CRISPR/Cas9 and targeted cancer therapy
Liu, Bin
DOI:
10.33612/diss.99103461
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CRISPR/Cas9 and targeted cancer
therapy
The research described in this thesis was carried out at the department of Chemical and Pharmcetical Biology at the University of Groningen.
The research was crried out according to the rquirements of the Graduate School of Science, Faculty of Science and Engineering, University of Groningen, The Netehrlands
Printing of this thesis was financially supported by the University Library and the Graduate School of Science, University of Groningen, The Netherlands.
Printing: Ridderprint BV | www.ridderprint.nl IBSN (ebook): 978-94-6375-611-2
IBSN (printed version): 978-94-6375-610-5
Copyright © 2019 Bin Liu. All rights are reserved. No part of this thesis may be reproduced or transmitted in any form or by any means without the prior permission in writing of the author.
therapy
PhD thesis
to obtain the degree of PhD at the University of Groningen
on the authority of the Rector Magnificus Prof. C. Wijmenga
and in accordance with the decision by the College of Deans. This thesis will be defended in public on Tuesday 29 October 2019 at 11.00 hours
by
Bin Liu
born on 16 February 1989 in Jiangsu, China
2
The research described in this thesis was carried out at the department of Chemical and Pharmcetical Biology at the University of Groningen.
The research was crried out according to the rquirements of the Graduate School of Science, Faculty of Science and Engineering, University of Groningen, The Netehrlands
Printing of this thesis was financially supported by the University Library and the Graduate School of Science, University of Groningen, The Netherlands.
IBSN (ebook): 978-94-6375-611-2 IBSN (printed version): 978-94-6375-610-5
Copyright © 2019 Bin Liu. All rights are reserved. No part of this thesis may be reproduced or transmitted in any form or by any means without the prior permission in writing of the author.
CRISPR/Cas9 and targeted cancer
therapy
PhD thesis
to obtain the degree of PhD at the University of Groningen
on the authority of the Rector Magnificus Prof. C. Wijmenga
and in accordance with the decision by the College of Deans. This thesis will be defended in public on Tuesday 29 October 2019 at 11.00 hours
by
Bin Liu
born on 16 February 1989 in Jiangsu, China
Prof. H.J. Haisma Prof. F.J. Dekker
Assessment Committee
Prof. M. Schmidt
Prof. V.W. van Beusechem Prof. F. Foijer
Chapter 1 Scope and outline of this thesis ... 7 Chapter 2 CRISPR/Cas9: A powerful tool for identification of new targets for cancer treatment... 17 Chapter 3 CRISPR/Cas9 for overcoming drug resistance in solid tumors... 75 Chapter 4 Transcriptional activation of cyclin D1 via HER2/HER3 contributes to cell survival and
tyrosine kinase inhibitor resistance in non-small cell lung carcinoma ... 101 Chapter 5 CX Chemokine Receptor 7 Contributes to Survival of KRAS-Mutant Non-Small Cell Lung
Cancer upon Loss of Epidermal Growth Factor Receptor... 135 Chapter 6 CRISPR-mediated ablation of overexpressed EGFR in combination with sunitinib significantly suppresses renal cell carcinoma proliferation ... 171 Chapter 7 Inhibition of Histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome
editing ... 195 Chapter 8 Summary and Future Perspectives ... 236 Appendices Acknowledgement
List of publication About the author
4
Supervisors
Prof. H.J. Haisma Prof. F.J. DekkerAssessment Committee
Prof. M. SchmidtProf. V.W. van Beusechem Prof. F. Foijer
5
Contents
Chapter 1 Scope and outline of this thesis ... 7 Chapter 2 CRISPR/Cas9: A powerful tool for identification of new targets for cancer treatment ... 17 Chapter 3 CRISPR/Cas9 for overcoming drug resistance in solid tumors ... 75 Chapter 4 Transcriptional activation of cyclin D1 via HER2/HER3 contributes to cell survival and
tyrosine kinase inhibitor resistance in non-small cell lung carcinoma ... 101 Chapter 5 CX Chemokine Receptor 7 Contributes to Survival of KRAS-Mutant Non-Small Cell Lung
Cancer upon Loss of Epidermal Growth Factor Receptor ... 135 Chapter 6 CRISPR-mediated ablation of overexpressed EGFR in combination with sunitinib significantly suppresses renal cell carcinoma proliferation ... 171 Chapter 7 Inhibition of Histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome
editing ... 195 Chapter 8 Summary and Future Perspectives ... 236 Appendices Acknowledgement
List of publication About the author
6 7
Chapter 1 Scope and outline of this thesis
Conventional cancer therapies, including resection surgery, chemotherapy, radiotherapy and their combinations have been the primary therapeutic options for cancers for decades1.
However, the improved survival of patients with advanced cancer, such as lung carcinoma and renal cell carcinoma, is usually months instead of years. Side effects of these conventional therapies significantly affect the living quality2.
Targeted therapy by small molecular compounds or antibodies seems better at least to reduce side effects. Targeted therapy is mainly based on the knowledge of tumor biology, such as targeting some essential proteins in oncogenic molecular pathways, for instance, EGFR-Tyrosine kinase inhibitors (TKIs) for lung cancer and EGFR-antibodies for colorectal cancer. Although small molecular inhibitors and antibodies have achieved some successes in cancer treatment, drug resistance inevitably occurs after a short term treatment, which limits the efficacy of targeted drugs3,4. Therefore, finding more novel targets, new combination therapeutic options
and alternative therapies are urgently required.
CRISPR/Cas-based technologies are revolutionizing every field of research in life sciences and medicine, cancer treatment research is certainly not an exception5. The applications of
CRISPR/Cas9 in cancer research and therapy are mainly focused on two aspects. One is screening and identifying therapeutic targets6, which may include a deeper understanding of old targets and finding novel targets using “CRISPR Way”. The other is directly using CRISPR/Cas9 as a therapy for cancer. Although a few clinical trials using CRISPR against cancer have been ongoing7, these clinical trials can only be regarded as first proof-of-concept studies due to many
unknowns.
Therefore, the aim for this thesis is exploring the potential of CRISPR/Cas9 in discovering therapeutic targets and in cancer therapy. Herein, three parts are presented in this thesis. Chapters 2, 3, 4, 5, and 6 present the applications of CRISPR/Cas9 in cancer target identification and treatment. Chapter 7 presents a strategy for improving CRISPR/Cas9 targeting efficiency for cancer treatment by HDAC inhibition. Finally a summary of the studies is presented in this thesis and future perspectives are drawn.
CHAPTER 1
8
Scope and Outline of This Thesis
9 Conventional cancer therapies, including resection surgery, chemotherapy, radiotherapy and their combinations have been the primary therapeutic options for cancers for decades1.
However, the improved survival of patients with advanced cancer, such as lung carcinoma and renal cell carcinoma, is usually months instead of years. Side effects of these conventional therapies significantly affect the living quality2.
Targeted therapy by small molecular compounds or antibodies seems better at least to reduce side effects. Targeted therapy is mainly based on the knowledge of tumor biology, such as targeting some essential proteins in oncogenic molecular pathways, for instance, EGFR-Tyrosine kinase inhibitors (TKIs) for lung cancer and EGFR-antibodies for colorectal cancer. Although small molecular inhibitors and antibodies have achieved some successes in cancer treatment, drug resistance inevitably occurs after a short term treatment, which limits the efficacy of targeted drugs3,4. Therefore, finding more novel targets, new combination therapeutic options
and alternative therapies are urgently required.
CRISPR/Cas-based technologies are revolutionizing every field of research in life sciences and medicine, cancer treatment research is certainly not an exception5. The applications of
CRISPR/Cas9 in cancer research and therapy are mainly focused on two aspects. One is screening and identifying therapeutic targets6, which may include a deeper understanding of old targets and finding novel targets using “CRISPR Way”. The other is directly using CRISPR/Cas9 as a therapy for cancer. Although a few clinical trials using CRISPR against cancer have been ongoing7, these clinical trials can only be regarded as first proof-of-concept studies due to many
unknowns.
