Cell-inflamed Tumor Microenvironment Research Articles

Dual nanoparticle immunostimulant hydrogel for synergistic combating “Cold” tumor

The immunosuppressive tumor microenvironment (TME) in “cold” triple-negative breast cancer (TNBC) leads to resistance to current immunotherapies. Here, we designed an in-situ hydrogel reservoir to deliver two types of nanoparticles (NPs) targeting 4T1 tumor cells and tumor-associated macrophages (TAMs) for cancer cell elimination and reversal of the immunosuppressive TME. The 4T1 tumor cell-targeted NPs could specifically deliver the mitochondria-targeted photosensitizer and catalase into 4T1 tumor cells to execute an enhanced photodynamic therapy and induce immunogenic cell death. The TAMs-targeted NPs delivered the stimulator of interferon genes (STING) agonist into macrophages to activate the STING pathway, triggering a type I interferon-driven innate immune response that induces a shift to a “hot” T cell-inflamed TME. This local synergistic therapy could effectively stimulate dendritic cell activation and the continuous T-cell infiltration, thereby reprograming the immunosuppressive TME, reinvigorating a powerful anti-tumor immune response and inhibiting tumor growth and metastasis in TNBC tumor model. In addition, this study combined with immune checkpoint blockade further achieves reinforced therapeutic outcome against “cold” tumor, providing a promising hydrogel-mediated platform for treatment of TNBC.

Tumor-targeted therapy with BRAF-inhibitor recruits activated dendritic cells to promote tumor immunity in melanoma

BackgroundTumor-targeted therapy causes impressive tumor regression, but the emergence of resistance limits long-term survival benefits in patients. Little information is available on the role of the myeloid cell network, especially...

Abstract 177: Targeting murine or human KLRG1 exerts potent anti-tumor efficacy involving both CD8T cells and NK cells

Abstract Clinical response to anti-PD-1 immunotherapy is correlated with patients who have a T cell-inflamed tumor microenvironment. Some patients with immune cell infiltration fail to respond, and some patients experiencing an initial clinical response develop acquired resistance. It is likely that additional immune-regulatory pathways need to be targeted to expand the efficacy of immunotherapies. To identify a broader array of therapeutic relevant immunotherapy molecules in T cell-inflamed tumors, our lab utilized gene expression profiling to characterize tumor antigen-specific dysfunctional CD8+ T cells in the tumor microenvironment. One molecule that was found to be overexpressed on these CD8+ T cells was C-type lectin inhibitory receptor, KLRG1. KLRG1 is an ITIM domain-containing receptor mostly explored on NK cells, and the known ligands are N- and E- cadherins which are broadly expressed in solid cancers. Analysis of single cell RNA-Seq data from tumor biopsies of melanoma patients classified as responders or non-responders to anti-PD-1 therapy revealed significantly higher expression of KLRG1 expression on tumor-infiltrating T cell subsets in non-responders, suggesting a possible salvage immune suppressive role. To determine the potential functional relevance of KLRG1 on tumor control, we generated KLRG1 knockout (KO) mice, then implanted tumors and assessed tumor growth rate in vivo. KLRG1 KO mice showed markedly slowed B16 melanoma tumor growth compared to WT mice, arguing it is a critical negative regulator of anti-tumor immunity. Both antigen-specific CD8+ T cells and NK cells were expanded in these tumors, and depletion of CD8+ T cells or NK cells eliminated this tumor control. Genetic deletion of N-cadherin from B16 cells also resulted in slowed tumor growth, suggesting that tumor cells are providing the functionally relevant ligand. To assess the degree of inhibitory function of human KLRG1, we generated a humanized KLRG1 knock-in mouse model. We confirmed that human KLRG1 can biochemically interact with murine cadherins. Knock-in mice with the humanized KLRG1 protein had significantly increased tumor growth compared to both WT and KLRG1 KO mice, indicating a stronger immunosuppressive effect of human KLRG1 compared to murine KLRG1. An antagonistic antibody blocking interaction of human KLRG1 with its ligands was generated, which potentiated human T cell activation in vitro. Administration of anti-human KLRG1 Ab to tumor-bearing humanized KLRG1 mice led to markedly reduced tumor growth. In summary, our data support KLRG1 as a functionally critical negative regulator of the anti-tumor immune response and that blocking KLRG1-cadherin interactions has potential as a therapeutic candidate. Citation Format: Tyler A. Jones, Stephen Sinicrope, Kei Yasuda, Jan Pinkas, Thomas F. Gajewski. Targeting murine or human KLRG1 exerts potent anti-tumor efficacy involving both CD8T cells and NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 177.

