Search

Isocitrate dehydrogenase 1 mutations (mIDH1) are common in cholangiocarcinoma. (R)-2-hydroxyglutarate generated by the mIDH1 enzyme inhibits multiple α-ketoglutarate–dependent enzymes, altering epigenetics and metabolism. Here, by developing mIDH1-driven genetically engineered mouse models, we show that mIDH1 supports cholangiocarcinoma tumor maintenance through an immunoevasion program centered on dual (R)-2-hydroxyglutarate–mediated mechanisms: suppression of CD8+ T-cell activity and tumor cell–autonomous inactivation of TET2 DNA demethylase. Pharmacologic mIDH1 inhibition stimulates CD8+ T-cell recruitment and interferon γ (IFNγ) expression and promotes TET2-dependent induction of IFNγ response genes in tumor cells. CD8+ T-cell depletion or tumor cell–specific ablation of TET2 or IFNγ receptor 1 causes treatment resistance. Whereas immune-checkpoint activation limits mIDH1 inhibitor efficacy, CTLA4 blockade overcomes immunosuppression, providing therapeutic synergy. The findings in this mouse model of cholangiocarcinoma demonstrate that immune function and the IFNγ–TET2 axis are essential for response to mIDH1 inhibition and suggest a novel strategy for potentiating efficacy. Significance: Mutant IDH1 inhibition stimulates cytotoxic T-cell function and derepression of the DNA demethylating enzyme TET2, which is required for tumor cells to respond to IFNγ. The discovery of mechanisms of treatment efficacy and the identification of synergy by combined CTLA4 blockade provide the foundation for new therapeutic strategies. See related commentary by Zhu and Kwong, p. 604. This article is highlighted in the In This Issue feature, p. 587

Syngeneic mouse models are tumors derived from murine cancer cells engrafted on genetically identical mouse strains. They are widely used tools for studying tumor immunity and immunotherapy response in the context of a fully functional murine immune system. Large volumes of syngeneic mouse tumor expression profiles under different immunotherapy treatments have been generated, although a lack of systematic collection and analysis makes data reuse challenging. We present Tumor Immune Syngeneic MOuse (TISMO), a database with an extensive collection of syngeneic mouse model profiles with interactive visualization features. TISMO contains 605 in vitro RNA-seq samples from 49 syngeneic cancer cell lines across 23 cancer types, of which 195 underwent cytokine treatment. TISMO also includes 1518 in vivo RNA-seq samples from 68 syngeneic mouse tumor models across 19 cancer types, of which 832 were from immune checkpoint blockade (ICB) studies. We manually annotated the sample metadata, such as cell line, mouse strain, transplantation site, treatment, and response status, and uniformly processed and quality-controlled the RNA-seq data. Besides data download, TISMO provides interactive web interfaces to investigate whether specific gene expression, pathway enrichment, or immune infiltration level is associated with differential immunotherapy response. TISMO is available at http://tismo.cistrome.org.

The characterization of cancer genomes has provided insight into somatically altered genes across tumors, transformed our understanding of cancer biology, and enabled tailoring of therapeutic strategies. However, the function of most cancer alleles remains mysterious, and many cancer features transcend their genomes. Consequently, tumor genomic characterization does not influence therapy for most patients. Approaches to understand the function and circuitry of cancer genes provide complementary approaches to elucidate both oncogene and non-oncogene dependencies. Emerging work indicates that the diversity of therapeutic targets engendered by non-oncogene dependencies is much larger than the list of recurrently mutated genes. Here we describe a framework for this expanded list of cancer targets, providing novel opportunities for clinical translation.

