Genome-wide CRISPR-Cas9 screens identify mechanisms of BET bromodomain inhibitor sensitivity

Abstract

Summary BET bromodomain inhibitors hold promise as therapeutic agents in diverse indications, but their clinical progression has been challenging and none have received regulatory approval. Early clinical trials in cancer have shown heterogeneous clinical responses, development of resistance, and adverse events. Increased understanding of their mechanism(s) of action and identification of biomarkers are needed to identify appropriate indication(s) and achieve efficacious dosing. Using genome-wide CRISPR-Cas9 screens at different concentrations, we report molecular mechanisms defining cellular responses to BET inhibitors, some of which appear specific to a single compound concentration. We identify multiple transcriptional regulators and mTOR pathway members as key determinants of JQ1 sensitivity and two Ca 2+ /Mn 2+ transporters, ATP2C1 and TMEM165, as key determinants of JQ1 resistance. Our study reveals new molecular mediators of BET bromodomain inhibitor effects, suggests the involvement of manganese, and provides a rich resource for discovery of biomarkers and targets for combination therapies.

A novel HERC4-dependent glue degrader targeting STING

“…The integrated gRNA sequences were PCR amplified using primers specific to the integrated lentiviral vector sequence and sequenced using the Illumina sequencing technology. Illumina library construction was performed as previously described 47 . Briefly, a total of 96 µg of DNA per sample was split into 24 PCR reactions each with the volume of 100 µl, containing a final concentration of 0.5 µM of each of the following primers (Integrated DNA Technologies, 5644 5 ‘-AATGATACGGCGACCACCGAGATCTACACTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGA-3 ‘ and INDEX 5 ‘-CAAGCAGAAGACGGCATACGAGATXXXXXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-3 ‘, where the Xs denote a 10 base PCR-sample specific barcode used for data demultiplexing following sequencing).…”

“…Sequencing analysis was performed as previously described 47 . In short, raw sequencing reads were converted to FASTQ format using bcl2fastq2 (version 2.17.1.14, retrieved from http://support.illumina.com/downloads/bcl2fastq-conversion-software-v217.html); trimmed to the guide sequence with the fastx-toolkit (version 0.0.13, retrieved from http://hannonlab.cshl.edu/fastx_toolkit/index.html) and aligned to the sgRNA sequences in the library using bowtie 49 with no mismatches allowed.…”

“…For genome-wide CRISPR knockout screening, THP1 cells constitutively expressing Cas9 were generated by lentiviral delivery of Cas9 protein gene in pNGx-LV-c004 and selected with 5 µg/ml blasticidin S HCl (Thermo Fisher) as previously described 46 . sgRNA library targeting 18,360 protein-coding genes with 5 sgRNA/gene, as previously described 47 . The sgRNA library was packaged into lentiviral particles using HEK293T cells as previously described 47,48 .…”

“…sgRNA library targeting 18,360 protein-coding genes with 5 sgRNA/gene, as previously described 47 . The sgRNA library was packaged into lentiviral particles using HEK293T cells as previously described 47,48 . Briefly, 2.1x10 7 HEK293T cells were seeded into CellSTACK (Corning) cell culture chambers and transfected with the sgRNA library plasmid mix together with lentiviral packaging mix (Cellecta, containing psPAX2 and pMD2 plasmids that encode Gag/Pol and VSV-G, respectively) using Trans-IT transfection reagent (Mirus) 24 hours after seeding.…”

“…The integrated gRNA sequences were PCR amplified using primers specific to the integrated lentiviral vector sequence and sequenced using the Illumina sequencing technology. Illumina library construction was performed as previously described 47 . Briefly, a total of 96 µg of DNA per sample was split into 24 PCR reactions each with the volume of 100 µl, containing a final concentration of 0.5 µM of each of the following primers (Integrated DNA Technologies, 5644 PCR samples were purified using 1.8x SPRI AMPure XL beads (Beckman Coulter) according to manufacturer's recommended protocol and the qPCR products quantified using primers specific to the Illumina sequences using standard methods.…”