Therefore, the aim for this thesis is exploring the potential of CRISPR/Cas9 in discovering therapeutic targets and in cancer therapy. Herein, three parts are presented in this thesis. Chapters 2, 3, 4, 5, and 6 present the applications of CRISPR/Cas9 in cancer target identification and treatment. Chapter 7 presents a strategy for improving CRISPR/Cas9 targeting efficiency for cancer treatment by HDAC inhibition. Finally a summary of the studies is presented in this thesis and future perspectives are drawn.
Selection of the right target is crucial for developing an effective treatment strategy for cancer8.
A candidate protein or molecule cannot be considered as an appropriate target only based on its expression level or mutation status. Recently, application of CRISPR/Cas9 have enabled greatly successful validation of potential therapeutic targets for cancer therapy7. Thus, in
Chapter 2, we provide a comprehensive overview of using CRISPR/Cas9 for identification of new targets for cancer treatment. Furthermore, we summarized the delivery of CRISPR/Cas9 and provided an approach for specifically disrupting cancer driver mutations9–11. In the end, we
discussed the challenges and the opportunities of CRISPR/Cas9 for cancer therapy.
However, resistance inevitably occurs in almost all cancer patients with drug treatment. The resistant mechanisms are complicated. CRISPR/Cas9 has been successfully used in elucidating some drug resistant mechanisms for cancer treatment. Understanding resistant mechanisms could provide useful guidance for personalized medicine in cancer12. Therefore, in chapter 3, we
focus specifically on the use of CRISPR/Cas9 for understanding drug resistance mechanisms and identification of resistance-related genes in solid tumors.
Lung cancer is the leading cause of cancer mortality worldwide13. Mechanisms of EGFR-TKI
resistance in NSCLC are complicated14. In Chapter 4, we aimed to characterize cellular and
molecular changes of HER family receptors upon EGFR ablation by CRISPR/Cas9 or acquisition of TKI resistance. We have characterized EGFR ablation by CRISPR/Cas9 and TKI resistance in lung cancer cell lines with different genotypes. We further reveal that HER2 and HER3 nuclear translocation mediated-cyclin D1 overexpression contributes to cell proliferation, survival and resistance in lung cancer cells upon EGFR targeting.
KRAS-driven non-small cell lung cancer patients have no effective targeted treatment15,16. EGFR
targeted therapy shows little or no efficacy in non-small-cell lung carcinoma (NSCLC) patients with KRAS mutation. We aimed to find a new therapeutic option for the treatment of NSCLC patients with KRAS mutation. To this end, in Chapter 5, we report that CXCR7 overexpression is a novel compensation and survival mechanism in EGFR targeted therapy (CRISPR/Cas9 and TKIs) in in KRAS-driven lung cancer cells. We show that EGFR and CXCR7 have a crucial interaction in
NSCLC. Dual EGFR and CXCR7 inhibition led to substantial reduction of MAPK (pERK) and synergistic inhibition of cell growth. Therefore, a combination therapy by dual inhibition EGFR and CXCR7 may be a potential therapeutic option for a subtype of patients with NSCLC. In addition, we observed that EGF-induced phosphorylation of Akt may be independent of EGFR, which to our knowledge has not been reported previously. One hypothesis is that EGF stimulates pAkt via CXCR7, because previous studies have shown that CXCR7 associated pathways can induce Akt phosphorylation.
Recently, CXCR7 has been classified as a novel receptor for human macrophage migration inhibitory factor (MIF)17–19. However, D-dopachrome tautomerase(D-DT), also called MIF2, is a
newly described cytokine, which is an analogue of MIF20. I hypothesized that D-DT may be a ligand of CXCR7. The new mechanism of DDT/CXCR7 signaling may deepen our understanding of some diseases development, such as COPD and cancer.
We concentrate on EGFR targeting and expand our study from lung cancer to renal cancer. RCC (Renal cell carcinoma) is one of the most aggressive malignant tumors. The 5-year survival rate of metastatic RCC is less than 10%. In Chapter 6, we show that ablation of EGFR by CRISPR/Cas9 significantly restrained tumor cell growth and activated the MAPK (pERK1/2) pathway. The VEGFR and PDGFR inhibitor, sunitinib, attenuated the expression of MAPK (pERK1/2) and pAKT induced by EGFR loss and further inhibited EGFR-/- cell proliferation.
Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency21,22, especially for cancer gene
therapy. Therefore, in Chapter 7, we provide a practical and clinically applicable approach for precise control of CRISPR/Cas9 mediated gene editing by modulation of HDAC and HAT activity in host cells. In this study, we hypothesized that regulation of chromatin compaction by inhibiting HAT and/or HDAC activity can modulate CRISPR-Cas9 based gene editing. We comprehensively investigated the impact of HDACs and HATs on CRISPR/Cas9 mediated gene editing. Our findings demonstrate that attenuation of HDAC1 and HDAC2 activity, but not other HDACs, leads to an open chromatin state, facilitates Cas9 access and binding to the targeted
CHAPTER 1
10
Selection of the right target is crucial for developing an effective treatment strategy for cancer8.
A candidate protein or molecule cannot be considered as an appropriate target only based on its expression level or mutation status. Recently, application of CRISPR/Cas9 have enabled greatly successful validation of potential therapeutic targets for cancer therapy7. Thus, in
Chapter 2, we provide a comprehensive overview of using CRISPR/Cas9 for identification of new targets for cancer treatment. Furthermore, we summarized the delivery of CRISPR/Cas9 and provided an approach for specifically disrupting cancer driver mutations9–11. In the end, we
discussed the challenges and the opportunities of CRISPR/Cas9 for cancer therapy.
However, resistance inevitably occurs in almost all cancer patients with drug treatment. The resistant mechanisms are complicated. CRISPR/Cas9 has been successfully used in elucidating some drug resistant mechanisms for cancer treatment. Understanding resistant mechanisms could provide useful guidance for personalized medicine in cancer12. Therefore, in chapter 3, we
focus specifically on the use of CRISPR/Cas9 for understanding drug resistance mechanisms and identification of resistance-related genes in solid tumors.
Lung cancer is the leading cause of cancer mortality worldwide13. Mechanisms of EGFR-TKI
resistance in NSCLC are complicated14. In Chapter 4, we aimed to characterize cellular and
molecular changes of HER family receptors upon EGFR ablation by CRISPR/Cas9 or acquisition of TKI resistance. We have characterized EGFR ablation by CRISPR/Cas9 and TKI resistance in lung cancer cell lines with different genotypes. We further reveal that HER2 and HER3 nuclear translocation mediated-cyclin D1 overexpression contributes to cell proliferation, survival and resistance in lung cancer cells upon EGFR targeting.
KRAS-driven non-small cell lung cancer patients have no effective targeted treatment15,16. EGFR
targeted therapy shows little or no efficacy in non-small-cell lung carcinoma (NSCLC) patients with KRAS mutation. We aimed to find a new therapeutic option for the treatment of NSCLC patients with KRAS mutation. To this end, in Chapter 5, we report that CXCR7 overexpression is a novel compensation and survival mechanism in EGFR targeted therapy (CRISPR/Cas9 and TKIs) in in KRAS-driven lung cancer cells. We show that EGFR and CXCR7 have a crucial interaction in
Scope and Outline of This Thesis
11 NSCLC. Dual EGFR and CXCR7 inhibition led to substantial reduction of MAPK (pERK) and synergistic inhibition of cell growth. Therefore, a combination therapy by dual inhibition EGFR and CXCR7 may be a potential therapeutic option for a subtype of patients with NSCLC. In addition, we observed that EGF-induced phosphorylation of Akt may be independent of EGFR, which to our knowledge has not been reported previously. One hypothesis is that EGF stimulates pAkt via CXCR7, because previous studies have shown that CXCR7 associated pathways can induce Akt phosphorylation.
