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PDF file 242K, Nkx2-1neg areas have a selective advantage in primary TMet tumors with significantly higher levels of proliferation and insignificant changes in levels of cell death

PDF file 91K, KPT-Late mice with lung tumor initiated with adenoviral or lentiviral Cre vectors have similar progression and metastasis

Supplementary Table S1, Gene sets that are enriched in Hmga2/GFP-positive and -negative PDAC cells; Supplementary Table S2, A stringent list of Blimp1-dependent genes that are either induced or repressed under hypoxia; Supplementary Table S3, GO terms enriched in hypoxia-induced, Blimp1-dependent genes by FuncAssociate 3.0.

PDF file 2560K, Titratable induction of early and late stage lung cancer in KrasLSLG12D/+;p53flox/flox;R26tdTomato/+ (KPT) mice

Supplementary Figure S1. Isolation of the Hmga2-GFPpos PDAC sub-population from the KPCcolors mice and GFPpos PDAC cells are a highly metastatic state;Supplementary Figure S2. Highly metastatic PDAC cells have a gene signature that is not enriched for CSC markers and distal PDAC metastases reveal minor gene expression changes related to glucose metabolism;Supplementary Figure S3. Identification of top candidate pro-metastatic genes and interrogation of Blimp1 function in PDAC metastasis;Supplementary Figure S4. Hmga2positive PDAC areas overlap with hypoxic areas and Hmga2 protein is slightly induced by hypoxia but not critical to the expression of hypoxia-induced target genes;Supplementary Figure S5. Hypoxia-induced Blimp1 expression is linked to functional HRE motifs 240 kb upstream of its transcription start site;Supplementary Figure S6. Blimp1 may contribute to migratory and clonal growth ability and is critical for a subset of hypoxia-induced gene expression changes that are independent of changes in chromatin accessibility;Supplementary Figure S7. Blimp1 regulates a subset of hypoxia-induced genes; Supplementary Figure S8. Blimp1 is required for hypoxia-induced cell cycle arrest and the expression of metastasis modulators.

PDF file 105K, Generation and validation of the multicolor Motley reporter allele

PDF file 128K, KT-Early and KT-Late mice have high total tumor burden

PDF file 320K, Similar total tumor burden and core genomic alterations in KPT-Early and KPT-Late mice

PDF file 91K, KPT-Late mice have a diverse numbers of disseminated tumor cells

PDF file 1080K, Molecular characterization of disseminated cancer cells is required to understand the stage of metastatic progression at which Nkx2-1 and genes associated with the Nkx2-1neg state function

Despite its clinical importance, very little is known about the natural history and molecular underpinnings of lung cancer dissemination and metastasis. Here, we used a genetically engineered mouse model of metastatic lung adenocarcinoma in which cancer cells are fluorescently marked to determine whether dissemination is an inherent ability or a major acquired phenotype during lung adenocarcinoma metastasis. We find very little evidence for dissemination from oncogenic KRAS-driven hyperplasias or most adenocarcinomas. p53 loss is insufficient to drive dissemination but rather enables rare cancer cells in a small fraction of primary adenocarcinomas to gain alterations that drive dissemination. Molecular characterization of disseminated tumor cells indicates that downregulation of the transcription factor Nkx2-1 precedes dissemination. Finally, we show that metastatic primary tumors possess a highly proliferative subpopulation of cells with characteristics matching those of disseminating cells. We propose that dissemination is a major hurdle during the natural course of lung adenocarcinoma metastasis.Significance: Because of its aggressively metastatic nature, lung cancer is the top cancer killer of both men and women in the United States. We show that, unlike in other cancer types, lung cancer dissemination is a major initial barrier to metastasis. Our findings provide insight into the effect of p53 deficiency and downregulation of Nkx2-1 during lung adenocarcinoma progression. Cancer Discov; 4(7); 781–9. ©2014 AACR.This article is highlighted in the In This Issue feature, p. 745

Supplementary Methods

OBJECTIVES: Pathologists have routinely observed distinct histologic patterns of growth in early-stage lung adenocarcinoma (LUAD), which have been suggested to be associated with prognosis. Herein, we investigated the relationship between LUAD patterns of growth, as defined by the updated international association for the study of lung cancer (IASLC) grading criteria, and differences in the tumor immune microenvironment to identify predictors of response to immunotherapy. METHODS: 174 resected stage I-III LUAD tumors were classified by histologic pattern of growth (i.e. solid, micropapillary, acinar, papillary, and lepidic) and then grouped as well differentiated, moderately differentiated, and poorly differentiated. Comprehensive multiplatform analysis including whole exome sequencing, gene expression profiling, immunohistochemistry, CIBERSORT, and T-cell receptor sequencing was performed and groups were compared for differences in genomic drivers, immune cell infiltrate, clonality, and survival. Finally, multivariate analysis was performed adjusting for pathologic stage and smoking status. RESULTS: Poorly differentiated tumors demonstrated a strong association with smoking relative to moderately differentiated or well differentiated tumors. However, unlike in prior reports, poorly differentiated tumors were not associated with a worse survival after curative-intent resection. Genomic analysis revealed that poorly differentiated tumors are associated with high tumor mutation burden but showed no association with oncogenic drivers. Immune analyses revealed that poorly differentiated tumors are associated with increased T-cell clonality, expression of PD-L1, and infiltration by cytotoxic CD8 T-cells, activated CD4 T-cells, and pro-inflammatory (M1) macrophages. Finally, multivariate analysis controlling for stage and smoking status confirmed independence of immune differences between IASLC grade groups. CONCLUSIONS: Poorly differentiated tumors, as defined by the updated IASLC grading criteria, are associated with a distinct immunogenic tumor microenvironment that predicts for therapeutic response to immune agents, including checkpoint inhibitors, and should be included in the clinical trial design of immunotherapy studies in early-stage lung adenocarcinoma.

