Genome-wide Clustered Regularly Interspaced Short Palindromic Repeats Research Articles

Imipridone Anticancer Compounds Ectopically Activate the ClpP Protease and Represent a New Scaffold for Antibiotic Development.

Systematic genetic interaction profiles can reveal the mechanisms-of-action of bioactive compounds. The imipridone ONC201, which is currently in cancer clinical trials, has been ascribed a variety of different targets. To investigate the genetic dependencies of imipridone action, we screened a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) knockout library in the presence of either ONC201 or its more potent analog ONC212. Loss of the mitochondrial matrix protease CLPP or the mitochondrial intermediate peptidase MIPEP conferred strong resistance to both compounds. Biochemical and surrogate genetic assays showed that impridones directly activate CLPP and that MIPEP is necessary for proteolytic maturation of CLPP into a catalytically competent form. Quantitative proteomic analysis of cells treated with ONC212 revealed degradation of many mitochondrial as well as nonmitochondrial proteins. Prompted by the conservation of ClpP from bacteria to humans, we found that the imipridones also activate ClpP from Escherichia coli, Bacillus subtilis, and Staphylococcus aureus in biochemical and genetic assays. ONC212 and acyldepsipeptide-4 (ADEP4), a known activator of bacterial ClpP, caused similar proteome-wide degradation profiles in S. aureus ONC212 suppressed the proliferation of a number of Gram-positive (S. aureus, B. subtilis, and Enterococcus faecium) and Gram-negative species (E. coli and Neisseria gonorrhoeae). Moreover, ONC212 enhanced the ability of rifampin to eradicate antibiotic-tolerant S. aureus persister cells. These results reveal the genetic dependencies of imipridone action in human cells and identify the imipridone scaffold as a new entry point for antibiotic development.

Genome-Wide CRISPR Screening Identifies JAK1 Deficiency as a Mechanism of T-Cell Resistance.

Somatic gene mutations play a critical role in immune evasion by tumors. However, there is limited information on genes that confer immunotherapy resistance in melanoma. To answer this question, we established a whole-genome knockout B16/ovalbumin cell line by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease technology, and determined by in vivo adoptive OT-I T-cell transfer and an in vitro OT-I T-cell-killing assay that Janus kinase (JAK)1 deficiency mediates T-cell resistance via a two-step mechanism. Loss of JAK1 reduced JAK-Signal transducer and activator of transcription signaling in tumor cells—resulting in tumor resistance to the T-cell effector molecule interferon—and suppressed T-cell activation by impairing antigen presentation. These findings provide a novel method for exploring immunotherapy resistance in cancer and identify JAK1 as potential therapeutic target for melanoma treatment.

Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation.

Glycosylation is a fundamental modification of proteins and membrane lipids. Toxins that utilize glycans as their receptors have served as powerful tools to identify key players in glycosylation processes. Here, we carried out Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9–mediated genome-wide loss-of-function screens using two related bacterial toxins, Shiga-like toxins (Stxs) 1 and 2, which use a specific glycolipid, globotriaosylceramide (Gb3), as receptors, and the plant toxin ricin, which recognizes a broad range of glycans. The Stxs screens identified major glycosyltransferases (GTs) and transporters involved in Gb3 biosynthesis, while the ricin screen identified GTs and transporters involved in N-linked protein glycosylation and fucosylation. The screens also identified lysosomal-associated protein transmembrane 4 alpha (LAPTM4A), a poorly characterized four-pass membrane protein, as a factor specifically required for Stxs. Mass spectrometry analysis of glycolipids and their precursors demonstrates that LAPTM4A knockout (KO) cells lack Gb3 biosynthesis. This requirement of LAPTM4A for Gb3 synthesis is not shared by its homolog lysosomal-associated protein transmembrane 4 beta (LAPTM4B), and switching the domains between them determined that the second luminal domain of LAPTM4A is required, potentially acting as a specific “activator” for the GT that synthesizes Gb3. These screens also revealed two Golgi proteins, Transmembrane protein 165 (TMEM165) and Transmembrane 9 superfamily member 2 (TM9SF2), as shared factors required for both Stxs and ricin. TMEM165 KO and TM9SF2 KO cells both showed a reduction in not only Gb3 but also other glycosphingolipids, suggesting that they are required for maintaining proper levels of glycosylation in general in the Golgi. In addition, TM9SF2 KO cells also showed defective endosomal trafficking. These studies reveal key Golgi proteins critical for regulating glycosylation and glycolipid synthesis and provide novel therapeutic targets for blocking Stxs and ricin toxicity.