Recently, CXCR7 has been classified as a novel receptor for human macrophage migration inhibitory factor (MIF)17–19. However, D-dopachrome tautomerase(D-DT), also called MIF2, is a
newly described cytokine, which is an analogue of MIF20. I hypothesized that D-DT may be a ligand of CXCR7. The new mechanism of DDT/CXCR7 signaling may deepen our understanding of some diseases development, such as COPD and cancer.
We concentrate on EGFR targeting and expand our study from lung cancer to renal cancer. RCC (Renal cell carcinoma) is one of the most aggressive malignant tumors. The 5-year survival rate of metastatic RCC is less than 10%. In Chapter 6, we show that ablation of EGFR by CRISPR/Cas9 significantly restrained tumor cell growth and activated the MAPK (pERK1/2) pathway. The VEGFR and PDGFR inhibitor, sunitinib, attenuated the expression of MAPK (pERK1/2) and pAKT induced by EGFR loss and further inhibited EGFR-/- cell proliferation.
Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency21,22, especially for cancer gene
therapy. Therefore, in Chapter 7, we provide a practical and clinically applicable approach for precise control of CRISPR/Cas9 mediated gene editing by modulation of HDAC and HAT activity in host cells. In this study, we hypothesized that regulation of chromatin compaction by inhibiting HAT and/or HDAC activity can modulate CRISPR-Cas9 based gene editing. We comprehensively investigated the impact of HDACs and HATs on CRISPR/Cas9 mediated gene editing. Our findings demonstrate that attenuation of HDAC1 and HDAC2 activity, but not other HDACs, leads to an open chromatin state, facilitates Cas9 access and binding to the targeted
DNA, and consequently enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 attenuates the ability of CRISPR/Cas9 mediated gene editing.
The studies presented in this thesis are summarized and discussed in Chapter 8 along with future perspectives.
1. Tannock, I. F. Conventional cancer therapy: promise broken or promise delayed? Lancet 351, SII9–SII16 (1998).
2. Baudino, T. A. Targeted Cancer Therapy: The Next Generation of Cancer Treatment. Curr. Drug Discov. Technol. 12, 3–20 (2015).
3. Holohan, C., Van Schaeybroeck, S., Longley, D. B. & Johnston, P. G. Cancer drug resistance: an evolving paradigm. Nat. Rev. Cancer 13, 714 (2013).
4. Saber, A., Liu, B., Ebrahimi, P. & Haisma, H. J. CRISPR/Cas9 for overcoming drug resistance in solid tumors. DARU J. Pharm. Sci. (2019). doi:10.1007/s40199-019-00240-z
5. Zhan, T., Rindtorff, N., Betge, J., Ebert, M. P. & Boutros, M. CRISPR/Cas9 for cancer research and therapy. Semin. Cancer Biol. (2018). doi:10.1016/J.SEMCANCER.2018.04.001 6. Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic screens in human cells using
the CRISPR-Cas9 system. Science 343, 80–84 (2014).
7. Liu, B., Saber, A. & Haisma, H. J. CRISPR/Cas9: a powerful tool for identification of new targets for cancer treatment. Drug Discov. Today (2019).
doi:10.1016/j.drudis.2019.02.011
8. Ahmad, G. & Amiji, M. Use of CRISPR/Cas9 gene-editing tools for developing models in drug discovery. Drug Discov. Today 23, 519–533 (2018).
9. Guernet, A. et al. CRISPR-Barcoding for Intratumor Genetic Heterogeneity Modeling and Functional Analysis of Oncogenic Driver Mutations. Mol. Cell 63, 526–538 (2016).
10. Kiessling, M. K. et al. Identification of oncogenic driver mutations by genome-wide CRISPR-Cas9 dropout screening. BMC Genomics 17, 723 (2016).
11. Gebler, C. et al. Inactivation of Cancer Mutations Utilizing CRISPR/Cas9. J. Natl. Cancer Inst. 109, (2017).
12. Fellmann, C., Gowen, B. G., Lin, P., Doudna, J. A. & Corn, J. E. Cornerstones of CRISPR–Cas in drug discovery and therapy. Nat. Publ. Gr. 4–7 (2016). doi:10.1038/nrd.2016.238 13. Park, K. et al. Afatinib versus gefitinib as first-line treatment of patients with EGFR
mutation-positive non-small-cell lung cancer (LUX-Lung 7): a phase 2B, open-label, randomised controlled trial. Lancet Oncol. 17, 577–589 (2016).
14. Soria, J.-C. et al. Gefitinib plus chemotherapy versus placebo plus chemotherapy in EGFR-mutation-positive non-small-cell lung cancer after progression on first-line gefitinib (IMPRESS): a phase 3 randomised trial. Lancet Oncol. 16, 990–998 (2015).
15. Mao, C. et al. KRAS mutations and resistance to EGFR-TKIs treatment in patients with non-small cell lung cancer: A meta-analysis of 22 studies. Lung Cancer 69, 272–278 (2010). 16. Riely, G. J., Marks, J. & Pao, W. KRAS mutations in non-small cell lung cancer. Proc. Am.
Thorac. Soc. 6, 201–205 (2009).
17. Chatterjee, M. et al. Macrophage migration inhibitory factor limits activation-induced apoptosis of platelets via CXCR7-dependent Akt signaling. Circ. Res. 115, 939–949 (2014). 18. Alampour-Rajabi, S. et al. MIF interacts with CXCR7 to promote receptor internalization,
ERK1/2 and ZAP-70 signaling, and lymphocyte chemotaxis. FASEB J. 29, 4497–511 (2015). 19. Alampour-Rajabi, S. et al. MIF interacts with CXCR7 to promote receptor internalization,
ERK1/2 and ZAP-70 signaling, and lymphocyte chemotaxis. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 29, 4497–4511 (2015).
CHAPTER 1
12
DNA, and consequently enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 attenuates the ability of CRISPR/Cas9 mediated gene editing.
The studies presented in this thesis are summarized and discussed in Chapter 8 along with future perspectives.
1. Tannock, I. F. Conventional cancer therapy: promise broken or promise delayed? Lancet 351, SII9–SII16 (1998).
2. Baudino, T. A. Targeted Cancer Therapy: The Next Generation of Cancer Treatment. Curr. Drug Discov. Technol. 12, 3–20 (2015).
3. Holohan, C., Van Schaeybroeck, S., Longley, D. B. & Johnston, P. G. Cancer drug resistance: an evolving paradigm. Nat. Rev. Cancer 13, 714 (2013).
4. Saber, A., Liu, B., Ebrahimi, P. & Haisma, H. J. CRISPR/Cas9 for overcoming drug resistance in solid tumors. DARU J. Pharm. Sci. (2019). doi:10.1007/s40199-019-00240-z
5. Zhan, T., Rindtorff, N., Betge, J., Ebert, M. P. & Boutros, M. CRISPR/Cas9 for cancer research and therapy. Semin. Cancer Biol. (2018). doi:10.1016/J.SEMCANCER.2018.04.001 6. Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic screens in human cells using
the CRISPR-Cas9 system. Science 343, 80–84 (2014).
7. Liu, B., Saber, A. & Haisma, H. J. CRISPR/Cas9: a powerful tool for identification of new targets for cancer treatment. Drug Discov. Today (2019).
doi:10.1016/j.drudis.2019.02.011
8. Ahmad, G. & Amiji, M. Use of CRISPR/Cas9 gene-editing tools for developing models in drug discovery. Drug Discov. Today 23, 519–533 (2018).
9. Guernet, A. et al. CRISPR-Barcoding for Intratumor Genetic Heterogeneity Modeling and Functional Analysis of Oncogenic Driver Mutations. Mol. Cell 63, 526–538 (2016).
Scope and Outline of This Thesis
13 10. Kiessling, M. K. et al. Identification of oncogenic driver mutations by genome-wide
CRISPR-Cas9 dropout screening. BMC Genomics 17, 723 (2016).
