Published OnlineFirst September 27, 2017; DOI: 10.1158/1535-7163.MCT-17-0130 Cancer Biology and Translational Studies Molecular Cancer Therapeutics CRISPR Genome-Wide Screening Identifies Dependence on the Proteasome Subunit PSMC6 for Bortezomib Sensitivity in Multiple Myeloma Chang-Xin Shi1, K. Martin Kortum€ 1,2, Yuan Xiao Zhu1, Laura A. Bruins1, Patrick Jedlowski1, Patrick G. Votruba3, Moulun Luo4, Robert A. Stewart1, Jonathan Ahmann1, Esteban Braggio1, and A. Keith Stewart1,5 Abstract Bortezomib is highly effective in the treatment of multiple validation. Of these 20 targets, the proteasome regulatory myeloma; however, emergent drug resistance is common. Con- subunit PSMC6 was the only gene validated to reproducibly sequently, we employed CRISPR targeting 19,052 human genes confer bortezomib resistance. We confirmed that inhibition of to identify unbiased targets that contribute to bortezomib chymotrypsin-like proteasome activity by bortezomib was sig- resistance. Specifically, we engineered an RPMI8226 multiple nificantly reduced in cells lacking PSMC6. We individually myeloma cell line to express Cas9 infected by lentiviral vector investigated other members of the PSMC group (PSMC1 to CRISPR library and cultured derived cells in doses of bortezo- 5) and found that deficiency in each of those subunits also mib lethal to parental cells. Sequencing was performed on imparts bortezomib resistance. We found 36 mutations in 19S surviving cells to identify inactivated genes responsible for drug proteasome subunits out of 895 patients in the IA10 release resistance. From two independent whole-genome screens, we of the CoMMpass study (https://themmrf.org). Our findings selected 31 candidate genes and constructed a second CRISPR demonstrate that the PSMC6 subunit is the most prominent sgRNA library, specifically targeting each of these 31 genes with target required for bortezomib sensitivity in multiple myeloma four sgRNAs. After secondary screening for bortezomib resis- cells and should be examined in drug-refractory populations. tance, the top 20 "resistance" genes were selected for individual Mol Cancer Ther; 16(12); 2862–70. Ó2017 AACR. Introduction have been identified frequently in multiple myeloma cell line but are not found in newly diagnosed patients (17, 18), while XBP1 Multiple myeloma is a plasma cell malignancy, which has seen mutations have also been seen but are also found in less than 1% significant improvements in outcomes following the introduction of newly diagnosed multiple myeloma cases. Indeed, the muta- of proteasome inhibitors (PI), specifically bortezomib, carfilzo- tion prevalence of these genes combined is only 2% in supposedly mib, and ixazomib into routine clinical practice (1–6). PIs used in PI-refractory patients (19). XBP1 knockdown experiments have combination with other compounds achieve initial response in shown correlation with bortezomib resistance (14, 20). Recently, nearly 90% to 100% of patients (7). Despite the high initial Acosta-Alvear and colleagues reported an shRNA screening and response rates, almost all patients develop drug resistance over found that genetic depletion of the vast majority of the 19S time and eventually becoming refractory to PIs. The underlying proteasomal regulator subunits, including PSMC1 and 6, con- mechanisms of such PI resistance are still incompletely under- ferred a marked resistance to PIs (21). In a separate study, Tsvetkov stood (8). The genes and pathways most commonly associated and colleagues using gene-trap screening identified that with bortezomib resistance are PSMB5 (9–13), the ERN1–XBP1 compromising the 19S proteasome including PSMC2 - 6 pro- pathway (14, 15) as well as the IGF-1 axis (16). PSBM5 mutations tected cells from proteasome inhibitors (22). Sheffer and collea- gues (23) using CRISPR technology also identified loss of PSMC6 as inducing resistance, although to date, results are only published 1Department of Hematology, Mayo Clinic in Arizona, Scottsdale, Arizona. in abstract form. 2Department of Hematology, University Hospital Wurzburg,€ Wurzburg,€ Ger- CRISPR-Cas9 knockout is a precise and efficient genome-edit- many. 3Bioinformatics Systems, Mayo Clinic in Arizona, Scottsdale, Arizona. ing technology (24, 25). This simple gene-engineering tool con- 4Mayo/ASU Center for Metabolic and Vascular Biology, Arizona State University, 5 sists of a 20-base pair single-guide RNA (sgRNA) and a Cas9 Scottsdale, Arizona. Center for Individualized Medicine, Mayo Clinic, Rochester, protein, which can specifically recognize and cut the sgRNA- Minnesota. binding region (24). Recently, a genome-scale CRISPR-Cas9 Note: Supplementary data for this article are available at Molecular Cancer library was developed (26, 27) including 19,052 human genes, Therapeutics Online (http://mct.aacrjournals.org/). each gene being targeted with six sgRNAs. We used this genome- Corresponding Author: A. Keith Stewart, Mayo Clinic in Arizona, 13400 East wide library to identify genes that are potentially associated Shea Blvd, Scottsdale, AZ 85259. Phone: 507-284-5511; Fax: 507-293-2379; with bortezomib resistance. Our screening identified absence of E-mail: [email protected] the proteasome-regulatory subunit PSMC6 as the gene most doi: 10.1158/1535-7163.MCT-17-0130 consistently conferring bortezomib resistance in human multiple Ó2017 American Association for Cancer Research. myeloma cells. 