Genome-wide CRISPR screen reveals SGOL1 as a druggable target of sorafenib-treated hepatocellular carcinoma

The genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screen is a powerful tool used to identify therapeutic targets that can be harnessed for cancer treatment. This study aimed to assess the efficacy of genome-wide CRISPR screening to identify druggable genes associated with sorafenib-treated hepatocellular carcinoma (HCC). A genome-scale CRISPR knockout (GeCKO v2) library containing 123,411 single guide RNAs (sgRNAs) was used to identify loss-of-function mutations conferring sorafenib resistance upon HCC cells. Resistance gene screens identified SGOL1 as an indicator of prognosis of patients treated with sorafenib. Of the 19,050 genes tested, the knockout screen identified inhibition of SGOL1 expression as the most-effective genetic suppressor of sorafenib activity. Analysis of the survival of 210 patients with HCC after hepatic resection revealed that high SGOL1 expression shortened overall survival (P = 0.021). Further, matched pairs analysis of the TCGA database revealed that SGOL1 is differentially expressed. When we used a lentivirus Cas9 vector to determine the effect of targeting SGOL1 with a specific sgRNA in HCC cells, we found that SGOL1 expression was efficiently inhibited and that loss of SGOL1 was associated with sorafenib resistance. Further, loss of SGOL1 from HCC cell decreased the cytotoxicity of sorafenib in vivo. We conclude that the CRISPR screen is a powerful tool for therapeutic target analysis of sorafenib treatment and that SGOL1 serves as a druggable target for HCC treated with sorafenib and an indicator of prognosis.

Next-Generation Sequencing of Genome-Wide CRISPR Screens.

Genome-wide functional genomic screens utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system have proven to be a powerful tool for systematic genomic perturbation in mammalian cells and provide an alternative to previous screens utilizing RNA interference technology. The wide availability of these libraries through public plasmid repositories as well as the decreasing cost and speed in quantifying these screens using high-throughput next-generation sequencing (NGS) allows for the adoption of the technology in a variety of laboratories interested in diverse biologic questions. Here, we describe the protocol to generate next-generation sequencing libraries from genome-wide CRISPR genomic screens.

In Vivo Genome-Wide Crispr Library Screen in a Xenograft Mouse Model of Tumor Growth and Metastasis of Multiple Myeloma

Introduction: Recent data show that Multiple Myeloma (MM) always progresses from a precursor state (monoclonal gammopathy of undetermined significance [MGUS]/smoldering multiple myeloma [SMM]) to overt MM indicating that there is continuous dissemination/clonal evolution of tumor cells from the original stages of tumor development to the time of clinical presentation. A major challenge in understanding the progression and metastasis of MM is to distinguish alterations driving the tumor growth and evolution from passenger mutations. Genetic screens are powerful tools for assaying phenotypes and identifying causal genes in various hallmarks of cancer progression. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged as a powerful technology to efficiently and simultaneously perform genome editing of multiple genes. Here we report a genome-wide CRISPR/Cas9-mediated loss of function screen in a xenograft mouse model to investigate the essential drivers of tumor growth and metastasis in MM.Methods: Lentiviral particles from 2 subpools of a human sgRNA library (Avana), each containing 1 sgRNA per gene were introduced into MM1.S (Cas9+/GFP+/Luc+) cell line with the pre-determined amount of virus to achieve 30-50% infection efficiency, corresponding to a multiplicity of infection (MOI) of ~0.5-1. Cells were selected with puromycin for 5-7 days following infection to remove uninfected cells. Selected cells were injected subcutaneously into SCID-Beige mice on both flanks. Genomic DNA from pre-transplantation cells, early primary tumors (~3 weeks post tumor cell injection), late stage primary tumors and metastatic bone marrow samples were extracted. gDNA was amplified following adaptor ligation and barcoding of the samples and PCR products were subsequently sequenced on a HiSeq2000 (Illumina).Results: To investigate the sgRNA library dynamics in different sample types (pre-transplantation cells, early primary tumor, late primary tumor, and bone marrow metastasis), we compared the overall distributions of sgRNAs from all sequenced samples. The early tumor sample replicates of both subpools on average retained 77.3% and 94.7% of the sgRNAs found in the pre-transplanted cell populations, while the late primary tumors retained 59.4% and 65.6% of the sgRNAs respectively, compared to early tumors. Interestingly, only a small fraction of sgRNAs (1.1% and 3.4% of sgRNAs in the pre-transplantation cells, 10.7% and 7.2% of sgRNAs in the late primary tumors for the 2 subpools respectively) were detected in the metastatic bone marrow samples. Using gene set enrichment analysis (GSEA), we found that the gene targets of the most enriched sgRNAs in the bone marrow samples were preferentially involved in important cellular processes, such as cell cycle regulation, protein translation, and several signaling pathways. Additionally we compared sgRNAs present in early primary tumor versus pre-transplantation cells and late primary tumor and found that many sgRNAs were depleted during tumor progression, indicating that their target genes were important for progression. These depleted sgRNAs in both stages mainly targeted genes involved in mTORC1 and DNA repair pathways, many of which are regulated by MYC and cell cycle related targets of E2F transcription factors.Conclusion: We established a platform for future in vivo Cas9 screens using the genome-wide CRISPR screening libraries to explore potential new targets in regulating tumor dissemination, colonization and metastasis in MM. In addition, this in vivo screening could potentially be used to investigate essential genes of response to targeted therapies or/and immunotherapies. Thus, CRISPR/Cas9-based in vivo screening is a powerful tool for functional genomics discoveries. DisclosuresRoccaro:Takeda Pharmaceutical Company Limited: Honoraria. Ghobrial:BMS: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria; Noxxon: Honoraria; Amgen: Honoraria.