11. Gebler, C. et al. Inactivation of Cancer Mutations Utilizing CRISPR/Cas9. J. Natl. Cancer Inst. 109, (2017).
12. Fellmann, C., Gowen, B. G., Lin, P., Doudna, J. A. & Corn, J. E. Cornerstones of CRISPR–Cas in drug discovery and therapy. Nat. Publ. Gr. 4–7 (2016). doi:10.1038/nrd.2016.238 13. Park, K. et al. Afatinib versus gefitinib as first-line treatment of patients with EGFR
mutation-positive non-small-cell lung cancer (LUX-Lung 7): a phase 2B, open-label, randomised controlled trial. Lancet Oncol. 17, 577–589 (2016).
14. Soria, J.-C. et al. Gefitinib plus chemotherapy versus placebo plus chemotherapy in EGFR-mutation-positive non-small-cell lung cancer after progression on first-line gefitinib (IMPRESS): a phase 3 randomised trial. Lancet Oncol. 16, 990–998 (2015).
15. Mao, C. et al. KRAS mutations and resistance to EGFR-TKIs treatment in patients with non-small cell lung cancer: A meta-analysis of 22 studies. Lung Cancer 69, 272–278 (2010). 16. Riely, G. J., Marks, J. & Pao, W. KRAS mutations in non-small cell lung cancer. Proc. Am.
Thorac. Soc. 6, 201–205 (2009).
17. Chatterjee, M. et al. Macrophage migration inhibitory factor limits activation-induced apoptosis of platelets via CXCR7-dependent Akt signaling. Circ. Res. 115, 939–949 (2014). 18. Alampour-Rajabi, S. et al. MIF interacts with CXCR7 to promote receptor internalization,
ERK1/2 and ZAP-70 signaling, and lymphocyte chemotaxis. FASEB J. 29, 4497–511 (2015). 19. Alampour-Rajabi, S. et al. MIF interacts with CXCR7 to promote receptor internalization,
ERK1/2 and ZAP-70 signaling, and lymphocyte chemotaxis. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 29, 4497–4511 (2015).
functional homolog of macrophage migration inhibitory factor (MIF). Proc. Natl. Acad. Sci. 108, E577–E585 (2011).
21. Chu, V. T. et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548 (2015). 22. Paix, A., Folkmann, A., Rasoloson, D. & Seydoux, G. High Efficiency, Homology-Directed
Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes. Genetics 201, 47–54 (2015).
CHAPTER 1
14
functional homolog of macrophage migration inhibitory factor (MIF). Proc. Natl. Acad. Sci. 108, E577–E585 (2011).
21. Chu, V. T. et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548 (2015). 22. Paix, A., Folkmann, A., Rasoloson, D. & Seydoux, G. High Efficiency, Homology-Directed
Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes. Genetics 201, 47–54 (2015).
Scope and Outline of This Thesis
Chapter 2 CRISPR/Cas9: A powerful tool for
identification of new targets for cancer treatment
Bin Liu, Ali Saber, Hidde J. Haisma
CHAPTER 1
16 17
Chapter 2 CRISPR/Cas9: A powerful tool for
identification of new targets for cancer treatment
Bin Liu, Ali Saber, Hidde J. Haisma
Abstract
CRISPR/Cas9, as a powerful genome editing tool, has revolutionized genetic engineering. It is widely used to investigate the molecular basis of different cancer types. In this review, we present an overview of recent studies in which CRISPR/Cas9 has been used for the identification of potential molecular targets. Based on the collected data, we suggest that CRISPR/Cas9 is an effective system to distinguish between mutant and wildtype alleles in cancer. We show that several new potential therapeutic targets such as CD38, CXCR2, MASTL, RBX2 as well as several non-coding RNAs have been identified using CRISPR/Cas9 technology. At the end, we discuss the obstacles and challenges that we face for using CRISPR/Cas9 as a therapeutic.
CHAPTER 2
18
CRISPR/Cas9 for Identification of New Targets for Cancer Treatment
19 Abstract
CRISPR/Cas9, as a powerful genome editing tool, has revolutionized genetic engineering. It is widely used to investigate the molecular basis of different cancer types. In this review, we present an overview of recent studies in which CRISPR/Cas9 has been used for the identification of potential molecular targets. Based on the collected data, we suggest that CRISPR/Cas9 is an effective system to distinguish between mutant and wildtype alleles in cancer. We show that several new potential therapeutic targets such as CD38, CXCR2, MASTL, RBX2 as well as several non-coding RNAs have been identified using CRISPR/Cas9 technology. At the end, we discuss the obstacles and challenges that we face for using CRISPR/Cas9 as a therapeutic.
1. Introduction
The accumulation of genetic mutations in cells over time leads to cancer. In addition, certain gene changes such as driver mutations in TP53, EGFR, KRAS, BRAF, HER2 and MET can make a cell cancerous. Treatment strategies are conventionally based on histological subtypes. However, today, beside the conventional histological classifications, each cancer type can be subdivided into various molecular subtypes which play a crucial role in treatment decision making process. Each molecular subtype is differently treated and clinicians may also predict treatment outcomes and patients’ survival. For instance, lung cancer patients with EGFR activating mutations are treated with different types of tyrosine kinase inhibitors (TKIs) such as gifitinib, erlotinib or afatinib, depending on the mutation. Yet, resistance to the TKIs inevitably
emerges either by DNA mutations or/and metabolic changes. As a result, treatment strategies
are modified based on the new molecular signature. However, eventually, the tumor cells do
not respond to any treatment [1]. Therefore, identification of new therapeutic targets to
improve patients’ survival and clinical outcomes is critical [2,3].
In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR
associated nuclease 9 (Cas9) has significantly influenced the field of molecular biology and gene therapy. Solid tumors are the most common type of tumors, but much little progress has been made for gene therapy-based treatment as compared to non-solid tumors such as leukemia. However, this situation is rapidly being changed with the developments of CRISPR/Cas9. There is a growing amount of promising pre-clinical data showing CRISPR/Cas9 is an effective tool to specifically target cancer cells and suppress tumor growth [4–6]. This may lead to the discovery of novel molecular targets for cancer treatment.
There is an increasing number of clinical trials utilizing CRISPR/Cas9 technology to treat cancers of different origin (Table 1). The majority of these trials are based on genetically engineered T cells for cancer immunotherapy, not targeting a specific gene in tumor cells themselves. One of the main problems in direct targeting of cancer is the lack of an effective and safe delivery method that can be used in patients. Tumor heterogeneity is another issue which might be a
challenge as tumors usually are comprised of different subclones (Intratumor heterogeneity) [7– 10]. Thus, even with the right delivery system, outgrowth of a minor subclone may emerge and the treatment would not be effective anymore. Nonetheless, identification of different major subclones prior to the treatment and recruitment of multiple Cas9/gRNA might be an option to minimize relapse in patients.
CHAPTER 2
20
1. Introduction
The accumulation of genetic mutations in cells over time leads to cancer. In addition, certain gene changes such as driver mutations in TP53, EGFR, KRAS, BRAF, HER2 and MET can make a cell cancerous. Treatment strategies are conventionally based on histological subtypes. However, today, beside the conventional histological classifications, each cancer type can be subdivided into various molecular subtypes which play a crucial role in treatment decision making process. Each molecular subtype is differently treated and clinicians may also predict treatment outcomes and patients’ survival. For instance, lung cancer patients with EGFR activating mutations are treated with different types of tyrosine kinase inhibitors (TKIs) such as gifitinib, erlotinib or afatinib, depending on the mutation. Yet, resistance to the TKIs inevitably
emerges either by DNA mutations or/and metabolic changes. As a result, treatment strategies
are modified based on the new molecular signature. However, eventually, the tumor cells do
not respond to any treatment [1]. Therefore, identification of new therapeutic targets to
improve patients’ survival and clinical outcomes is critical [2,3].
In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR
associated nuclease 9 (Cas9)has significantly influenced the field of molecular biology and gene therapy. Solid tumors are the most common type of tumors, but much little progress has been made for gene therapy-based treatment as compared to non-solid tumors such as leukemia. However, this situation is rapidly being changed with the developments of CRISPR/Cas9. There is a growing amount of promising pre-clinical data showing CRISPR/Cas9 is an effective tool to specifically target cancer cells and suppress tumor growth [4–6]. This may lead to the discovery of novel molecular targets for cancer treatment.
