Immune Checkpoint Blockade Resistance Research Articles

PD-1 and LAG-3 Checkpoint Blockade: Potential Avenues for Therapy in B-Cell Lymphoma.

The dependence of cancer on an immunotolerant tumor microenvironment (TME) is well established. Immunotherapies that overcome tumor-induced immune suppression have been central to recent advancements in oncology. This is highlighted by the success of agents that interrupt PD-1 mediated immune suppression in a range of cancers. However, while PD-1 blockade has been paradigm-shifting in many malignancies, the majority of cancers show high rates of primary resistance to this approach. This has led to a rapid expansion in therapeutic targeting of other immune checkpoint molecules to provide combination immune checkpoint blockade (ICB), with one such promising approach is blockade of Lymphocyte Activation Gene 3 (LAG-3). Clinically, lymphoproliferative disorders show a wide spectrum of responses to ICB. Specific subtypes including classical Hodgkin lymphoma have demonstrated striking efficacy with anti-PD-1 therapy. Conversely, early trials of ICB have been relatively disappointing in common subtypes of Non-Hodgkin lymphoma. In this review, we describe the TME of common lymphoma subtypes with an emphasis on the role of prominent immune checkpoint molecules PD-1 and LAG3. We will also discuss current clinical evidence for ICB in lymphoma and highlight key areas for further investigation where synergistic dual checkpoint blockade of LAG-3 and PD-1 could be used to overcome ICB resistance.

Tumor-Secreted Extracellular Vesicles Regulate T-Cell Costimulation and Can Be Manipulated To Induce Tumor-Specific T-Cell Responses

Colorectal cancer is a major cause of cancer-related deaths worldwide. Immune checkpoint blockade therapies are effective in 30%-60% of the microsatellite instable-high subtype. Unfortunately, most patients with colorectal cancer (>85%) have microsatellite stable tumors that do not respond. In this study, we aimed to decipher the underlying tumor-intrinsic mechanisms critical for improving immunotherapy in colorectal cancer. We used human and mouse tumor samples, cell lines, human colorectal cancer organoids, and various syngeneic orthotopic mouse models of late-stage colorectal cancer to define the effects of tumor cell-secreted extracellular vesicles (EVs) on antitumor immune response. Our analyses of human colorectal cancer immune profiles and tumor-immune cell interactions showed that tumor-secreted EVs containing microRNA miR-424 suppressed the CD28-CD80/86 costimulatory pathway in tumor-infiltrating T cells and dendritic cells, leading to immune checkpoint blockade resistance. Modified tumor-secreted EVs with miR-424 knocked down enhanced T-cell-mediated antitumor immune response in colorectal cancer tumor models and increased the immune checkpoint blockade response. Intravenous injections of modified tumor-secreted EVs induced tumor antigen-specific immune responses and boosted the immune checkpoint blockade efficacy in colorectal cancer models that mimic aggressively progressing, late-stage disease. Collectively, we show a critical role for tumor-secreted EVs in antitumor immune regulation and immunotherapy response, which could be developed as a novel treatment for immune checkpoint blockade-resistant colorectal cancer.

Immunotherapy Goes Local: The Central Role of Lymph Nodes in Driving Tumor Infiltration and Efficacy.

Immune checkpoint blockade (ICB) has changed the therapeutic landscape of oncology but its impact is limited by primary or secondary resistance. ICB resistance has been related to a lack of T cells infiltrating into the tumor. Strategies to overcome this hurdle have so far focused on the tumor microenvironment, but have mostly overlooked the role of tumor-draining lymph nodes (TDLN). Whereas for CTLA-4 blockade TDLN have long since been implicated due to its perceived mechanism-of-action involving T cell priming, only recently has evidence been emerging showing TDLN to be vital for the efficacy of PD-1 blockade as well. TDLN are targeted by developing tumors to create an immune suppressed pre-metastatic niche which can lead to priming of dysfunctional antitumor T cells. In this review, we will discuss the evidence that therapeutic targeting of TDLN may ensure sufficient antitumor T cell activation and subsequent tumor infiltration to facilitate effective ICB. Indeed, waves of tumor-specific, proliferating stem cell-like, or progenitor exhausted T cells, either newly primed or reinvigorated in TDLN, are vital for PD-1 blockade efficacy. Both tumor-derived migratory dendritic cell (DC) subsets and DC subsets residing in TDLN, and an interplay between them, have been implicated in the induction of these T cells, their imprinting for homing and subsequent tumor control. We propose that therapeutic approaches, involving local delivery of immune modulatory agents for optimal access to TDLN, aimed at overcoming hampered DC activation, will enable ICB by promoting T cell recruitment to the tumor, both in early and in advanced stages of cancer.