Autologous T cells genetically engineered to express a chimeric antigen receptor (CAR) targeting CD19 and/or CD22 have achieved high complete response rates in patients with hematologic malignancies, but >50% of patients progress following therapy. Here, we sought to understand key T cell intrinsic factors impacting efficacy: CAR T cell expansion, persistence, and homing to the tumor. Using an endogenous T cell receptor (TCR) sequence as a ‘barcode’, we followed individual T cell clonotypes at the single-cell level from pre-manufacture apheresis and infusion products to tumor-involved lymph node and blood at peak and late expansion in 22 adult patients with relapsed or refractory large B cell lymphoma (LBCL) or acute lymphoblastic leukemia (ALL) treated with axicabtagene ciloleucel, an FDA-approved CD19-CAR T cell immunotherapy, or bispecific CD19/CD22 CAR T cells on an investigator-initiated trial (NCT03233854). The resulting CAR T cell atlas comprises matched transcriptome (scRNA-seq) and surface protein expression (CITE-seq) for 846,344 cells from 97 samples, with 215,045 unique TCR clonotypes identified, including 8,747 clonotypes that could be traced across 2+ timepoints in CAR mRNA+ cells. This atlas enabled us to ask: “What were the phenotypes of ‘successful’ CAR T cell clonotypes with optimal homing, expansion, and persistence properties at the time of infusion or pre-manufacture apheresis?” We found that successful T cell clonotypes at apheresis had juvenile features, including IL7R expression. Conversely, successful clonotypes in the infusion product had elevated interferon pathway activity and effector signatures, including GZMB expression. Further, we built a cell-cell interactome using all live cells from on-treatment biopsies and identified a set of 149 specific ligand-receptor pairs significantly enriched in patients who progressed. Finally, we defined dynamics of TCR clonotypes with predicted specificities for viral and self-antigens. These analyses pinpoint the identities of source T cells and infusion CAR T cells with properties impacting efficacy, and also identify ligand-receptor pairs that could be modulated to enhance CAR T cell response in the tumor at the genetic or pharmacological level. This work was supported in part by the Parker Institute for Cancer Immunotherapy, California Institute for Regenerative Medicine, Kite Pharma, and Stanford Cancer Institute. Citation Format: Zinaida Good, Mark P. Hamilton, Jay Y. Spiegel, Sreevidya Kurra, Moksha H. Desai, Snehit Prabhu, Shin-Heng Chiou, Christine Y. Yeh, Yiyun Chen, Eric Yang, Michael G. Ozawa, Fang Wu, Matthew J. Frank, Lori Muffly, Gursharan K. Claire, Juliana Craig, Maria I. Iglesias, Sushma Bharadwaj, Katherine A. Kong, Dhananjay Wagh, John Coller, Mark M. Davis, Sylvia K. Plevritis, Bita Sahaf, David B. Miklos, Crystal L. Mackall. Lineage tracing of CAR T cells in patients with B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1128.

In this work, we find that CD8 + T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49 + CD8 + regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8 + T cells efficiently eliminated pathogenic gliadin-specific CD4 + T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIR + CD8 + T cells, but not CD4 + regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49 + CD8 + T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8 + T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases.

In this work, we find that CD8

e21004 Background: Whether peripheral blood T cell receptor (TCR) repertoire profiling can serve as a biomarker to predict clinical benefit from anti-PD-1/PD-L1 checkpoint immunotherapy is not well understood. Moreover, it is not known which methods for TCR repertoire analysis are most clinically meaningful. To address this, we have performed TCR repertoire analysis of patients with advanced/metastatic non-small cell lung cancer (NSCLC) receiving PD-1/PD-L1 treatment. Methods: We analyzed 29 patients receiving PD-1/PD-L1 monotherapy or combination therapy in any line of treatment, excluding patients with EGFR mutations or Alk alterations. Genomic DNA was extracted from peripheral blood examples, and CDR3 regions of the rearranged TCR beta genes were amplified and sequenced using the immunoSEQ platform (Adaptive Biotechnologies, Seattle, WA). Libraries were sequenced using Illumina HiSeq 2500. TCR clonality, diversity, evenness, and the percentage of high-frequency clones (> 0.1% of the repertoire) were calculated at pre-treatment and post-treatment (1 – 3 months) timepoints. Morisita’s overlap index was calculated for 25 paired pre- and post-treatment samples. Metrics were compared in patients with and without durable clinical response at 6 months using the Mann-Whitney test. Results: There were no statistically significant differences in TCR repertoire clonality, diversity, evenness, or high-frequency clones between patients with and without durable clinical benefit, either at pre-treatment or post-treatment time points (p > 0.05). Conclusions: TCR repertoire metrics including clonality, diversity, evenness, percentage of high-frequency clones, and Morisita’s overlap index do not predict immunotherapy responses in this cohort of advanced/metastatic NSCLC patients receiving PD-1/PD-L1 monotherapy or combination therapy. Limitations of this study include sample size and heterogeneity of the patient cohort. This highlights the need for advanced TCR repertoire metrics, for example, considering shared TCR specificities and clonal identity. In ongoing work, we are defining TCR specificity groups and TCR clones in NSCLC as biomarkers of immunotherapy responsiveness.

