Lentiviral Library Research Articles

Abstract 2916: The clonal evolution of glioblastoma

Abstract Background: Glioblastoma (GBM) is an aggressive cancer that often recurs despite multimodal therapy. Median survival is 12-15 months. Genomic profiling studies have shown marked tumor heterogeneity with distinct mutations among treated and untreated samples. The Cancer Genome Atlas (TCGA), revealed that mutations in TP53, CDKN2A, PTEN, EGFR and NF1 are predominant in untreated tumors. Of 19 patients who progressed on therapy, seven samples were hypermutated with mutations in mismatch repair proteins, mainly MSH6. We proposed to use the Cellecta Lentiviral-based tagging library, a novel model system to assess the role of intratumor heterogeneity (ITH) in the presumptive tumor-initiating fraction of primary GBM and in the development of resistance to temozolomide (TMZ). From a pool of 30,000,000 barcodes, the system enables the genetic integration of a unique barcode sequence into each cell. Each barcode can be quantitatively tracked via next-generation sequencing, allowing for dynamic monitoring of the subclonal architecture. Methods: GSC 272 and 627 (sensitive and resistant to TMZ) were derived from core biopsies of GBM patients. Cells were transduced with a lentiviral vector containing luciferase. Cells were expanded and infected with the Cellecta Lentiviral library. Multiplicity of infection was determined. Transduced cells were injected into B6.Cg-Foxn1nu/J mice brains using a guide screw system. Half of the mice were treated with TMZ via gavage at two weeks. Brain tumors were removed and total DNA was extracted. Barcode inserts were amplified and the Illumina Sequencing platform was used for barcode quantification. Additionally, whole exome sequencing was performed to assess mutation status. Results: Confirmation of luciferase signal has been performed using a Luciferase Reporter Assay. Multiplicity of infection was found to be 0.15 and 1 for GSC 6-27 and 272 respectively. Initial methylation studies showed GSC 272 to have methylation of MGMT while GSC 627 did not. There was no difference in OS in the TMZ Resistant (GSC 627) untreated vs. treated cohort (with a median OS of 55 days in both groups (N= 23 vs 25 respectively, P= 0.563). TMZ Sensitive (GSC 272) untreated mice did not survive as long as the treated cohort (N= 24 vs. 25, Median OS 43 vs. 206 days, P= 4.61e-09). Barcode and whole exome sequencing results is currently being analyzed. Conclusions: The Cellecta lentiviral tagging system is an innovative way to track ITH and clonal evolution in glioblastoma orthotopic models. We hope to discover novel insights into TMZ treatment response and resistance. Citation Format: Jennifer Brooke Goldstein, Ravesanker Ezhilarasan, Mona Jaffari, Alessandro Carugo, Giulio Draetta, Roeland Verhaak, Sahil Seth, Erik Sulman, Phillip Andrew Futreal. The clonal evolution of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2916. doi:10.1158/1538-7445.AM2017-2916

An Unbiased High-Throughput Screen to Identify Novel Effectors That Impact on Cardiomyocyte Aggregate Levels.

Postmitotic cells, such as cardiomyocytes, seem to be particularly susceptible to proteotoxic stimuli, and large, proteinaceous deposits are characteristic of the desmin-related cardiomyopathies and crystallin cardiomyopathic diseases. Increased activity of protein clearance pathways in the cardiomyocyte, such as proteasomal degradation and autophagy, has proven to be beneficial in maintaining cellular and cardiac function in the face of multiple proteotoxic insults, holding open the possibility of targeting these processes for the development of effective therapeutics. Here, we undertake an unbiased, total genome screen for RNA transcripts and their protein products that affect aggregate accumulations in the cardiomyocytes. Primary mouse cardiomyocytes that accumulate aggregates as a result of a mutant CryAB (αB-crystallin) causative for human desmin-related cardiomyopathy were used for a total genome-wide screen to identify gene products that affected aggregate formation. We infected cardiomyocytes using a short hairpin RNA lentivirus library in which the mouse genome was represented. The screen identified multiple candidates in many cell signaling pathways that were able to mediate significant decreases in aggregate levels. Subsequent validation of one of these candidates, Jak1 (Janus kinase 1), a tyrosine kinase of the nonreceptor type, confirmed the usefulness of this approach in identifying previously unsuspected players in proteotoxic processes.

