Research ArticleCell biologyTherapeutics Open Access | 10.1172/JCI165448

Ting-Yu Chang,1 Yan Yan,2 Zih-Yao Yu,1 Moeez Rathore,2,3 Nian-Zhe Lee,1 Hui-Ju Tseng,1 Li-Hsin Cheng,4,5 Wei-Jan Huang,6 Wei Zhang,2,3 Ernest R. Chan,7 Yulan Qing,2 Ming-Lun Kang,8 Rui Wang,2,3 Kelvin K. Tsai,4,5 John J. Pink,2 William E. Harte,9 Stanton L. Gerson,2,10 and Sung-Bau Lee1

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

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1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Yan, Y. in: JCI | PubMed | Google Scholar

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Yu, Z. in: JCI | PubMed | Google Scholar

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Rathore, M. in: JCI | PubMed | Google Scholar

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Lee, N. in: JCI | PubMed | Google Scholar

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Tseng, H. in: JCI | PubMed | Google Scholar |

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Cheng, L. in: JCI | PubMed | Google Scholar

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Huang, W. in: JCI | PubMed | Google Scholar |

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Zhang, W. in: JCI | PubMed | Google Scholar

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Chan, E. in: JCI | PubMed | Google Scholar |

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Qing, Y. in: JCI | PubMed | Google Scholar

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Kang, M. in: JCI | PubMed | Google Scholar

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Wang, R. in: JCI | PubMed | Google Scholar |

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Tsai, K. in: JCI | PubMed | Google Scholar

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Pink, J. in: JCI | PubMed | Google Scholar

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Harte, W. in: JCI | PubMed | Google Scholar

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Gerson, S. in: JCI | PubMed | Google Scholar |

1PhD Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2Case Comprehensive Cancer Center and

3Department of Surgery, Case Western Reserve University (CWRU) School of Medicine, Cleveland, Ohio, USA.

4Laboratory of Advanced Molecular Therapeutics, Graduate Institute of Clinical Medicine, College of Medicine,

5Core Laboratory of Organoids Technology, Office of R&D,

6Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

7Institute for Computational Biology, CWRU School of Medicine, Cleveland, Ohio, USA.

8Genomics Research Center, Academia Sinica, Taipei, Taiwan.

9Office of Translation and Innovation and

10Department of Medicine, CWRU School of Medicine, Cleveland, Ohio, USA.

Address correspondence to: Stanton L. Gerson, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Ave. Cleveland, Ohio 44016, USA. Phone: 1.216.368.2825; Email: slg5@case.edu. Or to: Sung-Bau Lee, Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886.2.27361661 ext. 6107; Email: sbl@tmu.edu.tw.

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Find articles by Lee, S. in: JCI | PubMed | Google Scholar |

Authorship note: TYC and YY contributed equally to this work and are listed alphabetically.

Published October 22, 2024 - More info

Published in Volume 134, Issue 23 on December 2, 2024
J Clin Invest. 2024;134(23):e165448. https://doi.org/10.1172/JCI165448.
© 2024 Chang et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published October 22, 2024 - Version history
Received: September 15, 2022; Accepted: October 8, 2024 View PDF Abstract

The elevated level of replication stress is an intrinsic characteristic of cancer cells. Targeting the mechanisms that maintain genome stability to further increase replication stress and thus induce severe genome instability has become a promising approach for cancer treatment. Here, we identify histone deacetylase 8 (HDAC8) as a drug target whose inactivation synergized with the inhibition of checkpoint kinases to elicit substantial replication stress and compromise genome integrity selectively in cancer cells. We showed that simultaneous inhibition of HDAC8 and checkpoint kinases led to extensive replication fork collapse, irreversible cell-cycle arrest, and synergistic vulnerability in various cancer cells. The efficacy of the combination treatment was further validated in patient tumor–derived organoid (PDO) and xenograft mouse (PDX) models, providing important insights into patient-specific drug responses. Our data revealed that HDAC8 activity was essential for reducing the acetylation level of structural maintenance of chromosomes protein 3 (SMC3) ahead of replication forks and preventing R loop formation. HDAC8 inactivation resulted in slowed fork progression and checkpoint kinase activation. Our findings indicate that HDAC8 guards the integrity of the replicating genome, and the cancer-specific synthetic lethality between HDAC8 and checkpoint kinases provides a promising replication stress–targeting strategy for treating a broad range of cancers.