Previous reports show a small subset of CD8+ T cells expressing Ly49 proteins in mice can suppress autoimmunity in a model of demyelinating disease. Here we find a markedly increased frequency of CD8+ T cells expressing inhibitory Killer cell Immunoglobulin like Receptors (KIR), the human equivalent of the Ly49 family, in the blood and inflamed tissues of various human autoimmune diseases. Increased KIR+ CD8+ T cells in the gut also correlate with disease activity in Celiac disease (CeD) patients. Moreover, KIR+ CD8+ T cells can efficiently eliminate pathogenic gliadin-specific CD4+ T cells from CeD patients’ leukocytes in vitro. Together with gene expression data, this shows that these cells are the likely equivalent of the mouse Ly49+ CD8+ T cells. Furthermore, we observe elevated levels of KIR+ CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 and influenza-infected patients, and this correlates with disease severity and vasculitis in COVID-19. Single-cell RNA and parallelized TCR sequencing reveals that expanded KIR+ CD8+ T cells from these different diseases and healthy subjects display shared phenotypes and similar T cell receptor sequences. Selective ablation of the murine counterpart in virus-infected mice leads to exacerbated autoimmunity developed after infection. These results characterize a regulatory CD8+ T cell subset in humans which we hypothesize functions to control pathogenic cells in autoimmune and infectious diseases, with important implications for the cellular dynamics and possible therapeutic approaches to suppress unwanted autoimmunity. Supported by National Institutes of Health U19-AI057229 Howard Hughes Medical Institute Stanford Diabetes Research Center (P30DK116074)

Individuals with narcolepsy suffer from abnormal sleep patterns due to loss of neurons that uniquely supply hypocretin (HCRT). Previous studies found associations of narcolepsy with the human leukocyte antigen (HLA)-DQ6 allele and T-cell receptor α (TRA) J24 gene segment and also suggested that in vitro-stimulated T cells can target HCRT. Here, we present evidence of in vivo expansion of DQ6-HCRT tetramer

Individuals with narcolepsy suffer from abnormal sleep patterns due to loss of neurons that uniquely supply hypocretin (HCRT). Previous studies found associations of narcolepsy with the human leukocyte antigen (HLA)-DQ6 allele and T-cell receptor α (TRA) J24 gene segment and also suggested that in vitro-stimulated T cells can target HCRT. Here, we present evidence of in vivo expansion of DQ6-HCRT tetramer+/TRAJ24+/CD4+ T cells in DQ6+ individuals with and without narcolepsy. We identify related TRAJ24+ TCRαβ clonotypes encoded by identical α/β gene regions from two patients and two controls. TRAJ24-G allele+ clonotypes only expand in the two patients, whereas a TRAJ24-C allele+ clonotype expands in a control. A representative tetramer+/G-allele+ TCR shows signaling reactivity to the epitope HCRT87–97. Clonally expanded G-allele+ T cells exhibit an unconventional effector phenotype. Our analysis of in vivo expansion of HCRT-reactive TRAJ24+ cells opens an avenue for further investigation of the autoimmune contribution to narcolepsy development., T cells from narcolepsy patients were recently reported to recognize hypocretin, a wakefulness-promoting neurohormone, suggesting autoimmune origin of the disease. Here the authors show that hypocretin-specific T cells expand both in healthy controls and in narcolepsy patients, and identify preliminary features that may distinguish them.

Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth. However, determining the functional significance of specific alterations remains a major bottleneck in our understanding of the genetic determinants of cancer. Here, we present a platform that integrates multiplexed AAV/Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple genomic alterations in de novo cancers in mice. Using this approach, we introduce a barcoded library of non-synonymous mutations into hotspot codons 12 and 13 of Kras in adult somatic cells to initiate tumors in the lung, pancreas, and muscle. High-throughput sequencing of barcoded Kras HDR alleles from bulk lung and pancreas reveals surprising diversity in Kras variant oncogenicity. Rapid, cost-effective, and quantitative approaches to simultaneously investigate the function of precise genomic alterations in vivo will help uncover novel biological and clinically actionable insights into carcinogenesis., Genome editing technologies enable the rapid interrogation of genetic alterations. Here, the authors present a CRISPR/Cas9-based platform to simultaneously investigate multiple activating point mutations in de novo cancers in mice; and generate panels of Kras-variants in different tissues to induce cancer.

Immunotherapy targeting T cells is increasingly utilized to treat solid tumors including non-small cell lung cancer (NSCLC). This requires a better understanding of the T cells in the lungs of patients with NSCLC. Here, we report T cell repertoire analysis in a cohort of 236 early-stage NSCLC patients. T cell repertoire attributes are associated with clinicopathologic features, mutational and immune landscape. A considerable proportion of the most prevalent T cells in tumors are also prevalent in the uninvolved tumor-adjacent lungs and appear specific to shared background mutations or viral infections. Patients with higher T cell repertoire homology between the tumor and uninvolved tumor-adjacent lung, suggesting a less tumor-focused T cell response, exhibit inferior survival. These findings indicate that a concise understanding of antigens and T cells in NSCLC is needed to improve therapeutic efficacy and reduce toxicity with immunotherapy, particularly adoptive T cell therapy., Relevant features of T cell repertoire in human cancer remain to be delineated. Here the authors show, by TCR sequencing in a large cohort of lung cancer patients, that while a majority of T cell clones are shared between tumor and adjacent lung tissue, less frequent tumor-unique T cell clones correlate with worse prognosis.

In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.