Abstract PO048: Loss of intra-tumoral RIG-I immune signaling is a potential microbiome-mediated mechanism underlying poor anti-tumor immunity and immunotherapy resistance in mucosal melanoma

Abstract Cutaneous melanoma (CM) has one of the highest response rates across cancers to immune checkpoint blockade (ICB) therapies, but mucosal melanoma (MM) responds poorly to ICB. MM patients have very few effective treatments options, making it critical to understand and improve anti-tumor immunity in these patients. MM is a rare subtype of melanoma, accounting for approximately 1% of all malignant melanoma diagnoses. MM occurs on mucosal surfaces such as nasal, vaginal, and anorectal, which provide a highly unique tumor microenvironment compared to CM arising in the skin. We compared the tumor immune microenvironment between CM and MM by multiplex immunofluorescence and discovered MM has decreased frequency of infiltrating immune cells. Using RNA sequencing, we investigated intra-tumoral differences in global gene expression between CM and MM that might explain the lack of immune cell infiltration and ICB response in MM. Compared to CM, MM had decreased expression of numerous innate immune genes in the RIG-I pathogen-sensing pathway. The RIG-I pathway produces an inflammatory response to destroy foreign pathogens, but is also important for ICB sensitivity and anti-tumor immunity in CM. Studies in CM demonstrate the RIG-I pathway enhances antigen presentation and promotes a favorable immune cell profile in the tumor microenvironment. Interestingly, the tumor microbiome, which is vastly different between CM and MM, has been shown to regulate the RIG-I pathway. We measured total fungal and bacterial load in MM and CM primary tumors and found increased levels of both bacteria and fungi in MM compared to CM. This raises the possibility that microbiome-mediated suppression of the RIG-I pathway underlies poor anti-tumor immunity and ICB response in MM, and that re-activation of this pathway may be a novel therapeutic strategy for overcoming ICB resistance in MM patients. Direct RIG-I agonists are still in clinical development, but FDA-approved hypomethylating agents, particularly 5’ aza-deoxycytidine (decitabine), can activate the RIG-I pathway and improve anti-tumor immunity by increasing the expression of RIG-I pathway activating genes, endogenous RNA retroviruses, tumor antigens, and natural killer (NK) cell ligands. We treated MM cell lines with decitabine and observed strong induction of RIG-I pathway genes, NK ligands, and tumor antigens. In conclusion, we have identified the loss of intra-tumoral RIG-I signaling as a potential microbiome-mediated mechanism underlying the poor tumor immune microenvironment and ICB response in MM. Combining decitabine, which is FDA-approved and now available orally, with ICB represents a novel treatment strategy for these difficult to treat MM patients that can be rapidly translated into the clinic. Citation Format: Morgan MacBeth, Richard Tobin, Robert Van Gulick, Martin D. McCarter, William A. Robinson, Kasey L. Couts. Loss of intra-tumoral RIG-I immune signaling is a potential microbiome-mediated mechanism underlying poor anti-tumor immunity and immunotherapy resistance in mucosal melanoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO048.

Targeting PIM1-Mediated Metabolism in Myeloid Suppressor Cells to Treat Cancer

There is a strong correlation between myeloid-derived suppressor cells (MDSC) and resistance to immune checkpoint blockade (ICB), but the detailed mechanisms underlying this correlation are largely unknown. Using single-cell RNA sequencing analysis in a bilateral tumor model, we found that immunosuppressive myeloid cells with characteristics of fatty acid oxidative metabolism dominate the immune-cell landscape in ICB-resistant subjects. In addition, we uncovered a previously underappreciated role of a serine/threonine kinase, PIM1, in regulating lipid oxidative metabolism via PPARγ-mediated activities. Enforced PPARγ expression sufficiently rescued metabolic and functional defects of Pim1 -/- MDSCs. Consistent with this, pharmacologic inhibition of PIM kinase by AZD1208 treatment significantly disrupted the myeloid cell-mediated immunosuppressive microenvironment and unleashed CD8+ T-cell-mediated antitumor immunity, which enhanced PD-L1 blockade in preclinical cancer models. PIM kinase inhibition also sensitized nonresponders to PD-L1 blockade by selectively targeting suppressive myeloid cells. Overall, we have identified PIM1 as a metabolic modulator in MDSCs that is associated with ICB resistance and can be therapeutically targeted to overcome ICB resistance.

Innate RIG-I signaling restores antigen presentation in tumors and overcomes T cell resistance.

