Recent clinical successes of cancer immunotherapy necessitate the investigation of the interaction between malignant cells and the host immune system. However, elucidation of complex tumor-immune ...interactions presents major computational and experimental challenges. Here, we present Tumor Immune Estimation Resource (TIMER; cistrome.shinyapps.io/timer) to comprehensively investigate molecular characterization of tumor-immune interactions. Levels of six tumor-infiltrating immune subsets are precalculated for 10,897 tumors from 32 cancer types. TIMER provides 6 major analytic modules that allow users to interactively explore the associations between immune infiltrates and a wide spectrum of factors, including gene expression, clinical outcomes, somatic mutations, and somatic copy number alterations. TIMER provides a user-friendly web interface for dynamic analysis and visualization of these associations, which will be of broad utilities to cancer researchers.
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Genome-wide screening using CRISPR coupled with nuclease Cas9 (CRISPR-Cas9) is a powerful technology for the systematic evaluation of gene function. Statistically principled analysis is needed for ...the accurate identification of gene hits and associated pathways. Here, we describe how to perform computational analysis of CRISPR screens using the MAGeCKFlute pipeline. MAGeCKFlute combines the MAGeCK and MAGeCK-VISPR algorithms and incorporates additional downstream analysis functionalities. MAGeCKFlute is distinguished from other currently available tools by its comprehensive pipeline, which contains a series of functions for analyzing CRISPR screen data. This protocol explains how to use MAGeCKFlute to perform quality control (QC), normalization, batch effect removal, copy-number bias correction, gene hit identification and downstream functional enrichment analysis for CRISPR screens. We also describe gene identification and data analysis in CRISPR screens involving drug treatment. Completing the entire MAGeCKFlute pipeline requires ~3 h on a desktop computer running Linux or Mac OS with R support.
Despite remarkable clinical efficacy of immune checkpoint blockade (ICB) in cancer treatment, ICB benefits for triple-negative breast cancer (TNBC) remain limited. Through pooled in vivo CRISPR ...knockout (KO) screens in syngeneic TNBC mouse models, we found that deletion of the E3 ubiquitin ligase Cop1 in cancer cells decreases secretion of macrophage-associated chemokines, reduces tumor macrophage infiltration, enhances anti-tumor immunity, and strengthens ICB response. Transcriptomics, epigenomics, and proteomics analyses revealed that Cop1 functions through proteasomal degradation of the C/ebpδ protein. The Cop1 substrate Trib2 functions as a scaffold linking Cop1 and C/ebpδ, which leads to polyubiquitination of C/ebpδ. In addition, deletion of the E3 ubiquitin ligase Cop1 in cancer cells stabilizes C/ebpδ to suppress expression of macrophage chemoattractant genes. Our integrated approach implicates Cop1 as a target for improving cancer immunotherapy efficacy in TNBC by regulating chemokine secretion and macrophage infiltration in the tumor microenvironment.
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•In vivo CRISPR screens identify new immune targets regulating the tumor microenvironment•Cop1 knockout in cancer cells enhances anti-tumor immunity•Cop1 modulates chemokine secretion and macrophage infiltration into tumors•Cop1 targets C/ebpδ degradation via Trib2 and influences ICB response
Large-scale in vivo CRISPR screens reveal that Cop1 knockout in cancer cells stabilizes C/ebpδ protein, suppressing macrophage infiltration and enhancing anti-tumor immunity.
Immune checkpoint blockade (ICB) therapy revolutionized cancer treatment, but many patients with impaired MHC-I expression remain refractory. Here, we combined FACS-based genome-wide CRISPR screens ...with a data-mining approach to identify drugs that can upregulate MHC-I without inducing PD-L1. CRISPR screening identified TRAF3, a suppressor of the NFκB pathway, as a negative regulator of MHC-I but not PD-L1. The
-knockout gene expression signature is associated with better survival in ICB-naïve patients with cancer and better ICB response. We then screened for drugs with similar transcriptional effects as this signature and identified Second Mitochondria-derived Activator of Caspase (SMAC) mimetics. We experimentally validated that the SMAC mimetic birinapant upregulates MHC-I, sensitizes cancer cells to T cell-dependent killing, and adds to ICB efficacy. Our findings provide preclinical rationale for treating tumors expressing low MHC-I expression with SMAC mimetics to enhance sensitivity to immunotherapy. The approach used in this study can be generalized to identify other drugs that enhance immunotherapy efficacy. SIGNIFICANCE: MHC-I loss or downregulation in cancer cells is a major mechanism of resistance to T cell-based immunotherapies. Our study reveals that birinapant may be used for patients with low baseline MHC-I to enhance ICB response. This represents promising immunotherapy opportunities given the biosafety profile of birinapant from multiple clinical trials.
