessandra Narcisi, Antonio Costanzo, Riccardo Borroni, Marco Ardigò Dermatology Practical & Conceptual.2024; 14(S1): e2024145S. CrossRef
Recent advancement in developing small molecular inhibitors targeting key kinase pathways against triple-negative breast cancer Rajibul Islam, Khor Poh Yen, Nur Najihah ’Izzati Mat Rani, Md. Selim Hossain Bioorganic & Medicinal Chemistry.2024; 112: 117877. CrossRef
Real-world experience with histological confirmation of clinical response of squamous cell carcinoma to topical tirbanibulin Angela Moore, Kara Hurley, Stephen A. Moore, Luke Moore JAAD Case Reports.2023; 40: 141. CrossRef
Synthesis and evaluation of tirbanibulin derivatives: a detailed exploration of the structure–activity relationship for anticancer activity Jaebeom Park, Minji Kang, Ahyoung Lim, Kyung-Jin Cho, Chong Hak Chae, Byumseok Koh, Hongjun Jeon RSC Advances.2023; 13(50): 35583. CrossRef
Tirbanibulina: revisión de su mecanismo de acción novedoso y de cómo encaja en el tratamiento de la queratosis actínica Y. Gilaberte, M.T. Fernández-Figueras Actas Dermo-Sifiliográficas.2022; 113(1): 58. CrossRef
Insights on Cancer Cell Inhibition, Subcellular Activities, and Kinase Profile of Phenylacetamides Pending 1H-Imidazol-5-One Variants Maan T. Khayat, Abdelsattar M. Omar, Farid Ahmed, Mohammad I. Khan, Sara M. Ibrahim, Yosra A. Muhammad, Azizah M. Malebari, Thikryat Neamatallah, Moustafa E. El-Araby Frontiers in Pharmacology.2022;[Epub] CrossRef
Identification of New Vulnerabilities in Conjunctival Melanoma Using Image-Based High Content Drug Screening Katya Nardou, Michael Nicolas, Fabien Kuttler, Katarina Cisarova, Elifnaz Celik, Mathieu Quinodoz, Nicolo Riggi, Olivier Michielin, Carlo Rivolta, Gerardo Turcatti, Alexandre Pierre Moulin Cancers.2022; 14(6): 1575. CrossRef
Targeting regulated cell death (RCD) with small-molecule compounds in triple-negative breast cancer: a revisited perspective from molecular mechanisms to targeted therapies Minru Liao, Rui Qin, Wei Huang, Hong-Ping Zhu, Fu Peng, Bo Han, Bo Liu Journal of Hematology & Oncology.2022;[Epub] CrossRef
SAR Probing of KX2-391 Provided Analogues With Juxtaposed Activity Profile Against Major Oncogenic Kinases Abdelsattar M. Omar, Maan T. Khayat, Farid Ahmed, Yosra A. Muhammad, Azizah M. Malebari, Sara M. Ibrahim, Mohammad I. Khan, Dhaval K. Shah, Wayne E. Childers, Moustafa E. El-Araby Frontiers in Oncology.2022;[Epub] CrossRef
Topical Tirbanibulin, a Dual Src Kinase and Tubulin Polymerization Inhibitor, for the Treatment of Plaque-Type Psoriasis: Phase I Results Jin-Bon Hong, Po-Yuan Wu, Albert Qin, Yi-Wen Huang, Kuan-Chiao Tseng, Ching-Yu Lai, Wing-Kai Chan, Jane Fang, David L. Cutler, Tsen-Fang Tsai Pharmaceutics.2022; 14(10): 2159. CrossRef
Tirbanibulin for Actinic Keratosis: Insights into the Mechanism of Action Todd Schlesinger, Eggert Stockfleth, Ayman Grada, Brian Berman Clinical, Cosmetic and Investigational Dermatology.2022; Volume 15: 2495. CrossRef
Dual Kinase Targeting in Leukemia Luca Mologni, Giovanni Marzaro, Sara Redaelli, Alfonso Zambon Cancers.2021; 13(1): 119. CrossRef
Recent Progress on Tubulin Inhibitors with Dual Targeting Capabilities for Cancer Therapy Wen Shuai, Guan Wang, Yiwen Zhang, Faqian Bu, Sicheng Zhang, Duane D. Miller, Wei Li, Liang Ouyang, Yuxi Wang Journal of Medicinal Chemistry.2021; 64(12): 7963. CrossRef
New FDA oncology small molecule drugs approvals in 2020: Mechanism of action and clinical applications Thais Cristina Mendonça Nogueira, Marcus Vinicius Nora de Souza Bioorganic & Medicinal Chemistry.2021; 46: 116340. CrossRef
Tirbanibulin: review of its novel mechanism of action and how it fits into the treatment of actinic keratosis Y. Gilaberte, M.T. Fernández-Figueras Actas Dermo-Sifiliográficas (English Edition).2021;[Epub] CrossRef
Recent progress in small molecule agents for the targeted therapy of triple-negative breast cancer Rajibul Islam, Kok Wai Lam European Journal of Medicinal Chemistry.2020; 207: 112812. CrossRef
Current advances of tubulin inhibitors as dual acting small molecules for cancer therapy Kinsie E Arnst, Souvik Banerjee, Hao Chen, Shanshan Deng, Dong‐Jin Hwang, Wei Li, Duane D Miller Medicinal Research Reviews.2019; 39(4): 1398. CrossRef
Reversible binding of the anticancer drug KXO1 (tirbanibulin) to the colchicine-binding site of β-tubulin explains KXO1's low clinical toxicity Lu Niu, Jianhong Yang, Wei Yan, Yamei Yu, Yunhua Zheng, Haoyu Ye, Qiang Chen, Lijuan Chen Journal of Biological Chemistry.2019; 294(48): 18099. CrossRef
Discovery of Novel Dual Mechanism of Action Src Signaling and Tubulin Polymerization Inhibitors (KX2-391 and KX2-361) Michael P. Smolinski, Yahao Bu, James Clements, Irwin H. Gelman, Taher Hegab, David L. Cutler, Jane W. S. Fang, Gerald Fetterly, Rudolf Kwan, Allen Barnett, Johnson Y. N. Lau, David G. Hangauer Journal of Medicinal Chemistry.2018; 61(11): 4704. CrossRef
KX2-361: a novel orally bioavailable small molecule dual Src/tubulin inhibitor that provides long term survival in a murine model of glioblastoma Michael J. Ciesielski, Yahao Bu, Stephan A. Munich, Paola Teegarden, Michael P. Smolinski, James L. Clements, Johnson Y. N. Lau, David G. Hangauer, Robert A. Fenstermaker Journal of Neuro-Oncology.2018; 140(3): 519. CrossRef