T cell dysfunction is a hallmark of many cancers, but the basis for T cell dysfunction and the mechanisms by which antibody blockade of the inhibitory receptor PD-1 (anti-PD-1) reinvigorates T cells are not fully understood. Here we show that such therapy acts on a specific subpopulation of exhausted CD8(+) tumor-infiltrating lymphocytes (TILs). Dysfunctional CD8(+) TILs possess canonical epigenetic and transcriptional features of exhaustion that mirror those seen in chronic viral infection. Exhausted CD8(+) TILs include a subpopulation of ‘progenitor exhausted’ cells that retain polyfunctionality, persist long term and differentiate into ‘terminally exhausted’ TILs. Consequently, progenitor exhausted CD8(+) TILs are better able to control tumor growth than are terminally exhausted T cells. Progenitor exhausted TILs can respond to anti-PD-1 therapy, but terminally exhausted TILs cannot. Patients with melanoma who have a higher percentage of progenitor exhausted cells experience a longer duration of response to checkpoint-blockade therapy. Thus, approaches to expand the population of progenitor exhausted CD8(+) T cells might be an important component of improving the response to checkpoint blockade.

Epigenetic dysregulation is a defining feature of tumorigenesis that is implicated in immune escape1,2. Here, to identify factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumour models treated with immune checkpoint blockade. We identified the H3K9 methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape3-5. We also found that amplification of SETDB1 (1q21.3) in human tumours is associated with immune exclusion and resistance to immune checkpoint blockade. SETDB1 represses broad domains, primarily within the open genome compartment. These domains are enriched for transposable elements (TEs) and immune clusters associated with segmental duplication events, a central mechanism of genome evolution6. SETDB1 loss derepresses latent TE-derived regulatory elements, immunostimulatory genes, and TE-encoded retroviral antigens in these regions, and triggers TE-specific cytotoxic T cell responses in vivo. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses tumour-intrinsic immunogenicity, and thus represents a candidate target for immunotherapy.

Summary Inflammation can support or restrain cancer progression and the response to therapy. Here, we searched for primary regulators of cancer-inhibitory inflammation through deep profiling of inflammatory tumor microenvironments (TMEs) linked to immune-dependent control in mice. We found that early intratumoral accumulation of interferon gamma (IFN-γ)-producing natural killer (NK) cells induced a profound remodeling of the TME and unleashed cytotoxic T cell (CTL)-mediated tumor eradication. Mechanistically, tumor-derived prostaglandin E2 (PGE2) acted selectively on EP2 and EP4 receptors on NK cells, hampered the TME switch, and enabled immune evasion. Analysis of patient datasets across human cancers revealed distinct inflammatory TME phenotypes resembling those associated with cancer immune control versus escape in mice. This allowed us to generate a gene-expression signature that integrated opposing inflammatory factors and predicted patient survival and response to immune checkpoint blockade. Our findings identify features of the tumor inflammatory milieu associated with immune control of cancer and establish a strategy to predict immunotherapy outcomes., Graphical Abstract, Highlights • NK cells drive broad inflammatory remodeling characteristic of T-cell-inflamed tumors • PGE2 acting on EP2 and EP4 on NK cells prevents the TME switch enabling immune escape • Opposing inflammatory profiles found in many human cancer types have prognostic value • A signature capturing pro- and anti-tumor factors predicts response to immunotherapy, Inflammation can fuel or suppress cancer. Bonavita et al. identify NK cells as primary orchestrators of T cell inflammation in murine models and devise an inflammatory signature that predicts patient survival and response to immunotherapy in multiple malignancies.

Both tumor- and host-derived PD-L1 can play critical roles in immunosuppression; differences in tumor immunogenicity appear to underlie their relative contributions. Juneja et al. show that in immunogenic MC38 tumors, PD-L1 on tumor cells dominates in suppressing tumor immunity by inhibiting CD8 T cell cytotoxicity., It is unclear whether PD-L1 on tumor cells is sufficient for tumor immune evasion or simply correlates with an inflamed tumor microenvironment. We used three mouse tumor models sensitive to PD-1 blockade to evaluate the significance of PD-L1 on tumor versus nontumor cells. PD-L1 on nontumor cells is critical for inhibiting antitumor immunity in B16 melanoma and a genetically engineered melanoma. In contrast, PD-L1 on MC38 colorectal adenocarcinoma cells is sufficient to suppress antitumor immunity, as deletion of PD-L1 on highly immunogenic MC38 tumor cells allows effective antitumor immunity. MC38-derived PD-L1 potently inhibited CD8+ T cell cytotoxicity. Wild-type MC38 cells outcompeted PD-L1–deleted MC38 cells in vivo, demonstrating tumor PD-L1 confers a selective advantage. Thus, both tumor- and host-derived PD-L1 can play critical roles in immunosuppression. Differences in tumor immunogenicity appear to underlie their relative importance. Our findings establish reduced cytotoxicity as a key mechanism by which tumor PD-L1 suppresses antitumor immunity and demonstrate that tumor PD-L1 is not just a marker of suppressed antitumor immunity.