In recent years, therapy with immune modulating antibodies, termed immune checkpoint blockade (ICB), has revolutionized the treatment of advanced metastatic melanoma, yielding long-lasting clinical responses in a subgroup of patients. But despite this remarkable progress, resistance to therapy represents a major clinical challenge. ICB efficacy is critically dependent on cytotoxic CD8+ T cells targeting tumor cells in an HLA class I (HLA-I) antigen-dependent manner. Transcriptional suppression of the HLA-I antigen processing and presentation machinery (HLA-I APM) in melanoma cells leads to HLA-I-low/-negative tumor cell phenotypes escaping CD8+ T cell recognition and contributing to ICB resistance. In general, HLA-I-low/-negative tumor cells can be re-sensitized to T cells by interferons (IFN), augmenting HLA-I APM expression. However, this mechanism fails when melanoma cells acquire resistance to IFN, which recently turned out as a key resistance mechanism in ICB, besides HLA-I APM suppression. Seeking for a strategy to overcome these barriers, we identified a novel mechanism that restores HLA-I antigen presentation in tumor cells independent of IFN (Such et al. (2020) J Clin Invest, doi: 10.1172/JCI131572). We demonstrated that tumor cell-intrinsic activation of the cytosolic innate immunoreceptor RIG-I by its synthetic ligand 3pRNA overcomes transcriptional HLA-I APM suppression in patient-derived IFN-resistant melanoma cells. De novo HLA-I APM expression is IRF1/IRF3-dependent and re-sensitizes melanoma cells to autologous cytotoxic CD8+ T cells. Notably, synthetic RIG-I ligands and ICB synergize in T cell activation, suggesting combinational therapy could be an efficient strategy to improve patient outcomes in melanoma.

Advances in the Study of Antitumour Immunotherapy for Newcastle Disease Virus.

This article reviews the preclinical research, clinical application and development of Newcastle disease virus (NDV) in the field of cancer therapy. Based on the distinctive antitumour properties of NDV and its positive interaction with the patient's immune system, this biologic could be considered a major breakthrough in cancer treatment. On one hand, NDV infection creates an inflammatory environment in the tumour microenvironment, which can directly activate NK cells, monocytes, macrophages and dendritic cells and promote the recruitment of immune cells. On the other hand, NDV can induce the upregulation of immune checkpoint molecules, which may break immune tolerance and immune checkpoint blockade resistance. In fact, clinical data have shown that NDV combined with immune checkpoint blockade can effectively enhance the antitumour response, leading to the regression of local tumours and distant tumours when injected, and this effect is further enhanced by targeted manipulation and modification of the NDV genome. At present, recombinant NDV and recombinant NDV combined with immune checkpoint blockers have entered different stages of clinical trials. Based on these studies, further research on NDV is warranted.

Anti-PD-1 antibody-mediated activation of type 17 T-cells undermines checkpoint blockade therapy.

Tumors that develop in the genetic LSL-K-rasG12D murine lung cancer model are resistant to anti-PD-1 antibody treatment. Analysis of tumor-bearing lungs from anti-PD-1-treated mice revealed an up to 2.5-fold increase in IL-17-producing T-cells, with minimal change in CD8+ T-cell activity. Neutralization of IL-17 concurrent with anti-PD-1 treatment on the other hand, resulted in robust CD8+ T-cell activation and a threefold reduction in tumor burden. Loss-of-function studies demonstrated that anti-PD-1 driven activation of CD4+ and γδTCR+ T-cells contributed to IL-17-mediated de-sensitization of CD8+ cytotoxic T-cells (CTL) to therapy; and that CTL activation was critical to tumor eradication. Importantly, post-therapy lung Th17 cell prevalence and activity prognosticated treatment efficacy. Consistent with the murine data, analysis of tumor biopsy samples from non-small cell lung cancer (NSCLC) patients revealed that pre-therapy intratumoral CD8+/RORc+ cell ratio correlated with response to immune checkpoint blockade (ICB). These findings provide the initial evidence for a new mechanism of ICB resistance in lung cancer.

Selective SIRPα blockade reverses tumor T cell exclusion and overcomes cancer immunotherapy resistance

T cell exclusion causes resistance to cancer immunotherapies via immune checkpoint blockade (ICB). Myeloid cells contribute to resistance by expressing signal regulatory protein-α (SIRPα), an inhibitory membrane receptor that interacts with ubiquitous receptor CD47 to control macrophage phagocytosis in the tumor microenvironment. Although CD47/SIRPα-targeting drugs have been assessed in preclinical models, the therapeutic benefit of selectively blocking SIRPα, and not SIRPγ/CD47, in humans remains unknown. We report a potent synergy between selective SIRPα blockade and ICB in increasing memory T cell responses and reverting exclusion in syngeneic and orthotopic tumor models. Selective SIRPα blockade stimulated tumor nest T cell recruitment by restoring murine and human macrophage chemokine secretion and increased anti-tumor T cell responses by promoting tumor-antigen crosspresentation by dendritic cells. However, nonselective SIRPα/SIRPγ blockade targeting CD47 impaired human T cell activation, proliferation, and endothelial transmigration. Selective SIRPα inhibition opens an attractive avenue to overcoming ICB resistance in patients with elevated myeloid cell infiltration in solid tumors.