Graphical Abstract Introduction

To maintain the integrity of the human genome, the genetic and epigenetic information in proliferating cells must be precisely duplicated during chromatin replication. Replication stress, defined as any impediment to genome replication, is considered a unique feature of cancer (1). Owing to uncontrolled cell proliferation driven by oncogenes, chromatin replication in cancer cells is often threatened in the face of numerous obstacles, such as deregulation of origin firing, shortage of replication building blocks, interference between replication and transcription, etc. (2). The common consequences of unresolved replication stress include stalling and collapse of DNA replication forks, leading to the accumulation of mutations and chromosomal instability (3, 4).

In response to high baseline levels of replication stress, cancer cells rely on checkpoint kinase signaling modules, which play a crucial role in cell survival by allowing time for stress resolution and genome maintenance (2). Molecularly, this signaling pathway becomes activated by extended single-stranded DNAs (ssDNAs) due to various mechanisms, such as uncoupling of DNA unwinding from strand synthesis, formation of R loops at regions of transcription-replication conflicts, generation of ssDNA gaps as a result of fork bypass or resection of DNA lesions, etc. (4, 5). The accumulated ssDNAs are avidly bound by replication protein A (RPA) and can undergo fork reversal to avoid fork collapse (6). RPA recruits the protein kinase ataxia telangiectasia and Rad3-related protein (ATR) via ATR-interacting protein (ATRIP). ATR subsequently phosphorylates a multitude of targets, including checkpoint kinase 1 (CHK1) (3). An important CHK1 target is WEE1, whose phosphorylation causes inhibition of cyclin-dependent kinase (CDK1/CDK2) activity (7). To this end, activation of this pathway is critical to cell-cycle arrest, fork stabilization, origin-firing suppression, and so on. This, in turn, promotes fork repair and restart to complete replication at stress-affected loci (5).

Mounting evidence in cancer cells has revealed that elevated replication stress and dependency on checkpoint kinases can be leveraged as a vulnerability for drug targeting (8–10). The triggering of suprathreshold replication stress by simultaneous inhibition of nucleotide synthesis and checkpoint kinases is an appealing approach to elicit replication catastrophe (11, 12). Many checkpoint kinase inhibitors have been developed and are under clinical evaluation in combination with chemotherapy or radiotherapy (2). However, highly proliferative tissues can also be susceptible to this damage, and several trials have failed due to intolerable side effects such as cardiotoxicity (10). Exacerbation of replication stress selectively in cancers by cotargeting different checkpoint kinases (13–15) highlights the potential for combinations with drugs that provoke replication stress through the same pathway. However, it remains unknown whether compounds that target different aspects of replication stress through an integrative network can work synergistically with checkpoint kinase inhibitors to specifically induce replication catastrophe in cancer cells.

In addition to DNA replication, the chromatin structure experiences global disturbances during genome duplication. Downstream from replication forks, nucleosomes are reassembled, epigenetic marks on DNA are reestablished, and sister chromatids are held together by cohesin complexes to prevent their segregation before mitosis (16, 17). A growing body of evidence indicates that perturbation of these processes by inhibiting key factors, such as histone chaperons, acetyltransferases/deacetylases, and methyltransferases/demethylases, often results in aberrant chromatin organization, dysregulation of gene expression, replication fork stalling, and genome instability (17–19). Moreover, interruption of histone supply or posttranslational modifications have also been shown to increase sensitivity to checkpoint kinase inhibitors (20–23). Therefore, epigenetic modifiers could be attractive pharmacologic targets to elicit replication stress and synergize with checkpoint kinase inhibitors for cancer treatment.