MicroRNA-200a-3p increases 5-fluorouracil resistance by regulating dual specificity phosphatase 6 expression

Acquisition of resistance to anti-cancer drugs is a significant obstacle to effective cancer treatment. Although several efforts have been made to overcome drug resistance in cancer cells, the detailed mechanisms have not been fully elucidated. Here, we investigated whether microRNAs (miRNAs) function as pivotal regulators in the acquisition of anti-cancer drug resistance to 5-fluorouracil (5-FU). A survey using a lentivirus library containing 572 precursor miRNAs revealed that five miRNAs promoted cell survival after 5-FU treatment in human hepatocellular carcinoma Hep3B cells. Among the five different clones, the clone expressing miR-200a-3p (Hep3B-miR-200a-3p) was further characterized as a 5-FU-resistant cell line. The cell viability and growth rate of Hep3B-miR-200a-3p cells were higher than those of control cells after 5-FU treatment. Ectopic expression of a miR-200a-3p mimic increased, while inhibition of miR-200a-3p downregulated, cell viability in response to 5-FU, doxorubicin, and CDDP (cisplatin). We also showed that dual-specificity phosphatase 6 (DUSP6) is a novel target of miR-200a-3p and regulates resistance to 5-FU. Ectopic expression of DUSP6 mitigated the pro-survival effects of miR-200a-3p. Taken together, these results lead us to propose that miR-200a-3p enhances anti-cancer drug resistance by decreasing DUSP6 expression.

CRISPR-Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome.

Large genome-mapping consortia and thousands of genome-wide association studies have identified non-protein coding elements in the genome as a having a central role in various biological processes. However, decoding the function of the millions of putative regulatory elements discovered in these studies remains challenging. CRISPR–Cas9-based epigenome editing technologies have enabled precise perturbation of the activity of specific regulatory elements. Here we describe CRISPR–Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity within the native genomic context. Using dCas9KRAB repressor and dCas9p300 activator constructs and lentiviral sgRNA libraries targeting DNase I hypersensitive sites surrounding a gene of interest, we perform both loss- and gain-of-function screens to identify regulatory elements for the β-globin and the HER2 loci in human cells. CERES readily identified known and novel regulatory elements, some of which were dependent on cell type or direction of perturbation. This technology allows the high-throughput functional annotation of putative regulatory elements in their native chromosomal context.

Single-minded CRISPR screening

Transcriptome-wide effects of gene knockouts are screened at the single-cell level by combining high-throughput, single-cell RNA-seq with lentiviral CRISPR libraries.

Abstract PR15: Cas9/RNA-based forward genetic screenings in mouse embryonic stem cells uncovered the role of genes mediating resistance to ATR inhibitors

Abstract Mouse embryonic stem cells (mESCs) represent an excellent platform to study processes of developmental biology, model disease in vitro and in vivo or perform drug discoveries. In the last few years, the development of precise genome engineering tools based on the RNA-guided Cas9 nuclease from the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system, has enabled efficient and easy to engineer targeted genome modifications in mammalian cells. Using this technology, we generated a highly controlled doxycycline-inducible Cas9 mESC line, which harbor one single copy of Cas9 located in the type I collagen gene. Lentiviral delivery of small guide RNAs (sgRNAs) in doxycycline-treated mESCs demonstrated to be a very efficient manner to generate specific knockout mESCs lines. Therefore, we generated mESC libraries of knockout cells by using a lentiviral library of 80000 sgRNAs designed against 20000 mouse genes to perform forward genetic screenings. As a proof of concept, we investigated the existence of genes providing resistance to the absence of the ataxia-telangiectasia and Rad3 related (ATR) function by incubating the mESC libraries with lethal doses of ATR inhibitor. We here report the identification of CDC25A as a major determinant of sensitivity to ATR inhibition. CDC25A deficient cells resist high doses of ATR inhibitors, which we show is due to their failure to prematurely enter mitosis in response to the drugs. Forcing mitotic entry with WEE1 inhibitors restores the toxicity of ATR inhibitors in CDC25A deficient cells. In addition, we have adapted the CRISPR-based SAM (Synergistic Activation Mediators) system to develop a doxycycline-inducible nuclease-dead Cas9 mESC line to induce gene expression at will. We generated mESC cell libraries by using a lentiviral library of 70000 sgRNAs designed against the proximal promoter of 20000 mouse genes to perform a gain-of-function screening with lethal doses of ATR inhibitor and results will be presented. With ATR inhibitors now entering the clinic, our work provides a better understanding of the mechanisms by which these compounds kill cells, and reveals genetic interactions that could be used for their rational use. Taking together, our loss-of-function and gain-of function cell libraries in mESCs represent an excellent platform to systematically identify genes involved in mediating resistance against chemotherapeutic agents. This abstract is also being presented as Poster A35. Citation Format: Sergio Ruiz, Cristina Mayor-Ruiz, Vanessa Lafarga, Matilde Murga, Maria Vega, Sagrario Ortega, Oscar Fernandez-Capetillo. Cas9/RNA-based forward genetic screenings in mouse embryonic stem cells uncovered the role of genes mediating resistance to ATR inhibitors [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR15.