Summary To identify disease-relevant T cell receptors (TCRs) with shared antigen specificity, we analyzed 778,938 TCRβ chain sequences from 178 non-small cell lung cancer patients using the GLIPH2 (grouping of lymphocyte interactions with paratope hotspots 2) algorithm. We identified over 66,000 shared specificity groups, of which 435 were clonally expanded and enriched in tumors compared to adjacent lung. The antigenic epitopes of one such tumor-enriched specificity group were identified using a yeast peptide-HLA A∗02:01 display library. These included a peptide from the epithelial protein TMEM161A, which is overexpressed in tumors and cross-reactive epitopes from Epstein-Barr virus and E. coli. Our findings suggest that this cross-reactivity may underlie the presence of virus-specific T cells in tumor infiltrates and that pathogen cross-reactivity may be a feature of multiple cancers. The approach and analytical pipelines generated in this work, as well as the specificity groups defined here, present a resource for understanding the T cell response in cancer., Graphical abstract, Highlights • The algorithm GLIPH2 enables analysis of shared TCR specificity and HLA prediction • Tumor-infiltrating T cells cross-react to EBV antigens and shared tumor antigens • EBV-specific T cells expanded in patients responding to immune checkpoint blockade • Cross-reactive CD8 T cells express GZMK, Chiou, Tseng, et al. analyze TCRβ chain sequences from 178 non-small cell lung cancer patients and identify shared specificity groups, which in turn enable antigen identification. One such antigenic epitope—a peptide from an epithelial protein—is cross-reactive to epitopes from Epstein-Barr virus and E. coli, suggesting that cross-reactivity may underlie the presence of pathogen-specific T cells in tumor infiltrates.

3087 Background: While there has been much attention on mutation-associated neoantigens in tumors, there is less known about non-mutated tumor antigens that are shared across individuals. Understanding tumor-infiltrating T cell recognition of shared tumor antigens is important for understanding cancer immune recognition and escape, and may reveal novel targets for therapy. Methods: We have established a novel approach for discovering shared tumor antigens in human lung cancer. This approach involves identifying candidate T cell receptor (TCR) alpha/beta pairs that are predicted to exhibit specificity for shared tumor antigens in the context of a given human leukocyte antigen (HLA). We then screen the T cell receptor for binding to yeast display libraries of peptide-HLA. The Mark Davis lab at Stanford has previously developed an algorithm that groups T cell receptors into antigen specificity groups based on shared motifs within the TCR complementarity-determining region 3 (CDR3) sequences. Leveraging a dataset of over 700K CDR3 sequences from 178 HLA-typed non-small cell lung cancer (NSCLC) patients, we have found up to 4,300 antigen specificity groups after applying stringent cutoffs. We sequenced TCR alpha/beta pairs from 15 patients with lung adenocarcinoma (n = 4,705). Results: We identified an antigen specificity group enriched in tumor compared to adjacent uninvolved lungs. Antigen screening of the T cell receptor belonging to this specificity group using an A02 yeast display libraries led to the identification of a dominant peptide after four rounds of enrichment. We functionally validated that the peptide derived from the protein TMEM161A stimulated Jurkat cells expressing the TCR alpha/beta receptor of interest. We show that full-length TMEM161A protein is processed and presented into a peptide that stimulates primary T cells expressing the TCR alpha/beta receptor. We observe that a peptide from Epstein-Barr virus (EBV) protein LMP2 also stimulated the same TCR alpha/beta receptor. We have show that TMEM161A RNA and protein are overexpressed in human lung cancer compared to adjacent uninvolved lungs. Conclusions: We have demonstrated a novel approach toward antigen discovery and identified a shared tumor antigen TMEM161A in human lung cancer.

Narcolepsy is a sleeping disorder, caused by selective loss of neurons secreting a wake-promoting hormone, hypocretin (HCRT). Increased narcolepsy incidence was noted in China after the peak of 2009 H1N1 pandemic influenza (pH1N1) cases and in Europe after Pandemrix vaccine administration. The causes of HCRT neuron depletion and its relationship with pH1N1 are unknown. As narcolepsy susceptibility is associated with HLA-DQ6 and with a TCR α gene (J24) polymorphism, we hypothesized that abnormally expanded J24+/CD4+ T cells in DQ6+ subjects mediate autoimmunity against HCRT neurons and that molecular mimicry (epitopes in pH1N1 and self-antigens) drives T cell expansion via DQ6 presentation. We reported the in vivo expansion of DQ6/HCRT87–97–reactive J24+/CD4+ clones using a J24 allele and an un-conventional phenotype in patients compared to related clones in DQ6+ controls. Here, DQ6/peptide binding and crystal structure identify an epitope, HA274–287 from pH1N1 hemagglutinin, with homology to HCRT87–97 and another peptide, HCRT1–13. DQ6/HA274–287 tetramers isolated >2000 tetramer+/CD4+ T cells in DQ6+ subjects. Single-cell sequencing of TCRs and 25 phenotypic transcripts identify a DQ6/HA274–287 tetramer+/J24+ clone with the identical TCR clonotype (TCR27) and phenotype as the expanded DQ6/HCRT87–97–reactive J24+ clones previously found in the same patient. Control-derived TCR27+ clones identified by DQ6/HCRT1–13 or DQ6/HCRT25–37 tetramers are unexpanded and lack expression of any of the phenotypic genes. Our findings show that DQ6+ subjects carry J24+/CD4+ cells, both public and cross-reactive (e.g TCR27). TCR27 expansion in patients argues for molecular mimicry as a factor in narcolepsy immunopathogenesis.