Abstract 164: Genetic elimination of β-catenin using a doxycycline-regulated GEM model fails to restore checkpoint blockade efficacy due to persistent M2-like macrophages

Abstract Response to checkpoint blockade immunotherapy (CBI) is not universal, partly due to a wide variation in baseline immune cell infiltration in the tumor microenvironment (TME). One mechanism contributing to this variability is tumor cell-intrinsic activation of the β-catenin signaling pathway, which has previously been shown to result in a non-T cell-inflamed TME. Constitutive β-catenin signaling resulted in reduced Batf3-lineage dendritic cell (cDC1) recruitment, leading to reduced T cell activation and infiltration and loss of anti-CTLA-4+anti-PD-L1 therapeutic efficacy. Efforts to target the Wnt/β-catenin pathway to enhance immune responses in β-catenin-active tumors have seen limited success. A genetic experiment eliminating β-catenin expression after establishment of a non-T cell-inflamed TME might illustrate the maximal biologic effect that could be expected with blockade of the pathway. We developed a genetically engineered mouse (GEM) model that allows for dynamic regulation of β-catenin on and off. This model employs tyrosinase-driven tamoxifen-regulated Cre-recombinase to activate the BRAF V600E oncogene and delete the tumor suppressor gene PTEN, along with a transactivator for doxycycline-regulated expression of a non-degradable β-catenin. Topical application of 4-hydroxytamoxifen resulted in melanoma formation, and administration of doxycycline resulted in robust nuclear expression of melanocyte-specific β-catenin compared to non-treated controls. This was accompanied by a significant reduction in CD3+ T cell infiltration within the TME, confirming the link between active β-catenin signaling and T cell exclusion. Discontinuing doxycycline treatment led to complete reduction of nuclear β-catenin expression in the tumor cells within seven days and a substantial return of CD3+ T cells into the TME. However, despite the re-infiltration of CD3+ T cells, the tumors previously expressing β-catenin (ex-β-catenin tumors) did not regain therapeutic responsiveness to anti-CTLA-4+anti-PD-L1. Single cell RNA sequencing of the ex-β-catenin tumors indicated the presence of an immunosuppressive-like macrophage population, characterized by the expression of Ccl8, Gas6 and Cd163. This finding suggests that the prior presence of β-catenin may induce long-lasting changes in the TME that suppress the immune response even after β-catenin levels are reduced. These data corroborate previous findings on the role of β-catenin in modulating the immunological landscape of melanoma and extend it by demonstrating the long-term persistence of immune evasion by suppressive myeloid cells even after β-catenin signaling is eliminated. These insights have implications for clinical strategies aiming to block Wnt/β-catenin signaling, which may additionally require myeloid cell modulation to restore immunotherapy efficacy. Citation Format: Alexandra Cabanov, Ruxandra Tonea, Jason Shapiro, Anna Martinez, Yuanyuan Zha, Thomas F. Gajewski. Genetic elimination of β-catenin using a doxycycline-regulated GEM model fails to restore checkpoint blockade efficacy due to persistent M2-like macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 164.

Abstract C016: Pre-existing T cell inflammation is a determinant of response to mesothelin chimeric antigen receptor T cell therapy in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a 12.5% 5-year survival rate and is predicted to become the 2nd leading cause of cancer-related death by 2030. PDAC is known to be resistant to immunotherapy. Thus, while chimeric antigen receptor (CAR) T cell therapy shows the potential to mediate durable responses against some hematologic and solid tumors, in PDAC, the efficacy of CAR T therapy has remained limited. PDAC tumors show heterogenous T cell infiltration with some tumors containing a high number of T cells. As T cell infiltration associates with prolonged survival, we hypothesized that the density of pre-existing T cell infiltration would predict response to CAR T therapy. Methods: Previously established clonal mouse PDAC cell lines, which show a T cell inflamed or non-inflamed tumor microenvironment upon in vivo implantation, were used. Mouse CAR T cells specific to mesothelin, a tumor antigen expressed by PDAC, were generated using retroviral transduction. Mice were implanted subcutaneously with T cell inflamed or non-inflamed tumor cell lines. Ten days later mesothelin CAR T cells were administered intravenously. Flow cytometry and multiplex immunohistochemistry was used to define the density and spatial location of CAR T cell infiltration. The impact of CAR T cells on tumor growth and survival was also measured. Results: CAR T cells were found to traffic to T cell inflamed tumors, which led to delayed tumor outgrowth. However, such results were not seen in T cell non-inflamed tumors. Rather in mice with T cell non-inflamed tumors, CAR T cells accumulated in the liver. Further, CAR T cells effectively eliminated T cell inflamed and non-inflamed tumor cells in vitro, demonstrating that tumor intrinsic mechanisms of resistance to T cell killing were not a major determinant of treatment efficacy. Conclusions: Our findings suggest the therapeutic potential of CAR T cells in PDAC but highlight mechanisms of T cell exclusion as a therapeutic barrier wherein CAR T cells accumulate in host organs. Citation Format: Jacqueline B. Plesset, Heather Coho, Kelly Markowitz, Dhruv Patel, Max M. Wattenberg, Meredith L. Stone, Devora Delman, Gregory L. Beatty. Pre-existing T cell inflammation is a determinant of response to mesothelin chimeric antigen receptor T cell therapy in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C016.

Uncovering the Potential of CD40 Agonism to Enhance Immune Checkpoint Blockade.

In this CCR Translations, we discuss the therapeutic potential of CD40 agonism, which stimulates antigen-presenting cells (APC) to activate effector T and NK cells. CD40 agonism may lead to development of an interferon-activated, T cell-inflamed tumor microenvironment and has the potential to facilitate long-term response with immune checkpoint blockade. See related article by Weiss et al., p. 74.

