Targeting TBK1 to overcome resistance to cancer immunotherapy

Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking1,2. Here we identify the innate immune kinase TANK-binding kinase 1 (TBK1)3 as a candidate immune-evasion gene in a pooled genetic screen4. Using a suite of genetic and pharmacological tools across multiple experimental model systems, we confirm a role for TBK1 as an immune-evasion gene. Targeting TBK1 enhances responses to PD-1 blockade by decreasing the cytotoxicity threshold to effector cytokines (TNF and IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumour models, with concordant findings in matched patient-derived organotypic tumour spheroids and matched patient-derived organoids. Tumour cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNF and IFNγ in a JAK-STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is an effective strategy to overcome resistance to cancer immunotherapy

Abstract 410: SPARC expression enhances cellular aggregation and resistance to gemcitabine in pancreatic cancer cells

Abstract Pancreatic cancer (PaCa) is a very aggressive disease with a high mortality rate, characterized by a survival time of only few months after diagnosis. Pancreatic cancers also demonstrate great resistance to chemotherapeutic regimes, associated with an extensive desmoplastic reaction. The matricelular protein SPARC is involved in the interaction between the cell and the extracellular matrix, and its expression has been associated with worse prognosis in general, and in patients treated with gemcitabine. To evaluate its role in pancreatic cancer and chemotherapeutic resistance, we overexpressed SPARC in the SPARC-negative pancreatic cancer cells lines Miapaca2, Sw1990, and Panc1. The in vitro growth of two stable clones obtained from the different cell lines was compared with eGFP-transduced controls. No significant differences were observed in the 2D in vitro growth or the clonogenic capacity between pancreatic cancer cells expressing or not expressing SPARC. However, cell growth in 3D spheroid culture was markedly different. Indeed, SPARC-expressing cells exhibited more compact structures, with 45% reduction in spheroid size compared to control eGFP-expressing cells. This could partially be explained by the fact that the SPARC-expressing cells themselves exhibited more than 50% reduction in their size compared to the eGFP cells. While SPARC expression in pancreatic cancer cells did not affect Gemcitabine EC50 in 2D culture, SPARC-positive Panc1 and SW1990 cells exhibited six to ten times increased EC50 in 3D spheroids. Because the cell-cell interactions could potentially affect drug efficacy, and because increased resistance to gemcitabine has been associated with epithelial-mesenchymal transition (EMT), we analyzed the expression of EMT-associated genes (SNAI1 and OCLN), and genes also involved in cellular junctions (CDH1 and OCLN). SPARC-positive pancreatic cancer cells down regulated the expression of SNAI1 and CDH2, while CDH1 and OCLN were up-regulated Taken together, these data indicate that SPARC expression in PaCa cell lines diminishes cells size and modulates gemcitabine resistance in 3D models of cell culture, possibly through the modulation of epithelial genes related to cell-cell connections and EMT. Citation Format: Edgardo Salvatierra, Elvia Rivas, Leandro Guttlein, Emily Robitschek, Andrea Llera, Osvaldo Podhajcer. SPARC expression enhances cellular aggregation and resistance to gemcitabine in pancreatic cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 410. doi:10.1158/1538-7445.AM2015-410</jats:p

