Cas9 system for drug screening

Accepted Manuscript Article The construction of drug-resistant cancer cell lines by CRISPR/Cas9 system for drug screening Lingmin Zhang, Ying Li, Qinghua Chen, Yong Xia, Wenfu Zheng, Xingyu Jiang PII: DOI: Reference:

S2095-9273(18)30484-5 https://doi.org/10.1016/j.scib.2018.09.024 SCIB 512

To appear in:

Science Bulletin

Received Date: Revised Date: Accepted Date:

30 March 2018 15 August 2018 31 August 2018

Please cite this article as: L. Zhang, Y. Li, Q. Chen, Y. Xia, W. Zheng, X. Jiang, The construction of drug-resistant cancer cell lines by CRISPR/Cas9 system for drug screening, Science Bulletin (2018), doi: https://doi.org/10.1016/ j.scib.2018.09.024

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The construction of drug-resistant cancer cell lines by

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CRISPR/Cas9 system for drug screening

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Lingmin Zhang1,3, Ying Li1, Qinghua Chen1, Yong Xia3, Wenfu Zheng1,* and Xingyu Jiang1,2,3*

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1CAS

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BioNanotechnology, CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety,

Center for Excellence in Nanoscience, Beijing Engineering Research Center for

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National Center for NanoScience and Technology, Beijing 100190, China

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2University

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3

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of Pharmaceutical Sciences and the Third & Fifth Affiliated Hospital, Guangzhou Medical

of Chinese Academy of Sciences, Beijing 100049, China

Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, School

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University, Guangzhou 511436, China

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* Corresponding authors.

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Email addresses: [email protected](X.Y. Jiang), [email protected] (W.F. Zheng)

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ABSTRACT

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Cancer therapy is often hampered by the rapid emergence of drug resistance. Drug-resistant

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cellular models are essential for understanding the drug resistance and developing new

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therapeutics. The low efficiency and long time required in creating these models are major

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obstacles hindering drug resistance research and drug screening. Herein, we report an

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approach that can accelerate (shortening the time from years to 3 weeks) the establishment of

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cancer cell line-based, inheritable drug resistance models by specific knockout of MED12

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gene using CRISPR/Cas9 system. The resultant MED12KO A375 (melanoma) cell line was

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resistant to inhibitors of B-Raf proto-oncogene, serine/threonine kinase (BRAF), whereas the

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resultant MED12KO PC9 (non-small cell lung cancer) cell line was resistant to inhibitors of

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epidermal growth factor receptor (EGFR). Evaluation of anti-cancer drugs and their

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combinations shows that certain combinations of BRAF inhibitors and TGF- receptor (TGF-

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R) inhibitors are active in suppressing the growth of MED12KO A375 cells, and a few

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combinations of EGFR inhibitors and TGF-R inhibitors were active in suppressing the growth

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of MED12KO PC9 cells. The drug-resistant models will be useful in screening novel drugs and

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drug combinations for multi-drug-resistant cancer therapy. 1

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Key Words:

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CRISPR/Cas9; Drug resistance; Cancer; Cell line; Gene editing

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1. Introduction

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The rapid emergence of drug resistance hampers the current cancer therapy. Both of the

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conventional chemotherapies and the emerging molecular targeted therapies suffer from a

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similar fate and show helplessness in the face of the drug resistance [1]. The generation of

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drug resistance concerns a diverse range of molecular mechanisms including elevated drug

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efflux [2-6], alterations in drug metabolism [7], mutations of drug targets [8], activation of

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survival signalling pathways [9], and the inactivation of downstream death signalling pathways

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[10]. Moreover, the epigenetic changes and the influence of the local tumor microenvironment

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also contribute to drug resistance. To improve cancer therapy efficiencies, strategies must be

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developed to combat the drug resistance. Better understanding of the underlying genetic

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diversity of tumor cells may help researchers to elucidate the mechanisms of drug resistance.

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Based on this, new ways to design drugs and concocting combination may better tackle drug

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resistance [11].

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Tractable and accurate drug resistance models are indispensable for unveiling the

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molecular mechanisms underlying drug resistance and providing novel strategies for effective

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cancer therapy. Presently, drug resistance models are mainly based on primary cancer cells

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from patients [12], animals [13] and transgenic cell lines [14]. However, models on primary

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cancer cells are usually unavailable due to the limited cell sources from patients. Models on

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animals are cumbersome in carrying out large-scale drug screening due to the tediously cell

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isolation processes [15]. The construction of drug-resistant cell lines by gradient dosage

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induction in vitro always spends at least months to years. Thus, a source-abundant, time-

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saving, and low-cost drug resistance model is highly demanding. Transformed cell lines

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engineered to edit a resistance-relevant gene of interest meet all the merits of an ideal model.