Immunotherapy has changed the treatment paradigm for patients with lung cancer and other solid tumors, but little is known about the antigen specificities of the T cell residing in tumors. In order to identify novel shared tumor antigens, we have previously developed an algorithm that allows the inference of T-cell specificities based on shared motifs within the T-cell receptor (TCR) CDR3 sequences. Leveraging with a dataset of 789K CDR3 sequences from 178 HLA-typed non-small cell lung cancer (NSCLC) patients, we have found up to 40K motifs after applying stringent cutoffs. Publicly available CDR3 sequences from various tetramer databases assisted with robust assignments of unique T-cell clones from the NSCLC cohorts to known specificities. For example, we have identified and validated multiple tumor-infiltrating T-cell clones that are specific to viral antigens. Furthermore, the availability of HLA genotype results for our lung cancer cohorts facilitated prediction of motifs that are potentially “restricted” to a given HLA allele or supertype. Together with single cell-sorted T-cell clones from our Stanford NSCLC cohort (n = 5,448), we have narrowed down to 60 tumor-specific CDR3 motifs (wherein at least one Stanford sequence could be found) that are potentially restricted to the A02 allele. Within these top A02-enriched motifs, 49 candidates TCR alpha/beta pairs were identified. In an effort to find novel tumor antigens, we prioritized four top T-cell candidates and screened A02 yeast display libraries with distinct peptide length (8-11mer). Four rounds of repeated screens led to the enrichment of a few dominant mimotopes with similar sequences. Database search identified a near-perfect match to a peptide 9mer from the TMEM161A locus that is predicted to bind HLA-A02 with high affinity. Functionally, the synthetic TMEM161A peptide 9mer stimulated Jurkat cells expressing the specific TCR alpha/beta chains as well as the top mimotopes. In conclusion, our results highlight the robustness of our current pipeline for the identification of novel shared tumor antigens. The ongoing work involves further characterizing the functional state of these specific T cells within the tumor microenvironment. Citation Format: Shin-Heng Chiou, Diane Tseng, Xinbo Yang, Alexandre Reuben, Alana McSween, Julie Wilhelmy, Chunlin Wang, Joseph Shrager, Jianjun (Jay) Zhang, K. Christopher Garcia, Crystal Mackall, Mark M Davis. Towards the identification of novel tumor antigens in human lung cancer [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 A26.

Expression of the chromatin-associated protein HMGA2 correlates with progression, metastasis and therapy resistance in pancreatic ductal adenocarcinoma (PDAC). Hmga2 has also been identified as a marker of a transient subpopulation of PDAC cells that has increased metastatic ability. Here, we characterize the requirement for Hmga2 during growth, dissemination, and metastasis of PDAC in vivo using conditional inactivation of Hmga2 in well-established autochthonous mouse models of PDAC. Overall survival, primary tumour burden, presence of disseminated tumour cells in the peritoneal cavity or circulating tumour cells in the blood, and presence and number of metastases were not significantly different between mice with Hmga2-wildtype or Hmga2-deficient tumours. Treatment of mice with Hmga2-wildtype and Hmga2-deficient tumours with gemcitabine did not uncover a significant impact of Hmga2-deficiency on gemcitabine sensitivity. Hmga1 and Hmga2 overlap in their expression in both human and murine PDAC, however knockdown of Hmga1 in Hmga2-deficient cancer cells also did not decrease metastatic ability. Thus, Hmga2 remains a prognostic marker which identifies a metastatic cancer cell state in primary PDAC, however Hmga2 has limited if any direct functional impact on PDAC progression and therapy resistance.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most metastatic and deadly cancers. Despite the clinical significance of metastatic spread, our understanding of molecular mechanisms that drive PDAC metastatic ability remains limited. By generating a genetically engineered mouse model of human PDAC, we uncover a transient subpopulation of cancer cells with exceptionally high metastatic ability. Global gene expression profiling and functional analyses uncovered the transcription factor BLIMP1 as a driver of PDAC metastasis. The highly metastatic PDAC subpopulation is enriched for hypoxia-induced genes, and hypoxia-mediated induction of BLIMP1 contributes to the regulation of a subset of hypoxia-associated gene expression programs. These findings support a model in which upregulation of BLIMP1 links microenvironmental cues to a metastatic stem cell character. Significance: PDAC is an almost uniformly lethal cancer, largely due to its tendency for metastasis. We define a highly metastatic subpopulation of cancer cells, uncover a key transcriptional regulator of metastatic ability, and define hypoxia as an important factor within the tumor microenvironment that increases metastatic proclivity. Cancer Discov; 7(10); 1184–99. ©2017 AACR. See related commentary by Vakoc and Tuveson, p. 1067. This article is highlighted in the In This Issue feature, p. 1047

Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-β-catenin signaling. Constitutive activation of Wnt-β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.

Lung cancer is the leading cause of cancer deaths worldwide, with the majority of mortality resulting from metastatic spread. However, the molecular mechanism by which cancer cells acquire the ability to disseminate from primary tumors, seed distant organs, and grow into tissue-destructive metastases remains incompletely understood. We combined tumor barcoding in a mouse model of human lung adenocarcinoma with unbiased genomic approaches to identify a transcriptional program that confers metastatic ability and predicts patient survival. Small-scale in vivo screening identified several genes, including Cd109, that encode novel pro-metastatic factors. We uncovered signaling mediated by Janus kinases (Jaks) and the transcription factor Stat3 as a critical, pharmacologically targetable effector of CD109-driven lung cancer metastasis. In summary, by coupling the systematic genomic analysis of purified cancer cells in distinct malignant states from mouse models with extensive human validation, we uncovered several key regulators of metastatic ability, including an actionable pro-metastatic CD109-Jak-Stat3 axis.