Here, we performed an epigenetic compound screen to uncover synthetically lethal interactions with replication checkpoint kinases as an unexplored vulnerability in cancer cells. We identify histone deacetylase 8 (HDAC8) as a promising druggable candidate whose inhibition gave rise to extensive replication stress, robust DNA damage, and persistent S-phase arrest when combined with checkpoint kinase inhibitors. The synthetic lethality of the dual inhibition was validated in various in vitro and in vivo models, including patient tumor–derived xenograft (PDX) and organoid (PDO) models, indicating its potential for clinical applications. Moreover, this synergistic vulnerability is specific to cancer cells, suggesting a strong therapeutic index. We also showed that HDAC8 inactivation led to hyperacetylation of the cohesin subunit structural maintenance of chromosomes protein 3 (SMC3) on unreplicated chromatin and exacerbated the formation of DNA-RNA hybrids. These data reveal a critical function of HDAC8 in the regulation of genome stability during chromatin replication, and the cancer-specific synthetic lethality by inhibiting HDAC8 and checkpoint kinases supports a promising strategy for replication stress–targeting cancer therapy.

Results

Coinhibition of HDAC8 and replication checkpoints elicits severe replication stress, culminating in replication-dependent DNA double-stranded breaks. To search for compounds that could work in tandem with checkpoint kinase inhibitors to disrupt replication fork stability, we performed rigorous high-throughput compound screening (Supplemental information and Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/JCI165448DS1). This involved evaluation of the cellular DNA damage response using H2AX phosphorylation at Ser139 (γH2AX) as a readout and measuring ssDNA accumulation at replication forks by determining RPA protein loading on replicating chromatin marked with proliferating cell nuclear antigen (PCNA) after compound treatment. This screen led to the discovery of HDAC8 inhibitors that potentially induce replication stress and enhance genotoxicity from checkpoint kinase inhibition (Supplemental Methods and Supplemental Figure 1).

In light of the high levels of RPA and γH2AX on replicating chromatin observed from our screen results, we sought to determine whether dual inhibition of HDAC8 and checkpoint kinases induces a lethal push of replication stress that triggers fork collapse, resulting in DNA double-stranded breaks (DSBs). As single regimens, treatment with either HDAC8 inhibitors or AZD-7762 induced a mild replication stress response and ATR activation, as evidenced by phosphorylation of CHK1 at S345 (pS345) and RPA2 at S33 (pS33) (Figure 1A). Strikingly, the combination of HDAC8 inhibitor and AZD-7762 induced a strong DSB signal, reflected by phosphorylation of ATM (pS1981), KAP1 (pS824), CHK2 (pT68), and RPA2 (pS4/S8) (Figure 1A). The accumulation of DSBs in cotreated cells was further validated by pulsed-field gel electrophoresis (PFGE) (Figure 1B). This synergistic genotoxicity was also observed in cells treated with combinations of AZD-7762 and other top hits identified from the screen (resminostat, scriptaid, SP2509, and ITF 2357) (Supplemental Figure 2A). In addition, gene-specific knockdown of HDAC8 by an siRNA showed the enhanced DNA damage response from checkpoint inhibition (Supplemental Figure 2B), further corroborating the pharmacological effect of HDAC8 inhibitors.

Figure 1

Coinhibition of HDAC8 and replication checkpoints elicits severe replication stress, culminating in replication-dependent DNA double-stranded breaks. (A) Western blot analysis of the DNA damage response in U-2 OS cells treated with the indicated compounds for 4 hours. Representative results from 1 of 2 biological replicates are shown. The lanes were run on the same gel but were noncontiguous. (B) PFGE analysis of DNA breaks in U-2 OS cells treated with the indicated compounds for 15 hours. Relative intensities of broken DNAs were obtained by normalizing individual values to the corresponding untreated control group values. Representative results (upper) and quantification of broken DNAs from 3 biological replicates (lower; n = 3) are shown. Triangles represent the relative intensities of each biological replicate; lines indicate the