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Functional Genomics Identifies Metabolic Vulnerabilities in Pancreatic Cancer [CRISPR Screen]

ABSTRACT: Purpose: Modeling microenvironmental influences in cell culture has been challenging, and technical limitations have hampered the comprehensive study of tumor-specific metabolism in vivo. To systematically interrogate metabolic vulnerabilities in PDA, we employed parallel CRISPR-Cas9 genetic screens using both in vivo and in vitro model systems. Methods: A custom library of lentiviral vectors encoding ~18,000 sgRNAs targeting ~3,000 mouse metabolic genes and performed CRISPR-Cas9 screens using a C/57BL6 (B6) mouse pancreatic cancer cell line derived from a KrasG12D-driven autochthonous PDA model. After transduction and selection, parallel screens were performed by in vitro passage or tumor implantation subcutaneously into the flanks of syngeneic hosts. Genomic DNA was then harvaested in vitro after 7 passages (21 days in culture) and in vivo 21 days after implantation. Results: This work revealed striking overlap of in vivo metabolic dependencies with those in vitro, validating that standard 2D culture conditions are a reliable system for studying cancer metabolism. Moreover, we identified that intercellular nutrient sharing can mask cancer dependencies in pooled screening experiments, highlighting an important limitation of this approach to study tumor metabolism Conclusions:Our work demonstrates the power of genetic screening approaches to define the compendium of in vivo metabolic dependencies in PDA and highlights critical metabolic pathways that may have therapeutic utility.

ORGANISM(S): Mus musculus

PROVIDER: GSE158789 | GEO | 2020/10/01

REPOSITORIES: GEO

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Functional Genomics Identifies Metabolic Vulnerabilities in Pancreatic Cancer [RNA-Seq]

Project description:Purpose: Modeling microenvironmental influences in cell culture has been challenging, and technical limitations have hampered the comprehensive study of tumor-specific metabolism in vivo. To systematically interrogate metabolic vulnerabilities in PDA, we employed parallel CRISPR-Cas9 genetic screens using both in vivo and in vitro model systems. Methods: 2D culture collected 3 days after plating grown in DMEM, 10%fbs, 1%p/s. 3D culture collected 3 days after plating grown in DMEM, 10%fbs, 1% p/s, 5% matrigel. Tumors were collected from orthotopic injections collected at endpoint of experiment. Results: This work revealed striking overlap of in vivo metabolic dependencies with those in vitro, validating that standard 2D culture conditions are a reliable system for studying cancer metabolism. Moreover, we identified that intercellular nutrient sharing can mask cancer dependencies in pooled screening experiments, highlighting an important limitation of this approach to study tumor metabolism Conclusions: Our work demonstrates the power of genetic screening approaches to define the compendium of in vivo metabolic dependencies in PDA and highlights critical metabolic pathways that may have therapeutic utility.

2020-10-01 | GSE158657 | GEO

eIF4A supports an oncogenic translation program in pancreatic ductal adenocarcinoma

Project description:Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with limited treatment options. Although metabolic reprogramming is a hallmark of many cancers, including PDA, previous attempts to target metabolic changes therapeutically have been stymied by drug toxicity and tumour cell plasticity. Here, we show that PDA cells engage an eIF4F-dependent translation program that supports redox and central carbon metabolism. Inhibition of the eIF4F subunit, eIF4A, using the synthetic rocaglate CR-1-31-B (CR-31) reduced the viability of PDA organoids relative to their normal counterparts. In vivo, CR-31 suppresses tumour growth and extends survival of genetically-engineered murine models of PDA. Surprisingly, inhibition of eIF4A also induces glutamine reductive carboxylation. As a consequence, combined targeting of eIF4A and glutaminase activity more effectively inhibits PDA cell growth both in vitro and in vivo. Overall, our work demonstrates the importance of eIF4A in translational control of pancreatic tumour metabolism and as a therapeutic target against PDA.