Metastases are the main cause of cancer deaths, but the mechanisms underlying metastatic progression remain poorly understood. We isolated pure populations of cancer cells from primary tumors and metastases from a genetically engineered mouse model of human small cell lung cancer (SCLC) to investigate the mechanisms that drive the metastatic spread ofthis lethal cancer. Genome-wide characterization of chromatin accessibility revealed the opening of large numbers of distal regulatory elements across the genome during metastatic progression. These changes correlate with copy number amplification of the Nfib locus, and differentially accessible sites were highly enriched for Nfib transcription factor binding sites. Nfib is necessary and sufficient to increase chromatin accessibility at a large subset of the intergenic regions. Nfib promotes pro-metastatic neuronal gene expression programs and drives the metastatic ability of SCLC cells. The identification of widespread chromatin changes during SCLC progression reveals an unexpected global reprogramming during metastatic progression.

The activation of innate and adaptive immunity is always balanced by inhibitory signalling mechanisms to maintain tissue integrity. We have identified the E3 ligase c-Cbl--known for its roles in regulating lymphocyte signalling--as a modulator of dendritic cell activation. In c-Cbl-deficient dendritic cells, Toll-like receptor-induced expression of proinflammatory factors, such as interleukin-12, is increased, correlating with a greater potency of dendritic-cell-based vaccines against established tumours. This proinflammatory phenotype is accompanied by an increase in nuclear factor (NF)-κB activity. In addition, c-Cbl deficiency reduces both p50 and p105 levels, which have been shown to modulate the stimulatory function of NF-κB. Our data indicate that c-Cbl has a crucial, RING-domain-dependent role in regulating dendritic cell maturation, probably by facilitating the regulatory function of p105 and/or p50.

Cancer genome sequencing has been instrumental in identifying the genomic alterations that occur in human tumors. However, the functional importance of the vast majority of these alterations, both alone and in combination, remains unknown. I will describe methods that integrate tumor barcoding with CRISPR/Cas9-mediated genome editing and ultradeep barcode sequencing to interrogate multiple tumor genotypes simultaneously in autochthonous mouse models of human cancer. We initially used this method to quantify the effects of eleven of the most frequently inactivated genes in human lung adenocarcinoma on tumor growth in vivo. We also investigated the in vivo fitness advantage conferred by inactivation of each of these eleven genes in combination with inactivation of p53 or Lkb1. This map of tumor-suppressive effects for >30 common lung adenocarcinoma genotypes revealed a complex landscape of context dependence and variability in effect strength. I will also describe a platform that integrates multiplexed Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple oncogenic alterations in parallel. Using this approach, we introduced a library of nonsynonymous mutations into endogenous Kras in adult somatic cells to initiate tumors. High-throughput sequencing of barcoded Kras HDR alleles from bulk tumor-bearing lung uncovered surprising diversity in Kras variant oncogenicity. These in vivo approaches may redefine our ability to understand how diverse genomic alterations impact tumor initiation, growth, malignant transformation, and therapy responses. Citation Format: Ian P. Winters, Zoë N. Rogers, Christopher D. McFarland, Pranav V. Lalgudi, Shin-Heng Chiou, Mark A. Kay, Dmitri Petrov, Monte M. Winslow. Functional lung cancer genomics through in vivo genome editing [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr IA03.

Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.

Tumor-infiltrating lymphocytes (TILs) develop as manifestations of the recognition and defense against malignant cells by the host immune system. TILs were literally defined as "tumor-infiltrating lymphocytes", which a posteriori locate within the tumor tissues. Although such cells can be found, they fail to control the growth of tumor. Many have proposed diverse mechanisms for dysfunction of TILs with regard to the roles of immunosurveillance against cancer. However, only a few cancer types, e.g. melanoma, have seen the benefits brought by activating these cells for immunotherapy. Functional defects of TILs have been linked to abnormalities of signaling molecules; however, there is conflicting data. The death of TILs was attributed to expression of cancer-derived FasL, PD-1 and RCAS1, and cancer-induced activation-induced cell death (AICD). Confirmed by studies using TILs and animal models, the compromise of tumor-specific immune responses was thought to result from not only mechanisms of clonal anergy but also exhaustion and/or deletion. Furthermore, functional cytotoxic CD8 + TILs might be rendered incompetent by cancer-induced up-regulation of inhibitory NK receptors or proximal signaling abnormalities. Additionally, immune privilege was partly attributed to recruitment of regulatory T cells to the tumor sites. The failure of IL-2 signaling, which stands at the center of T cell functionalities, had been linked to the enzymatic activity of cancer-derived matrix metalloproteinases (MMPs). Finally, the exploitation of IDO expression, an important enzyme in pregnancy-related immunosuppression, by cancer cells might play a role in tumor immunity. The disparity of cancer types, origin, developmental stages and individual genetic backgrounds likely account for differences, or even contradictions, which might be the reason why immunotherapy works only on a few cancer types. Delineating the mechanisms behind functional defects of TILs can help not only boost chances of the development of a successful cure but understand the not fully identified roles played by immune system in the face of malignancies.

In human cervical cancer (CC), local immunity against this human papilloma virus (HPV)-associated neoplasia has been signified. To stratify the possibility of HPV integration on HLA mutations, we measured the genotypic and phenotypic integrity of all available HLA class I loci in 30 cases of CC. Paired normal and cancer genomic DNA was analyzed with DNA typing trays, including 57 subtypes of HLA-A, 120 subtypes of HLA-B, and 60 subtypes of HLA-C. We demonstrated significant mutations of HLA genotype with reduced HLA molecule expression in CC. HPV coincide in >70% cases of aberrant HLA genes. Southern blot analysis revealed the presence of HPV DNA within the mutated HLA foci. Our study reveals a plausible role of HPV integration in the contexts of aberrant HLA genotypes in CC cells. Disruptions of the HLA genes can be possible tactics of HPV to attain the potential carcinogenetic purposes, and thus the cancer immune escape.