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Immune checkpoint blockade (ICB) therapy has improved patient survival in a variety of cancers, but only a minority of cancer patients respond. Multiple studies have sought to identify general ...biomarkers of ICB response, but elucidating the molecular and cellular drivers of resistance for individual tumors remains challenging. We sought to determine whether a tumor with defined genetic background exhibits a stereotypic or heterogeneous response to ICB treatment.
We establish a unique mouse system that utilizes clonal tracing and mathematical modeling to monitor the growth of each cancer clone, as well as the bulk tumor, in response to ICB. We find that tumors derived from the same clonal populations showed heterogeneous ICB response and diverse response patterns. Primary response is associated with higher immune infiltration and leads to enrichment of pre-existing ICB-resistant cancer clones. We further identify several cancer cell-intrinsic gene expression signatures associated with ICB resistance, including increased interferon response genes and glucocorticoid response genes. These findings are supported by clinical data from ICB treatment cohorts.
Our study demonstrates diverse response patterns from the same ancestor cancer cells in response to ICB. This suggests the value of monitoring clonal constitution and tumor microenvironment over time to optimize ICB response and to design new combination therapies. Furthermore, as ICB response may enrich for cancer cell-intrinsic resistance signatures, this can affect interpretations of tumor RNA-seq data for response-signature association studies.
BRAF is a serine/threonine kinase that harbors activating mutations in ∼7% of human malignancies and ∼60% of melanomas. Despite initial clinical responses to BRAF inhibitors, patients frequently ...develop drug resistance. To identify candidate therapeutic targets for BRAF inhibitor resistant melanoma, we conduct CRISPR screens in melanoma cells harboring an activating BRAF mutation that had also acquired resistance to BRAF inhibitors. To investigate the mechanisms and pathways enabling resistance to BRAF inhibitors in melanomas, we integrate expression, ATAC-seq, and CRISPR screen data. We identify the JUN family transcription factors and the ETS family transcription factor ETV5 as key regulators of CDK6, which together enable resistance to BRAF inhibitors in melanoma cells. Our findings reveal genes contributing to resistance to a selective BRAF inhibitor PLX4720, providing new insights into gene regulation in BRAF inhibitor resistant melanoma cells.
BackgroundCancer immunotherapy, especially immune checkpoint blockade (ICB) therapy, is leading to a paradigm shift in cancer treatment, as a small percentage of cancer patients have obtained durable ...remission following ICB treatment. Successful ICB responses rely on cancer cells presenting antigens to the cell surface via the major histocompatibility complex (MHC), which activates antigen-specific T-lymphocytes to kill cancer cells. Type-I MHC (MHC-I) is wildly expressed in all cell types and mediates the interaction with cytotoxic CD8 T cells. However, over 65% of cancer patients are estimated to show defects in MHC-I-mediated antigen presentation, including downregulation of its expression that can lead to primary or acquired resistance to ICB therapy, and therapeutic strategies to effectively restore or boost MHC-I are limited.MethodsHere, we employed a CRISPR screening approach with dual-marker FACS sorting to identify factors that decouple the regulation of MHC-I and PD-L1. The experimentally validated target was used to generate a KO differential expression signature. Using this signature, we analyzed transcriptome data from drug perturbation studies to identify drugs that regulate MHC-I but not PD-L1. Finally, we validated the effect of the identified drug to enhance ICB response in a T-cell-dependent manner in vivo.ResultsCRISPR screens identified TRAF3, a suppressor of the NF-κB pathway, as a negative regulator of MHC-I but not PD-L1. The Traf3-knockout (Traf3-KO) gene expression signature is associated with better survival in ICB-naive cancer patients and better ICB response. We then screened for drugs with similar transcriptional effects as this signature and identified SMAC mimetics. We experimentally validated that the SMAC mimetic birinapant upregulates MHC-I, sensitizes cancer cells to T-cell-dependent killing, and adds to ICB efficacy. However, in cancer cells with high NF-κB activity, the effect of birinapant on MHC-I is weak, indicating context-dependent MHC-I regulation.ConclusionsIn summary, Traf3 deletion specifically upregulates MHC-I without inducing PD-L1 in response to various cytokines and sensitizes cancer cells to T-cell-driven cytotoxicity. The SMAC mimetic birinapant phenocopies Traf3-knockout and sensitizes MHC-I-low melanoma to ICB therapy. Further studies are needed to elucidate the context-dependencies of MHC-I regulation. Our findings provide preclinical rationale for treating some tumors expressing low MHC-I with SMAC mimetics to enhance sensitivity to immunotherapy. The approach used in this study can be generalized to identify other drugs that enhance immunotherapy efficacy.AcknowledgementsThis study was supported by grants from the NIH (R01CA234018 to XSL, R01AI137337 to BEG, P50CA101942-12 and P50CA206963 to GJF), Breast Cancer Research Foundation (BCRF-19-100 to XSL), Burroughs Wellcome Career Award in Medical Sciences (to BEG), and Sara Elizabeth O'Brien Trust Fellowship (to SG).We thank Drs. Kai Wucherpfennig and Deng Pan for their insightful suggestions on this study.Ethics ApprovalAll mice were housed in standard cage in Dana-Farber Cancer Institute Animal Resources Facility (ARF). All animal procedures were carried out under the ARF Institutional Animal Care and Use Committee (IACUC) protocol and were in accordance with the IACUC standards for the welfare of animals.