Targeting immune-checkpoint inhibitor resistance mechanisms by MEK inhibitor and agonist anti-CD40 antibody combination therapy.

The widespread application of immune-checkpoint blockade (ICB) has resulted in unprecedented response rates in patients with immunogenic cancers, such as melanoma and lung cancer. However, sub-groups of patients with these indications do not respond to ICB, and the same applies to patients with other cancer types. Mechanisms of resistance to ICB include low tumor immunogenicity associated with low T cell infiltration (‘cold' tumors), suppression of anti-tumor immunity by immunosuppressive cells in the tumor microenvironment (TME), lack of antigen-presentation and immune escape (e.g. by downregulation of MHC-I on tumor cells) as well as oncologic pathways that suppress immune responses. Combination strategies, involving cytostatic drugs, harbor the potential to overcome refractoriness to immunotherapy. However, suppression of immune cell function by cytostatic drugs may limit the efficacy. In our study, we show that combination treatment of targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) and agonist immunostimulatory anti-CD40 antibody (Ab) is particularly suitable in counteracting aforementioned ICB resistance mechanisms (Fig. 1).

Abstract 3863: CODEX high-multiplex imaging reveals distinct tumor microenvironment in mouse melanoma models associated with response to immunotherapy

Abstract In order to identify the determinants of melanoma resistance to immunotherapies and predictive biomarkers, we developed a series of immunocompetent syngeneic mouse models that represent diverse subtypes of human melanoma exhibiting a range of sensitivity to immune checkpoint blockade (ICB) therapies (Pérez-Guijarro et al, BioRxiv 2019). Comparative genomic and immunological analysis identified that melanoma plasticity as well as T cell dysfunction and exclusion programs strongly correlated with ICB resistance. However, how interactions between tumor and immune cells influence therapeutic efficacy is still unknown. To address this question, we used CODEX high-multiplex imaging technology that enables quantitative detection of 20+ markers at single cell level resolution preserving spatial context. CODEX does not require cell isolation that may result in variable cell loss. Furthermore, the whole tissue imaging provides insights into tumor/stroma and cellular heterogeneity. Fresh frozen tissues (n=3 for each model) were stained with a panel of 16 antibodies to identify major immune cell phenotypes including cytotoxic and helper T cells, B cells, and subsets of myeloid cells. Quantitative single cell level analysis of images was performed with HALO (Indica Labs) and multiplex analysis viewer (MAV) (Akoya Biosciences) software. Preliminary analysis identified unique tumor architecture, immune cell densities and distribution in each model. Although we found high diversity in the vasculature (CD31+), proliferation (Ki67+) and number of infiltrating leukocytes (CD45+) between the models, these were not associated with resistance. In contrast, high infiltration of cytotoxic T cells and dendritic cells was associated with response to ICBs. These results validated tumor analyses by FACS and gene signatures of immune cells. In addition, CODEX imaging revealed heterogeneity and complex spatial organization in the tumor microenvironment. Importantly, ICB-resistant tumors exhibited compact tumor structure with less stroma infiltration and lack of melanin expression, while ICB-sensitive tumors had complex, nodular and pigmented structures, indicating different differentiation status. Our data also suggested that myeloid phenotypes and functional compartment interactions may contribute to the response to ICBs. The study is expected to provide a higher resolution profiling of tumor-immune cell interactions and facilitate mechanistic understanding of resistance to immune checkpoint therapy. Citation Format: Noemi Kedei, Eva E. Pérez-Guijarro, Jin-Qiu Chen, Chi-Ping Day, Mariam Q. Malik, David J. Goldstein, Glenn T. Merlino. CODEX high-multiplex imaging reveals distinct tumor microenvironment in mouse melanoma models associated with response to immunotherapy [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 3863.