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So, it would be a distinct advantage to develop drug resistance model using genetically

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transformed cell lines. 2

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There are genome editing techniques, such as zinc-finger nucleases and transcription

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activator-like effector nucleases (TALENs) capable of introducing site-specific modifications in

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the genomes of cells and organisms [16-19]. However, the difficulty of protein design,

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synthesis, and validation remains a huge barrier to wide spread adoption of these techniques

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for routine use. The type II bacterial CRISPR (clustered regularly interspaced short palindrome

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repeats)-Cas9 (CRISPR-associated protein) system provides an effective means for gene

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editing (20). Compared to previous gene editing tools, the target specificity of CRISPR/Cas9 is

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dictated by a synthetic single-guide RNA (sgRNA), being much facile and time-saving for the

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construction of gene editing reagents (Fig. 1a) [21,22]. Guided by a sgRNA, Cas9 can induce

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DNA double-strand breaks (DSBs) or single-strand “nicks” at specific genomic loci [23]. The

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ease of programmability of the Cas9-mediated genetic perturbation provides researchers

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powerful tool to construct various cell lines for research of interest [24-27].

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In this study, we established drug-resistance cancer model cell lines using CRISPR/Cas9

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system. Previous reports indicated that the suppression of MED12 (a key component of the

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transcriptional MEDIATOR complex) confers resistance to chemotherapy in treatment of non-

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small-cell lung carcinoma (NSCLC), colon cancer, and liver cancer [1,28]. Furthermore, the

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loss of MED12 gene confers the resistance to molecular targeting drugs such as vemurafenib

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[29]. Thereby, using CRISPR/Cas9 system, we constructed B-Raf proto-oncogene,

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serine/threonine kinase (BRAF)-resistant MED12KO A375 (melanoma) cell model and

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epidermal growth factor receptor (EGFR)-resistant MED12KO PC9 (NSCLC) cell model. A

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proof-of-concept small scale drug screening revealed that the combinations of BRAF inhibitors

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and TGF-R inhibitors, and the combination of EGFR inhibitors and TGF-R inhibitors

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effectively suppressed the proliferation of MED12KO A375 cells and MED12KO PC9 cells,

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respectively. The CRISPR/Cas9-edited drug resistance model, thus, is a facile platform for

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proof-of-concept evaluation of the anti-cancer drugs and discovery of new drug cocktails.

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2. Materials and methods

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2.1 Regents 3

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LY2157299, LY2109761, SB431542, SB525334, GW788388, PLX-4032, Selumetibib

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(AZD6244), Sorafenib Tosylate, PLX-4720, RAF265 (CHIR-265), SB590885, ZM336372,

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SCH772984, Gefitinib (ZD1839), Erlotinib HCl (OSI-744), PD168393, CNX-2006, AG-490

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(Turphostin B42), Canertinib (CI-1033), AZD8931 (Sapitinib), Afatinib (BIBW2992), BI2536,

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Volasertib (BI 6727), GSK461364, MLN0905 and Rigosertib were from Selleck Chemicals.

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Antibody against MED12 (A300-774A) was from Bethyl Laboratories.

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2.2 Cell culture and CRISPR/Cas9 treatment

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A375 and PC9 cells were obtained from the Institute of Basic Medical Sciences (Beijing,

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China). The cells were grown in Dulbecco's modified Eagle’s medium (DMEM) supplemented

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with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin in 5% CO2 at 37 C. A375

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or PC9 cells were seeded into 24-well plates (Corning) one day prior to transfection at a

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density of 1×105 cells per well. Cells were transfected using Lipofectamine LTX (Life

13

Technologies) following the recommended protocol of the manufacturer. For each well of cells,

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a total of 1 µg plasmids were used in the 24-well plate.

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2.3 Flow cytometry screen

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Cells were collected 72 h after transduction and analysed on a flow cytometry (BD Facs ARIA).

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EGFP fluorescence was measured using a 488 nm laser for excitation and a 530/30 filter for

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detection. For sorting experiments, the GFP-positive cells were sorted into 96 well plates with

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single cell per well.

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2.4 Genomic DNA sequencing

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Genomic DNA from wild type cells (A375 or PC9 cells) and mutated cells (A375-M1~M3 or

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PC9-M1~M3 cells) was extracted with a Blood & Cell Culture Midi kit (Qiagen) following the

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protocol recommended by the manufacturer.

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Genomic region surrounding the CRISPR target site for the MED12 gene was amplified by polymerase chain reaction (PCR) with Q5® High-Fidelity DNA Polymerase (New England

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Biolabs) according to the instruction of the manufacturer. The amplified products were purified

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using QiaQuick Spin Column (Qiagen) following the protocol provided by the manufacturer.