Genome-wide, CRISPR-Cas9-based genetic screening for regulators of interferon-induced PD-L1 expression in AML cells.

116 Background: Expression of PD-L1 in AML cells is induced by pro-inflammatory signals, but how the induction is regulated is incompletely known. Genome-wide screening using the CRISPR-Cas9 technology could provide a strategy to interrogate the regulators of PD-L1 expression. Methods: A lentiviral CRISPR-Cas9 library, containing 3 sgRNA targeting each of 19,050 human genes (Nat Methods 2014, 11:783), was used to transduce THP-1 AML cells. Transduced cells were treated with IFN-γ and stained with an anti-PD-L1 antibody. Two subsets of cells were isolated by cell sorting according to their levels of PD-L1 expression. One subset consisted of cells that showed the median 5% of PD-L1 expression and the other subset, the lowest 5%. sgRNA integrated in the sorted subsets were amplified and their frequencies quantified by deep sequencing. Results: As expected, sgRNA targeting the PD-L1 gene itself were highly enriched in the subset that expressed a low level of PD-L1. sgRNA that targeted IFNGR2, β-chain of the IFN-γ receptor, were also enriched in this subset, whereas those for IFNGR1 were not. sgRNA targeting both JAK1 and JAK2 were enriched. By contrast, those for JAK3 and TYK2 were not. Enrichment was seen for sgRNA that targeted STAT1, a transcriptional activator downstream of JAK1/2, whereas sgRNA targeting the other STAT activators (STAT-2, 3, 4, 5A, 5B and 6) showed no enrichment. sgRNA targeting several serine kinases that regulate STAT1 phosphorylation (PKCδ, PKCε, PKCα, CAMKII, ERK, JNK, and CDK8) were not enriched. Nor were sgRNA targeting several phosphatases that modulate STAT1 activity (TC-PTP, PTPN1, SHP1 and SHP2). sgRNA targeting IRF1 (IFN regulatory transcription factor 1) were highly enriched, whereas sgRNA for IRF-2 to IRF-9 were not. In each of the affected genes, at least 2 out of the 3 sgRNA targeting the gene were enriched, indicating a generality of the findings. Conclusions: A CRISPR-Cas9-based screening strategy provided an unbiased interrogation of genes essential for PD-L1 expression on AML cells. It confirmed the importance of the IFNGR2-JAK1/2-STAT1-IRF1 axis, which could serve as targets for the development of drugs to suppress the ability of AML cells to evade host immunity.

Mechanisms of iPS cell generation and beyond.

The generation of induced pluripotent stem cells (iPSCs) achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc, transformed our classical views of the cellular epigenetic landscape and delivered a new concept for cell and tissue engineering. In addition to iPSCs, several other cell types have also been generated by master transcription factor (TF)-mediated transdifferentiation. However, the critical molecular mechanisms amongst diverse cellular identity changes are not well understood. Through the investigation of reprogramming mechanisms, we recently revealed that over-expression of constitutive active Smad3 boosted not only iPSC generation, but also 3 other master TF-mediated conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons. This demonstrated that there were common mechanisms underlying different master TF-mediated cell conversions. To illuminate such mechanisms further, we have recently performed CRISPR/Cas9-mediated genome-wide knockout screening during reprogramming with a lentiviral gRNA library containing 90,000 gRNAs. This screening provided us with ~15 novel reprogramming roadblock genes as well as ~20 candidate genes essential for the reprogramming process but not for ES cell self-renewal. This data set will be a valuable resource to further understand how overexpression of master TFs alters cellular identity, and to achieve more faithful, efficient cell conversions for regenerative medicine.(Presented at the 1934th Meeting, March 17, 2017).

Genome-Wide CRISPR Screen Identifies Regulators of Mitogen-Activated Protein Kinase as Suppressors of Liver Tumors in Mice