BET Bromodomain Degradation As a Therapeutic Strategy in Multiple Myeloma

Multiple myeloma (MM) remains an incurable malignancy with a clear need for novel therapeutic modalities. Moreover, acquired or de novo resistance to established or novel therapeutics remains a major challenge in this, and other, neoplasias. BET Bromodomain inhibitors (BBIs), including JQ1, have potent anti-MM activity in vitro and in in vivo, but do not provide curative outcome and do not induce apoptosis in many tumor cell types. Recently, a "next-generation" BBI, dBET, that causes degradation of BET Bromodomains (BRDs) through CRBN-mediated ubiquitination has been demonstrated to have potent activity in leukemia and myeloma. Here we sought to compare the mechanistic differences between BRD inhibition with BRD degradation in treatment-naive and drug-resistant MM. Additionally, we posited that resistance to dBET treatment could emerge through genetic perturbations and wished to uncover potential mechanisms prior to its clinical utilization. To address this, we compared effects of JQ1 with lead optimized compound dBET6, in a panel of human MM cell lines (± stromal cells), including clones resistant to JQ1, bortezomib and IMIDs, and assessed viability using CS-BLI/CTG assay. RNAseq and reverse phase protein arrays (RPPA) were employed to compare the transcriptional and translational effects of BRD degradation vs. inhibition. Using an open-ended unbiased genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)-associated Cas9 approach, we examined whether we could uncover genes associated with resistance to dBET6. MM1.S cells were transduced with Cas9 and pooled lentiviral particles of the GeCKO library, consisting of 2 pooled sgRNA sub-libraries (~120,000 sgRNAs; targeting ~19,000 genes and ~1800 miRNAs). Using this CRISPR/Cas9-based approach we sought to expedite the isolation of MM cells resistant to dBET6. We treated the pool of cells thrice with dBET (250nM), allowing regrowth between treatments and maintaining a coverage of 1000 cells/sgRNA. dBET6-resistant cells were processed to quantify sgRNA enrichment or depletion, using deep sequencing. We observed dBET6 to have significantly greater potency against MM cells than JQ1, or its combination with lenalidomide, and that MM1S.CRBN-/- cells were resistant to dBET6. Resistance to neither JQ1 nor bortezomib conferred resistance to dBET6. We observed dBET6 to induce rapid and robust (<4hrs) degradation of BRD2, BRD3 and BRD4 and loss of c-MYC protein, compared with JQ1 which caused an apparent increase in BRD4 protein and significantly less c-MYC down-regulation. Interestingly, while dBET6 caused a time-dependent reduction in pro-survival Mcl-1 protein (among others) and increased cleavage of caspase-3/7, JQ1 caused Mcl-1 upregulation and did not induce cleavage of caspase-3/7. As predicted, our CRISPR/Cas9 screen identified significant enrichment of sgRNAs targeting CRBN, as well as several members of the Cullin-RING ligase (CRL) complex, known to play a critical role in E3 ubiquitin ligase activity. Preliminary experiments using individual sgRNAs appear to validate the role the CRL complex in dBET resistance. In summary, our data strongly support the development of dBET for the treatment of treatment-naive and drug-resistant MM. We demonstrate overlapping and distinct mechanisms of action between BRD inhibition vs. degradation and suggest that differential potencies of JQ1 vs. dBET is, at least in part, due to far greater loss of c-MYC and Mcl-1 expression, however further analysis is warranted. Additionally, our results demonstrate that loss of function of CRBN or the CRL complex induces dBET resistance by perturbing dBET-mediated BRD4 degradation. However, it is plausible that additional CRBN/CRL-independent mechanisms of dBET resistance exist that allow cells to survive despite complete degradation of BRDs and this will be a key question to be answered in future studies. DisclosuresBradner:Novartis Institutes for BioMedical Research: Employment.