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Primers sequences to amplify the target site for the PCR are:

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Forward, 5’-CTCGGCTGTTCGCAAGA-3’;

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Reverse, 5’-TCACCAGCCAGAAGTTATCCT-3’.

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2.5 T7 Endonuclease I (T7EI) mutation detection assays

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Targeted genomic loci were amplified from genomic DNA from wild type or the MED12

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mutated cells using primers designed to anneal approximately 200 to 250 base pairs upstream

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and downstream from the expected cut site (all of the primers in this assay was shown in Table

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S2). The purification of the PCR products is described as above. T7E1 assays were performed

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according to the protocol of the manufacturer as follows: 200 ng of purified PCR products were

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denatured for 5 min at 95 C, re-annealed at room temperature and reacted with T7

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Endonuclease I (New England BioLabs) for 30 min at 37 C. The resulting products were

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analysed by electrophoresis in 2% agarose gel, stained with ethidium bromide, and imaged by

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a Gel imaging system (GE Healthcare).

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2.6 TA cloning

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The target locus was amplified by PCR from pooled genomic DNA. The resulting PCR

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products were cloned into a plasmid using the pGEM-T kit (Promega). Following

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transformation of these reactions, plasmid DNAs isolated from overnight cultures of single

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colonies were sequenced and identified by comparison to the wild-type unmodified sequence.

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Single base substitutions, deletions, or insertions were not designated as mutant alleles

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because we could not exclude the possibility that these alterations might also be generated by

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the PCR amplification process.

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2.7 Western blot

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In Western blot analysis, transfected cells were first washed twice with cold PBS and then

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resuspended in 50 μL of lysis buffer (50 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 1% Triton

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X-100, 10% glycerol, 1.5 mmol/L MgCl2, 1 mmol/L EGTA) freshly supplemented with Roche’s

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Complete Protease Inhibitor Cocktail Tablets. All lysates were freshly prepared and resolved

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by SDS gel electrophoresis and followed by Western blotting with primary antibody against

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MED12 (Rabbit mAb A300-774A, Bethyl Laboratories, Inc.) at dilution of 1:1000. The

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quantitative analysis of the protein expression was performed by Image J software. The strips

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of the blots were plotted. The basal background was subtracted from the total pixels. The ratio

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of the MED12 versus GAPDH was calculated.

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2.8 Immunofluorescence staining

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A375 or PC9 monoclonal cells were cultured in the confocal dishes 24 h prior to the treatment.

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The cells were washed thrice with the pre-warmed PBS and then were fixed with 4%

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paraformaldehyde in PBS for 15 min at ambient temperature. The cells were washed with PBS

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twice. Next, the cells were permeabilized with 0.1% Triton X-100 in PBS, and washed again in

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PBS. The cells were cultured with MED12 Rabbit mAb (Bethyl Laboratories, Inc.) at a dilution

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of 1:200 in 1% BSA/PBS for 1h at 37 C. Following washing twice with 0.05% Tween 20 in

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PBS, the cells were incubated with rhodamine red labelled goat anti-rabbit IgG (H+L) (Life

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Technologies, Inc.) at a dilution of 1:200 in 1% BSA/PBS for 30 min at ambient temperature

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followed by washing twice with 0.05% Tween 20 in PBS. When staining with F-actin, cells

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were incubated in a 50 µg/mL Alexa488-phalloidin conjugate solution in PBS for 40 min at

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ambient temperature. The cells were washed several times with PBS to remove the unbound

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phalloidin conjugate. The cells were then stained with DAPI in a concentration of 1 µg/mL

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followed with washing with PBS thrice. The cells were examined using a confocal laser

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scanning microscopy (LSM 710; CarlZeiss, Germany). For excitation of DAPI fluorescence, a

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wavelength of 405 nm laser was used. The Alexa488-phalloidin was excited with a wavelength

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of 488 nm laser. The excitation of Rhodamine Red was performed with a helium-neon laser at

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555 nm.

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2.9 Cell viability assay

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Single clone A375 or PC9 cells were plated into 96-well plates at a density of 5×103 cells per

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well. Either PLX or vehicle (DMSO) was added 1 day after plating. PLX was added at the

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following concentrations: 25 nmol/L, 250 nmol/L, 2.5 mol/L, 25 mol/L, and 250 mol/L;

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Gefitinib was added at the following concentrations: 28 nmol/L, 280 nmol/L, 2.8 mol/L, 28

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mol/L, and 280 mol/L. The medium was replaced with freshly prepared medium containing

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drug/vehicle every 2-3 days. After 5 days of drug/vehicle treatment, serum-free medium with

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10% CCK-8 and the cells were incubated for another 2 h. Then, the optical density readings

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were performed using an Ultra Multifunctional Microplate Reader (Tecan, Switzerland) at a

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wavelength of 450 nm. The absorbance was read relative to the blank well. Cell viability (%) in

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each well was calculated by OD450 test/OD450 control×100%. The IC50 was calculated in SPSS.