It has been a challenge to identify liver tumor suppressors or oncogenes due to the genetic heterogeneity of these tumors. We performed a genome-wide screen to identify suppressors of liver tumor formation in mice, using CRISPR-mediated genome editing. We performed a genome-wide CRISPR/Cas9-based knockout screen of P53-null mouse embryonic liver progenitor cells that overexpressed MYC. We infected p53-/-;Myc;Cas9 hepatocytes with the mGeCKOa lentiviral library of 67,000 single-guide RNAs (sgRNAs), targeting 20,611 mouse genes, and transplanted the transduced cells subcutaneously into nude mice. Within 1 month, all the mice that received the sgRNA library developed subcutaneous tumors. We performed high-throughput sequencing of tumor DNA and identified sgRNAs increased at least 8-fold compared to the initial cell pool. To validate the top 10 candidate tumor suppressors from this screen, we collected data from patients with hepatocellular carcinoma (HCC) using the Cancer Genome Atlas and COSMIC databases. We used CRISPR to inactivate candidate tumor suppressor genes in p53-/-;Myc;Cas9 cells and transplanted them subcutaneously into nude mice; tumor formation was monitored and tumors were analyzed by histology and immunohistochemistry. Mice with liver-specific disruption of p53 were given hydrodynamic tail-vein injections of plasmids encoding Myc and sgRNA/Cas9 designed to disrupt candidate tumor suppressors; growth of tumors and metastases was monitored. We compared gene expression profiles of liver cells with vs without tumor suppressor gene disrupted by sgRNA/Cas9. Genes found to be up-regulated after tumor suppressor loss were examined in liver cancer cell lines; their expression was knocked down using small hairpin RNAs, and tumor growth was examined in nude mice. Effects of the MEK inhibitors AZD6244, U0126, and trametinib, or the multi-kinase inhibitor sorafenib, were examined in human and mouse HCC cell lines. We identified 4 candidate liver tumor suppressor genes not previously associated with liver cancer (Nf1, Plxnb1, Flrt2, and B9d1). CRISPR-mediated knockout of Nf1, a negative regulator of RAS, accelerated liver tumor formation in mice. Loss of Nf1 or activation of RAS up-regulated the liver progenitor cell markers HMGA2 and SOX9. RAS pathway inhibitors suppressed the activation of the Hmga2 and Sox9 genes that resulted from loss of Nf1 or oncogenic activation of RAS. Knockdown of HMGA2 delayed formation of xenograft tumors from cells that expressed oncogenic RAS. In human HCCs, low levels of NF1 messenger RNA or high levels of HMGA2 messenger RNA were associated with shorter patient survival time. Liver cancer cells with inactivation of Plxnb1, Flrt2, and B9d1 formed more tumors in mice and had increased levels of mitogen-activated protein kinase phosphorylation. Using a CRISPR-based strategy, we identified Nf1, Plxnb1, Flrt2, and B9d1 as suppressors of liver tumor formation. We validated the observation that RAS signaling, via mitogen-activated protein kinase, contributes to formation of liver tumors in mice. We associated decreased levels of NF1 and increased levels of its downstream protein HMGA2 with survival times of patients with HCC. Strategies to inhibit or reduce HMGA2 might be developed to treat patients with liver cancer.