There is an increasing number of clinical trials utilizing CRISPR/Cas9 technology to treat cancers of different origin (Table 1). The majority of these trials are based on genetically engineered T cells for cancer immunotherapy, not targeting a specific gene in tumor cells themselves. One of the main problems in direct targeting of cancer is the lack of an effective and safe delivery method that can be used in patients. Tumor heterogeneity is another issue which might be a
CRISPR/Cas9 for Identification of New Targets for Cancer Treatment
21 challenge as tumors usually are comprised of different subclones (Intratumor heterogeneity) [7– 10]. Thus, even with the right delivery system, outgrowth of a minor subclone may emerge and the treatment would not be effective anymore. Nonetheless, identification of different major subclones prior to the treatment and recruitment of multiple Cas9/gRNA might be an option to minimize relapse in patients.
Table 1. List of phase I/II clinical trials that use CRISPR/Cas9 genome editing technologies
Condition Target Interventions Phase Status Clinical Trials Gov Identifier Solid Tumor, Adult PD-1 and TCR anti-mesothelin
CAR-T cells I Active NCT03545815 Melanoma Synovial Sarcoma Liposarcoma TCRendo and PD-1 NY-ESO-1 Drug: Cyclophosphamide Fludarabine I Active NCT03399448
Cancer, Gastrointestinal CISH
inactivated TIL Cyclophosphamide Drug: Fludarabine Aldesleukin
I – NCT03538613
Human
Papillomavirus-Related Malignant Neoplasm HPV-related Cervical Biological: TALEN Biological: CRISPR/Cas9
I – NCT03057912
Gastrointestinal Infection Host Factors
of Norovirus Duodenal biopsy Saliva – Active NCT03342547 B Cell Leukemia
B Cell Lymphoma CD20 or CD22 CD19 and CAR-T Universal Dual Specificity CD19 and CD20 or CD22 CAR-T Cells I/II Active NCT03398967
Leukemia and Lymphoma Relapsed or Refractory
CD19+
UCART019 I/II Active NCT03166878
Stage IV Gastric Carcinoma; Stage IV Nasopharyngeal
Carcinoma; T-Cell Lymphoma Stage IV
? Drug: Fludarabine Cyclophosphamide
Interleukin-2
I/II Active NCT03044743
In this review, we provide an overview of the current studies in which CRISPR/Cas9 has been utilized for the identification of potential therapeutic targets in some of the most frequent solid tumors including lung, breast, brain, liver and colorectal cancer. We discuss potential candidates for therapy which are highly expressed or activated in different cancer types, as they are more convenient to inhibit or disrupt. Finally, recent advances and different delivery methods of CRISPR/Cas9 are presented.
2. CRISPR/Cas9 gene editing technology
CRISPR/Cas9 is a newly discovered, powerful gene editing tool which is derived from a
prokaryotic defense system [11–14]. This technology has enabled researchers to edit the genome eukaryotic cells more precisely and efficiently as compared to previous methods such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) [15].
2.1 The structure of CRISPR/Cas9
The structure of CRISPR/Cas9 has comprehensively been described elsewhere [15–17]. CRISPR/Cas9 system consists of three components; a single guide RNA (sgRNA) which is specific to a target sequence of DNA, Cas9 protein with DNA endonuclease activity and a tracrRNA that interacts with Cas9 (Figure 1). The gRNA (approximately 20 bp length) binds to the target site in the genome and directs the Cas9 protein. The Cas9 protein is a RNA-guided nuclease which was discovered in the CRISPR type II adaptive immunity system of Streptococcus pyogenes and it is responsible for cleaving double strand DNA [17].
CHAPTER 2
22
Table 1. List of phase I/II clinical trials that use CRISPR/Cas9 genome editing technologies
Condition Target Interventions Phase Status Clinical Trials Gov Identifier Solid Tumor, Adult PD-1 and TCR anti-mesothelin
CAR-T cells I Active NCT03545815 Melanoma Synovial Sarcoma Liposarcoma TCRendo and PD-1 NY-ESO-1Drug: Cyclophosphamide Fludarabine I Active NCT03399448
Cancer, Gastrointestinal CISH
inactivated TIL CyclophosphamideDrug: Fludarabine Aldesleukin
I – NCT03538613
Human
Papillomavirus-Related Malignant Neoplasm HPV-relatedCervical Biological: TALENBiological: CRISPR/Cas9
I – NCT03057912
Gastrointestinal Infection Host Factors
of Norovirus Duodenal biopsySaliva – Active NCT03342547 B Cell Leukemia
B Cell Lymphoma CD20 or CD22 CD19 and CAR-T Universal Dual Specificity CD19 and CD20 or CD22 CAR-T Cells I/II Active NCT03398967
Leukemia and Lymphoma Relapsed or Refractory
CD19+
UCART019 I/II Active NCT03166878
Stage IV Gastric Carcinoma; Stage IV Nasopharyngeal
Carcinoma; T-Cell Lymphoma Stage IV
? Drug: Fludarabine Cyclophosphamide
Interleukin-2
I/II Active NCT03044743
CRISPR/Cas9 for Identification of New Targets for Cancer Treatment
23 In this review, we provide an overview of the current studies in which CRISPR/Cas9 has been utilized for the identification of potential therapeutic targets in some of the most frequent solid tumors including lung, breast, brain, liver and colorectal cancer. We discuss potential candidates for therapy which are highly expressed or activated in different cancer types, as they are more convenient to inhibit or disrupt. Finally, recent advances and different delivery methods of CRISPR/Cas9 are presented.
2. CRISPR/Cas9 gene editing technology
CRISPR/Cas9 is a newly discovered, powerful gene editing tool which is derived from a
prokaryotic defense system [11–14]. This technology has enabled researchers to edit the genome eukaryotic cells more precisely and efficiently as compared to previous methods such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) [15].
2.1 The structure of CRISPR/Cas9
The structure of CRISPR/Cas9 has comprehensively been described elsewhere [15–17]. CRISPR/Cas9 system consists of three components; a single guide RNA (sgRNA) which is specific to a target sequence of DNA, Cas9 protein with DNA endonuclease activity and a tracrRNA that interacts with Cas9 (Figure 1). The gRNA (approximately 20 bp length) binds to the target site in the genome and directs the Cas9 protein. The Cas9 protein is a RNA-guided nuclease which was discovered in the CRISPR type II adaptive immunity system of Streptococcus pyogenes and it is responsible for cleaving double strand DNA [17].
Figure 1. Schematic picture of genome editing mediated by CRISPR/Cas9 and DNA repairing. The Cas9 protein, which is guided by a desired single strand guide RNA (gRNA), cuts the double stranded DNA and makes a double stand break (DSB). Subsequently, DNA repair occurs either through the non-homologous end joining (NHEJ) or the homology directed repair (HDR) pathways.
2.1.1 gRNAs and its specificity
Several factors such as sequence, length and secondary structure of the gRNAs can influence its efficiency and specificity [18,19]. In addition, the efficiency of CRISPR/Cas9 complex can be influenced by other factors including genomic locus of the target, chromatin accessibility, nucleosomes and other components around gRNA binding sites [19]. The gRNA sequence plays
a crucial role in theefficiency, specificity and accuracy of CRISPR/Cas9-mediated genome editing.
The first 10-12 nucleotides at the 3’ end of gRNA, immediately adjacent to a protospacer
adjacent motif (PAM), called “seed sequence’’, bind to the target sequence and determine the specificity [18,20]. Truncated gRNAs with shorter complementary nucleotides (less than 20) can
reduce off-target effects by 5000-fold without sacrificing on-target efficiency [21].Moreover,
extending gRNA duplex by 5 bp can significantly improve the knockout efficiency [22].