Abstract B086: CRISPR-Cas9 engineering and screening in primary human T cells

Abstract Recent clinical data indicate immunotherapy can be an effective treatment for cancer patients, yielding dramatically increased survival times in some cases. As not all patients benefit from treatments such as anti-CTLA4 and anti-PD1 antibodies, a major focus in immuno-oncology research is finding new immuno-oncology targets, including those that alter the character and frequency of T-cell-mediated anti-tumor responses. We have had great success using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 mediated genome editing to probe gene function in cancer cells via the generation of knock-out and knock-in mutants. We are now applying this approach to primary immune cells and are deploying both CRISPR–Cas9 cell engineering and CRISPR–Cas9 screens to better understand T cell biology and to find new therapeutic targets. We use T cells negatively purified by magnetic sorting from peripheral blood mononuclear cells taken from healthy donors. Our initial work in primary human T cells has focused on the capacity to knock out and knock in genes using the Neon™ transfection system. Our data indicate that primary T cells are amenable to gene editing, and the capacity to rapidly modify loci, such as PDCD1, which encodes PD-1 enables generation of primary T cell models suitable for comprehending the function of modified receptor–ligand pairs involved in an immune checkpoint response. Our pooled sgRNA–Cas9 screens have used our in-house sgRNA libraries, which include a modified tracrRNA component improving Cas9 affinity and subsequently the performance of a typical sgRNA for promoting gene editing and modifying phenotype. Briefly, isolated CD3+ T cells are stimulated in vitro with anti-CD3 and anti-CD28 antibodies in the presence of recombinant IL-2. Next, the proliferating T cells are co-cultured with a GFP expressing Lentivirus that directs expression of Cas9 and an sgRNA drawn from a 3900 member sgRNA library targeting genes involved in the regulation of metabolism and a control library of 2442 guide RNAs. After prolonged Lentivirus and T cell co-culture, transduced T cells are sorted based on their GFP expression and periodically re-stimulated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 to allow their proliferation and expansion over several weeks. Representative cell pellets are taken after GFP sorting and then at specific time points throughout the screen. For each time point, gDNA is extracted and PCR is carried out to isolate the gRNAs that are present in each cell and these are analysed using NGS. NGS results are interpreted using algorithms from the previously published model-based analysis of genome-wide CRISPR–Cas9 knockout (MAGeCK) and Bayesian normalisation of gene expression levels (BAGEL) approaches. These screens are currently ongoing and are being run in T cells isolated from five independent donors to assess the impact of donor variability. We will present results from these screens assessing guide drop-out kinetics and reproducibility by comparing the performance of specific guides over multiple time points in each of the donors. We anticipate that these data will be useful in building more complex screens that assess T cell biology in the presence of additional cells, such as myeloid derived suppressor cells, involved in regulating the immune response to tumor development and progression. Citation Format: Cristina Ghirelli, Thibault Laurent, Kim Hoenderdos, Chris Lowe, Nicola McCarthy, Jonathan Moore. CRISPR-Cas9 engineering and screening in primary human T cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B086.