Abstract 400: Targeting myeloid cell-cycle related kinase signaling amplifies anti-tumor T cell responses via inducing myeloid derived suppressor cell differentiation

Abstract Key words: CDK20, E4BP4, MDSC, Immunosuppression, T cell immunity The breakthrough in understanding the inhibition of negative immune regulation has paved way for cancer immunotherapy. The immune-checkpoint blockade (ICB) therapy has produced promising and yet modest objective response rates in some solid cancers such as hepatocellular carcinoma (HCC), which have been attributable to the strong immunosuppressive tumor microenvironment (TME). Accumulating evidence has demonstrated that myeloid-derived suppressor cell (MDSC), an immature myeloid population with potent T cell-suppressive activity, is remarkably associated with poor prognosis and ICB resistance of cancer patients. While targeting MDSC can blunt T cell activity, new approach is directed towards driving differentiation of MDSC into antigen presentation cell crucial for T cell priming and activation. Therefore, decoding the molecular pathways that sustain the immunosuppressive MDSCs will be instrumental in deriving strategies for greater anti-tumor immune responses. Given their pivotal roles in cell cycle and transcriptional regulation, deregulation of cyclin-dependent kinases (CDKs) has become a hallmark of several cancer types. We have recently shown that hepatoma-intrinsic CDK20, or more commonly known as cell cycle-related kinase (CCRK), mitigates anti-tumor T cell responses by expanding MDSCs within TME, while tumoral CCRK depletion diminishes MDSC-mediated immunosuppression leading to improved ICB efficacy. As emerging evidence highlights the key roles of CDKs in immune cell signaling and identity, our new findings from tumor-bearing mice and healthy human blood-derived cell models uncovered specific over-expression of CCRK in MDSCs but not lymphocytes. Notably, blockade of MDSC-intrinsic CCRK induced its differentiation into mature macrophage which amplified T cell responses in vitro and in vivo, resulting in reduced tumorigenicity. Mechanistically, CCRK inhibition suppressed signal transducer and activator of transcription 3 (STAT3) signaling to revert E4-binding protein 4 (E4BP4)-dependent interleukin-10 (IL-10)/IL-12 imbalance and arginase I expression for maintenance of immunosuppression. As we also showed CCRK over-expression in HCC patient-derived MDSCs, the findings of this study not only unravel mechanistic insights in MDSC maintenance and differentiation, but also offer a novel therapeutic kinase-target to resolve ICB resistance, thereby conferring durable eradication of solid tumors. Acknowledgement: This project was supported by the University Grants Committee through the General Research Fund 14108219 and the Collaborative Research Fund C4045-18W. The authors declare no conflict of interests. Citation Format: Jingying Zhou, Alfred Sze Lok Cheng. Targeting myeloid cell-cycle related kinase signaling amplifies anti-tumor T cell responses via inducing myeloid derived suppressor cell differentiation [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 400.

Abstract 799: CDKN2A mutation predicts immunotherapy resistance in stage III/IV NSCLC

Abstract Purpose: Immune checkpoint blockade (ICB) has improved outcomes for patients with non-small cell lung cancer (NSCLC). However, most patients experience disease progression. There is limited data regarding molecular predictors of progression, particularly in those patients predicted to respond most favorably. We used a next-generation sequencing (NGS) platform to identify genomic aberrations associated with clinical outcomes following ICB in NSCLC. Methods: We retrospectively reviewed stage III/IV NSCLC patients who received radiotherapy (RT) in our department and underwent NGS with OncoPlus, a 1212-gene hybrid capture genomic sequencing assay. We characterized tumor mutations and insertions/deletions for patients who received ICB as well as other systemic therapies and investigated their associations with clinical outcome. Results: 149 patients with stage III/IV NSCLC received RT from 2011-2019 and underwent OncoPlus. Median age was 65, median ECOG was 1, and 125 had a smoking history. 87% had adenocarcinoma. AJCC 8th edition staging: IIIA (n=24), IIIB (n=14), IIIC (n=3), IVA (n=16), and IVB (n=92). 79% had PD-L1 staining: 0% in 45 patients (38%), 1-49% in 34 (29%), and ≥50% in 38 (32%). All received RT to a median of 2 lesions (range 1-15); 48% received palliative RT and 52% received ≥1 course of definitive RT (conventional or ablative). The majority received systemic therapy: 68% (n=102) received chemotherapy, 55% (n=82) received ICB, and 30% (n=44) received targeted therapies. Median follow-up was 18 months (range 1-95); most experienced isolated distant failure (n=64, 43%) or regional/distant failure (n=35, 23%) following their first RT course. The most frequent pathogenic mutations/indels were TP53 (68%, n=101), KRAS (31%, n=45), CDKN2A (27%, n=40), STK11 (26%, n=39), EGFR (19%, n=28), and TP53/KRAS co-mutation (17%, n=26). We found CDKN2A mutation (25%)/deletion (75%) was associated with inferior median recurrence-free survival (RFS; 3 vs. 6 mo., p=0.013) and more markedly median OS (13 vs. 38 mo., p=0.015) compared to wild-type tumors. On multivariate Cox proportional hazards analysis including stage, ECOG, PD-L1 %, TMB, type of RT, and # of RT sites, CDKN2A loss was independently associated with 2.5-fold risk of progression (p=0.018) and 3.3-fold risk of death (p=0.05). In high TMB (top 25th%; n=21) or high PD-L1 (>50%; n=30) NSCLCs, CDKN2A loss was negatively associated with RFS (TMB p=0.005; PD-L1 p=0.034) and in the high PD-L1 cohort with a trend towards inferior OS (p=0.08). Conclusions: In a large cohort of stage III/IV NSCLC patients treated with systemic therapy and RT, NGS identified CDKN2A mutation/deletion as a predictor of ICB resistance and poor outcome. In the context of recent evidence for sensitivity to CDK4/6 inhibition of CDKN2A-deficient tumors, our findings raise the possibility of utilizing currently available targeted agents in the treatment of CDKN2A-deficient NSCLC patients progressing on ICB. Citation Format: Stanley I. Gutiontov, Sean Pitroda. CDKN2A mutation predicts immunotherapy resistance in stage III/IV NSCLC [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 799.