Abstract 3276: Tumor cell-intrinsic PGE2-EP4 signaling is a major immunosuppressive pathway in pancreatic cancer

Abstract Pancreatic adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the US partly because of its resistance to conventional chemotherapies. Immunotherapies that improve survival in many cancers, almost universally fail in PDA. The resistance to immune checkpoint blockade is at least partially explained by the lack of T cells in the tumor microenvironment (TME) in PDA. We have previously demonstrated that tumor cell-derived prostaglandin E2 (PGE2) is one of the tumor intrinsic factors that creates/supports immunosuppressive TME in PDA presumably through its paracrine effect on the stromal cells. In the current study, we investigate the role of autocrine PGE2 signaling through its cognate EP4 receptor in shaping the T cell low, myeloid cell rich PDA TME. The knockdown (EP4 KD) or knockout (EP4 KO) of the receptors on KrasG12D/p53R172H/YFP/PdxCre (KPCY) tumor cells was sufficient to suppress or abolish the growth of implanted tumors, respectively (control vs EP4 KD, p=0.01, and control vs EP4 KO, p<0.0001). The rejection of the EP4 KO PDA cell lines was T cell dependent as it did not happen in hosts depleted of CD4+ and CD8+ T cells. Upon rechallenge, 80-90% of mice that rejected the primary EP4 KO tumors were able to reject the control, EP4-sufficient tumors in a T cell-dependent manner. While EP4 KO tumors were universally rejected by wild-type (WT) or EP4 global KO (KOgl) hosts, control cell lines formed tumors and grew in both hosts albeit with significantly slower growth in EP4 KOgl hosts (p<0.0001). The TME analysis revealed changes in the T cell presence, subtypes, and functional state in EP4 KO tumors demonstrating more T cell inflamed TME with increased proportions of central memory CD4+ and CD8+ cells (p=0.02 both) along with downregulation of exhaustion markers PD-1 (p=0.02) and CD39 (p=0.009) on CD4+ and CD8+ cells, respectively. Deletion of EP4 on tumor cells changed the systemic anti-tumor immunity by inducing T cell proliferation in lymph nodes (EP4 KO vs control, Ki67+CD8+ p<0.0001 and Ki67+CD4+ p=0.003) and depleting gMDSC population in the spleens of EP4 KO tumor bearing mice (p<0.0001). RNAseq analysis of tumor cells (cultured and in vivo) as well as bulk tumors identified TNFa, IFNa, and IFNg pathways as top upregulated ones in EP4 KO tumors while TGFb and Myc pathways were among the significantly downregulated pathways. The study demonstrates a primary role of tumor cell PGE2-EP4 signaling in creating immunosuppressive TME in PDA suggesting EP4 blockade as a novel approach in treatment of pancreatic cancer. Citation Format: Nune Markosyan, Charu Arora, Liz Quinones, Il-Kyu Kim, Nikhil Joshi, Noah Cheng, John Tobias, Ben Stanger, Robert Vonderheide. Tumor cell-intrinsic PGE2-EP4 signaling is a major immunosuppressive pathway in pancreatic cancer [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 3276.

Abstract 5170: STING inflames NF1 malignancies for immunotherapy

Abstract Introduction: Neurofibromatosis Type 1 (NF1) is caused by mutations in the NF1 gene that encodes neurofibromin, a RAS GTPase-Activating Protein. Inactivating NF1 mutations result in hyperactivation of RAS-mediated signaling. NF1 patients develop benign tumors on the skin and along nerves in the spinal cord and brain, and ~10% of these progress to Malignant Peripheral Nerve Sheath Tumors (MPNST). Attempts to inhibit RAS-signaling to treat MPNST have not worked in clinical trials. Surgical removal is the only cure for MPNST, which is challenging due to large tumor size and/or proximity to nerves. MPNSTs often recur, metastasize, and respond poorly to chemo- and radiotherapy. As a result, MPNSTs are the leading cause of death in NF1 patients. Immune Checkpoint Blockade (ICB) is an approach to treat inoperable, undruggable cancers. Immune checkpoint proteins (ICPs) terminate normal immune responses to prevent collateral tissue damage. Cancer cells hijack this mechanism and express ICPs to avoid immune detection. ICB disrupts this process, enhancing antitumor immunity. Monoclonal antibodies (mABs) that disrupt the interaction between Programmed Cell Death Receptor 1 (PD-1) and its ligand Programmed Cell Death Ligand 1 (PD-L1) are a widely used ICB therapy. Having a T cell-enriched hot tumor microenvironment (TME) is required for successful ICB. However, MPNSTs lack a T cell-inflamed TME and respond poorly to ICB therapy. Activation of the protein “stimulator of interferon genes” (STING) enhances anti-tumor immunity via induction of pro-inflammatory cytokines, thus increasing T cell infiltration into the TME. We hypothesized that reprogramming the MPNST TME by STING pathway activation would turn cold MPNSTs into hot tumors amenable to ICB. Methods: We treated mouse MPNSTs with STING agonist or STING agonist + anti-PD-1/anti-PD-L1 mAb. Tumor size was measured throughout treatment. The mice were sacrificed and tumor tissue was analyzed for STING pathway activation, T cell infiltration, cell proliferation, and apoptosis. MPNST allograft tumors in athymic mice were controls. Results: STING pathway activation and increased T cell presence was observed in STING agonist-treated MPNSTs compared to vehicle-treated. MPNSTs treated with STING agonist alone or STING agonist + PD-1/PD-L1 mAbs showed less tumor growth relative to vehicle. Importantly, STING agonist + PD-1/PD-L1 mAb-treated tumors showed significant cell death increase while cell proliferation remained unchanged. Interestingly, tumors in athymic mice continued to grow despite treatment suggesting the antitumor effects seen in immunocompetent mice were mediated by T cells. Conclusions: Our data show that MPNSTs can become T cell-rich tumors by activating STING signaling. Reprogramming the TME made MPNSTs susceptible to immune destruction by ICB therapy. Our studies support this novel treatment strategy for MPNST, an aggressive, deadly tumor for which no molecular therapy currently exists. Citation Format: Nipunika Somatilaka, Laasya Madana, Ali Sadek, Renee M. McKay, Lu Q. Le. STING inflames NF1 malignancies for immunotherapy. [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 5170.