Identification of CCND2 As a RUNX1/ETO Target Required for Leukaemic Propagation

The translocation t(8;21) is the most common chromosomal rearrangement in AML and generates the RUNX1/ETO fusion protein. RUNX1/ETO is required for maintaining the leukaemic phenotype influencing both leukaemic clonogenicity and proliferation and is traditionally thought of as a transcriptional repressor through its actions with histone deacetylases and transcriptional co-repressors. Intriguingly our RNAi experiments have also identified many RUNX1/ETO target genes that are transcriptionally upregulated by this fusion protein.To delve deeper into the involvement of these upregulated genes in RUNX1/ETO driven leukaemia, targeted RNAi screens were performed both in vitro and in vivo, directed at genes found directly bound by RUNX1/ETO and that were found differentially expressed upon RUNX1/ETO knockdown. For the RNAi screens, we used a doxycycline (dox)-inducible lentiviral RNAi library covering each gene with 3 shRNAs. We transduced two t(8;21)-positive AML cell lines, Kasumi-1 and SKNO-1, with this library and performed parallel screens employing colony formation and long-term suspension culture assays in the in vitro arm, and intrafemoral xenotransplantation of immunodeficient NSG mice for the in vivo arm. For comparative purposes, both arms of the RNAi screen were performed in the presence and absence of dox. DNA was isolated throughout both screens and was analysed by Next Generation Sequencing (NGS). Comparison of the changes in level of integrated shRNA coding sequences between dox and no dox groups were made using DESEQ and those genes that are required for RUNX1/ETO driven leukaemia identified.As expected, RUNX1/ETO shRNA constructs diminished upon induction of shRUNX1/ETO expression by dox, therefore implicating RUNX1/ETO dependency in cell survival. Furthermore non-targeting control shRNA (shNTC) levels were unaffected. Out of the many hits identified (which include previously reported hits such as Pontin (RUBVL1), SKP2 and KIT, Cyclin D2 (CCND2) stood out significantly, since levels of shCCND2 were depleted in all dox samples from both arms of the screen.CCND2 is a cell cycle regulator whose activity is dependent on its binding to CDK4/6 in G1 phase. Phosphorylation of Rb (Retinoblastoma), by CDK4/6-CCND2, uncouples Rb from E2F allowing transcription of essential S phase genes. Here we show CCND2 knockdown downregulates both cell proliferation and colony formation in t(8;21) positive cells by causing G1 phase cell cycle arrest via a reduction in Rb phosphorylation, which is a phenotype copied by our RUNX1/ETO knockdown. Moreover, inhibition of CDK4/6-CCND2 by palbociclib (PD-0332991) in t(8;21) positive cells similarly reduces cell proliferation and colony formation via a G1 cell cycle arrest. Interestingly, primary CD34+ human stem/progenitor cells (HSPCs) containing an activated form of KIT (N822K) and the RUNX1/ETO 9a isoform are likewise responsive to palbociclib treatment. In addition to in vitro studies, palbociclib was tested in vivo in two separate experimental models: firstly, mice were intravenously injected with murine CD34+ cKit+ HSPCs expressing the RUNX1/ETO9a isoform and secondly, immunodeficient mice were intrahepatically injected with Kasumi-1. Mice were dosed with palbociclib upon confirmation of engraftment. Engraftment was detected using bioluminescence and survival rates recorded. Both studies showed significant increased survival with palbociclib treatment with the first giving an increased median survival of 59 versus 29 days (p<0.005) and the second giving a median survival of 87 and 67 days for palbociclib treated and control mice (p<0.005), respectively.Finally, Gene Set Enrichment Analysis (GSEA) shows the correlations between palbociclib treatment, CCND2 knockdown and RUNX1/ETO knockdown and shows common gene sets shared between them and other gene sets which include those targeted by MYC and E2F. Interestingly, gene sets that only correlate with knockdown of CCND2 and RUNX1/ETO and evidentially unaffected by CDK4/6 activity will be of interest, in order to target other RUNX1/ETO dependent proteins and pathways alongside of CDK4/6 inhibition. We are currently investigating the use of palbociclib with other drugs in order to develop new drug combinations with reduced toxicity and minimal risk of developing resistance. DisclosuresNo relevant conflicts of interest to declare.

CRISPR/Cas9 Technique for Identification of Genes Regulating Oxaliplatin Resistance of Pancreatic Cancer Cell Line

Genome editing approach based on prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) system is a simple and useful way to investigate gene functions on a genome-wide scale. It is especially important for cancer research because of genetic contribution to tumor development. We applied this technique in a high-throughput screening format to find genes that could be involved in chemotherapy resistance of pancreatic cancer. We used AsPC1 cell line expressing doxycycline-inducible Cas9 to screen two sgRNA lentiviral libraries: (1) cell cycle genes (CC, 983 genes, ∼12,000 sgRNA) and (2) genome-wide (GW, ∼90,000 sgRNA). These sets of cells with different gene knockouts were treated with oxaliplatin to identify knockouts which increase sensitivity to the drug. We have performed screening both in vitro and in vivo settings. For the in vivo arm of our experiments, peritoneal carcinomatosis model in severe combined immunodeficiency (SCID) mice was created by intraperitoneal injection of AsPC1/Cas9 cells infected with sgRNA library. Genomic DNA from cells and animal tumor material was analyzed using next generation sequencing (NGS) to obtain data about representation of sgRNA. Preliminary data allowed us to identify genes potentially modulating oxaliplatin sensitivity.

Clonal characteristics of mesenchymal stem cells able to transfer microenvironment

Mesenchymal stem cells (MSCs) can transfer hematopoietic microenvironment in vivo. After implantation of murine femur bone marrow (BM) plug or adherent cell layer (ACL) of long-term bone marrow culture (LTBMC) established from the same BM equivalent, ectopic hematopoietic focus is formed under the renal capsule of syngeneic recipient. Stromal cells in those foci are derived from donor MSCs, while hematopoietic cells are of recipient origin. Cellularity of individual foci is proportional to the number of transplanted femur equivalents. Consequently, this assay can be used to quantitatively assess the number of MSCs in BM. Based on the self-renewal potential of MSCs, ectopic foci can be serially retransplanted. The aim of this study was to characterize the clonal structure of MSC populations. LTBMCs were obtained from F1 (CBA x C57Black/J6) mice. Cultures were transduced 2 weeks after establishment with a lentiviral library containing genetic barcodes (32 variable nucleotides). After additional 2 weeks, the proportion of marked stromal clonogenic cells (colony forming units fibroblast /CFU-F/) in ACLs was 73.7±5.1%. ACLs were implanted under the renal capsules of syngeneic recipients. Six weeks later the developed ectopic foci were partially retransplanted. DNA was isolated from LTBMCs, initial and secondary ectopic foci. Barcode sequences were determined on Illumina platform. In initial ectopic foci 278±40 (n = 7) individual barcodes were identified. The secondary ectopic foci contained 247±23 (n = 4) barcodes. Only 12±1 common barcodes were found in both initial and secondary foci. Since the secondary were descendants of the initial foci, we calculated that the initial foci contained about 500 barcoded MSCs, meaning that at least 2.3% of MSCs took part in the formation of hematopoietic microenvironment in the initial and the secondary foci. All other MSCs formed microenvironment only once. Taking into account the proportion of marked stromal clonogenic cells in ACLs, initial foci contained at least 600 MSCs. As each LTBMC was established from one femur, a femur contains no less than 600 MSCs. Presuming a linear relationship between the number of implanted MSCs and the size of ectopic foci, our data suggest that not all MSCs, but only part of them is involved in formation of hematopoietic microenvironment.