Heterogeneity of response to immune checkpoint blockade in hypermutated experimental gliomas

Intrinsic malignant brain tumors, such as glioblastomas are frequently resistant to immune checkpoint blockade (ICB) with few hypermutated glioblastomas showing response. Modeling patient-individual resistance is challenging due to the lack of predictive biomarkers and limited accessibility of tissue for serial biopsies. Here, we investigate resistance mechanisms to anti-PD-1 and anti-CTLA-4 therapy in syngeneic hypermutated experimental gliomas and show a clear dichotomy and acquired immune heterogeneity in ICB-responder and non-responder tumors. We made use of this dichotomy to establish a radiomic signature predicting tumor regression after pseudoprogression induced by ICB therapy based on serial magnetic resonance imaging. We provide evidence that macrophage-driven ICB resistance is established by CD4 T cell suppression and Treg expansion in the tumor microenvironment via the PD-L1/PD-1/CD80 axis. These findings uncover an unexpected heterogeneity of response to ICB in strictly syngeneic tumors and provide a rationale for targeting PD-L1-expressing tumor-associated macrophages to overcome resistance to ICB.

Blockade of myeloid-derived suppressor cell function by valproic acid enhanced anti-PD-L1 tumor immunotherapy

Regardless of the remarkable clinical success of immune checkpoint blockade (ICB) against PD-1/PD-L1 pathway, this approach has encountered drawbacks in most patients due to the activation of tumor immunosuppressive factors such as myeloid-derived suppressor cells (MDSCs). Histone deacetylase (HDAC) inhibitors combat ICB resistance by attenuating the immunosuppressive function of MDSCs and increasing PD-L1 expression on tumor cells. However, whether an HDAC inhibitor - valproic acid (VPA) suppression of MDSCs function could enhance PD-L1 blockade-mediated tumor immunotherapy remains unknown. Here we report that VPA and anti-PD-L1 antibody combined treatment promoted the polarization of bone marrow-derived precursor cells into M-MDSCs. Interestingly, the combination treatment of VPA and anti-PD-L1 antibody activated IRF1/IRF8 transcriptional axis in MDSCs leading to blockade of their immunosuppressive function by downregulating the expression of IL-10, IL-6, and ARG1 while re-activating CD8+ T-cells for the production of TNFα to further enhance anti-tumor immunity. These observations provide further rationale for the combination therapy of VPA with anti-PD-L1 antibody in preclinical settings.

Immuno-subtyping of breast cancer reveals distinct myeloid cell profiles and immunotherapy resistance mechanisms

Cancer-induced immune responses affect tumor progression and therapeutic response. In multiple murine models and clinical datasets, we identified large variations of neutrophils and macrophages, which define “immune subtypes” of triple negative breast cancer (TNBC) including neutrophil-enriched (NES) and macrophage-enriched subtypes (MES). Different tumor-intrinsic pathways and mutual regulation between macrophages/monocytes and neutrophils contribute to the development of dichotomous myeloid compartment. MES contains predominantly macrophages that are CCR2-dependent and exhibit variable responses to immune checkpoint blockade (ICB). NES exhibits systemic and local accumulation of immunosuppressive neutrophils (or granulocytic myeloid-derived suppressor cells (gMDSCs), is resistant to ICB, and contains a minority of macrophages that appear to be unaffected by CCR2 knockout. A MES-to-NES conversion mediated acquired ICB resistance of initially sensitive MES models. Our results demonstrate diverse myeloid cell frequencies, functionality, and potential roles in immunotherapies, and highlight the need to better understand the inter-patient heterogeneity of the myeloid compartment.