Summary CRISPR-Cas9 genome engineering has increased the pace of discovery for immunology and cancer biology, revealing potential therapeutic targets and providing insight into mechanisms underlying resistance to immunotherapy. However, endogenous immune recognition of Cas9 has limited the applicability of CRISPR technologies in vivo. Here, we characterized immune responses against Cas9 and other expressed CRISPR vector components that cause antigen-specific tumor rejection in several mouse cancer models. To avoid unwanted immune recognition, we designed a lentiviral vector system that allowed selective CRISPR antigen removal (SCAR) from tumor cells. The SCAR system reversed immune-mediated rejection of CRISPR-modified tumor cells in vivo and enabled high-throughput genetic screens in previously intractable models. A pooled in vivo screen using SCAR in a CRISPR-antigen-sensitive renal cell carcinoma revealed resistance pathways associated with autophagy and major histocompatibility complex class I (MHC class I) expression. Thus, SCAR presents a resource that enables CRISPR-based studies of tumor-immune interactions and prevents unwanted immune recognition of genetically engineered cells, with implications for clinical applications.

Epigenetic alterations are defining features of many tumor types and have recently been implicated in tumor immunity. However, the epigenetic mechanisms that mediate immune sensitivity or resistance in cancer cells are poorly characterized. To systematically identify epigenetic regulators of immune evasion in cancer, we performed in vivo loss of function CRISPR screens against 936 chromatin regulator genes in syngeneic murine tumor models treated with immune checkpoint blockade. These screens identified SETDB1, an H3K9-methyltransferase, and associated members of the HUSH and KAP1 complexes as intrinsic mediators of immune evasion in cancer cells. We also found that amplification of SETDB1 (1q21) in certain human tumors is associated with immune exclusion and resistance to immune checkpoint blockade. Mechanistically, we find that SETDB1 targets broad domains, hundreds of kilobases in size, that are predominantly located within the open genome compartment “A” (i.e., euchromatin). These SETDB1 domains show strong enrichment for transposable elements (TEs) of the LTR family, and gene loci that arose through segmental duplication events, a key driver of mammalian genome evolution. Setdb1 KO derepresses latent regulatory elements at TEs within these regions and leads to the transcriptional up-regulation of nearby immune genes, including canonical stimulatory ligands of the NKG2D receptor. SETDB1 loss also triggers the activation of hundreds of TEs with the potential to encode retroviral proteins (Gag, Pol, Env), and promotes immune responses dependent on CD8+ T cells and tumor expression of MHC Class I. Our study establishes SETDB1 as an epigenetic checkpoint that represses intrinsic immunogenicity in cancer cells, and thus represents a novel target to enhance the efficacy and scope of immunotherapy. Citation Format: Jingyi Wu, Arvin Iracheta-Vellve, James Patti, Jeffrey Hsu, Thomas Davis, Deborah Dele-Oni, Peter Du, Jeffrey Ishizuka, Sarah Kim, Susan Klaeger, Nelson Knudsen, Brian Miller, Tung Nguyen, Emily Robitschek, Emily Schneider, Margaret Zimmer, Jacob Jaffe, John Doench, W. Nicholas Haining, Kathleen Yates, Robert Manguso, Bradley Bernstein, Gabriel K. Griffin. Epigenetic silencing by SETDB1 represses tumor-cell intrinsic immunogenicity [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO009.