2019-10-03 | GSE125380 | GEO

Homo sapiens

Project description:PARALLEL IN VIVO AND IN VITRO MELANOMA RNAi DROPOUT SCREENS

| PRJNA262511 | ENA

CRISPR Screening for CALM-AF10 interactome and transcriptional targets

Project description:We performed pooled CRISPR screens to determine the whether the CALM-AF10 target genes and protein interactors identified by RNA-Seq and proteomics methods are functional dependencies for CALM-AF10-driven AML. We constructed a pooled CRISPR library of CALM-AF10 mouse target genes and interactors, and used it to transduce CALM-AF10 mouse AMLs. The cells were sampled at "Day 0" and kept in culture to determine in vitro dependencies at day 12. For in vivo screens, the input cell pool from day 0 was injected in mice and mice where sacrificed upon signs of AML disease, and the AML cells were harvested.

2021-01-05 | GSE164183 | GEO

Transcriptional responses of autophagy proficient and deficient pancreatic tumors and cells treated with TNFα

Project description:Pancreatic ductal adenocarcinoma (PDAC) cells require substantial metabolic rewiring to overcome nutrient limitations and immune surveillance. However, the metabolic pathways necessary for pancreatic tumor growth in vivo are poorly understood. To address this, we performed metabolism-focused CRISPR screens in PDAC cells grown in culture or engrafted in immunocompetent mice. While most metabolic gene essentialities are unexpectedly similar under these conditions, a small fraction of metabolic genes are differentially required for tumor progression. Our screens identify autophagy as a metabolic requirement for pancreatic tumor immune evasion. Mechanistically, autophagy protects cancer cells from CD8+ T cell killing through TNFα-induced cell death in vitro. This data set contains RNA-seq data of KP pancreas Atg7_KO cells expressing empty vector or Atg7 cDNA. These cells were grown as subcutaenous tumors and also cultured in vitro with and without TNFα.

2020-09-30 | GSE158707 | GEO

PARALLEL IN VIVO AND IN VITRO MELANOMA RNAi DROPOUT SCREENS

Project description:To identify factors preferentially necessary to drive tumor expansion we performed parallel in vitro and in vivo negative selection shRNA screens. Melanoma cells harboring shRNAs targeting several DNA Damage Response (DDR) kinases had a greater selective disadvantage in vivo than in vitro, indicating an essential contribution of these factors during tumor expansion. In growing tumors, DDR kinases were activated following hypoxia. Correspondingly, depletion or pharmacologic inhibition of DDR kinases was toxic to melanoma cells, including those that were resistant to BRAF inhibitor, and this could be enhanced by angiogenesis blockade. These results reveal that hypoxia sensitizes melanomas to targeted inhibition of the DDR and illustrate the utility of in vivo shRNA dropout screens for identification of pharmacologically tractable targets. A lentivirus-based kinome shRNA library (four pools) was used to transduce 888mel cells (MOI<0.2). After puromycin selection (1μg/ml), two reference samples were collected as controls. Next, tumor cells (5x105 per injection) were either injected s.c. into 6 NSG mice or plated into 6 independent plates (5x105) for in vitro culture. Tumors were removed from the mice and genomic DNA used to recover shRNAs by PCR amplification followed by deep sequencing. Three analyses were performed independently in parallel: (1) Tumors versus cultured cells (Log2 Fold Change < -1); (2) Tumors versus references (Log2 Fold Change < -2.5) and (3) Cultured cells versus references (Log2 Fold Change < -2.5). Genes targeted with at least 2 shRNAs in each of the analysis were considered hits with an enhanced in vivo effect.

2014-09-30 | E-GEOD-61826 | biostudies-arrayexpress

MYC dependency in GLS1 and NAMPT is a therapeutic vulnerability in multiple myeloma

Project description:Multiple myeloma is an incurable hematological malignancy evolving from precursor states to advanced phases of the disease. MYC abnormalities play a critical role in the disease progression. Nevertheless, MYC lacks therapeutic drugability, thereby necessitating the exploration of alternative strategies aimed at circumventing the challenges associated with targeting MYC. In this study, we hypothesized that MYC upregulation induces genomic dependencies in tumor cells, creating vulnerabilities that can be exploited therapeutically. We discovered a differential dependency on glutamine metabolism in MYC overexpressing cells. We functionally explored these dependencies as a selective targetable vulnerability in vitro and in vivo. Furthermore, we uncovered a potential synergistic combination that can exacerbated this metabolic vulnerability, Collectively, our in vitro and in vivo results revealed an effective therapeutic combinatory strategy in the context of MYC overexpressing MM.