2862 Mol Cancer Ther; 16(12) December 2017 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst September 27, 2017; DOI: 10.1158/1535-7163.MCT-17-0130 PSMC6 Deficiency Causing Bortezomib Resistance in MM Materials and Methods hour later, 3 mL of fresh medium was added. Next morning, the medium was changed to 6 mL fresh medium. Cell lines and plasmids Human multiple myeloma cell lines RPMI8226, FR4, JJN3, CRISPR sgRNA screening KMS11, KMS18, MM1.R, OPM2, and U266 were maintained in RPMI8226Cas9 cells were infected with the CRISPR library. The m RPMI1640 with 5% FBS, 100 U/mL penicillin and 100 g/mL infected cells were treated with bortezomib at 7 nmol/L for 7 days streptomycin. All multiple myeloma cells were from Dr. Leif (changed medium with 7 nmol/L bortezomib at days 2 and 4). Bergsagel's laboratory. These cells were tested negative for myco- Residual cells surviving bortezomib treatment were expanded fi plasma and validated by ngerprinting developed by Dr. Bergsa- over 15 days without bortezomib treatment and then subjected gel's laboratory (unpublished data). All experiments were per- to DNA sequencing formed on multiple myeloma cells less than 20 passages after thawing from liquid nitrogen. Plasmids pCDHpuroCr expressing DNA sequencing and data analysis selection marker puromycin and pCDHgfpCr expressing a green We screened the abundance of sgRNA targeting the approxi- fl uorescent protein selection marker (GFP) for cloning sgRNAs mately 19K genes in cells showing resistance to bortezomib by were derived from pCDH-CMV-MCS-EF1-Puro and pCDH-CMV- next-generation sequencing (Ion Torrent PGM, Thermo Fisher MCS-EF1-copGFP (SBI), respectively, by cloning the sgRNA Scientific). An in-house algorithm was used to clean the gene- expression cassette from plasmid lentiGuide-Puro (Addgene specific sgRNA by eliminating the surrounding adapters #52963; ref. 28). Plasmid pCDHBlastCas9HA was derived from sequences. A script written in the Perl programming language lentiCas9-blast (#52962; ref. 28) by cloning Cas9 gene (Flag tag was used to identify all instances of the "tag" sequence was replaced with HA tag) into pCDHBlast under the control of AAAGGACGAAACACC? that occur in the fasta files. The "?" can CMV promoter, which was constructed by Shi and colleagues denote A, C, G, or T. The script also captures the next "target" (C.X. Shi; unpublished observations). sequence of 20 nucleotides downstream from the tag. The targets are stored in a hash table, which counts the frequency with which Small molecules each target appears adjacent to the tag. The gene-specific sgRNA Tunicamycin was from Abcam (cat. Ab120296) and its struc- were quantified, and the 100 most abundant hits were subse- ture was published by Takatsuki and colleagues (29). Stauros- quently blasted against the human genome. porine was from ApexBio (cat. A8192) and its structure was published by Furusaki and colleagues (30, 31). Bortezomib was Proteasome chymotrypsin-like activity, caspase-3 and 7 activity from LC Laboratories (cat. B-1408). Carfilzomib was from MCE assay, and 26S proteasome assay Medchem Express (cat. HY10455). Dexamethasone (cat. D4902- Proteasome chymotrypsin-like activity was measured by cell- 25MG) and melphalan (cat M2011-100MG) were from Sigma. based assay, which was performed according to the manufac- fl Utilization of sgRNA turer's description (cat no. G8660, Promega). Brie y, 2,000 cells The GeCKO V2 library (Addgene #100000049; ref. 28) was were plated into 384-well plates. Cells were treated with different expanded by transforming the library into DH10B cells by elec- concentration (3, 4, 5, 6, 7, 8, and 9 nmol/L) of bortezomib for trophoresis with a total of 2.5 Â 107 clones produced. A sgRNA 24 hours. Then, proteasome-Glo reagent was added into the cells. library for the second round screening was created by cloning Luciferase activity was assayed by luciferase reader Cytation 3 sgRNAs synthesized by IDT technology individually, mixed man- imaging reader. Caspase-3 and 7 activity assay was similar to ually, and finally cloned into the vector pCDHgfpCr.