Genome-wide gain-of-function screen for genes that induce epithelial-to-mesenchymal transition in breast cancer.

Epithelial to mesenchymal transition (EMT) is a developmental program that has been implicated in progression, metastasis and therapeutic resistance of some carcinomas. To identify genes whose overexpression drives EMT, we screened a lentiviral expression library of 17000 human open reading frames (ORFs) using high-content imaging to quantitate cytoplasmic vimentin. Hits capable of increasing vimentin in the mammary carcinoma-derived cell line MDA-MB-468 were confirmed in the non-tumorigenic breast-epithelial cell line MCF10A. When overexpressed in this model, they increased the rate of cell invasion through Matrigel™, induced mesenchymal marker expression and reduced expression of the epithelial marker E-cadherin. In gene-expression datasets derived from breast cancer patients, the expression of several novel genes correlated with expression of known EMT marker genes, indicating their in vivo relevance. As EMT-associated properties are thought to contribute in several ways to cancer progression, genes identified in this study may represent novel targets for anti-cancer therapy.

Abstract B43: PILOT: a patient-oriented in vivo functional platform to identify new lethalities and optimize cancer treatment

Abstract B43: PILOT: a patient-oriented in vivo functional platform to identify new lethalities and optimize cancer treatment

Abstract 4368: High-throughput phenotyping of lung cancer somatic mutations

Abstract Recent cancer genome sequencing and analysis has identified millions of somatic mutations in cancer. However, the functional impact of most variants is poorly understood, limiting the use of this genetic knowledge for clinical decision-making. Here we describe a new high-throughput approach, expression-based variant impact phenotyping (eVIP), which uses gene expression changes to infer somatic mutation impact. We generated a lentiviral expression library representing 53 genes and 194 somatic mutations identified in primary lung adenocarcinomas. Next, we introduced this library into A549 lung adenocarcinoma cells and 96 hours later performed gene expression profiling using Luminex-based L1000 profiling. We built a computational pipeline, eVIP, to compare mutant and wild-type expression signatures to infer whether variants were gain-of-function, change-of-function, loss-of-function, or neutral. Overall, eVIP identified 69% of mutations as impactful whereas 31% appeared functionally neutral. A very high rate, 92%, of missense mutations in the KEAP1 and STK11 tumor suppressor genes were found to inactivate or diminish protein function. As a complementary approach, we assessed which mutations are epistatic to EGFR or capable of initiating xenograft tumor formation in vivo. A subset of the impactful mutations identified by eVIP could induce xenograft tumor formation in mice and/or confer resistance to cellular EGFR inhibition. Among these mutations were 20 rare or non-canonical somatic variants in clinically-actionable or -relevant oncogenes including EGFR S645C, ARAF S214C and S214F, ERBB2 S418T, and PIK3CA E600K. eVIP can, in principle, characterize any genetic variant, independent of prior knowledge of gene function. Further application of eVIP should significantly advance the pace of functional characterization of mutations identified from genome sequencing. Citation Format: Alice H. Berger, Angela N. Brooks, Xiaoyun Wu, Yashaswi Shrestha, Candace Chouinard, Federica Piccioni, Mukta Bagul, Atanas Kamburov, Marcin Imielinski, Larson Hogstrom, Cong Zhu, Xiaoping Yang, Sasha Pantel, Ryo Sakai, Nathan Kaplan, David Root, Rajiv Narayan, Ted Natoli, David Lahr, Itay Tirosh, Pablo Tamayo, Gad Getz, Bang Wong, John Doench, Aravind Subramanian, Todd R. Golub, Matthew Meyerson, Jesse S. Boehm. High-throughput phenotyping of lung cancer somatic mutations. [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 4368.