Summary Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer characterized by complex metabolic adaptations that promote survival in a severely hypoxic and nutrient-limited tumor microenvironment (TME). 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 screens using in vivo and in vitro systems. This work revealed striking overlap of in vivo metabolic dependencies with those in vitro. Moreover, we identified that intercellular nutrient sharing can mask dependencies in pooled screens, highlighting a limitation of this approach to study tumor metabolism. Furthermore, metabolic dependencies were similar between 2D and 3D culture, although 3D culture may better model vulnerabilities that influence certain oncogenic signaling pathways. Lastly, our work demonstrates the power of genetic screening approaches to define in vivo metabolic dependencies and pathways that may have therapeutic utility.

Intrahepatic cholangiocarcinoma (ICC) is a cancer of the liver bile ducts, which has been rising in incidence and carries a 5-year survival rate of less than 10%. Mutations in isocitrate dehydrogenase 1 and 2 (mIDH1/2) are among the most common genetic alterations in ICC, present in 20% patients. The hot-spot point mutations of IDH result in aberrant enzymatic function, reducing alpha-ketoglutarate (alphaKG) to the oncometabolite, 2-hydroxyglutarate (2-HG), which accumulates to millimolar concentrations within the cell. 2-HG competitively inhibits alphaKG-dependent histone and DNA demethylating enzymes, thereby altering chromatin regulation. The first-in-class small molecule selective IDH1m inhibitor, ivosidenib, has shown benefit in phase I and III trials for patients with IDH1m ICC. In order to gain insight into the mechanisms underlying the response to IDH1m inhibition, we developed the first genetically engineered mouse model (GEMM) of IDH1m ICC. We found that ivosidenib reduced the overall tumor burden and increased overall survival of syngeneic allografts of ICCs derived from the GEMM. The positive response to ivosidenib was associated with an induction of mature hepatocyte lineage markers and a reduction of tumor produced 2-HG >85%, supporting observations from the ivosidenib phase I data in ICC patients that mIDH1 inhibition may promote ICC tumor cell differentiation. In addition, there was pronounced induction of immune stimulatory interferon signaling, increased recruitment of CD8+ T cells to the tumors and strong PD-L1 upregulation. Thus, ivosidenib treatment leads to a sustained inhibition of 2-HG production, which correlated with an increased immune stimulation compared to vehicle treatment. We will present these data as well as ongoing studies interrogating the immune and transcriptomic changes with further resolution via fluorescence-activated cell sorting and single-cell RNA-sequencing analysis, direct functional testing of contributions of specific immune populations to the therapeutic efficacy of ivosidenib, and testing ivosidenib in combination with checkpoint inhibition. These studies highlight the impact of ivosidenib on hepatocyte differentiation and immune stimulation in IDH1m ICC and support the key role of IDHm in ICC tumor maintenance. Citation Format: Meng-Ju Wu, Lei Shi, Juan Dubrot, Christine Hudson, Joshua Merritt, Ramzi Adil, Qibiao Wu, Sebastien Ronseaux, Rohini Narayanaswamy, Robert Manguso, Brandon Nicolay, Nabeel Bardeesy. The mutant IDH1 inhibitor ivosidenib promotes tumor cell differentiation and anti-tumor immunity in intrahepatic cholangiocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1810.

The ability to make rapid genetic alterations to tumor cells with CRISPR for screens and single-gene studies has been transformative for studying cancer biology. Recently, we and others have used CRISPR in murine tumor models in vivo to identify targets and resistance mechanisms for immunotherapy. However, the use of these techniques in immunocompetent mice is limited by endogenous immune recognition of CRISPR components such as Cas9. Here we report the design of a screening-compatible lentiviral vector system which allows selective CRISPR antigen removal (SCAR) from tumor cells before they are implanted in vivo. The SCAR system enables highly efficient genome engineering and reverses CD8+ T cell-mediated rejection of CRISPR modified tumor cells in vivo. Using this technology, we conducted a pooled, in vivo screen designed to probe tumor-immune interactions in the highly immunogenic CT26 colon carcinoma model and discovered that these tumors require intact interferon signaling to evade immune destruction. The SCAR system enables the study of tumor-immune interactions in any cancer model and can be more broadly applied to generate non-immunogenic lentivirally-engineered cells. Citation Format: Juan Dubrot, Sarah Kate Lane-Reticker, Emily Kessler, Clara Wolfe, Animesh Mahapatra, Peter Du, Robert T. Manguso. SCAR (Selective CRISPR Antigen Removal) vector system expands cancer immunology discovery with in vivo functional genomics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5866.

