The LKS Faculty of Medicine of The University of Hong Kong (HKUMed), in collaboration with School of Life Sciences of The Chinese University of Hong Kong, Faculty of Health Sciences of University of Macau, and the Ming Wai Lau Centre for Reparative Medicine of Karolinska Institute (Hong Kong node), have developed a new platform to systemically study the knockout effects of all possible two-way or three-way combinations in a given set of genes, with simple steps. By applying the platform to the druggable genes, which have existing drugs to inhibit their proteins, the team has successfully identified effective double or triple regimen combinations suppressing ovarian cancer cell growth and against Parkinson’s disease toxicity. Their work is now published in Cell Reports [link to the publication] and a patent application has been filed based on this work.

 

Background

“No man is an island”, the same is also true for genes. Multiple genes co-operate to regulate almost all biological processes, including those driving diseases. Therefore, combination therapy, targeting multiple genetic elements, is developed, especially for complicated diseases, such as cancers and neurodegenerative diseases. Although it is promising, the traditional way to find the synergistic regimen combinations is always prohibitedly expensive and labour-intensive, which demands of testing all the possible combinations individually. Due to the limited manpower, the testing is hardly to be sufficient.

 

Findings

The team developed the CombiGEM-CRISPR v2.0 system to guide selection of drug combinations to be tested. Only a single set of gRNA oligos is required to build screening libraries, which drastically reduced the cost. Further, only one-pot sequencing of short barcode sequences is enough to guide the selection of effective two- and three- way combinations in this system. The merit of this platform is simplicity and extensibility. “Technically, there is only one step of cloning to make a one-way gRNA expression library to two-way, and another step to make it a three-way” stated by Dr Zhou, the first author of the work.   

 

Gene knockout could mimic the effects of the drug inhibitors on its protein. With the new platform in hand, the team decided to identify novel drug combinations for therapeutics of ovarian cancer, the 6^th most common cancer among women in Hong Kong, and Parkinson’s disease, which has a prevalence of more than 1% in people over 60-year old in Hong Kong. Screening in the cancer cells, the research group found that knocking out DNMT1 + POLA1 + EGFR or ERBB2, as well as CDK4 + MAP2K1 + POLA1 in three-way combinations, and PARP1 + DNMT1 and CDK2 + FKBP1A in two-way combinations significantly suppress the cancer cell growth. Then it comes to the most exciting part. The researchers applied the drug inhibitors of each gene by the combinatorial way, the synergistic effects of suppressing the growth of multiple ovarian cancer cell lines are valid. In another screen to find the drug pairs against Parkinson’s disease toxicity, the researchers  identified double knockout of HSP90B1 + HDAC2 to be an effective combination for protecting cell death from induced PD-associated toxicity. The combination of matching drugs for these two gene products are also found to be effective and synergistic.

 

Significance of the study

With the new platform, researchers could rapidly identify effective combinations within three months from constructing complex gRNA libraries, estimated by the team lead Dr Alan Wong Siu-lun.  “It is hard to imagine how much effort and money would be spent to identify all those drug combinations by testing all the possibilities one by one. And we hope the drug combinations we discovered could eventually benefit those patients,” said Becky, the second author of the work. Indeed, PARP inhibitors are widely used as frontline therapeutics against ovarian cancer. One of our identified combination (PARP1 + DNMT1) has also been discovered by other groups for treating multiple cancers including ovarian cancer. Finding drug combinations is one important application of CombiGEM-CRISPR v2.0. “We hope our system could help identifying more unanticipated drug combinations that could benefit patients”, wished by the team.   

 

Previous study results

Dr Alan Wong Siu-lun has been developing various high throughput-screening technologies to systematically dissect the complex genetics underlying diseases including cancers and from there devising new therapeutic strategies. Previous work, published in Nature Biotechnology in 2015 and Proceedings of the National Academy of Sciences in 2016, focused on developing CombiGEM technology to screen genetic interactions, as the foundation of this current study. The team also published a review article in the Annual Review of Genetics in 2016 highlighting the potential of that technology in addressing practical challenges in biomedical research. Other than that, the team developed a new CombiSEAL technology published in Nature Methods in 2019, which extends the scope and impact of combinatorial genetics to engineer proteins for gene-editing applications.

 

About the research team

This study was led by Dr Alan Wong Siu-lun, Assistant Professor of the School of Biomedical Sciences, HKUMed. Dr Peng Zhou, Post-doctoral Fellow of the School of Biomedical Sciences, HKU, was the first author of this work. Ms Becky Chan Ka-ching, Ms Wan Yuk-kei, Ms Chaya Yuen Tsz-lo, Dr Gigi Choi Ching-gee, Ms Xinran Li, Ms Cindy S.W. Tong, Ms Sophia S.W. Zhong, Mr Jeiran Sun, and Mr Jien Vei Khaw (from HKUMed School of Biomedical Sciences and the HKU Biomedical Engineering Programme) were co-authors and they also performed the experiments. Other researchers were Prof Suet Yi Leung from Department of Pathology and Dr Lydia W.T. Cheung from School of Biomedical Sciences at HKUMed, Dr Zongli Zheng, Ms Yufan Bao, Ms Silvia Y.L. Mak, and Ms Maggie Z.Y. Chow from the Ming Wai Lau Centre for Reparative Medicine, Karolinska Institute in Hong Kong, Dr Wong Koon-ho and Dr Kaeling Tan from the Faculty of Health Sciences, University of Macau in Macau, and Prof CHAN Ho Yin Edwin from School of Life Sciences, CUHK in Hong Kong.

 

Acknowledgement

This work was supported by funds for Dr Alan Wong Siu-lun’s team including The University of Hong Kong Start-up and Internal funds, Hong Kong Research Grants Council (ECS-27105716, GRF-17102218, and TRS-T12-710/16-R), Innovation and Technology Commission (ITS/133/18), and Food and Health Bureau (HMRF-04151416). Other supports came from CUHK Gerald Choa Neuroscience Centre (7105306) (for Prof CHAN Ho Yin Edwin), the Research Services and Knowledge Transfer Office (MYRG2018-00017-FHS) and Faculty of Health Sciences (FHS collaboration and innovation grants) of the University of Macau (for Dr Wong Koon-ho’s team), and the Swedish Research Council (2016-02830) and the National Natural Science Foundation of China (81672098) (for Dr Zongli Zheng’s team). This project has been developed as part of the Ming Wai Lau Centre for Reparative Medicine Associate Member Programme, and Dr Alan Wong Siu-lun is a Ming Wai Lau Centre for Reparative Medicine Associate Member.

 

Media enquiries

School of Biomedical Sciences, HKUMed
Dr Wong Siu Lun Alan (Tel: 3917 9208 | Email: aslw@hku.hk)

School of Biomedical Sciences (General Office), HKUMed
Christina Chan (Tel: 3917 6334 | Email: slcchan@hku.hk)