Abstract 2059: Identification of novel paclitaxel sensitivity genes via lentiviral CRISPR/Cas9 screening in haploid human cells

Abstract Some cancers lack clearly druggable driver mutations and might also have too few neo-antigen generating mutations to provoke a tumor-specific immune response, even in the presence of antibodies targeting immune checkpoint mechanisms. Synthetic lethal/non-oncogene addiction represents an alternative approach to the therapy of such cancers, as exemplified by the recent approval of olaparib. RNA interference has identified several putative synthetic lethal targets, but few, if any, of these have have withstood vigorous validation. The recent application of RNA-guided gene editing to mammalian cells offers new prospects for target identification. The complete ablation of gene expression, which this technology permits allows the identification of targets that have no phenotype when partially depleted by RNA interference. Horizon has recently established a capability for CRISPR-Cas9 KO screening. Screens are performed by transducing a pool of cells with a complex lentiviral library. Next generation sequencing is then used to identify which sgRNAs are accumulating or being depleted from the population. As there are multiple sgRNAs per gene, we can rank genes for their likelihood of having a phenotype under the conditions used in the screen. Our proof-of-principle small molecule resistance and sensitivity screens using both whole-genome libraries and custom subset libraries have successfully identified previously published hits and also yielded novel hits. Horizon has also developed a fully haploid cell line (eHAP), which has the potential to provide additional clarity in genetic screening. To evaluate the power of CRISPR-Cas9 KO screening in haploid cells, we have used our screening platform to identify genes required for survival in low doses of paclitaxel. Whole-genome screening using the GeCKOv2 library identified three kinesins amongst the most prominent of the hits whose depletion sensitised cells to this microtubule stabilising agent. Intriguingly, two of these kinesins, KIF2A and KIF2C are thought to destabilise mitotic microtubules, providing an attractive explanation for the synthetic lethality. We will also present results from a second genome-wide CRISPR KO screen in haploid cells to identify genes required for survival under glucose starvation. Prominent hits included components of the electron transport chain and the tuberous sclerosis complex. Citation Format: Jessica R. Hunt, Steffen Lawo, David Walter, Isabelle Nett, Joanne Yarker, Benedict C S Cross, Jonathan D. Moore. Identification of novel paclitaxel sensitivity genes via lentiviral CRISPR/Cas9 screening in haploid human cells. [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 2059.

Practical Considerations for Using Pooled Lentiviral CRISPR Libraries.

CRISPR/Cas9 technology is ideally suited for genome-wide screening applications due to the ease of generating guide RNAs (gRNAs) and the versatility of Cas9 or Cas9 derivatives to knockout, repress, or activate expression of target genes. Several pooled lentiviral CRISPR libraries have been developed and are now publicly available, but while using CRISPR/Cas9 for genetic experiments has become widely adopted, genome-wide screening experiments remain technically challenging. This review covers the basics of CRISPR/Cas9, describes several publicly available CRISPR libraries, and provides a general protocol for conducting genome-wide screening experiments using CRISPR/Cas9. © 2016 by John Wiley & Sons, Inc.

Investigation of the Mesenchymal Stem Cell Compartment by Means of a Lentiviral Barcode Library.

The hematopoietic bone marrow microenvironment is formed by proliferation and differentiation of mesenchymal stem cells (MSCs). The MSC compartment has been less studied than the hematopoietic stem cell compartment. To characterize the structure of the MSC compartment, it is necessary to trace the fate of distinct mesenchymal cells. To do so, mesenchymal progenitors need to be marked at the single-cell level. A method for individual marking of normal and cancer stem cells based on genetic "barcodes" has been developed for the last 10 years. Such approach has not yet been applied to MSCs. The aim of this study was to evaluate the possibility of using such barcoding strategy to mark MSCs and their descendants, colony-forming units of fibroblasts (CFU-Fs). Adherent cell layers (ACLs) of murine long-term bone marrow cultures (LTBMCs) were transduced with a lentiviral library with barcodes consisting of 32+ 3 degenerate nucleotides. Infected ACLs were suspended, and CFU-F derived clones were obtained. DNA was isolated from each individual colony, and barcodes were analyzed in marked CFU-F-derived colonies by means of conventional polymerase chain reaction and Sanger sequencing. Barcodes were identified in 154 marked colonies. All barcodes appeared to be unique: there were no two distinct colonies bearing the same barcode. It was shown that ACLs included CFU-Fs with different proliferative potential. MSCs are located higher in the hierarchy of mesenchymal progenitors than CFU-Fs, so the presented data indicate that MSCs proliferate rarely in LTBMCs. A method of stable individual marking and comparing the markers in mesenchymal progenitor cells has been developed in this work. We show for the first time that a barcoded library of lentiviruses is an effective tool for studying stromal progenitor cells.