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2.10 Long-term cell proliferation assays

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Cells were seeded into 6-well plates (2-5×105 cells/well) and cultured both in the absence and

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presence of drugs as indicated. The concentrations of inhibitors were showed in table S1.

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A375-WT or mutated clones cells were cultured in 50 nmol/L PLX-4032, 2.5 µmol/L or 0.5

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µmol/L Selumetibib (AZD6244). PC9-WT or mutated clones were cultured in 100 nmol/L

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Gefitinib (ZD1839) or 50 nM Erlotinib HCl (OSI-744). The cells cultured in the same complete

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medium with an equal volume of DMSO (Sigma Aldrich) were used as control. The cells were

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fixed, stained, and photographed after 14-days of culture. At the endpoints of colony formation

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assays, cells were fixed, stained with crystal violet and photographed. All relevant assays were

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performed independently at least three times.

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2.11 Drug screen

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We screened the drugs from a small library, which included 8 kinds of BRAF inhibitors, 5 kinds

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of TGF-R inhibitors, and 8 kinds of EGFR inhibitors. There are 40 BRAF inhibitor/TGF-R

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inhibitor combinations for drug screening on A375 cells and 40 EGFR inhibitor/TGF-R

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inhibitor combinations for drug screening on PC9 cells. Cells were seeded into 6-well plates (2-

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5×105 cells/well) and incubated for 24 h prior to drug treatments. Next, the cells were cultured 7

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in different combinations as indicated. Briefly, for the single drug screen, the A375 mutated

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monoclone 3 (A375-M3) or PC9 mutated monoclone 3 (PC9-M3) cells were treated with RAF,

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EGFR, or TGF-R inhibitors alone; for the combination screen, the A375-M3 or PC9-M3 cells

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were treated with the combination of RAF/TGF-R inhibitors or EGFR/TGF-R inhibitors. The

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working concentration of each inhibitor was showed in table S1. Cells were either passaged or

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fresh media was added every 2-3 days, lasting at least two weeks. To avoid systematic error,

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each reagent used in this study was newly purchased and had the consistent lot number

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throughout the experimental period. The cell lines were from the same clones and at the same

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generations during the experimental period. All the instruments used in this study maintained

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the consistency during the experimental period. To avoid stochastic errors, the experiments

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were performed with 3 parallel samples for each assay and each experiment was repeated at

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least for 3 times. The cell coverage was calculated with Image J software according to

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guidance of the software supplier.

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3. Results

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3.1 Construction of the MED12KO cell lines

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We designed sgRNAs targeting MED12 and fused its gene into the human codon optimized

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Cas9 (hCas9)-expressing plasmid which also fused with gene of nuclear localization signals

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(NLSs) and green fluorescent protein (GFP) (Fig. 1b). We used Lipofectamine LTX to transfect

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the MED12-targeting CRISPR/hCas9 plasmids into cancer cell lines, including A375 and PC9

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cells. To select plasmids with optimized specificity, we constructed 11 alternative plasmids

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encoding the same hCas9 but different sgRNAs (sgRNA 1-11, Fig. S1 online) for transfection.

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We amplified the genomic DNA isolated from the transfected cells at the targeted site and

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obtained 500 bp products of polymerase chain reaction (PCR). The PCR products were

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analyzed using T7 endonuclease I (T7E1), an endonuclease that specifically cleaves

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heteroduplexes formed by the hybridization of wild-type and mutant DNA sequences. The

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successful gene editing in MED12 gene usually results in two cleavable DNA fragments 8

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through T7E1 assay. By comparing the cleavage efficiency of various sgRNA encoded

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CRISPR/hCas9 plasmids, we found that sgRNA-2 had relatively higher cleavage efficiency in

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both cell lines (8.7% in A375 and 19.1% in PC9 cells, Figure S2 and Figure S3). Hence, we

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used sgRNA-2-encoded plasmid (Fig. 1c) to edit the genes in the following experiments. After

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transfection, the GFP positive cells (Fig. S4 online) were screened and separated by flow

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cytometry (FCM) (Fig. S5 online) and expanded into clones. We next selected and analyzed

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A375 clones resistant to BRAF protein kinase inhibitor vemurafenib (PLX4032) or PC9 clones

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resistant to EGFR inhibitor Gefitinib (ZD1839). Briefly, parts of the individual clones were

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plated in 96 wells and treated with vemurafenib in A375-derived cells or Gefitinib in PC9-

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derived cells for two weeks. The significant drug-resistant clones including 3 A375-derived

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clones (A375-M1, A375-M2, A375-M3) and 3 PC9-derived clones (PC9-M1, PC9-M2, PC9-M3)

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were selected and further analyzed.