220P HDAC1 inhibition sensitizes refractory tumor cells to anti-PD-1 therapy by upregulation chemokine expression and stimulating T cells infiltration into tumor beds

Immune checkpoint inhibitors (ICIs), exemplified by anti-PD-1 are promising treatments for many tumors. However, the majority of patients lack effective responses because of the emergence of immune-refractory tumors that disrupt the amplification of antitumor immunity. In many cases, one of the major causes of resistance to these agents is the limited tumor penetrance of effector T cells. Durable clinical responses using anti–PD-1 have been associated with T cell–inflamed tumor microenvironment (TME) favoring the infiltration of functional cytotoxic T lymphocytes (CTLs).

Inhibition of stearoyl-CoA desaturase 1 (SCD1) enhances the antitumor T cell response through regulating β-catenin signaling in cancer cells and ER stress in T cells and synergizes with anti-PD-1 antibody

BackgroundUnderstanding the mechanisms of non-T cell inflamed tumor microenvironment (TME) and their modulation are important to improve cancer immunotherapies such as immune checkpoint inhibitors. The involvement of various immunometabolisms has...

Abstract 5018: A comprehensive analysis of genomic determinants of response to immune checkpoint inhibitor-based immunotherapy

Abstract A low response rate is the major challenge for existing cancer immunotherapies. A number of biomarkers have been reported to be associated with the likelihood of patient responses to immune checkpoint inhibitors (ICI), including the expression of PD-L1, tumor mutational burden (TMB), tumor infiltration lymphocytes, and T cell repertoire. However, these biomarkers showed variable performance even conflicting conclusions in different cohorts. And a large proportion of patients exhibited an immune-exclusion or immune-desert phenotype that cannot be explained by current biomarkers. In addition, establishing a cohort with adequate sample size requires multiple years of multi-center efforts. Therefore, it is still a challenge and a lack for the evaluation of efficacy of known biomarkers and the discovery of new signature(s) in a large-scale study. We hypothesized that a data-driven meta-analysis approach integrating multi-cohorts with multi-omics data will help to characterize the predictive genomic features and discover the mechanisms of antitumor immune responses. In this work, we collected 3,037 ICI therapy samples with genetic and transcriptomics data for 14 cancer types. Then a data-driven strategy was designed to explore each type of genomic feature which included gene mutations, pathway mutations, TMB, mutational signatures, gene expression, pathway expression, interaction among checkpoint blockade, and immune cell components. Then a comprehensive analysis, which contained a meta-analysis for the identification of consistent biomarkers across multiple cohorts and a data aggregation-analysis for the identification of rare biomarkers on aggregated data, was performed to explore the associations between ICI response and omics features. Furthermore, we validated these signatures as biomarkers in three independent cohorts. Finally, we identified five types of biomarkers, including 1) two biomarkers regarding source of antigen (TMB and mutations of MYO7B), 2) one feature of responding to antigen (cytotoxic T lymphocytes pathway), 3) three biomarkers indicating macrophages M1 infiltrations (macrophages M1 component, and two marker genes of macrophages M1, CXCL11 and CCL19), 4) one biomarker regarding immune interactions (expression relations among immune checkpoint genes), and 5) two biomarkers about T cell-inflamed tumor microenvironment (IFNG and ligands genes of chemokine receptor CXCR3). In addition, a predictive module was built based on these signatures and outperformed than TMB or PD-L1 expression which were approved molecular biomarkers by FDA. We found high TMB patients had more likelihood to active the cytotoxic T lymphocytes which secret IFNG to kill tumor cell and promote the recruitment of macrophage M1. And the T cells were stimulated by macrophage M1 via the binding of CXCR3 of T cell surface and receptor of CXCR3 of macrophage M1 surface. Citation Format: Jing Yang, Qi Liu, Yu Shyr. A comprehensive analysis of genomic determinants of response to immune checkpoint inhibitor-based immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5018.