Abstract LB-218: High-dimensional modeling of single-cell-based CD8+ T cell exhaustion predicts response to immune checkpoint blockade

Abstract INTRODUCTION Accurate prediction of response to existing immune checkpoint blockade (ICB) and development of more efficacious ICB strategies are needed for cancer immunotherapy. The single-cell-level characterization of tumor-infiltrating CD8+ T lymphocytes (TILs) has presented new opportunities. However, the phenotypic variability of TILs, including tumor-specificity and CD8+ TIL exhaustion that regulate response to ICB, is poorly understood. Computationally modeling a large number of CD8+ TILs with high-dimensional T cell functional markers can link high-dimensional single-cell data with fundemental T cell functionalities. METHODS Here we develop a computational method (called HD-scMed) to predict response to ICB at the single-cell level. Our high-dimensional single-cell method, HD-scMed, models the phenotypic variability of CD8+ TILs directly within the original high-dimensional expression space, defined by k-dimensional exhaustion markers and other T cell functional markers. Within the high-dimensional expression space, HD-scMed identifies Pareto-optimal Frontier (PF) TILs representing a subset of exhausted TILs, by a mathematical model, Pareto Optimization. A quantitative criterion, D-value, defined as the Euclidean distance from PF TILs to the baseline TILs excluded by the PF, is used to indicate the extent to which the PF TILs are exhausted. RESULTS D-value accurately predicts clinical response to the ICB, i.e., αPD-1, αCTLA-4, and αPD-1+αCTLA-4, with performance of AUC=100% by single-cell flow cytometry (CyTOF) data with 101,401 CD8+ TILs from human tumors after ICBs, and AUC=95% and 86% by single-cell RNA-seq (scRNA-seq) data including 9,405 CD8+ TILs from human tumors before and after ICBs, respectively. Mechanistically, “D-high” TILs are associated with high tumor-specificity, enhanced exhaustion, and inactivated effector and cytotoxic signatures in non-responders; whereas “D-low” TILs are enriched in responders demonstrating tumor-specific T cells with T cell activation and cytolytic effector T cell signatures. We also observed “D negative” bystander T cells irrelevant to response. Notably, LAG3 shows significantly higher contribution to TIL D values in non-responders than responders. To reverse the functionality of the LAG3-high TILs after receiving αPD-1+αCTLA-4, we tested the effects of αLAG3 with αPD-1+αCTLA-4 in murine tumor models. Remarkably, the combination, αLAG3+αPD-1+αCTLA-4, showed extraordinary antitumor efficacy in eradicating advanced tumors, which was associated with burst GZMBhi TIL populations; whereas αPD-1+αCTLA-4 or αPD-1+αCTLA-4 plus other ICBs as control showed only moderate inhibition of tumor growth. CONCLUSION Our study has important implications for cancer immunotherapy regarding both accurate prediction of ICB response and development of better strategies to reverse existing ICB resistance. Citation Format: Guangxu Jin, Gang Xue, Rui-Sheng Wang, Ling-Yun Wu, Lance Miller, Yong Lu, Wei Zhang. High-dimensional modeling of single-cell-based CD8+ T cell exhaustion predicts response to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-218.

Blood-based genomic profiling of cell-free DNA (cfDNA) to identify microsatellite instability (MSI-H), tumor mutational burden (TMB) and Wnt/B-Catenin pathway alterations in patients with gastrointestinal (GI) tract cancers.

3552 Background: MSI-H cancers are responsive to immune checkpoint blockade (ICB), but nearly half of all patients experience primary or early treatment resistance. Activation of the WNT/B-Catenin pathway can lead to immune exclusion and may drive resistance to ICB. Methods: 12 patients had stage III (N = 1) or IV (N = 11) MSI-H GI tract (small bowl, colon, or rectal) cancers. Blood samples were obtained after (N = 5) or during (N = 5) ICB. 2 patients did not receive ICB. Blood samples from 8 patients with microsatellite stable (MSS) metastatic colorectal cancer were included as controls. The Guardant Health (Redwood City, CA) Omni 2.0 mb panel was used to analyze cfDNA. We analyzed MSI-H status, TMB, and mutations within the WNT/B-Catenin pathway, including APC, RNF43 and CTNNB1. Results: Of 12 patients with MSI-H GI cancers, 1 sample failed enrichment due to hemolysis. MSI-H was not detected in 2 patients with a history of MSI-H in tissue; however these patients had a complete response to ICB at the time of blood collection. The Omni panel identified MSI-H in the remaining 9 patients with MSI-H disease in tissue. Among 8 control patients with MSS disease in tissue, MSI-H was not detected. Median TMB (mutations/Mb) was greater for MSI-H specimens (109; range 30-807) than for MSS specimens (13; range 6-24). All 8 patients with MSS GI cancers were identified to have APC mutations, and none were found to have CTNNB1 or RNF43 mutations. Of 9 evaluable MSI-H GI cancers, 2 had APC mutations alone. The remaining 7 carried RNF43 mutations (G659fs). All patients with RNF43 mutations were found to have disease progression while on ICB. Among these 7 patients with RNF43 mutations, 6 had additional mutations in APC or CTNNB1. Conclusions: Blood based genomic profiling can identify MSI-H cancers. Patients with MSI-H cancers resistant to ICB in this cohort have mutations in RNF43 as well as additional mutations in APC or CTNNB1, suggesting that co-activation of the WNT/B-Catenin pathway may be biologically important. Further study of the role of WNT/B-Catenin pathway activation in ICB resistance will be pursued using tumor tissue from this cohort.