Despite its clinical importance, very little is known about the natural history and molecular underpinnings of lung cancer dissemination and metastasis. Here, we used a genetically engineered mouse model of metastatic lung adenocarcinoma in which cancer cells are fluorescently marked to determine whether dissemination is an inherent ability or a major acquired phenotype during lung adenocarcinoma metastasis. We find very little evidence for dissemination from oncogenic KRAS-driven hyperplasias or most adenocarcinomas. p53 loss is insufficient to drive dissemination but rather enables rare cancer cells in a small fraction of primary adenocarcinomas to gain alterations that drive dissemination. Molecular characterization of disseminated tumor cells indicates that downregulation of the transcription factor Nkx2-1 precedes dissemination. Finally, we show that metastatic primary tumors possess a highly proliferative subpopulation of cells with characteristics matching those of disseminating cells. We propose that dissemination is a major hurdle during the natural course of lung adenocarcinoma metastasis. Significance: Because of its aggressively metastatic nature, lung cancer is the top cancer killer of both men and women in the United States. We show that, unlike in other cancer types, lung cancer dissemination is a major initial barrier to metastasis. Our findings provide insight into the effect of p53 deficiency and downregulation of Nkx2-1 during lung adenocarcinoma progression. Cancer Discov; 4(7); 781–9. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 745

Introduction: Intra-tumoral generation of adenosine (ADO), a potent inhibitor of T-cell activation, requires the coordinated and sequential cleavage of extracellular adenosine triphosphate (ATP) by the ecto-nucleotidases CD39 (which produces adenosine monophosphate, AMP) and CD73 (which hydrolyzes AMP to ADO). For this reason, various anti-CD73 antibodies are being advanced into clinical trials; however, there are few reports of potent, selective, small-molecule CD73 inhibitors, such as those described here. Methods: Enzymatic assays: Ecto-nucleotidase activity was calculated based on the amount of inorganic phosphate (malachite green assay; absorbance at 620 nm) produced after 50-min incubation with 25μM AMP or ATP, in the presence of varying concentrations of test compound(s). The following systems were used. Endogenous expression: hCD73/SKOV-3 cells; mCD73/E771 cells. Stable over-expression: hCD73/CHO; hCD39/CHO. Transient expression: mCD73/CHO; NTPDase2/CHO; NTPDase3/CHO; NTPDase8/CHO. CD8+ T cell functional assays: Human (enriched from buffy coats or leukopaks) and mouse (isolated from spleen of C57BL/6 mice) CD8+ T cells were pretreated with CD73 inhibitor or buffer control. One hour later, cells were stimulated (anti-CD3/CD28; +/- 50 μM AMP) in the presence of ADO deaminase inhibitor (10 μM). 3 days after stimulation, cell activation (CD25), proliferation (CFSE) and effector function (IFN-γ and granzyme B) were quantified by flow cytometry. Tumor model: CT26 cells were subcutaneously implanted into Balb/c mice. When tumor volumes were ∼100 mm3, mice were enrolled into cohorts (1: vehicle control; 2: A000830; 3: PD-1 Ab; 4: A000830 + PD-1 Ab) of ≥13 mice each. Interim immune phenotyping was performed when Group 1 tumor volumes were 800-1,000 mm3. Single-cell suspensions were generated from tumors and spleens (gentleMACS). 106 cells were blocked using purified CD16/32 antibodies (clone 2.4G2) and stained with the appropriate antibody cocktails. Results: We have designed a series of potent and specific small-molecule inhibitors of human and mouse CD73, represented by A000830 (IC50 against human and mouse CD73 of 1.0 nM and 3 nM, respectively). A000830 blocked ADO generation from AMP by human ovarian cancer cells (SKOV-3) with IC50: 0.2 nM and >10,000-fold selectivity relative to other ecto-nucleotidases (CD39, NTPDase2, NTPDase3, NTPDase8) and a large panel of unrelated enzymes, receptors and ion channels. In in vitro models of ADO-driven inhibition of human and mouse CD8+ T-cell activation, A000830 showed robust rescue of proliferation, CD25 expression, and IFNγ and granzyme B production. A000830 was easily administered (and well tolerated) to mice, resulting in sustained plasma concentrations at least 1,000 times higher than its potency against mouse CD73. In the spleen of CT26 tumor-bearing Balb/c mice, CD39 expression was found mainly on natural killer (NK) cells and myeloid-lineage cells, while in the tumor CD39 was highly expressed on myeloid cells (myeloid-derived suppressor cells (MDSC) and dendritic cells (DC)) as well as CD4+ and CD8+ T cells. CD73 expression was limited (spleen and tumor) to NK cells and CD4+ and CD8+ T cells. Therapeutic dosing of A000830 to these mice (tumors ∼100 mm3 in size) in combination with anti-PD1 antibody produced robust tumor growth inhibition, greater than either treatment alone. This effect was associated with increased CD8:Treg ratios in tumors (but not spleens). Conclusions: We describe a novel class of potent and selective small-molecule CD73 inhibitors that effectively block the generation of ADO from extracellular ATP, reverse the ADO-driven inhibition of human and mouse T-cell activation, and display promising anti-tumor activity when dosed in combination with PD-1 blockade. Citation Format: Juan C. Jaen, Jay P. Powers, Ada Chen, Manmohan R. Leleti, Jarek Kalisiak, Ulrike Schindler, Joanne Bl Tan, Steve Young, Shin-Heng Chiou, Laurent Debien. Small-molecule inhibitors of CD73 promote activation of human CD8+ T cells and have profound effects on tumor growth and immune parameters in experimental tumors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR10.