CASTING: A Potent Supramolecular Strategy to Cytosolically Deliver STING Agonist for Cancer Immunotherapy and SARS-CoV-2 Vaccination

CASTING: A Potent Supramolecular Strategy to Cytosolically Deliver STING Agonist for Cancer Immunotherapy and SARS-CoV-2 Vaccination

Which angiosarcoma subtypes may benefit from immunotherapy?

11572 Background: Angiosarcomas (AS) are aggressive mesenchymal tumors arising from cells with endothelial properties. They include de novo primary AS (pAS), and secondary AS (sAS) due to prior radiotherapy, UV exposure or chronic lymphedema. Treatment options are limited and their prognosis is poor. Development of new treatment strategies is difficult due to the heterogeneity and rarity of AS. We hypothesize that immunological and genomic profiles are significantly different between pAS and sAS and may result in different immune checkpoint inhibition (ICI) based treatment strategies. Methods: Tumor samples from AS patients were retrospectively collected. Patients were categorized as pAS or sAS. Lymphocytes were analyzed using multiplex immunohistochemistry on tissue microarrays. Genomic profiling was performed in a selected subgroup with “TruSight Oncology 500”, a Next Generation Sequencing panel containing 523 cancer related genes. Results: Immunological data were analyzed from 257 AS patients. The cohort comprised 80 pAS patients and 177 sAS patients. The median density of CD3+ T cells was 250 cells/mm2 in pAS vs 452 cells/mm2 in sAS (p< 0.001). Median CD4+ T helper cell density was 128 cells/mm2 in pAS vs 246 cells/mm2 in secondary AS (p< 0.001). The median density of CD8+ cytotoxic T cells was 85 cells/mm2 in pAS vs 111 cells/mm2 in sAS ( p= 0.057). Density of FoxP3+ T regulatory cells was higher in sAS (median 42 cells/mm2) compared to pAS (median 23 cells/mm2) (p< 0.001). The median count of CD20+ B cells in pAS was 24 cells/mm2 compared to 32 cells/mm2 in sAS ( p= 0.533). Genomic analysis was performed on tumor DNA from 51 patients (25 pAS and 26 sAS). Median tumor mutational burden (TMB) was 3.2 (range 0.8-11.9) mutations per megabase (mut/Mb) in pAS vs 3.9 (range 0.0-99.6) in sAS ( p= 0.485). No microsatellite instability was detected. A pathogenic mutation, gene amplification or gene loss was identified in 82% of all patients (n = 42, 70% of pAS vs 100% of sAS ( p< 0.01)). In 36 patients (71%) at least one (likely) pathogenic mutation was detected (54% pAS vs 88% sAS, ( p= 0.013)). In 20 patients (39%) mutations in the DNA damage response (DDR) pathway were detected (12% pAS vs 68% sAS ( p< 0.01)). The most frequently found mutated genes were TP53 (10%), BRAF (6%), ERCC4 (6%), PTPRD (6%), WETD2 (6%) and SETD2 (6%). Amplifications were found in 49% (n = 25) of all patients (15% pAS vs 84% sAS, ( p< 0,01)). MYC amplifications were most common and were detected in 15% of pAS and 68% of sAS. Immune profiles of the 51 genomically characterized patients are currently under further investigation. Conclusions: We showed a clear distinction in immunological and genomic profiles between pAS and sAS. The potential benefit of ICI seems to be most promising in sAS with a T cell inflamed tumor microenvironment, frequent MYC amplifications, DDR mutations, and high mutational load, while in pAS boosting strategies to enhance susceptibility to ICI might be interesting for further investigation.

Abstract P5-13-18: Upregulation of immune response biomarkers by ribociclib plus endocrine therapy (ET) in paired tumor samples from phase I studies