2.1.2 CRISPR-associated nucleases
Different versions of CRISPR-associated nucleases are currently under development, which greatly expand the CRISPR-based toolbox for genome editing (Table 2). Cpf1 is an RNA guided
endonuclease which belongs to class 2 CRISPR-Cas system, the same as Cas9 [23]. However,
Cpf1 has different featuresas compared to Cas9.For example, it has only one single nuclease
domain and a shorter gRNA. Creating staggered cuts is one of the main characteristic features of Cpf1. This type of cut is of great importance for introducing exogenous DNA into the genome by the homology directed repair (HDR) pathway. Besides, Cpf1 has both endoribonuclease and endonuclease activities, which is a very unique feature for a nuclease [24]. These properties make Cpf1 a complex and effective genome editing tool for both gene targeting and gene silencing. C2C2 is another member of class 2 type VI-A CRISPR-Cas and was found in Leptotrichia
shahii, where it protects the bacteria against RNA phages. C2C2 can be used to target and
regulate RNAs. It has two RNase catalytic pockets with the function of dual RNase activities
whichcan be recruited for identification of cellular transcripts [25]. The structure and function
of C2C2 is very unique and provides a novel tool forRNA manipulation [25–27].
Precisely editing a single base in the genome without introducing DSBs was a dream for decades, and with engineered Cas9 base editors, it is possible now. The “base editors” consist of fusions of a dead Cas9 domain and a cytidine deaminase enzyme which is able to convert GC to AT without introducing DSBs [28]. Recently, researchers created a Cas9 fused with a transfer RNA adenosine deaminase which can mediate conversion of AT to GC [29]. These base editors are undoubtedly valuable tools for repairing disease related mutations.
CHAPTER 2
24
Figure 1. Schematic picture of genome editing mediated by CRISPR/Cas9 and DNA repairing. The Cas9 protein, which is guided by a desired single strand guide RNA (gRNA), cuts the double stranded DNA and makes a double stand break (DSB). Subsequently, DNA repair occurs either through the non-homologous end joining (NHEJ) or the homology directed repair (HDR) pathways.
2.1.1 gRNAs and its specificity
Several factors such as sequence, length and secondary structure of the gRNAs can influence its efficiency and specificity [18,19]. In addition, the efficiency of CRISPR/Cas9 complex can be influenced by other factors including genomic locus of the target, chromatin accessibility, nucleosomes and other components around gRNA binding sites [19]. The gRNA sequence plays a crucial role in the efficiency, specificity and accuracy of CRISPR/Cas9-mediated genome editing. The first 10-12 nucleotides at the 3’ end of gRNA, immediately adjacent to a protospacer
CRISPR/Cas9 for Identification of New Targets for Cancer Treatment
25 adjacent motif (PAM), called “seed sequence’’, bind to the target sequence and determine the specificity [18,20]. Truncated gRNAs with shorter complementary nucleotides (less than 20) can
reduce off-target effects by 5000-fold without sacrificing on-target efficiency [21]. Moreover,
extending gRNA duplex by 5 bp can significantly improve the knockout efficiency [22].
2.1.2 CRISPR-associated nucleases
Different versions of CRISPR-associated nucleases are currently under development, which greatly expand the CRISPR-based toolbox for genome editing (Table 2). Cpf1 is an RNA guided
endonuclease which belongs to class 2 CRISPR-Cas system, the same as Cas9 [23]. However,
Cpf1 has different features as compared to Cas9. For example, it has only one single nuclease
domain and a shorter gRNA. Creating staggered cuts is one of the main characteristic features of Cpf1. This type of cut is of great importance for introducing exogenous DNA into the genome by the homology directed repair (HDR) pathway. Besides, Cpf1 has both endoribonuclease and endonuclease activities, which is a very unique feature for a nuclease [24]. These properties make Cpf1 a complex and effective genome editing tool for both gene targeting and gene silencing. C2C2 is another member of class 2 type VI-A CRISPR-Cas and was found in Leptotrichia
shahii, where it protects the bacteria against RNA phages. C2C2 can be used to target and
regulate RNAs. It has two RNase catalytic pockets with the function of dual RNase activities
which can be recruited for identification of cellular transcripts [25]. The structure and function
of C2C2 is very unique and provides a novel tool for RNA manipulation [25–27].
Precisely editing a single base in the genome without introducing DSBs was a dream for decades, and with engineered Cas9 base editors, it is possible now. The “base editors” consist of fusions of a dead Cas9 domain and a cytidine deaminase enzyme which is able to convert GC to AT without introducing DSBs [28]. Recently, researchers created a Cas9 fused with a transfer RNA adenosine deaminase which can mediate conversion of AT to GC [29]. These base editors are undoubtedly valuable tools for repairing disease related mutations.
Table 2: CRISPR-associated nucleases.
Name advantages applications Ref
Naturally Cas9
SpCas9 Most common used Gene editing
23–26
SaCas9 1 kb smaller than SpCas9 StCas9
Differ in the PAM sequence CjCas9
FnCas9
CasX most compact naturally
CRISPR variant potentially useful untapped nucleases CasY
Cas12a
(Cpf1) guide RNA shorter thansmaller than SpCas9; SpCas9
more economical to
produce Engineered Cas9
Variants Nickases Cas9 variants nicked a single DNA strand rather than DSB high fidelity 27,28
eSpCas9 an enhanced nuclease High specificity and low
off-target rate Base Editors BE1/2/3
ABEs nucleotide exchanges a complete range of without DSBs
Gene writing 29,30 23,31,32
RNA Editors Cas13a/C as13b/ Cas13d
targeting RNA rather than
DNA RNA editing
2.2 The advantages of CRISPR/Cas9 over ZFN and TALENs
CRISPR/Cas9 system has several advantages over ZFN and TALENs with respect to simplicity,
flexibility and affordability. The most important difference is that CRISPR system relies on RNA-DNA recognition, rather than on the protein-RNA-DNA binding mechanism [11,17,30]. Thus, it is more doable and easier to construct a customized CRISPR/Cas9 complex by only changing the gRNA sequence instead of engineering a new protein. It is important to take into account that the target sequence needs to be immediately upstream of a PAM [17]. The PAM sequence (5’-NGG-3’) is essential for target recognition by Cas9. This short sequence occurs approximately once every eight base pairs in the human genome which makes it possible to design several gRNAs for one specific target gene [31].
3. Targeting cancer-related genes and identification of potential therapeutic targets in solid tumors
Nowadays, CRISPR/Cas9 is routinely used in research laboratories because of its simplicity and efficiency. In addition, the price of this gene editing tools continuous to decrease over time. CRISPR/Cas9 gene editing technology helped researchers to identify the role of different genes in cancer; for instance whether they function as oncogene or tumor gene [32–36]. Several groups generated in vitro and in vivo knockout models to study molecular basis of different cancer types [37–39]. CRISPR/Cas9 is also widely used for inducing specific mutations in certain genes to explore potential causative role of these mutations in disease development. In addition, CRISPR barcoding technology can be used to investigate tumor heterogeneity [40,41]. Moreover, genome wide CRISPR/Cas9 screening is frequently used to identify potential therapeutic targets in different cancers. Below, we mainly focus on new potential molecular targets which have been identified by using CRISPR/Cas9in some of the most frequent solid tumors including lung, breast, brain, liver and colorectal cancers.
3.1 Lung cancer
Lung cancer is the leading cause of cancer related death worldwide. Non-small cell lung cancer (NSCLC) subtypes accounts for approximately 85% of all lung cancer [42]. Identification of specific genetic aberrations is important to choose the appropriate treatment strategy, specifically in adenocarcinoma patients. Currently, several molecular targets such as EGFR, BRAF, ALK-EML4 and cMET are clinically available for the treatment of NSCLC. However, treatment options are limited firstly by the number of molecular targets and secondly by emerging resistance in the patients [1,42]. In recent years, CRISPR/Cas9-based in vitro and in vivo studies on lung cancer have identified new treatment strategies and potential therapeutic targets.
CHAPTER 2
26
Table 2: CRISPR-associated nucleases.