Single cell analysis of urothelial carcinoma (UC) liver metastases identifies epithelial-mesenchymal transition (EMT) as a potential mechanism of resistance to immunotherapy.

e16018 Background: UC liver metastases are associated with a low response rate to immune checkpoint blockade (ICB) and short survival. We sought to identify tumor specific factors promoting resistance to ICB. Methods: DEPArray is a dielectrophoresis based cell sorting platform for isolating immunohistochemically-defined cell populations from formalin-fixed paraffin-embedded (FFPE) tissue. Available FFPE tissue blocks of mUC liver metastases from patients (pts) treated with ICB were selected for analysis. Following histologic evaluation, tissue blocks were digested to yield cellular suspensions for immunophenotypic DEPArray sorting by keratin (K) and vimentin (V) to identify carcinoma cells and stromal cells, respectively. Cell pools were further analyzed for recurrent mutations and copy number alterations by a targeted OncoSeek panel and by MiSeq. Results: 12 unique patient samples were profiled. Median age was 73 (range 65-89), 11/12 male, 7 (58%) were platinum-refractory, and 4 (33%) received only ICB. 7 (58%) biopsies were obtained prior to initiation of ICB (1PR, 1SD, 5PD). Routine H&E sections generally revealed tumors completely devoid of immune cells, but admixed with atypical mesenchymal cells ranging from 5% to 60% of cellular composition. DEPArray sorting by K and V identified three discrete cell populations: K+/V- carcinoma cells, K-/V+ stromal cells, and K+/V+ cells that exhibited mesenchymal morphology. Of 8 samples with adequate cell counts (range 75-154 cells), next-generation sequencing (NGS) identified that K+/V- carcinoma cells and K+/V+ double positive cells shared unique somatic alterations in 7 (88%) pts, including point mutations in FGFR3, ERBB2, FBXW7, PIK3CA, FLT3 and STK11, and amplification of CDK4. In one liver responder to ICB, V+ stromal cells carried a putative germline TP53 R248W mutation. Conclusions: Archival FFPE tissue from mUC liver metastases can be digitally sorted by DEPArray to yield pools of ~100 cells suitable for NGS. UC liver metastases exhibit a population of K+/V+ cells with clonal somatic alterations suggestive of an EMT cancer cell phenotype that may be playing a role in ICB resistance.

Abstract B077: Signatures of T-cell dysfunction and exclusion predict cancer immunotherapy response

Abstract Cancer treatment by immune checkpoint blockade (ICB) can bring long-lasting clinical benefits, but only a fraction of patients responds to treatment. To predict ICB response we developed TIDE, a computational method to model two primary mechanisms of tumor immune evasion: inducing T-cell dysfunction in tumors with high infiltration of cytotoxic T lymphocytes (CTL) and preventing T-cell infiltration in tumors with low CTL level. We identified signatures of T-cell dysfunction from large tumor cohorts by testing how the expression of each gene in tumors interacts with the CTL infiltration level to influence patient survival. We also modeled factors that exclude T-cell infiltration into tumors using expression signatures from immunosuppressive cells. Using this framework and pre-treatment RNA-Seq or NanoString tumor expression profiles, TIDE predicted the outcome of melanoma patients treated with first-line anti-PD1 or anti-CTLA4 more accurately than other biomarkers such as PD-L1 level and mutation load. TIDE also revealed new candidate ICB resistance regulators, such as SERPINB9, demonstrating utility for immunotherapy research. TIDE source code and a web application are available at http://tide.dfci.harvard.edu. Citation Format: Peng Jiang, Shengqing Gu, Deng Pan, Jingxin Fu, Avinash Sahu, Xihao Hu, Ziyi Li, Nicole Traugh, Xia Bu, Bo Li, Jun Liu, Gordon J Freeman, Myles A Brown, Kai W. Wucherpfennig, Xiaole Shirley Liu. Signatures of T-cell dysfunction and exclusion predict cancer immunotherapy response [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B077.