Abstract Background: In addition to inhibiting cell cycle progression, CDK4/6 inhibitors have been found to modulate anti-cancer immune response (Goel S, et al. Nature. 2017;548:471-475). Data from preclinical studies suggested multiple mechanisms—involving changes in both cancer cells and immune cells—by which CDK4/6 inhibition can enhance anti-cancer immunity. These mechanisms include increased antigen presentation, interferon response triggering, and regulatory T-cell suppression. Clinical correlates to preclinical observations have so far been obtained using paired biopsies from neoadjuvant breast cancer trials (Johnston S, et al. J Clin Oncol. 2019;37:178-189; Hurvitz S, et al. Clin Cancer Res. 2020; ;26:566-80). Here, we describe a gene expression analysis of paired pre- and on-treatment biopsies from CLEE011A2115C and CLEE011X2107, two phase I clinical trials evaluating ribociclib in combination with ET in the metastatic breast cancer (MBC) setting. Methods: The nCounter PanCancer IO 360 Panel (NanoString) was used to quantify expression of 770 genes in 7 pairs of tumor samples (baseline vs. cycle 1 day 15) from two phase I trials in the MBC setting (5 patients from CLEE011A2115C and 2 from CLEE011X2107). All patients were treated with ribociclib and an ET. Pairwise differential gene expression analysis of individual genes and previously published immune-related gene signatures was conducted (Bedognetti D, et al. Curr Opin Oncol. 2015;27:433-444). Results: Many genes that were markedly suppressed with treatment (eg, MKI67, MYC, CDK2, CCNB1, TYMS, and DNMT1) were related to proliferation, DNA replication, and G1/S transition of the cell cycle, as expected. Interestingly, expression of numerous genes involved in immune response was increased. These were mostly interferon-regulated genes; particularly, a gene signature indicative of a T cell-inflamed tumor microenvironment (Ayers M, et al. J Clin Invest. 2017;127:2930-2940) was upregulated. Immune-related genes tended to preferentially increase with treatment in patients who later experienced a clinical response. Since bulk RNA from tumor biopsies was analyzed, we could not distinguish whether the observed expression changes reflected increased infiltration by immune cells or an endogenous interferon response in cancer cells. The latter could be triggered by DNMT1 suppression (as previously described), which then promotes T cell-mediated immunity by increasing antigen presentation and chemokine production in cancer cells. Conclusions: This analysis was conducted with a small sample size and is considered hypothesis generating; further investigation is needed. Cell cycle and proliferation markers showed robust suppression by ribociclib-based treatment regimens, as expected. Conversely, a notable increase in immune-related genes was detected that tended to occur preferentially in patients who later experienced more favorable clinical outcomes. Both changes occurred early (cycle 1 day 15). To our knowledge, this is the first clinical biomarker report of immune activation mediated by CDK4/6 inhibition in MBC. Citation Format: Dejan Juric, Chong Ma, Ralph Tiedt, Yoon-Sim Yap, Joanne Chiu, Pamela Munster, Roohi Ismail-Khan, Laura Garcia-Estevez, Ingrid A. Mayer, Carlos Becerra, Nadia Solovieff, Agnes Lteif, Faye Su, Yen-Shen Lu. Upregulation of immune response biomarkers by ribociclib plus endocrine therapy (ET) in paired tumor samples from phase I studies [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-18.

The Prognostic and Predictive Role of Xeroderma Pigmentosum Gene Expression in Melanoma.

BackgroundAssessment of immune-specific markers is a well-established approach for predicting the response to immune checkpoint inhibitors (ICIs). Promising candidates as ICI predictive biomarkers are the DNA damage response pathway genes. One of those pathways, which are mainly responsible for the repair of DNA damage caused by ultraviolet radiation, is the nucleotide excision repair (NER) pathway. Xeroderma pigmentosum (XP) is a hereditary disease caused by mutations of eight different genes of the NER pathway, or POLH, here together named the nine XP genes. Anecdotal evidence indicated that XP patients with melanoma or other skin tumors responded impressively well to anti-PD-1 ICIs. Hence, we analyzed the expression of the nine XP genes as prognostic and anti-PD-1 ICI predictive biomarkers in melanoma.MethodsWe assessed mRNA gene expression in the TCGA-SKCM dataset (n = 445) and two pooled clinical melanoma cohorts of anti-PD-1 ICI (n = 75). In TCGA-SKCM, we applied hierarchical clustering on XP genes to reveal clusters, further utilized as XP cluster scores. In addition, out of 18 predefined genes representative of a T cell inflamed tumor microenvironment, the TIS score was calculated. Besides these scores, the XP genes, immune-specific single genes (CD8A, CXCL9, CD274, and CXCL13) and tumor mutational burden (TMB) were cross-correlated. Survival analysis in TCGA-SKCM was conducted for the selected parameters. Lastly, the XP response prediction value was calculated for the two pooled anti-PD-1 cohorts by classification models.ResultsIn TCGA-SKCM, expression of the XP genes was divided into two clusters, inversely correlated with immune-specific markers. A higher ERCC3 expression was associated with improved survival, particularly in younger patients. The constructed models utilizing XP genes, and the XP cluster scores outperformed the immune-specific gene-based models in predicting response to anti-PD-1 ICI in the pooled clinical cohorts. However, the best prediction was achieved by combining the immune-specific gene CD274 with three XP genes from both clusters.ConclusionOur results suggest pre-therapeutic XP gene expression as a potential marker to improve the prediction of anti-PD-1 response in melanoma.

Checkpoint protein expression in the tumor microenvironment defines the outcome of classical Hodgkin lymphoma patients

AbstractEmerging evidence indicates a major impact for the tumor microenvironment (TME) and immune escape in the pathogenesis and clinical course of classical Hodgkin lymphoma (cHL). We used gene expression profiling (n = 88), CIBERSORT, and multiplex immunohistochemistry (n = 131) to characterize the immunoprofile of cHL TME and correlated the findings with survival. Gene expression analysis divided tumors into subgroups with T cell-inflamed and -noninflamed TME. Several macrophage-related genes were upregulated in samples with the non–T cell-inflamed TME, and based on the immune cell proportions, the samples clustered according to the content of T cells and macrophages. A cluster with high proportions of checkpoint protein (programmed cell death protein 1, PD-1 ligands, indoleamine 2,3 dioxygenase 1, lymphocyte-activation gene 3, and T-cell immunoglobulin and mucin domain containing protein 3) positive immune cells translated to unfavorable overall survival (OS) (5-year OS 76% vs 96%; P = .010) and remained an independent prognostic factor for OS in multivariable analysis (HR, 4.34; 95% CI, 1.05-17.91; P = .043). cHL samples with high proportions of checkpoint proteins overexpressed genes coding for cytolytic factors, proposing paradoxically that they were immunologically active. This checkpoint molecule gene signature translated to inferior survival in a validation cohort of 290 diagnostic cHL samples (P < .001) and in an expansion cohort of 84 cHL relapse samples (P = .048). Our findings demonstrate the impact of T cell- and macrophage-mediated checkpoint system on the survival of patients with cHL.