Name advantages applications Ref
Naturally Cas9
SpCas9 Most common used Gene editing
23–26
SaCas9 1 kb smaller than SpCas9 StCas9
Differ in the PAM sequence CjCas9
FnCas9
CasX most compact naturally
CRISPR variant potentiallyuseful untapped nucleases CasY
Cas12a
(Cpf1) guide RNA shorter thansmaller than SpCas9; SpCas9
more economical to
produce Engineered Cas9
Variants NickasesCas9 variants nicked a single DNA strand rather than DSB high fidelity 27,28
eSpCas9 an enhanced nuclease High specificity and low
off-target rate Base Editors BE1/2/3
ABEs nucleotide exchanges a complete range of without DSBs
Gene writing 29,30 23,31,32
RNA Editors Cas13a/C as13b/ Cas13d
targeting RNA rather than
DNA RNA editing
2.2 The advantages of CRISPR/Cas9 over ZFN and TALENs
CRISPR/Cas9 system has several advantages over ZFN and TALENs with respect to simplicity, flexibility and affordability. The most important difference is that CRISPR system relies on RNA-DNA recognition, rather than on the protein-RNA-DNA binding mechanism [11,17,30]. Thus, it is more doable and easier to construct a customized CRISPR/Cas9 complex by only changing the gRNA sequence instead of engineering a new protein. It is important to take into account that the target sequence needs to be immediately upstream of a PAM [17]. The PAM sequence (5’-NGG-3’) is essential for target recognition by Cas9. This short sequence occurs approximately once every eight base pairs in the human genome which makes it possible to design several gRNAs for one specific target gene [31].
CRISPR/Cas9 for Identification of New Targets for Cancer Treatment
27 3. Targeting cancer-related genes and identification of potential therapeutic targets in solid tumors
Nowadays, CRISPR/Cas9 is routinely used in research laboratories because of its simplicity and efficiency. In addition, the price of this gene editing tools continuous to decrease over time. CRISPR/Cas9 gene editing technology helped researchers to identify the role of different genes in cancer; for instance whether they function as oncogene or tumor gene [32–36]. Several groups generated in vitro and in vivo knockout models to study molecular basis of different cancer types [37–39]. CRISPR/Cas9 is also widely used for inducing specific mutations in certain genes to explore potential causative role of these mutations in disease development. In addition, CRISPR barcoding technology can be used to investigate tumor heterogeneity [40,41]. Moreover, genome wide CRISPR/Cas9 screening is frequently used to identify potential therapeutic targets in different cancers. Below, we mainly focus on new potential molecular targets which have been identified by using CRISPR/Cas9 in some of the most frequent solid tumors including lung, breast, brain, liver and colorectal cancers.
3.1 Lung cancer
Lung cancer is the leading cause of cancer related death worldwide. Non-small cell lung cancer (NSCLC) subtypes accounts for approximately 85% of all lung cancer [42]. Identification of specific genetic aberrations is important to choose the appropriate treatment strategy, specifically in adenocarcinoma patients. Currently, several molecular targets such as EGFR, BRAF, ALK-EML4 and cMET are clinically available for the treatment of NSCLC. However, treatment options are limited firstly by the number of molecular targets and secondly by emerging resistance in the patients [1,42]. In recent years, CRISPR/Cas9-based in vitro and in vivo studies on lung cancer have identified new treatment strategies and potential therapeutic targets.
Targeting mutant version of certain genes using CRISPR/Cas9 gene editing system can specifically target mutant cancer cells, but not normal cells. Targeting mutant version of the EGFR gene (L858R) resulted in the selective elimination of mutant cells and reduced cell proliferation both in vitro and in vivo [43]. In another study, Koo et al (2017) selectively abrogated EGFR mutant alleles (L858R) in a NSCLC cell line (H1975) using AdV vector which resulted in cancer cells death and significantly reduced tumor size in vivo [4]. These findings underscore the potential of CRISPR-based therapeutics in tumor-specific targeted therapy and in distinguishing normal and mutant tumor cells.
Approximately 30% of patients diagnosed with lung cancer have somatic activating KRAS mutations. Currently, there is no effective treatment for KRAS mutant lung tumors which are considered as undrugable. CRISPR-based deletion of murine Kras in two different KrasG12D/+/p53−/− lung cancer cell lines resulted in a significant reduction in cell proliferation, but the cells were still viable and sustained their ability to form tumor in vivo. Transcriptome sequencing revealed a substantially higher expression of Fas receptor in the knockout cells. Interestingly, an activating Fas receptor antibody selectively induced apoptosis in these Kras -/-lung cancer cells [44]. Oncogenic Ras can inhibit Fas ligand-mediated apoptosis through downregulation of Fas [45]; therefore, simultaneous inhibition of KRAS and activation of FAS might be an effective therapeutic approach against KRAS-driven lung cancer tumors in the future.
CD38 is a glycoprotein that functions as both an ADP-ribosyl cyclase and a NAD glycohydrolase. Its expression level is negatively associated with poor prognosis in chronic lymphocytic leukemia patients and it is used as a therapeutic target in multiple myeloma [46,47]. However, its role is solid tumors such as lung cancer is not clear. CRISPR-based deletion of CD38 in a lung adenocarcinoma cell line (A549) resulted in substantial suppression of cell growth and invasion in vitro and in xenografts in mice suggesting CD38 as a potential target in lung cancer. Further investigations unraveled an elevated level of CD38 in 93% (27/29) of lung cancer cell lines and 40% (11/27) of NSCLC primary tumors. Hence, direct disruption of this target through
monoclonal antibodies such as daratumumab might be effective in NSCLC patients with CD38 overexpression [48].
It has been shown that disruption of focal adhesion kinase (FAK), a non-receptor tyrosine kinase which is frequently amplified in lung cancer cell lines, results in DNA damage and sensitivity to ionizing radiation. It has also been revealed that the presence of FAK is crucial for oncogenic and clonogenic abilities of the KRAS-mutant in tumor xenografts using CRISPR/Cas9 approaches [49,50]. In addition, FAK is significantly overexpressed in NSCLC patients and it is associated with poorer clinical outcomes [51–53] which make it an attractive target to treat NSCLC patients and prevent distant metastasis.
TAZ is a co-activator of Hippo pathway and it has been shown to be upregulated in lung cancer. Dual inactivation of TAZ and YAP (a transcriptional activator) suppressed cell proliferation as well as cancer stem cell (CSC) sphere formation in lung cancer, suggesting them as potential molecular targets [54]. CRISPR-based disruption of oncogenic MUC1-C hindered the growth of KRAS-dependent lung adenocarcinoma cells, i.e. A549 and A460 [55]. In addition, overexpression of MUC1 has been shown in more than 80% of NSCLCs and it is associated with poor prognosis [56,57]. It also plays a role in epithelial-mesenchymal transition (EMT) and self-renewal ability each of which are known as drug resistance mechanisms in cancer [58,59]. Thus, suppression of MUC1-C might delay resistance or even prevent tumor recurrence. Likewise, it has been reported that overexpression of TNC, an extracellular matrix (ECM) protein, is associated with lung cancer recurrence [60]. CRISPR-based transcriptional activation of Tnc led to metastatic dissemination of lung adenocarcinoma cells in vivo. This finding highlights the central role of EMC-related proteins in metastasis and its potential to be used as recurrence and metastasis biomarkers as well as therapeutic target in NSCLCs [61].
Chromatin remodeling genes are frequently mutated, mostly inactivating mutations, in lung adenocarcinoma [62–64]. A recent study used CRISPR technology to knockout Smarca4, Arid1a or Setd2 to investigate their role in lung tumorigenesis. Loss of Arid1a and Setd2 resulted in the
CHAPTER 2
28
Targeting mutant version of certain genes using CRISPR/Cas9 gene editing system can specifically target mutant cancer cells, but not normal cells. Targeting mutant version of the EGFR gene (L858R) resulted in the selective elimination of mutant cells and reduced cell proliferation both in vitro and in vivo [43]. In another study, Koo et al (2017) selectively abrogated EGFR mutant alleles (L858R) in a NSCLC cell line (H1975) using AdV vector which resulted in cancer cells death and significantly reduced tumor size in vivo [4]. These findings underscore the potential of CRISPR-based therapeutics in tumor-specific targeted therapy and in distinguishing normal and mutant tumor cells.