During the last decade, there has been notable progress in cancer immunotherapy, including promising clinical success of immune checkpoint inhibitors. Moreover, there is an increasing number of new potential targets for cancer immunotherapy that are currently being developed both as monotherapy or in combination. However, the lack of durable clinical responses due in part to the resistance mechanisms that tumors exhibit in a significant proportion of patients urges novel approaches to find the right therapeutic strategies. The understanding of such tumor evasion mechanisms and the complex interactions that take place in the tumor microenvironment is essential to obtain successful treatments. Functional genomics has emerged as a powerful tool that can help to reveal some of these unknown processes, with CRISPR-Cas9 technology the most prominent example. However, in vivo genetic screens require stable expression of several components that in most cases are not derived from murine cells, which represents a major obstacle in the context of cancer immunology. The inoculation of engineered tumor cell lines in immunocompetent mice results in either tumor rejection or an aberrant response to immunotherapy, making the experiments technically impossible in the first case or providing inconsistent results in the latter. We have designed new vector strategies to overcome the altered immunogenicity of Cas9-expressing tumor cell lines using the Cre-lox system. Expression and effective removal of Cas9 and vector components are monitored using fluorescent reporter proteins. Both genome-editing capacity and normal in vivo behavior of all tested cell lines remain largely unaltered using this novel strategy. This technology represents a major advance for preclinical functional genomics, allowing studies in fully competent animals looking for better combination treatments and resistance mechanism in particular cancer models, and ultimately will help in the design of clinical trials. Citation Format: Juan Dubrot, Robert T. Manguso, Sarah Kate Lane-Reticker, Austin Ayer, Emily Kessler, Clara Wolfe, Animesh Mahapatra. Novel CRISPR/Cas9 vectors for in vivo tumor functional genomics [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B10.

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

T-cell exhaustion is a heterogeneous state, with recent work in chronic viral infections revealing at least two subtypes with different functional properties: "stem-like" and "terminal" exhausted cells. Whether these two populations exist in tumors and have different roles to control tumor growth remains unknown. We performed single-cell RNA-seq on exhausted CD8+ T cells from chronic virally infected mice, generating unique transcriptional signatures of both exhausted populations. By transcriptional enrichment and flow cytometry, we discovered both the stem-like and terminal exhausted populations in the B16 mouse melanoma model and confirmed their presence in human melanoma samples with multiplex immunofluorescence. ATAC-seq revealed that these two subpopulations are defined by state-specific changes in chromatin accessibility. These states are shared between exhausted CD8+ T cells from both viral and tumor models, demonstrating a common epigenetic program of T-cell dysfunction. The two populations have different functional properties, with the stem-like cells having a more polyfunctional cytokine response whereas terminal cells are more cytotoxic. When transferred into new tumor-bearing mice, stem-like cells were better able to control tumor growth, suggesting superior long-term functionality. Checkpoint blockade with anti-PD-1 is known to increase CD8+ T cell numbers in the tumor microenvironment, but it is not known which subpopulation responds to therapy. Treatment with anti-PD-1 antibody resulted in proliferation of the stem-like population, which differentiate into the more cytotoxic terminal exhausted cells. We have shown that two subpopulations of exhausted CD8+ T cells exist in mouse and human tumors, and anti-PD-1 therapy activates the stem-like CD8+ T cells to promote tumor control. Citation Format: Brian C. Miller, Debattama R. Sen, Rose Al-Abosy, Kevin Bi, Kathleen B. Yates, Evisa Gjini, Kristen Felt, Robert T. Manguso, Scott J. Rodig, Arlene H. Sharpe, Nicholas Haining. Distinct subsets of dysfunctional CD8+ T cells underlie response to checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4683.