Anlotinib Induces a T Cell-Inflamed Tumor Microenvironment by Facilitating Vessel Normalization and Enhances the Efficacy of PD-1 Checkpoint Blockade in Neuroblastoma.

Anlotinib has achieved good results in clinical trials of a variety of cancers. However, the effects of anlotinib on the tumor microenvironment (TME) and systemic immunity have not been reported. There is an urgent need to identify the underlying mechanism to reveal new opportunities for its application in neuroblastoma (NB) and other cancers. Understanding the mechanism will hopefully achieve the goal of using the same method to treat different cancers. This study used bioinformatics, NB syngeneic mouse models, flow cytometry, RNA-seq, and immunofluorescence staining to explore the mechanisms of anlotinib on the TME, and further explored anlotinib-containing combination treatment strategies. We proved that anlotinib facilitates tumor vessel normalization at least partially through CD4+ T cells, reprograms the immunosuppressive TME into an immunostimulatory TME, significantly inhibits tumor growth, and effectively prevents systemic immunosuppression. Moreover, the combination of anlotinib with a PD-1 checkpoint inhibitor counteracts the immunosuppression caused by the upregulation of PD-L1 after monotherapy, extends the period of vascular normalization, and finally induces NB regression. To our knowledge, this study is the first to dynamically evaluate the effect of a multitarget antiangiogenic tyrosine kinase inhibitor on the TME. These findings have very important clinical value in guiding the testing of related drugs in NB and other cancers. Based on these findings, we are conducting a phase II clinical study (NCT04842526) on the efficacy and safety of anlotinib, irinotecan, and temozolomide in the treatment of refractory or relapsed NB, and hopefully we will observe patient benefit.

918 Doxycycline regulatable ß-catenin demonstrates inducible immune evasion in a melanoma GEM model

BackgroundLack of response to checkpoint blockade immunotherapy has been linked to a deficiency of immune cell infiltration within the tumor microenvironment (TME). One demonstrated mechanism sufficient for the non-T cell inflamed TME is tumor cell-intrinsic activation of the β-catenin signaling pathway. Using genetically engineered mouse models (GEMMs), tumors constitutively expressing active β-catenin lack a robust endogenous T cell infiltrate and fail to respond to immunotherapies. In support of these mouse studies, human melanoma metastases with increased active β-catenin signaling exhibit decreased numbers of tumor infiltrating Batf3-driven cDC1 and CD3+ T cells. However, whether temporal activation and inactivation of β-catenin within the same developing tumor would alter immune cell infiltration is not known.MethodsA model was created in which tamoxifen-regulated Cre-recombinase mediates BRAFV600E oncogene activation and PTEN tumor suppressor gene deletion as well as expression of a doxycycline regulatable reverse transactivator. Upon administration of doxycycline via the drinking water to these animals, a non-degradable form of nuclear β-catenin becomes expressed. Immunofluorescence assays were performed assessing the β-catenin expression status in the tumor cells as well as immune cell infiltration within the TME. Additionally, immunotherapy efficacy experiments were performed.ResultsWe observed that administration of doxycycline to these animals drove expression of an active form of nuclear β-catenin. Activation of nuclear β-catenin resulted in a 2-fold decrease in the overall CD3+ T cells infiltration into the TME. Moreover, this decrease in immune infiltration also resulted in loss of anti-PD-L1 + anti-CTLA-4 therapy efficacy. We next performed studies assessing the kinetics with which β-catenin levels diminish upon doxycycline removal. Switching animals to regular drinking water resulted in rapid reduction of nuclear β-catenin levels, including 50 percent reduction after two days of doxycycline removal and almost complete reduction of nuclear β-catenin after four days.ConclusionsWe describe a novel mouse model in which we induce autochthonous melanoma tumors in mice along with inducible expression of a non-degradable, nuclear β-catenin modulated by doxycycline in the drinking water. Activation of β-catenin signaling in melanoma tumors resulted in reduction of immune cells in the TME as well as loss of checkpoint blockade immunotherapy efficacy. This activation can be rapidly reversed by removing doxycycline, allowing for future studies evaluating the consequences of turning off β-catenin once it has already driven a non-T cell-inflamed TME.AcknowledgementsThis work was supported by the Wissler Fellowship from the University of Chicago (SS) K99/R00 (NCI; SS), and R35CA210098 (TG).