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3.2 T7E1 assay confirms the induced mutation in targeting sites of cell clones

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To corroborate the targeted disruption of endogenous MED12 genes in both cell lines by the

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CRISPR/hCas9 system, we also analyzed genomic DNA isolated from transfected cells using

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T7E1 assay. A 670 bp sequence flanking the target site treated by sgRNA2-encoded plasmids

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was amplified by PCR. As expected, the PCR products were cleaved into two fragments

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(about 260 bp and 410 bp) in the MED12 mutated cell clones (Fig. 2). Thus, the T7E1 assay

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confirmed that we have successfully produced mutations in A375 and PC9 cells.

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3.3 Sanger sequencing confirmed targeting indels in MED12 gene

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We performed DNA sequencing analysis on 3 MED12 gene-edited A375 or PC9 cell clones to

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confirm the induction of CRIPSR/hCas9-mediated mutations at the endogenous site. The

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mutated monoclone-3 of A375 cells (A375-M3) showed 11-bp deletions (the base position in

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815..825) from the MED12 gene with 23899 bp (Fig. 3a). There were multiple peaks

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overlapping in PC9 monoclone-3 (PC9-M3) (Fig. 3a) and this is attributed to different

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mutations in each allele. We further determined the result of the gene editing through TA

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cloning technology (Fig. 3b). Sequencing analysis of the PCR products from 5 bacteria clones 9

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revealed that all the clones contained a 11-bp deletions in A375 M3 cells (Figure 3B). For PC9-

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M3 cells, seven bacteria clones revealed that four clones contained 1-bp deletions (genome

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base position in 825) and three clones contained 5-bp deletions precisely at the cleavage site

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(genome base position in 823-827), a pattern that was also observed at the MED12 locus (Fig.

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3b). Thus, we can conclude that two kinds of mutations took place in the PC9-M3 cells. The

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indels in other monoclonal cell lines are showed in Fig. S6-S9 (A375-M1, A375-M2, PC9-M1,

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and PC9-M2). These results indicate that the CRISPR/hCas9 systems have cleaved

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chromosomal DNA at the expected positions.

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3.4 Immunofluorescence and Western blot indicate the down-regulation of MED12

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protein

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We further verified the MED12 mutation on protein expression level. To demonstrate the

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down-regulation of MED12 protein expression of the MED12KO cells, we applied

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immunofluorescence staining to analyze the change. The combinations of MED12 antibody

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and rhodamine red-labelled secondary antibodies were used to show the level and distribution

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of MED12 protein in the cells. As expected, the fluorescence of rhodamine red decreased

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significantly in A375- or PC9-mutated cells, which indicated the decrease of MED12 protein

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(Fig. 4a). Furthermore, Western blot analysis indicated significant down-regulation of MED12

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protein in the mutated cell clones. The statistical data indicated that the MED12 expression of

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the MED-12-mutated A375 clones (A375-M3) decreased about 97% that of the wild type A375

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cells, and the MED12-mutated PC9 clones (PC9-M3) decreases about 99% that of the wild

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type PC9 cells, which was in good agreement with the immunofluorescence analysis (Fig. 4b).

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3.5 MED12KO cell lines were resistant to BRAF or EGFR inhibitors

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To analyze the resistance of MED12KO cells to drugs of interest, we tested the IC50 of the cell

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clones and found that the IC50 of A375-M3 was about 93.9 times larger than that of the wild

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type A375 for BRAF inhibitor Vemurafenib, while the IC50 of PC9-M3 was about 336.4 times

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larger than that of the wild type PC9 for the EGFR inhibitor Gefitinib (Fig. S10 online). Further,

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we cultured both wild type and MED12KO A375 cells in the absence and presence of BRAF 10

1

inhibitor Vermurafenib, Selumetibib (AZD6244) or TGF-R inhibitor LY2157299, while cultured

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both wild type and MED12KO PC9 cells with EGFR inhibitor Gefitinib, Erlotinib HCl (OSI-744),

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or TGF-R inhibitor LY2157299. After 14 days of treatment, the MED12KO cells were

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significantly different when compared with the wild type cells. Vermurafenib or Selumetibib

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alone potently inhibited the proliferation of the wild-type A375 cells, but did not affect the

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proliferation of the MED12KO cells, indicating the successful establishment of the BRAF

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inhibitor-resitant cell lines by the gene editing (Fig. 5a). Gefitinib or Erlotinib HCl alone could

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effectively inhibit the growth of the wild PC9 cells, but could not inbibit the MED12KO PC9 cells,

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demonstrating the successful establishment of EGFR inhibitor-resistant PC9 cell lines.