OVERVIEW: Despite the impact of cancer immunotherapy, expanding its clinical utility requires a rational method for identifying combination therapies and resistance mechanisms. This is pertinent to glioblastoma (GBM), where initial trials show uncertain response rates. Functional genomic screens used to identify new therapies and escape mechanisms were generally conducted in vitro, where interaction with the immune system is absent. Here we present the development of a high-throughput, in vivo, loss-of-function screening platform for GBM immune-escape mechanisms. METHODS: CT2A- and GL261-astrocytoma cells bearing Cas9-endonuclease were engineered to express a library of barcoded single guide RNAs (sgRNAs). These cells form tumors when implanted intracranially in immunocompetent mice. Tumor-bearing mice were treated with vaccination or PD-1 checkpoint blockade. Dropout of sgRNAs targeting putative immune evasion molecules or enrichment of sgRNAs mimicking resistance mechanisms were detected using next-generation sequencing at the time of tumor implantation and harvest post-immunotherapy. RESULTS: We first developed an in vivo, pooled, loss-of-function genetic screen using Cas9/CRISPR genome editing in mouse transplantable tumors subjected to titratable, selective immune-pressure. Screening 2,400 genes expressed by melanoma cells for those that synergize with or cause resistance to checkpoint blockade recovered known immune-evasion molecules PD-L1 and CD47. Novel immunotherapy targets validated individually, identifying essential pathways of immune-evasion. We then sought to recapitulate this approach in the CNS, and showed that 500-1000 genes can be functionally screened under graded immune-pressure. In optimized immune-competent CT2A and GL261 GBM models, tumors derived from cancer stem cell CD133hi-rich neurospheres were sensitive to immunotherapy and more aggressive and infiltrative than tumors derived from adherent tumor cells. A neurosphere based immune-competent model can be scaled up to a whole-genome screen due to a shorter experimental time requirement and improved engraftment allowing for functional screening of >1000 genes. We curated a GBM-specific library based on differential in vitro and in vivo gene expression profiles of CT2A and GL261 cells exposed to graded immune pressure. We are now screening this GBM-specific library in our optimized in vivo pooled loss-of-function genetic screen using immune-pressure titration to identify novel immunotherapy targets in GBM. CONCLUSIONS: This assay provides the first high-throughput method for systematically identifying resistance mechanisms and new candidate targets for immunotherapy in CNS tumors. Our optimized model could be scaled up to whole-genome loss of function screens, serving as an important tool for identification of next-generation and combination immunotherapies. Citation Format: Martha R. Neagu, Robert T. Manguso, Hans Pope, Maria C. Speranza, Gordon J. Freeman, John Doench, Arlene H. Sharpe, William Nicholas Haining. Defining molecular mechanisms of resistance to glioblastoma immunity using a novel CRISPR/Cas9 in vivo loss-of-function screening platform [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 417. doi:10.1158/1538-7445.AM2017-417

Recent success of immune checkpoint blockade solidifies the importance of the immune system in the defense against cancer. The clinical impact of the immune response is, however, very heterogeneous, with some patients achieving dramatic responses while others fail to respond. Known genomic correlates of response to immunotherapy are not perfectly predictive of clinical outcome, supporting the existence of unknown mechanisms of resistance to tumor immunity. I hypothesize somatic acquired mutations of individual tumors may account for heterogeneity in the spontaneous response to tumors and response to immunotherapy. I have undertaken a systematic in vivo screen to identify mechanisms of resistance to tumor immunity in order to discover new mechanisms of immune resistance, define a comprehensive set of therapeutic targets and provide biomarkers of sensitivity to immunotherapeutic strategies. Mouse tumor cell lines (MC38 colon carcinoma or B16 melanoma) were engineered to express a library of barcoded open reading frames (ORFs) mutagenized to encode known cancer-associated somatic mutations from the Pan Cancer analysis within The Cancer Genome Atlas (TCGA). These cell lines form tumors when implanted subcutaneously in immunocompetent animals. Tumor-bearing animals were then subjected to immunotherapy with either therapeutic vaccination or checkpoint blockade with anti-PD-1. Barcode relative representation was measured by next generation sequencing at the time of tumor implantation and at the time of tumor harvest post-immunotherapy. Barcoded mutant ORFs that confer immune resistance increased in representation under immune pressure in comparison to untreated or immunodeficient animals. A mutation in Phospho-Inositol 3 Kinase (PI3K), PIK3CA c.3140A>G, consistently increased in representation in both B16 and MC38 immunotherapy-treated tumors. This mutation encodes a constitutively active mutant catalytic domain of PI3K, PIK3CA H1047R. MC38 tumors homogenously expressing PIK3CA H1047R and implanted into wild type mice failed to respond to anti-PD-1 therapy, while tumors expressing a control gene or non-scoring mutation in PI3K regressed after treatment with anti-PD-1. Pharmacologic PI3K inhibition resensitized tumors to treatment with anti-PD-1. PD-1-treated PIK3CA H1047R tumors had fewer infiltrating CD8+ T cells as measured by immunohistochemistry and flow cytometry of tumor infiltrating lymphocytes. I conclude that PI3K has, in addition to its well-described oncogenic role, a role in tumor immune evasion. As such, activation of PI3K may be useful as a predictor of resistance to immunotherapy. Importantly, these findings also provide a rationale for therapeutic combination trials of immune checkpoint blockade and PI3K inhibition. Citation Format: Natalie B. Collins, Robert Manguso, Hans Pope, W. Nicholas Haining. Defining molecular mechanisms of resistance to tumor immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A16.