Approximately 30% of patients diagnosed with lung cancer have somatic activating KRAS mutations. Currently, there is no effective treatment for KRAS mutant lung tumors which are considered as undrugable. CRISPR-based deletion of murine Kras in two different KrasG12D/+/p53−/−lung cancer cell lines resulted in a significant reduction in cell proliferation, but the cells were still viable and sustained their ability to form tumor in vivo. Transcriptome sequencing revealed a substantially higher expression of Fas receptor in the knockout cells. Interestingly, an activating Fas receptor antibody selectively induced apoptosis in these Kras -/-lung cancer cells [44]. Oncogenic Ras can inhibit Fas ligand-mediated apoptosis through downregulation of Fas [45]; therefore, simultaneous inhibition of KRAS and activation of FAS might be an effective therapeutic approach against KRAS-driven lung cancer tumors in the future.
CD38 is a glycoprotein that functions as both an ADP-ribosyl cyclase and a NAD glycohydrolase. Its expression level is negatively associated with poor prognosis in chronic lymphocytic leukemia patients and it is used as a therapeutic target in multiple myeloma [46,47]. However, its role is solid tumors such as lung cancer is not clear. CRISPR-based deletion of CD38 in a lung adenocarcinoma cell line (A549) resulted in substantial suppression of cell growth and invasion in vitro and in xenografts in mice suggesting CD38 as a potential target in lung cancer. Further investigations unraveled an elevated level of CD38 in 93% (27/29) of lung cancer cell lines and 40% (11/27) of NSCLC primary tumors. Hence, direct disruption of this target through
CRISPR/Cas9 for Identification of New Targets for Cancer Treatment
29 monoclonal antibodies such as daratumumab might be effective in NSCLC patients with CD38 overexpression [48].
It has been shown that disruption of focal adhesion kinase (FAK), a non-receptor tyrosine kinase which is frequently amplified in lung cancer cell lines, results in DNA damage and sensitivity to ionizing radiation. It has also been revealed that the presence of FAK is crucial for oncogenic and clonogenic abilities of the KRAS-mutant in tumor xenografts using CRISPR/Cas9 approaches [49,50]. In addition, FAK is significantly overexpressed in NSCLC patients and it is associated with poorer clinical outcomes [51–53] which make it an attractive target to treat NSCLC patients and prevent distant metastasis.
TAZ is a co-activator of Hippo pathway and it has been shown to be upregulated in lung cancer. Dual inactivation of TAZ and YAP (a transcriptional activator) suppressed cell proliferation as well as cancer stem cell (CSC) sphere formation in lung cancer, suggesting them as potential molecular targets [54]. CRISPR-based disruption of oncogenic MUC1-C hindered the growth of KRAS-dependent lung adenocarcinoma cells, i.e. A549 and A460 [55]. In addition, overexpression of MUC1 has been shown in more than 80% of NSCLCs and it is associated with poor prognosis [56,57]. It also plays a role in epithelial-mesenchymal transition (EMT) and self-renewal ability each of which are known as drug resistance mechanisms in cancer [58,59]. Thus, suppression of MUC1-C might delay resistance or even prevent tumor recurrence. Likewise, it has been reported that overexpression of TNC, an extracellular matrix (ECM) protein, is associated with lung cancer recurrence [60]. CRISPR-based transcriptional activation of Tnc led to metastatic dissemination of lung adenocarcinoma cells in vivo. This finding highlights the central role of EMC-related proteins in metastasis and its potential to be used as recurrence and metastasis biomarkers as well as therapeutic target in NSCLCs [61].
Chromatin remodeling genes are frequently mutated, mostly inactivating mutations, in lung adenocarcinoma [62–64]. A recent study used CRISPR technology to knockout Smarca4, Arid1a or Setd2 to investigate their role in lung tumorigenesis. Loss of Arid1a and Setd2 resulted in the
development of higher grade tumors and strong tumor progression in both early and late stage lesions, respectively. On the other hand, ablation of Smarca4 led to tumor development, whereas it attenuated disease progression in vivo over time [34]. Previously, it has been shown that SMARCA4 inactivation promotes NSCLC aggressiveness [65]. Nevertheless, loss of function mutations in SMARCA4 have been reported to increase tumors cells sensitivity to Aurora kinase A inhibitor VX-680 both in vitro and in xenograft mouse models [66]. All these discrepancies make it challenging to decide whether SMARCA4 is an appropriate molecular candidate for therapy. However, genetic disruption or chemical-based inhibition of SMARCA4 may be of benefit for more advanced NSCLC patients.
MicroRNAs play important roles in cells and their dysregulation has been shown in various types of cancers [67]. A recent study exploited CRISPR/Cas9-based gene activation technology to investigate the role of miRNAs located on 14q32 in lung cancer cells. They showed that overexpression of those miRNAs significantly elevated cell migration and invasion. Moreover, higher expression levels of mir-323b, mir-487a and mir-539 were associated with metastasis and poorer prognosis in patients with lung adenocarcinoma, especially in never-smokers [68]. Thus, these 14q32 miRNAs might be potential targets to prevent tumor cell dissemination and distant metastasis in lung adenocarcinoma patients.
Overall, application of CRISPR/Cas9 genome editing system in lung cancer has led to the identification of several potential therapeutic targets. Both in vitro and in vivo studies have shown very promising results, especially in the suppression of distant metastasis which is the main cause of death in patients. In addition, CRISPR/Cas9 can target specific oncogenic alleles of certain genes such as EGFR, which is an important step towards cancer gene therapy.
3.2 Breast cancer
Breast cancer is the most common type of cancer and the leading cause of cancer-related death in women in the world [69]. It is divided into various subtypes with distinct morphologies. Based on the expression of estrogen receptor (ER), progesterone receptor (PR), ERBB2 (HER2), p53 and Ki-67, it can be classified into four main molecular subtypes; i.e. triple-negative/basal-like, the
Her2-enriched, luminal A and luminal B subtypes [70]. ER-positive luminal subtypes are the most common types of breast cancer (almost 70%) and resistance to endocrine therapies occurs in approximately in 30% of these patients [71]. Therefore, finding new treatment options is crucial, especially in the case of recurrence. Recent CRISPR-mediated studies have led to the identification of potential therapeutic targets in different breast cancer subtypes.
Distant metastasis is one of the main characteristics of late stage cancers and the main reason for cancer mortality. Identification of new therapeutic targets may help to prolong patients’ survival and improve their life quality. MLK3 is a member of MAP3K family which involves in signal transduction and activation of MAPK pathway. Abrogation of Mlk3 in murine triple negative breast cancer (TNBC) 4T1 cells, which are highly metastatic, led to suppression of cell invasion and migration [72]. In another study, CRISPR-mediated depletion of CX3CR1, a protein involved in dissemination of tumor cells into blood vessels, in breast cancer cells impaired lodging of the cancer cells to bone and reduction in the number of cancerous lesions in mice [73].
In a recent study by Liao and colleagues, deletion of Ubr5, a member of the E3 ligase family, in a murine mammary TNBC model resulted in the inhibition of tumor growth and distant metastasis in vivo as well as promoted apoptosis and necrosis through impairment of angiogenesis. The authors also showed high expression levels of UBR5 in TNBC patients which make this protein interesting for further investigation for targeted therapy [74]. CRISPR-mediated knockout of CXCR2 (IL-8 receptor) in breast cancer cells, showed a significant reduction in cell migration in vitro as well as lower rate of lung metastasis in vivo [75]. CXCR2 is recognized as a stem-like cell marker for TNBC and it shows significantly lower expression in this subtype as compared to non-TNBC [76]. It is also known that targeting CXCR2 improves the chemotherapeutic response in lung cancer [77]. Thus, treatment of advanced non-TNBC patients with anti-CXCR2 drugs might be beneficial for this subgroup of patients. In addition, MARK4 and FERMT2 are other potential targets related to breast cancer cell migration and metastasis that have been identified by CRISPR/Cas9 genome editing system [78–80].