Wnt signaling is crucial for a variety of biological processes, including body axis formation, planar polarity, stem cell maintenance and cellular differentiation. Therefore, targeted manipulation of Wnt signaling in vivo would be extremely useful. By applying chemical inducer of dimerization (CID) technology, we were able to modify the Wnt co-receptor, low-density lipoprotein (LDL)-receptor-related protein 5 (LRP5), to generate the synthetic ligand inducible Wnt switch, iLRP5. We show that iLRP5 oligomerization results in its localization to disheveled-containing punctate structures and sequestration of scaffold protein Axin, leading to robust β-catenin-mediated signaling. Moreover, we identify a novel LRP5 cytoplasmic domain critical for its intracellular localization and casein kinase 1-dependent β-catenin signaling. Finally, by utilizing iLRP5 as a Wnt signaling switch, we generated the Ubiquitous Activator of β-catenin (Ubi-Cat) transgenic mouse line. The Ubi-Cat line allows for nearly ubiquitous expression of iLRP5 under control of the H-2K(b) promoter. Activation of iLRP5 in isolated prostate basal epithelial stem cells resulted in expansion of p63(+) cells and development of hyperplasia in reconstituted murine prostate grafts. Independently, iLRP5 induction in adult prostate stroma enhanced prostate tissue regeneration. Moreover, induction of iLRP5 in male Ubi-Cat mice resulted in prostate tumor progression over several months from prostate hyperplasia to adenocarcinoma. We also investigated iLRP5 activation in Ubi-Cat-derived mammary cells, observing that prolonged activation results in mammary tumor formation. Thus, in two distinct experimental mouse models, activation of iLRP5 results in disruption of tissue homeostasis, demonstrating the utility of iLRP5 as a novel research tool for determining the outcome of Wnt activation in a precise spatially and temporally determined fashion.

Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly fatal malignancy of the exocrine pancreas. Although several factors contribute to the poor prognosis of this disease, the ability of cancer cells to disseminate and establish inoperable metastases before diagnosis is a major impediment to successful therapy. Unfortunately, strategies that enable efficacious treatment or early detection of the disease do not exist, highlighting an urgent need for a critical understanding of PDAC progression and dissemination. To identify novel molecular mechanisms that drive metastasis, we compared the transcriptomes of “invasive” and “non-invasive” cancer cells isolated from tumors that are derived from a previously described autochthonous PDAC model (KrasLSL-G12D;p53LSL-R172H;Pdx1-cre or KPC mice). This mouse model was chosen because it recapitulates many aspects of the human disease, including cellular heterogeneity, abundant stromal responses, and great propensity for metastasis. To isolate pure PDAC cells, we first excluded the infiltrating, non-cancer cells by incorporating a Cre-reporter allele (R26LSL-Tomato) into the KPC mice. Our strategy to distinguish “invasive” from “non-invasive” cancer cells was based on our previous observations that the high mobility group AT-hook 2 protein (Hmga2), which is otherwise not expressed in most of adult tissues, is specifically expressed in cancer cells with mesenchymal phenotype. To label cancer cells that express Hmga2, we generated a conditional Hmga2-GFP reporter mouse. When a single Hmga2GFP allele is present in the KPC;R26LSL-Tom mice, we were able to sort both Hmga2-positive (GFP+Tomato+) and Hmga2–negative (GFP-Tomato+) cancer cells. Intravenous transplantations indicated that the GFP+ cancer cells have significantly higher metastatic ability. Furthermore, transcriptomic profiling of the sorted, un-passaged cells through RNA-Seq indicated that Hif1α activity is highly enriched in the invasive GFP+ cancer cells. Consistent with the RNA-Seq data, the expression of the Hif1α target Glut1 correlates with Hmga2 under immunohistochemical staining. We hypothesized that during PDAC progression, hypoxia induces the both up- and down-regulation of a set of genes, including both Hif1α-dependent and –independent ones, that are necessary to drive cancer metastasis. Future experiments will functionally interrogate, both in vitro and in vivo, the candidate genes selected based on presence of human orthologs, cross-examination with co-purified myofibroblast transcriptomic profiles, fold change, and novelty. Our study will help identify important molecular mechanisms that drive PDAC metastasis. Citation Format: Shin-Heng Chiou, Monte Winslow. Molecular dissection of pancreatic cancer invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A20.

To investigate the expression of natural killer receptors (NKRs) within the human tumor milieu, we directly examined the in vivo expressions of various NKRs on tumor-infiltrating lymphocytes (TILs) derived from human endometrial carcinoma (EC). In total, 22 patients with stage IA-IIIA EC were enrolled. TILs were isolated from tissue specimens by means of a mechanical dispersal technique. The subpopulations of immunocytes were quantified, and expressions of NKRs on CD8+ T cells were analyzed by triple-color flow cytometry. CD8+ T cells express higher ratios of CD94 and NKG2A in TILs than in peripheral blood mononuclear cells (PBMCs) in human EC. Flow cytometry reveals that 15.90% of CD3+CD8+ TILs compared with 2.10% of CD3+CD8+ PBMCs express the NKG2A molecules (P < 0.001). The percentage expressions of CD94 are 8.40% in CD3+CD8+ TILs and 3.80% in CD3+CD8+ PBMCs (P= 0.013). The numbers of CD8+ T cells expressing CD158b and NKB1 are higher in CD3+CD8+ PBMCs in EC than in normal (CD158b: 10.70% vs 2.60%, P < 0.001; NKB1: 2.20% vs 0.40%, P= 0.018, respectively). Increased expression of CD94/NKG2A restricted to tumor-infiltrating CD8+ T cell subsets may shape the cytotoxic responses, which indicate a possible role of tumor escape from host immunity in human EC.