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LY2157299 had little effect on all cell lines (A375-WT, MED12KO A375, PC9-WT, MED12KO

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PC9) (Fig. 5a, b), which is consistent with previous reports that the suppression of MED12

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results in activation of TGF-R signaling [1, 30].

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3.6 Drug screening identified effective drug combinations for suppression of MED12KO

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cells

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Previous works indicate that MED12 suppression results in multidrug resistance (1,30). In this

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work, we constructed the MED12KO cell lines which bear the property of inherited resistance to

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BRAF (A375) or EGFR (PC9) inhibitors (Fig. 5), demonstrating the successful formation of

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resistance models to perform the drug screening, for their significant down-regulation of

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MED12 protein and potent drug resistance (Fig. 4, Fig. 5). Based on this, we were able to

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evaluate drug efficiencies for effective cancer therapy. Compared with previous work [1], we

21

screened more drug combinations to evaluate their synergistic effects on drug resistant cells

22

(Table S3 online). Interestingly, the MED12KO A375 cells showed resistance to BRAF inhibitors

23

and TGF-R inhibitors individually, which may be attributed to the Tyrosine Kinase mediated

24

signaling pathway (such as BRAF or EGFR) is not blocked completely. However, they were

25

suppressed by the combined administration of BRAF inhibitors and TGF-R inhibitors (Fig. 6a,

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Fig. S11 online). For instance, the combinations of Vemurafenib & LY2157299 showed strong

27

synergy to suppress the proliferation of the MED12KO A375 cells, while each of them

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individually could not suppress the cells. This is the same for other combinations, such as 11

1

Vemurafenib & LY2109761 and Vemurafenib & GW788388 (Fig. 6a, Fig. S11 online). Thus,

2

the combination of BRAF inhibitors with TGF-R inhibitors can successfully reverse the drug

3

resistance of MED12KO A375 cells which are resistant to the individual inhibitors. We thus

4

supposed that other BRAF inhibitors combined with TGF-R inhibitors will also suppress the

5

tumor cell growth. The proof-of-concept drug screening showed that more unreported

6

combinations of BRAF and TGF-R inhibitors, such as Selumetibib & LY2157299, Sorafenib &

7

SB431542, PLX-4720 & LY2109761, SB590885 & SB525334, ZM336372 & GW788388, and

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SCH772984 & SB431542 had strong inhibitory effect in MED12KO A 375 cells (Fig. 6a, Fig.

9

S11 online). Most of the effective combinations that can inhibit MED12KO A 375 cells, to our

10 11

best knowledge, are the first time reported in this study. Inspired by the findings in the synergistic drug combination effects on MED12KO A375 cells,

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we explored if the same synergistic drug effect is true for the MED12KO PC9 cells. When

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Gefitinib (EGFR inhibitor) was combined with LY2157299 (TGF-R inhibitor), they showed

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strong inhibitory effects on the proliferation of the MED12KO PC9 cells (Fig. 6b, Fig. S12 online),

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this synergistic drug effect also worked for unreported combinations of Gefitinib with other

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TGF-R inhibitors including LY2109761, SB431542, SB525334, and GW788388 (Fig. 6b, Fig.

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S12 online). The small-scale drug screening showed that more unreported combinations of

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EGFR inhibitor & TGF-R inhibitor, such as Erlotinib HCl & LY2109761, PD168393 &

19

SB431542, AG-490 & GW788388, Canertinib & GW788388, AZD8931 & SB525334, and

20

Afatinib & LY2157299) showed strong inhibitory effect in the MED12KO PC9 cells.

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Unexpectedly, by this screening, we also found that the BRAF inhibitors (PLX-4032, Sorafenib

22

Tosylate, or SCH772984), combined with TGF-R inhibitors, were active in the suppression of

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MED12KO PC9 cell clones (Fig. S13, S14 online). To our knowledge, these effective drug

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combinations for treating MED12KO PC9 cells are reported for the first time in this study.

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Hence, with the CRISPR/hCas9-edited cell clones, we found new solutions for combating

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drug-resistant cancers by combining drugs which are resisted individually by the cells. Our

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study provides alternatives for clinical treatment of multidrug-resistant tumors. Also, the small-

12

1

scale screening platform in this study is useful for new drug discovery and validation, such as

2

PD168393 and CNX-2006 evaluated in our research.