2024-04-09 | PXD050010 | Pride

RNA-Seq Samples of siTFE3 in 8988T PDA Cell Line to Investigate Transcriptional Control of the Autophagy-Lysosome System

Project description:The activation of cellular quality control pathways to maintain metabolic homeostasis and mitigate diverse cellular stresses is emerging as a critical growth and survival mechanism in many cancers. Autophagy, a highly conserved cellular self-degradative process, is a key player in the initiation and maintenance of pancreatic ductal adenocarcinoma (PDA). However, the regulatory circuits that activate autophagy, and how they enable reprogramming of PDA cell metabolism are unknown. We now show that autophagy regulation in PDA occurs as part of a broader program that coordinates activation of lysosome biogenesis, function and nutrient scavenging, through constitutive activation of the MiT/TFE family of bHLH transcription factors. In PDA cells, the MiT/TFE proteins - MITF, TFE3 and TFEB - override a regulatory mechanism that controls their nuclear translocation, resulting in their constitutive activation. By orchestrating the expression of a coherent network of genes that induce high levels of lysosomal catabolic function, the MiT/TFE factors are required for proliferation and tumorigenicity of PDA cells. Importantly, unbiased global metabolite profiling reveals that MiT/TFE-dependent autophagy-lysosomal activation is specifically required to maintain intracellular AA pools in PDA. This AA flux is part of a program that is essential for metabolic homeostasis and bioenergetics of PDA but not for their non-transformed counterparts. These results identify the MiT/TFE transcription factors as master regulators of the autophagy-lysosomal system in PDA and demonstrate a central role of the autophagosome-lysosome compartment in maintaining tumor cell metabolism through alternative amino acid acquisition and utilization. Examination of mRNA levels in pancreatic ductal adenocarcinoma (PDA) cell line 8988T after treatment with siRNA for control or TFE3

2015-04-03 | E-GEOD-62077 | biostudies-arrayexpress

MYC dependency in GLS1 and NAMPT is a therapeutic vulnerability in multiple myeloma

Project description:Multiple myeloma is an incurable hematological malignancy evolving from precursor states to advanced phases of the disease. MYC abnormalities play a critical role in the disease progression. Nevertheless, MYC lacks therapeutic drugability, thereby necessitating the exploration of alternative strategies aimed at circumventing the challenges associated with targeting MYC. In this study, we hypothesized that MYC upregulation induces genomic dependencies in tumor cells, creating vulnerabilities that can be exploited therapeutically. We discovered a differential dependency on glutamine metabolism in MYC overexpressing cells. We functionally explored these dependencies as a selective targetable vulnerability in vitro and in vivo. Furthermore, we uncovered a potential synergistic combinations that can exacerbated this metabolic vulnerability, Collectively, our in vitro and in vivo results revealed an effective therapeutic combinatory strategy in the context of MYC overexpressing MM.

2023-09-04 | GSE241948 | GEO

Enhancer reprogramming promotes pancreatic cancer progression and metastasis [ATAC-seq]

Project description:Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human malignancies, owing in part to its propensity for metastasis. Here, we used an organoid culture system to investigate how transcription and the enhancer landscape become altered during each stage of PDA progression. This approach revealed that the metastatic transition is accompanied by massive, and recurrent alterations in enhancer activity. We implicate the transcription factors FOXA1 and GATA5 as drivers of enhancer activation in this system, a mechanism that we show renders PDA cells more invasive and less anchorage-dependent for growth in vitro, as well as more metastatic in vivo. FOXA1 and GATA5 were found to activate a foregut endoderm transcriptional program in PDA cells, without altering genes associated with the epithelial-to- mesenchymal transition. Collectively, our study implicates FOXA1/GATA5 upregulation, enhancer reprogramming, and a novel retrograde developmental transition in PDA progression and metastasis.

2017-07-31 | GSE99275 | GEO

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