Abstract 3044: The unexplored immune landscape of high-risk pediatric cancers

Abstract In adult cancer, immune signatures such as the T cell-inflamed gene expression profile (GEP) have been developed to predict which patients are likely to respond to immune checkpoint inhibitors (ICIs) beyond high tumor mutation burden (TMB) and PD-L1 expression. The GEP infers T cell infiltration and activation in the tumor microenvironment (TME) from transcriptomic data. However, it is not known whether tools such as GEP are applicable in pediatric cancer, as the TME in childhood cancers is largely unexplored and response to ICIs are rare. We have undertaken an integrated analysis of the pediatric TME using RNA-sequencing (RNA-seq) and immunohistochemistry (IHC). Our goal is to identify patients with T cell-inflamed or “hot” tumors who may benefit from ICIs. Through Australia's ZERO childhood cancer precision medicine program we performed RNA-seq on 347 high-risk pediatric cancers (estimated &amp;lt;30% chance of survival) and performed IHC for CD4, CD8, CD45 and PD-L1 on 112 matching samples. Using both informatic assessments and IHC as independent measures of immune infiltration, we mapped the immune landscape of the TME across a broad range of high-risk pediatric cancers. As RNA-seq is increasingly used in the analysis of patient tumors, we investigated numerous molecular correlates of immune infiltration, tailored specifically to pediatric patients. RNA-seq was used to generate the GEP and map expression profiles of immune checkpoint genes, and deconvolution algorithms were used to extract the immune cell composition for every tumor. The correlation analysis between IHC, deconvolution of cell mixture composition and GEP were assessed, including PD-L1 protein and mRNA expression. We observed significant correlation between PD-L1 protein and mRNA expression and a weak correlation of CD8+ T cells with GEP. Deconvoluted TME estimates were most tightly correlated with the presence of T cell infiltrates (CD4 and CD8) with IHC. TMB and tumor purity estimates were derived from whole genome sequencing for each case. No correlation was observed between TMB and immune infiltration, however, tumor purity was negatively correlated with immune infiltration. Using IHC as an independent marker of a T cell-inflamed TME, we have identified a novel pediatric immune signature that includes markers of CD4 and CD8 T cells, T cell cytotoxicity, T and NK cell recruitment and activation, MHC Class II molecules and immune checkpoints. This is the first study to comprehensively analyze the pediatric TME in a cohort of this size and diversity, with matching IHC for orthogonal validation. Through the combination of RNA-seq and IHC, we have devised a novel immune signature specific to pediatrics and these techniques have identified a subset of patients that are immune “hot” and may potentially respond to ICIs. Conversely, we also highlight the potential of identifying immune “cold” patients who may need immunomodulatory combination strategies to maximize immune response. Citation Format: Chelsea Mayoh, Rachael L. Terry, Marie Wong, Loretta M. Lau, Dong Anh Khuong-Quang, Marion K. Mateos, Vanessa Tyrrell, Michelle Haber, David S. Ziegler, Mark J. Cowley, Joseph A. Trapani, Paul J. Neeson, Paul G. Ekert. The unexplored immune landscape of high-risk pediatric cancers [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 3044.

Distinct Stromal and Immune Features Collectively Contribute to Long-Term Survival in Pancreatic Cancer

Background: The aggressive biology and treatment refractory nature of pancreatic ductal adenocarcinoma (PDAC) significantly limits long-term survival. Examining the tumor microenvironment (TME) of long-term survivors (LTS) of PDAC offers the potential of unveiling novel biological insights and therapeutic targets.Methods: We performed an integrated approach involving immunophenotyping, stromal scoring and histomorphological profiling of a cohort of 112 PDAC-cases, including 25 long-term survivors (LTSs, OS ≥ 60 months). Mutational frequencies were assessed using targeted next generation sequencing. Finally, we validated our findings in silico using an external cohort of microarray data from PDAC patients.Results: LTS cases exhibit a largely quiescent population of cancer-associated fibroblasts (CAFs). Immune profiling revealed key differences between LTS and NON-LTS cases in the intratumoral and stromal compartments. In both compartments, LTS cases exhibit a T cell inflamed profile with higher density of CD3+ T cells, CD4+ T cells, iNOS+ leukocytes and strikingly diminished numbers of CD68+ total macrophages, CD163+ (M2) macrophages and FOXP3+ Tregs. A large proportion of LTS cases exhibited tertiary lymphoid tissue (TLT) formation, which has been observed to be a positive prognostic marker in a number of tumor types. Using a Random-Forest variable selection approach, we identified the density of stromal iNOS+ cells and CD68+ cells as strong positive and negative prognostic variables, respectively. In an external cohort, computational cell-type deconvolution revealed a higher abundance of T cells, B lymphocytes and dendritic cells (DCs) in patients with long-term OS compared to short-term survivors. Thus, in silico profiling of long-term survivors in an external cohort, strongly corroborated the T cell-inflamed TME observed in our LTS group.Conclusions: Collectively, our findings highlight the prognostic importance of TME profiles in PDAC, underlining the crucial role of tumor associated macrophages (TAMs) and the potential interdependence between immunosuppressive TAMs and activated CAFs in pancreatic cancer. Additionally, our data has potential for precision medicine and patient stratification. Patients with a T cell inflamed TME might derive benefit from agonistic T cell antibodies (e.g., OX40 or CD137 agonists). Alternately, patients with activated CAFs and high infiltration of immunosuppressive TAMs are highly likely to exhibit therapeutic responses to macrophage targeted drugs (e.g., anti-CSF1R) and anti-CAF agents.