3

4. Discussion

4

Cas9-mediated genome editing has enabled accelerated generation of drug-resistant cell

5

models beyond traditional drug induction by gradient dosage induction, for the previous

6

methods will spend months to years to yield a stable cell line, which will delay the drug test

7

with great probability. Moreover, the previous methods are difficult to establish a cell line with

8

unified evaluation criterion, which is subject to the drug dosage, induction time, and heritable

9

traits. In the view of technology, the CRISPR/Cas9 system is convenient in the construction of

10

gene-edited cell line, for CRISPR/Cas9 contains only two components: i) Cas9 endonuclease,

11

ii) single guide RNA (sgRNA), which is convenient for construction and delivery of the editing

12

toolbox. Compared with the conventional technology, such as siRNA or shRNA, the CRISPR-

13

Cas9 system can inactivate genes at the DNA level and the resulting phenotypes showing

14

complete loss of gene function. Furthermore, the CRISPR-Cas9 technology enables functional

15

interrogation at the introns coding sequence, which is unavailable in manner of RNAi-induced

16

silence. In the view of the time for cell line construction by CRISPR/Cas9 system, the whole

17

process including transfection, monoclonal cell growth, and mutational identification usually

18

could be completed within 3 weeks. Thus, CRISPR/Cas9 system is an excellent tool for editing

19

inheritable cell properties of interest. Herein we presented an exemplary case of generating

20

drug-resistance models through CRISPR/hCas9 system targeting MED12 in cancer cell lines.

21

A major advantage of the drug-resistant suppressor gene MED12 is its ability to conveniently

22

create the required drug-resistant cell models in various cancer cells, for use in drug screening

23

or in vivo tumor modelling. By sequencing of single-cell derived clones, specific

24

immunostaining, and Western blot, we confirmed that the MED12 gene was successfully

25

mutated and the MED12 protein was down-regulated in A375 cells and PC9 cells which were

26

resistant to BRAF and EGFR inhibitors, respectively. This strategy could be extended to the

27

establishment of multi-mutation cell models and can be further combined with microfluidics to

28

realize the high-throughput, large-scale drug screening. 13

1

The MED12KO drug-resistant cell models provide excellent platform for large-scale drug

2

screening. For example, we not only confirmed that the previously reported combination of

3

targeted drugs (e.g. Vemurafenib & LY2157299) can reverse the drug resistance in tumor cells,

4

but found that many unreported combinations were effective in drug-resistant cells (Figure 6A,

5

B). Furthermore, by drug screening, we also found that not all of the Tyrosine Kinase inbihitor

6

+ TGF-R inhibitor combinations are effective (Figure 6). It is encouraging to find these

7

unreported drugs or drug-combinations for their great potential to cure real cancer patients

8

who suffering multi-drug resistance.

9

MED12 is a component of the MEDIATOR transcriptional mediator complex that mediates

10

the basal transcription machinery and its upstream activators. In the TGF-βR signalling

11

pathway, TGF-βR activates Smads (29), which translocate to cell nucleus and regulate the

12

expression of TGF-β targeted genes (Fig. S15 online). MED12 negatively regulates TGF-β

13

signalling by inhibiting the glycosylation of immature forms of TGF-βR. Thus, the loss of

14

MED12 results in activation of TGF-R signalling, which was reported to be necessary and

15

sufficient for drug resistance. The loss of MED12 also accompanies activating TGF-βR-

16

induced collateral signalling through the RAS-RAF-MEK-ERK pathway, which causes

17

resistance to EGFR and BRAF inhibitors in oncogene-driven tumors (Fig. S15) [1, 29]. Wild-

18

type A375 cell line is BRAF-mutated and is sensitive to BRAF inhibitors such as Vemurafenib.

19

In this study, we mutated MED-12 of A375 cells by the CRISPR/hCas9 system, which will

20

induce the acquisition of drug resistance to BRAF inhibitors. At the same time, the MED-12

21

mutation also activated TGF-βR signalling pathway and the inhibition of TGF-R signalling can

22

restore drug responsiveness in MED12KO cells [1]. Thus, the combination of the TGF-β

23

inhibitors and BRAF inhibitors showed strong synergy in suppressing the acquired drug

24

resistance to BRAF inhibitors in the MED12KO A375 cells. Similarly, wild-type PC9 cell line is

25

EGFR-mutated and is sensitive to EGFR inhibitors such as Gefitinib. We mutated MED-12 of

26

PC9 cells by the CRISPR/hCas9 system, which will induce the acquisition of drug resistance to

27

EGFR inhibitors. The MED-12 mutation also activated TGF-βR signalling pathway and the

28

inhibition of which will restore drug responsiveness in MED12KO PC9 cells. Thus, the 14

1

combination of the TGF-β inhibitor and EGFR inhibitors could synergistically suppress the

2

acquired drug resistance to EGFR inhibitors in MED12KO PC9 cells.