Abstract 4713: BET bromodomain degradation as a therapeutic strategy in drug-resistant multiple myeloma

Abstract Multiple myeloma (MM) is an incurable malignancy with an unmet need for novel therapeutic modalities. Moreover, acquired or de novo resistance to established or novel therapeutics remains a major challenge in this, and other, neoplasias. BET Bromodomain inhibitors (BBIs), including JQ1, have potent anti-MM activity in vitro and in in vivo, but do not provide curative outcome and do not induce apoptosis in most cell types. We sought to investigate dBET, a class of BBIs that induce degradation of BET Bromodomains (BRDs) through CRBN-mediated ubiquitination and proteasomal degradation, in drug-resistant MM. Additionally, we posited that resistance to dBET treatment would emerge through genetic perturbations and wished to uncover potential mechanisms prior to its clinical utilization. To address this, we compared effects of optimized lead compound, dBET6, with JQ1 on a panel of MM cell lines, including clones resistant to JQ1 or bortezomib and assessed viability using CS-BLI/CTG assay and BRD/c-MYC expression by western blot. Using an open-ended unbiased genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)-associated Cas9 approach, we examined whether we could uncover genes associated with resistance to dBET6. MM1.S cells were transduced with Cas9 and pooled lentiviral particles of the GeCKO library, consisting of 2 pooled sgRNA sub-libraries (∼120,000 sgRNAs; targeting ∼19,000 genes and ∼1800 miRNAs). Using this CRISPR/Cas9-based approach we sought to expedite the isolation of MM cells resistant to dBET6. We treated the pool of cells thrice with dBET (≥IC80), allowing regrowth between treatments and maintaining a coverage of 1000 cells/sgRNA. dBET6-resistant cells were processed to quantify sgRNA enrichment or depletion, using deep sequencing. We observed dBET6 to have significantly greater potency against MM cells than JQ1, or its combination with lenalidomide, and that MM1S.CRBN-/- cells were resistant to dBET6. Resistance to neither JQ1 nor Bortezomib conferred resistance to dBET6. We observed dBET6 to induce rapid (&amp;lt;4hrs) degradation of BRD2, BRD3 and BRD4 and complete absence of c-MYC protein, in contrast to JQ1 which caused dose-dependent down-regulation of c-MYC, and apparent upregulation of BRD4. As predicted, our CRISPR/Cas9 screen identified significant enrichment of sgRNAs targeting CRBN, as well as the Cullin-RING ligase (CRL) complex, known to play a critical role in E3 ubiquitin ligase activity. In summary, our data support the development of dBET for the treatment of drug-resistant MM. Additionally, our results demonstrate that loss of function of CRBN or the CRL complex induces dBET resistance by perturbing dBET-mediated BRD4 degradation. However, it is plausible that additional CRBN/CRL-independent mechanisms of dBET resistance exist that allow cells to survive despite complete degradation of BRDs and this will be a key question to be answered in future studies. Citation Format: Geoffrey M. Matthews, Yiguo Hu, Michal Sheffer, Paul J. Hengeveld, Dennis L. Buckley, Megan A. Bariteau, Haley Poarch, James E. Bradner, Constantine S. Mitsiades. BET bromodomain degradation as a therapeutic strategy in drug-resistant multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4713.

A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation

Inflammasomes are high molecular weight protein complexes that assemble in the cytosol upon pathogen encounter. This results in caspase-1-dependent pro-inflammatory cytokine maturation, as well as a special type of cell death, known as pyroptosis. The Nlrp3 inflammasome plays a pivotal role in pathogen defense, but at the same time, its activity has also been implicated in many common sterile inflammatory conditions. To this effect, several studies have identified Nlrp3 inflammasome engagement in a number of common human diseases such as atherosclerosis, type 2 diabetes, Alzheimer disease, or gout. Although it has been shown that known Nlrp3 stimuli converge on potassium ion efflux upstream of Nlrp3 activation, the exact molecular mechanism of Nlrp3 activation remains elusive. Here, we describe a genome-wide CRISPR/Cas9 screen in immortalized mouse macrophages aiming at the unbiased identification of gene products involved in Nlrp3 inflammasome activation. We employed a FACS-based screen for Nlrp3-dependent cell death, using the ionophoric compound nigericin as a potassium efflux-inducing stimulus. Using a genome-wide guide RNA (gRNA) library, we found that targeting Nek7 rescued macrophages from nigericin-induced lethality. Subsequent studies revealed that murine macrophages deficient in Nek7 displayed a largely blunted Nlrp3 inflammasome response, whereas Aim2-mediated inflammasome activation proved to be fully intact. Although the mechanism of Nek7 functioning upstream of Nlrp3 yet remains elusive, these studies provide a first genetic handle of a component that specifically functions upstream of Nlrp3.