Cancer treatment by immune checkpoint blockade (ICB) can bring long-lasting clinical benefits, but only a fraction of patients respond to treatment. To predict ICB response, we developed TIDE, a ...computational method to model two primary mechanisms of tumor immune evasion: the induction of T cell dysfunction in tumors with high infiltration of cytotoxic T lymphocytes (CTL) and the prevention of T cell infiltration in tumors with low CTL level. We identified signatures of T cell dysfunction from large tumor cohorts by testing how the expression of each gene in tumors interacts with the CTL infiltration level to influence patient survival. We also modeled factors that exclude T cell infiltration into tumors using expression signatures from immunosuppressive cells. Using this framework and pre-treatment RNA-Seq or NanoString tumor expression profiles, TIDE predicted the outcome of melanoma patients treated with first-line anti-PD1 or anti-CTLA4 more accurately than other biomarkers such as PD-L1 level and mutation load. TIDE also revealed new candidate ICB resistance regulators, such as SERPINB9, demonstrating utility for immunotherapy research.
Abstract
Background: Cancer-Associated Fibroblasts (CAFs) are a major cell type within breast tumor microenvironment (TME) which have emerged as a new target for cancer therapy given their ...contributions to tumor growth, metastasis, and suppression of the anti-tumor immune response. In the breast, CAFs are predominantly formed from conversion of tissue-resident mammary fibroblasts through mechanisms including increasing extracellular matrix (ECM) stiffness, ECM composition, hypoxia, and activating signals TGFβ and IL-6. Domain Discoidin Receptor 1 (DDR1) is a cell surface tyrosine kinase receptor expressed by epithelial and stromal cells which is activated by collagen within the TME. DDR1 expression and activity has been implicated in the development of fibrosis within the breast as well as chemotherapy resistance in several tumor types. Thus, we set out to examine whether selective inhibition of DDR1 would affect CAF immunomodulatory function to generate an immune-permissive breast microenvironment and result in reduction in stromal desmoplasia. Results: Using a human cell model of CAFs, we studied the effects of DDR1 inhibition (DDR1i) on the expression of immune modulating factors and CAF functions and features. Using a multiplex cytokine array, we observed high expression levels of key immunomodulatory cytokines IL-6, IL-8, and MCP-1 in conditioned media collected from CAF-like cells cultured in 3D collagen gels which were consequently downregulated in the presence of DDR1i. Downregulation of IL-6, IL-8, and MCP-1 induced by DDR1i was also by observed by RT-qPCR, with enhanced downregulation seen in cells cultured with increasing concentrations of collagen. Downregulation of CAF markers αSMA and FAP as well as Collagen type 1 were observed with selective DDR1 inhibition, an effect seen also with CAF-like cells stimulated with recombinant TGFβ1. Additionally, we observed reduced contraction of CAF-like cells in 3D collagen gels in the presence of DDR1i. In vivo DDR1i resulted in tissue remodeling and reduced collagen deposition as well as changes in immunomodulatory cytokine release within the mammary gland in a murine model. We observed downregulation of IL-6, as well as downregulation of Collagens type I, type IV, and MMP9. RT-qPCR also revealed reduced expression of potent chemokines CXCL1 and CXCL12 in mammary gland tissue of mice treated with DDR1i. Consistent with this, multiplexed flow cytometry demonstrated trends of increased CD45.2+ immune cell infiltration within the mammary glands and reduction of Ly6G+/Ly6C- neutrophil infiltration within the mammary gland and spleens in mice treated with DDR1i. Conclusions: Collectively, these findings suggest that breast fibroblast-specific DDR1 mediates collagen deposition and immunomodulatory function within the mammary gland and warrants further investigation as a potential target for CAF-modulating therapy in breast cancer.
Citation Format: Daniel EC Fein, Nicole Traugh, Gat Rauner, Youssof Mal, Colin Trepicchio, Charlotte Kuperwasser. Selective inhibition of fibroblast-specific domain discoidin receptor 1 (DDR1) results in reduced collagen deposition and immunomodulatory cytokine release: A potential target to modulate the activity of breast cancer associated fibroblasts abstract. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-07.
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