3 4

5. Conclusion

5

In this study, we screened drugs with the gene-edited cells after they became fully established

6

cell lines within 3 weeks, much less than the time for construction of animal models and

7

resistant cell lines prepared by gradient dosage induction or patient derivation. Furthermore,

8

the mutations can be genomically stable during passaging. When this technology is applied in

9

editing potential resistance-induced genes, such as neurofibromin 1 (NF1), neurofibromin 2

10

(NF2), Cullin 3 E3 ligase (CUL3), Transcriptional Adaptor 1 (TADA1), Transcriptional Adaptor

11

2B (TADA2B genes), kinesin-5, and hypoxanthine phosphoribosyltransferase (HPRT1), we

12

can obtain the drug resistant-cell clones on purpose. We believe that such functional screens

13

performed on cells induced from genome editing by CRISPR/Cas9 might reform the choice of

14

experimental therapies, advancing toward a future of truly diversified and accurate cancer

15

therapy.

16

Conflict of interest

17

The authors declare that they have no conflict of interest.

18

Acknowledgments

19

This work was supported by NSFC (81361140345, 31470911, and 81700382), CAS

20

(XDA09030305, XDA09030307), CAS/SAFEA International Partnership Program for Creative

21

Research Teams.

22

Appendix A. Supplementary data

23

Appendix data associated with this article can be found in the online version, at xxxxx.

24

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3 4 5

TABLE AND FIGURES LEGENDS

6

Figure 1. Schematic diagram of the working mechanism of the induction, selection and

7

separation of drug-resistant cancer cell lines. (a) Schematic of the structure and working

8

process of the CRISPR/hCas9 system; (b) Plasmid map of the CRISPR/hCas9 system; (c)

9

The location of the sgRNA targeted locus of human MED12 indicated by blue lines with the

10

corresponding PAM. The small caps represented the intron sequence.

11 12

Figure 2. T7E1 assay for CRISPR/Cas9-mediated indels. (a) Mutated A375 cell clones; (b)

13

Mutated PC9 cell clones. Mutations resulted from cleavage of the targeting sites were

14

detected through PCR amplification of a 670 bp amplicon flanking the target sequence, re-

15

annealing of the PCR product and selective digestion of mismatched heteroduplex fragments.

16

+ indicates presence of the PCR product; - indicates absence of the PCR product.

17 18

Figure 3. Targeted indel mutations induced by the CRISPR/hCas9 system at the MED12 gene

19

in A375 and PC9 cell lines. (a) Sanger sequencing of wild type A375 (A375-WT) and mutated

20

A375 monoclone-3 (A375-M3), wild type PC9 (A375-WT) and mutated PC9 monoclone-3

21

(PC9-M3) (The indels appear behind the underlined sequence); (b) TA cloning analysis of

22

A375-M3 and PC9-M3 cells (Deletions are shown as black dots; –, deletion).

23 24

Figure 4. Immunofluorescence and Western blot analysis of the expression of MED12 protein

25

in MED12KO cell monoclones. (a) Immunofluorescence analysis demonstrates MED12 protein

26

levels in the MED12KO cells. The MED12KO cells were stained with Alexa 488-Phalloidin, 18

1

Rabbit anti-MED12 Antibody/Rhodamine red-labeled goat anti-rabbit IgG (H+L), and DAPI.

2

Excitation wavelength: DAPI, 405 nm; Alexa-Phalloidin, 488 nm; rhodamine red, 555 nm. (b)

3

Western blot analysis indicates MED12 protein levels in the MED12KO cells.

4 5

Figure 5. The MED12KO confers multidrug resistance to BRAF inhibitors (mutated A375 clones)

6

or EGFR (mutated PC9 clones) inhibitors. (a) A375-WT or MED12KO A375 cells were cultured

7

in 2.5 µmol/L Vemurafenib, 0.5 µmol/L Selumetibib, or 5 µmol/L LY2157299. The cells were

8

fixed, stained, and photographed after the drug treatments for 14 days. (b) PC9-WT or

9

MED12KO PC9 cells were cultured in 100 nmol/L Gefitinib, 50 nmol/L Erlotinib HCl, or 5 µmol/L

10

LY2157299. The cells were fixed, stained, and photographed after the drug treatments for 14

11

days. NC, the cells treated with no drugs.

12 13

Figure 6. Comparative analysis of BRAF or EGFR inhibitor-resistant clones treated with

14

various inhibitor combinations. (a) A375-M3 cells were treated with various BRAF and TGF-R

15

inhibitor combinations. (b) PC9-M3 cells were treated with various EGFR and TGF- inhibitor

16

combinations. The cells were fixed, stained, and photographed after 14 days of drug treatment.

17

Colors indicate different drug efficiencies: no significant inhibitory effect (white, cell coverage

18

≥80%), mild inhibitory effect (yellow, 60%≤cell coverage＜80% ), moderate effect (orange,

19

30%≤cell coverage＜60% ), and strong inhibitory effect (red, cell coverage＜30%).

20 21 22 23 24

19

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Figure 1

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Figure 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Figure 3 21

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Figure 4

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Figure 5

4 5 6 7

Figure 6

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