Abstract 448: The role of the Nanog transcription factor in tumor immune escape

Abstract The emergence of cancer stem cells and escape from immune surveillance play major roles in the progression of many cancer types. We recently established a link between these two “hallmarks” of cancer by demonstrating that they are both driven by overexpression of Nanog, a pivotal transcription factor in the self-renewal of pluripotent stem cells. In particular, we found that tumor cells acquire Nanog expression in the course of immune selection, and that Nanog confers a stem-like and immune-resistant phenotype to these cells. However, the way in which Nanog causes this to occur remains largely unknown. We reasoned that Nanog-driven immune escape may be in part due to the effect of this transcription factor on the tumor microenvironment. Therefore, here we investigated the relationship between Nanog, immune selection, and the properties of the tumor microenvironment. We found that mice bearing tumor cells that underwent immune selection, compared to those bearing parental tumor cells (without immune selection), had markedly elevated levels of the cytokine IL-6 as well as of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment; both IL-6 and MDSCs have been well-documented to promote cancer immune escape. We further investigated the role of Nanog in setting up this microenvironment. To this end, we have devised an in vivo system that enables us to examine the relationship between Nanog expression in the tumor microenvironment and tumor growth in real-time. Taken together, we infer from these data that not only is Nanog induction a consequence of immune selection, but Nanog also plays an essential role in tumor immune escape. In fact, we found in multiple mouse and human tumor models that Nanog is necessary and sufficient for immune escape. Additionally, these results suggest that Nanog mediates immune escape by converting the tumor microenvironment into an immune-inhibitory site, through the induction of IL-6 and MDSCs. These studies authenticate the role of Nanog in cancer progression as a central instigator of tumor immune escape, and demonstrate its potential both as an important diagnostic/prognostic index and as a therapeutic target. Citation Format: Emily Robitschek, Chih-Ping Mao, Shiwen Peng, Chien-Fu Hung, TC Wu. The role of the Nanog transcription factor in tumor immune escape. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 448. doi:10.1158/1538-7445.AM2015-448</jats:p

Abstract 2846: Stereotypic patterns and genomic correlates of organotropism in metastatic melanoma

Abstract Despite the major role that metastasis plays in the morbidity and mortality of melanoma, stereotyped patterns of metastasis and drivers of its organotropism in melanoma are still not well characterized, limited by a dearth of sequencing data in well-annotated clinical melanoma samples. To address these open questions, we performed an integrative analysis of clinical and genomic features from 243 patients with metastatic melanoma treated at Dana Farber Cancer Institute (DFCI). Tumor biopsies were sequenced with OncoPanel, a next-generation sequencing panel that identifies mutations in 331 cancer genes. Presence of site metastases was evaluated radiographically pre-treatment for each patient; sites include lymph node (64% of cohort), soft tissue (59%), lung (57%), liver (32%), brain (24%), bone (22%), mesentery (12%), adrenal gland (9%), spleen (7%), and other metastatic sites (13%). Metastases showed significant co-occurrences (e.g. bone and lung, OR 2.8, 95% CI = [2.3, 3.3], p &amp;lt; 0.01; adrenal and mesentery, OR 4.4, 95% CI = [3.8, 5.0], p &amp;lt; 0.01) and exclusions (lymph node and brain, OR 0.5, 95% CI = [0.2, 0.8], p = 0.02). We performed unsupervised hierarchical clustering of patients with cutaneous melanoma (n = 203) by metastatic site pattern using a Euclidean distance metric weighted to favor uncommon metastatic sites, yielding five stereotypic patterns of metastasis, characterized by: (1) co-occurrence of adrenal, mesenteric, and abdominal metastases (n=19); (2) liver metastases (n=33); (3) low metastatic burden (n=80); (4) co-occurrence of lung, brain, and mesentery metastases (n=42); and (5) co-occurrence of bone and lung metastases (n=29). Clustering is stable, with highly concordant cluster assignments in repeated subsampling of the data. Patients with cutaneous melanoma (n=203) exhibited both site-specific and pattern-specific genomic correlates of metastatic organotropism that persist after correction for mutational burden. Tumors from patients with liver metastases showed significantly higher prevalence (p &amp;lt; 0.05) of mutation compared to patients without liver metastases in KMT2D (56% vs 18%), BCL6 (22% vs 0%), TMPRSS2 (22% vs 0%), ARID1B (33% vs 4%), MET (33% vs 4%), and AXL (44% vs 11%), with similar enrichment in the liver met-predominant metastatic cluster, implicating dysregulation of histone and protein deacetylation pathways in liver metastatic organotropism (p &amp;lt; 0.01). Numerous additional mutational correlates were found for the remaining nine metastatic sites and all five metastatic patterns, and validation in an orthogonal dataset is ongoing. We present robust stereotypic patterns of metastasis and both site- and pattern-specific genomic correlates of organotropism in metastatic melanoma. By leveraging a valuable clinical/genomic data set, we nominate genetic correlates of organotropism for functional validation and potential therapeutic targets. Citation Format: William H. Ge, Giuseppe Tarantino, Emily Robitschek, Michael P. Manos, Lauren Eastman, Olivia Ouyang, Patrick Ott, Ann W. Silk, Osama E. Rahma, Alexander Gusev, Rizwan Haq, Elizabeth I. Buchbinder, Megan L. Insco, Stephen Hodi, Eliezer Van Allen, David Liu. Stereotypic patterns and genomic correlates of organotropism in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2846.</jats:p