Abstract P3-06-04: Investigating clonal dynamics in triple negative breast cancer chemoresistance

Abstract Approximately 50% of triple-negative breast cancer (TNBC) patients have extensive residual disease following neoadjuvant chemotherapy (NAC). These patients have a four-fold increase in mortality risk and an increased risk of distant metastases within three years (1). Understanding the molecular basis of resistance to NAC is expected to provide opportunities to better treat patients in the primary setting. Extensive intratumoral subclonal heterogeneity has been well documented in primary, treatment-naïve TNBC (2). Subclonal populations harboring distinct molecular profiles may confound targeted therapy strategies, yet the functional impact of subclonal heterogeneity in TNBC resistance to therapy is unknown. We are implementing DNA barcoding to quantitatively track changes in subclonal architecture pre- and post-treatment in patient-derived xenograft (PDX) models of TNBC in order to design novel combination therapies. Such barcoding strategies have been used to monitor clonal dynamics in breast cancer PDXs with great sensitivity (3). We have established an orthotopic PDX from a treatment-naïve TNBC patient (PIM1, procured from a patient later found to have chemoresistant disease). In order to model chemoresistance, we treated PIM1 with Adriamycin and cyclophosphamide (AC), standard of care NAC for TNBC patients, which resulted in partial response but left residual disease. To characterize subclonal dynamics in response to NAC, we transduced freshly isolated PIM1 cells with a lentiviral library expressing 25 million unique DNA barcodes (Cellecta) using conditions to ensure each transduced cell contained a single unique barcode. Transduced cells were selected with puromycin, then orthotopically implanted into immuno-compromised mice. High-throughput barcode sequencing revealed reproducible maintenance of greater than 60,000 unique barcodes in PDX tumors. Comparison of barcode distribution in tumors treated with vehicle or NAC will reveal whether NAC selects for a subpopulation of cells during the development of resistance. Future directions will include whole-exome and RNA sequencing to characterize genomic changes associated with alterations in barcode distribution in response to NAC treatment. Our ultimate goal is to identify novel combination therapies to eliminate subclones that contribute to chemoresistance in primary TNBC.

Abstract A27: An in vivo gain-of-function screening identified the Williams-Beuren syndrome gene GTF2IRD1 as a mammary tumor promoter

Abstract Precision medicine has shown great efficacy in oncology, but its broad application requires a full understanding of the cancer genome. We have performed the first gain-of-function screening in mice to functionally identify candidates from a “druggable” gene set for their roles in breast cancer. We transduced mammary gland stem/progenitor cells isolated from MMTV-ErbB2 transgenic mice with a lentiviral cDNA library and implanted them into the cleared fat pads of syngeneic recipients. The transplanted mammary gland cells formed a normal mammary ductal tree within 6 weeks. After another 8 months latency, small lesions were detected along the mammary ducts in mice harboring cells that had been transduced with the lentiviral library. We isolated genomic DNA from these lesions to amplify any lentiviral cDNA sequences within the lesions and identified individual genes by sequencing. Gain-of-function of five genes, including RET, GTF2IRD1, ADORA1, LARS2, and DPP8, was found to promote mammary tumor growth consistently. We further studied one novel tumor promoting gene, transcription factor GTF2IRD1. GTF2IRD1 regulates the expression of multiple genes in the mammary gland system, including OPN, TβR2, and BMPR1b. In tissue culture, high levels of GTF2IRD1 inhibited cell proliferation but increased the colony formation ability of the un-transformed mammary gland cell line MCF10A. In mice, the mis-regulation of GTF2IRD1, OPN, TβR2, and BMPR1b, also individually promoted mammary cancer development. Silencing of TβR2 delayed the incidence of tumor formation by GTF2IRD1. We also found that GTF2IRD1 is highly expressed in human breast tumors, correlating with high tumor grades and poor prognosis. In conclusion, we performed the first gain-of-function screening in vivo for breast cancer promoting genes. All candidate genes from this study are potential drug targets to treat cancer. We showed that GTF2IRD1 promotes breast cancer development in mice through regulation of its downstream targets, and correlates clinically with high tumor grades and poor prognosis. We believe our in vivo screening method is readily expandable to whole genome annotation of cancer genes. Citation Format: Yongliang Huo, Yinghao Su, Qiuyin Cai, Ian Macara. An in vivo gain-of-function screening identified the Williams-Beuren syndrome gene GTF2IRD1 as a mammary tumor promoter. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A27.