CRISPR Genome-Wide Screening Identifies Dependence on the Proteasome Subunit PSMC6 for Bortezomib Sensitivity in Multiple Myeloma

Abstract

Bortezomib is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. Consequently, we employed CRISPR targeting 19,052 human genes to identify unbiased targets that contribute to bortezomib resistance. Specifically, we engineered an RPMI8226 multiple myeloma cell line to express Cas9 infected by lentiviral vector CRISPR library and cultured derived cells in doses of bortezomib lethal to parental cells. Sequencing was performed on surviving cells to identify inactivated genes responsible for drug resistance. From two independent whole-genome screens, we selected 31 candidate genes and constructed a second CRISPR sgRNA library, specifically targeting each of these 31 genes with four sgRNAs. After secondary screening for bortezomib resistance, the top 20 "resistance" genes were selected for individual validation. Of these 20 targets, the proteasome regulatory subunit PSMC6 was the only gene validated to reproducibly confer bortezomib resistance. We confirmed that inhibition of chymotrypsin-like proteasome activity by bortezomib was significantly reduced in cells lacking PSMC6. We individually investigated other members of the PSMC group (PSMC1 to 5) and found that deficiency in each of those subunits also imparts bortezomib resistance. We found 36 mutations in 19S proteasome subunits out of 895 patients in the IA10 release of the CoMMpass study (https://themmrf.org). Our findings demonstrate that the PSMC6 subunit is the most prominent target required for bortezomib sensitivity in multiple myeloma cells and should be examined in drug-refractory populations. Mol Cancer Ther; 16(12); 2862-70. ©2017 AACR.

Figure 1

Infecetion efficiency of lentivirus and targeting efficiency of sgRNA against ERN1 in MM cells. (A) Lenti virus infection efficiency to MM cells detected by flow cytometry. (B). Cas9 with HA tag was detected by Western Blot with HA antibody. (C) ERN1 knocking out efficiency by CRISPR technology in MM cells. Cr=ERN1 sgRNA. NS= non-specific sgRNA control.

Figure 2

PSMC6 was the only gene producing resistance to BTZ after knocking out by CRISPR sgRNA. (A) Schematic diagram for genome wide sgRNA screen. (B) Top gene list for first round screen. (C) Top gene list for second screen. (D) Three independent experiments (MTT assay) of RPMI8226 cells knocked out by a mix of four PSMC6 sgRNAs (A+B+C+D). (E) PSMC6 knock out by three independent sgRNAs produces resistance to bortezomib (BTZ).

Figure 3

PSMC6 was validated as a BTZ-sensitive gene. (A) A pair of sgRNAs targeting intron region flanking Exon 1. (B) PSMC6 exon 1 deletion was validated by PCR for two cloned cells. (C) PSMC6 expression levels detected by quantitative PCR. (D) Sanger sequencing analyze cloned RPMI8226 cells deleted PSMC6. PCR amplified PSMC6 exon 1 region and cloned into T-vector. Then purified plasmid was analyzed by Sanger sequencing. (E) MTT assay for cloned RPMI8226 cells (clone A and B) deleted PSMC6. The cells were treated with BTZ for 72 hours. Control=RPMI8226 cells infected with lenti-vector with non-specific sgRNA. (F) Over expression of PSMC6 with HA tag in cloned PSMC6 deficiency cells. (G) Over expression of PSMC6 wild type or with HA tag rescues the phenotype of PSMC6 deficiency. NS= non-specific sgRNA control. bortezomib (BTZ). Vector=empty lentivirus vector. PSMC6HA=lentivirus expressing PSMC6 with HA tag.

Figure 4

MM cells without PSMC6 have no significant difference in sensitivity to others chemicals tested. Cloned RPMI8226 deleted for PSMC6 by CRISPR sgRNA (PSMC6Cr5+6) were tested for response to different chemicals assayed by MTT (72 hours treatment). (A) Dexmethasone. (B) Melphalan. (C) Tunicamycin. (D) Staurosporine.

Figure 5

MM cells without PSMC6 showed resistance to bortezomib (BTZ) induced apoptosis. (A) Cell-based, chymotrypsin-like activity assay on cell with or without PSMC6 without treatment with BTZ. (B) Cell-based, chymotrypsin-like activity assay. The cells were treated with BTZ for 24 hours. (C) Caspase 3/7 activity detected by luminescent assay. The cells were treated with BTZ for 24 hours. (D) Caspase 8 p55/53 and cleaved caspase 8 p43/41 detection by Western blot. The cells were treated with BTZ for 24 hours.

Figure 6

Expression of SQTM1/p62, VCP/p97, and UFD1 in RPMI8226 and KMS 11 cells with or without PSMC6 treated with BTZ. Cloned RPMI8226 or KMS11 cells (clone A and B) deleted PSMC6 and Control cells infected with non-specific sgRNA (NS) treated with BTZ at 10 or 50 nM for 4 hours. The proteins expression was detected by western blot and analyzed by ImageJ software (numbers below the Figure 6 blots are normalized to GAPDH and untreated control).

Figure 7

Deficiency of PSMC1-PSMC5 also results in resistance to bortezomib (BTZ) treatment. The cells were infected with lentivirus including three sgRNAs for each gene. The infected cells were treated with BTZ at concentration of 4, 5, and 6 nM 72 hours after infection. The cells that survived to the highest concentration for drug treatment were tested for response to BTZ assayed by MTT (72 hours treatment). (A) RPMI8226 cells. (B) KMS11 cells.