Background Ovarian cancer is the fourth leading cause of cancer-related deaths among women in Denmark, largely due to the advanced stage at diagnosis in most patients. Approximately 90% of ovarian cancers originate from the single-layered ovarian surface epithelium (OSE). Defects in the primary cilium, a solitary sensory organelle in most cells types including OSE, were recently implicated in tumorigenesis, mainly due to deregulation of ciliary signaling pathways such as Hedgehog (Hh) signaling. However, a possible link between primary cilia and epithelial ovarian cancer has not previously been investigated. Methods The presence of primary cilia was analyzed in sections of fixed human ovarian tissue as well as in cultures of normal human ovarian surface epithelium (OSE) cells and two human OSE-derived cancer cell lines. We also used immunofluorescence microscopy, western blotting, RT-PCR and siRNA to investigate ciliary signaling pathways in these cells. Results We show that ovarian cancer cells display significantly reduced numbers of primary cilia. The reduction in ciliation frequency in these cells was not due to a failure to enter growth arrest, and correlated with persistent centrosomal localization of aurora A kinase (AURA). Further, we demonstrate that ovarian cancer cells have deregulated Hh signaling and platelet-derived growth factor receptor alpha (PDGFRα) expression and that promotion of ciliary formation/stability by AURA siRNA depletion decreases Hh signaling in ovarian cancer cells. Lastly, we show that the tumor suppressor protein and negative regulator of AURA, checkpoint with forkhead-associated and ring finger domains (CHFR), localizes to the centrosome/primary cilium axis. Conclusions Our results suggest that primary cilia play a role in maintaining OSE homeostasis and that the low frequency of primary cilia in cancer OSE cells may result in part from over-expression of AURA, leading to aberrant Hh signaling and ovarian tumorigenesis.

The microtubule (MT) plus-end-tracking protein EB1 is required for assembly of primary cilia in mouse fibroblasts, but the mechanisms involved and the roles of the related proteins EB2 and EB3 in ciliogenesis are unknown. Using protein depletion experiments and expression of dominant-negative constructs we show here that EB1 and EB3, but not EB2, are required for assembly of primary cilia in cultured cells. Electron microscopy and live imaging showed that cells lacking EB1 or EB3 are defective in MT minus-end anchoring at the centrosome and/or basal body, and possess abnormally short cilia stumps surrounded by vesicles. Further, GST pull-down assays, mass spectrometry and immunoprecipitation indicated that EB1 and EB3 interact with proteins implicated in MT minus-end anchoring or vesicular trafficking to the cilia base, suggesting that EB1 and EB3 promote ciliogenesis by facilitating such trafficking. In addition, we show that EB3 is localized to the tip of motile cilia in bronchial epithelial cells and affects the formation of centriole-associated rootlet filaments. Collectively, our findings indicate that EBs affect biogenesis of cilia by several centrosome-related mechanisms and support the idea that different EB1–EB3 dimer species have distinct functions within cells.