Epigenetic silencing by SETDB1 suppresses tumour intrinsic immunogenicity

Epigenetic dysregulation is a defining feature of tumorigenesis that has been implicated in immune escape1,2. To identify epigenetic factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumor models treated with immune checkpoint blockade (ICB). We identified the H3K9-methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape in tumor cells3–5. We also found that amplification of SETDB1 (1q21.3) in human tumors is associated with immune exclusion and resistance to ICB. SETDB1 represses broad genomic domains, many of which reside within the open genome compartment. These domains are enriched for transposable elements (TEs) and immune gene 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 tumor cell immunogenicity by silencing TEs and evolving genomic loci, and thus represents a candidate target for immunotherapy.Accepted Manuscrip

Mature and Migratory Dendritic Cells Promote Immune Infiltration and Response to Anti-Pd-1 Checkpoint Blockade in Metastatic Melanoma

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy, yet most patients fail to achieve durable responses. To better understand the tumor microenvironment (TME), we analyze single-cell RNA-seq (~189 K cells) from 36 metastatic melanoma samples, defining 14 cell types, 55 subtypes, and 15 transcriptional hallmarks of malignant cells. Correlations between cell subtype proportions reveal six distinct clusters, with a mature dendritic cell subtype enriched in immunoregulatory molecules (mregDC) linked to naive T and B cells. Importantly, mregDC abundance predicts progression-free survival (PFS) with ICIs and other therapies, especially when combined with the TCF7 + /- CD8 T cell ratio. Analysis of an independent cohort (n = 318) validates mregDC as a predictive biomarker for anti-CTLA-4 plus anti-PD-1 therapies. Further characterization of mregDCs versus conventional dendritic cells (cDC1/cDC2) highlights their unique transcriptional, epigenetic (single-nucleus ATAC-seq data for cDCs from 14 matched samples), and interaction profiles, offering new insights for improving immunotherapy response and guiding future combination treatments

Evolution of Delayed Resistance to Immunotherapy in a Melanoma Responder

Despite initial responses, most melanoma patients develop resistance to immune checkpoint blockade (ICB). To understand the evolution of resistance, we studied 37 tumor samples over 9 years from a metastatic melanoma patient with exceptional response followed by delayed recurrence and death. Phylogenetic analysis revealed co-evolution of 7 lineages with multiple convergent, but independent resistance-associated alterations (RAAs). All recurrent tumors emerged from a lineage characterized by loss of chromosome 15q, with post-treatment clones acquiring additional genomic driver events. Deconvolution of bulk RNAseq and highly-multiplexed immunofluorescence (t-CyCIF) revealed differences in immune composition amongst different lineages. Imaging revealed a vasculogenic mimicry phenotype in NGFR-High tumor cells with high PD-L1 expression in close proximity to immune cells. Rapid autopsy demonstrated 2 distinct NGFR spatial patterns with high polarity and proximity to immune cells in subcutaneous tumors versus a diffuse spatial pattern in lung tumors, suggesting different roles of this neural crest-like program in different tumor microenvironments. Broadly, this study establishes a high-resolution map of the evolutionary dynamics of resistance to ICB, characterizes a de-differentiated, neural crest tumor population in melanoma immunotherapy resistance, and describes site specific differences in tumor-immune interactions via longitudinal analysis of a melanoma patient with an unusual clinical course.Other Research UnitAccepted Manuscrip