Changes In Immune Cell Infiltration Research Articles

EGFR-mutant NSCLC may remodel TME from non-inflamed to inflamed through acquiring resistance to EGFR-TKI treatment

BackgroundEGFR-TKI represent the standard first-line therapy for advanced NSCLC harboring EGFR mutations. However, resistance to EGFR-TKI inevitably develops in nearly all patients. Previous clinical study have demonstrated that, some patients that failed EGFR-TKI therapy show a benefit outcome from immunotherapy. Our objective is to explore the immune microenviroment remodeling induced by EGFR-TKI treatment in EGFR mutant lung cancer patients and to investigate the immune cell types and potential molecular signatures involved. MethodsA cohort of 37 EGFR mutant advanced-stage NSCLC patients, who are resistant to at least one type of TKI treatment, was retrospectively established. Both pre-treatment and TKI resistance tumor FFPE samples of each pairs were collected. Transcriptional profiling and bioinformatics analysis were employed to evaluate the change of immune associated hallmarks before and after EGFR-TKI therapy. ResultsTumor samples after EGFR-TKI treatment displayed enrichment of proinflammatory signaling like interferon-γ, allograft rejection and inflammatory response. Of note, cytotoxic factor granzyme A as well as PD-L1 were found to be more expressed in EGFR-TKI resistance samples. Approximately 33.3 % (11/33) of EGFR-TKI treated samples were classified as “hot” tumor, especially for EGFR L858R mutated NSCLC patients (46.7 %,7/15). Effector cells were significantly overexpressed in ‘hot’ tumors feature following TKI resistance. In addition, we found that four effector genes (CD8A, CDB8, GZMB, GZMK) showed higher expression in ‘hot’ tumors post-TKI resistance, and its 4-gene effector cell signature was found to have a good correlation with survival benefit in external immunotherapy database. ConclusionsTKI treatment may initiate immune activation in EGFR mutant NSCLC, leading to changes in immune cell infiltration following TKI resistance. We mechanistically explored that this might be due to an increased immune response caused by the rise in effector cells post-TKI resistance.

Abstract CT218: Phase II open label trial of Minnelide™ in patients with chemotherapy refractory metastatic pancreatic cancer

Abstract Background: Triptolide and its water-soluble prodrug, Minnelide™, have shown promising activity against pancreatic ductal adenocarcinoma (PDAC) in both in vitro and in vivo experiments. Triptolide induces apoptosis in pancreatic cancer cells by different pathways, including caspase-dependent apoptotic death and caspase -independent autophagic death. It inhibits the activity of the TFIIH general transcription factor complex and modulates the transcriptional landscape of both the stroma and cancer cell compartments. In the epithelial tumour compartment, triptolide down-regulates key transcription factors such as c-MYC. Preclinical investigations indicate significant activity against super enhancer targets. In the phase I clinical trial of Minnelide™ activity in highly refractory metastatic pancreatic carcinoma was observed. Hence, we undertook this study in patients with chemotherapy refractory metastatic PDAC. Our aims were to determine clinical anticancer effects, and to define effects of the drug on super enhancer expression and downstream effects. Methods: Eligibility criteria included stage IV PDAC progressing on standard chemotherapy, Karnofsky performance status ≥ 70%, and measurable disease by RECIST 1.1. Planned sample size was up to 35 patients with interim analysis after the first 18 evaluable patients, aiming for at least a 20% difference in DCR (10% v 30%). Minnelide™ 0.67 mg/m2 was administered intravenously over 30 minutes on days 1-21, followed by a 7-day rest period in a 28-day cycle. Exploratory objectives included: analyses of paired tumour biopsies for changes in immune cell infiltration, stromal activation, chromatin accessibility, proteomics and metabolism. Results: 17 patients were treated. There were no RECIST responses or stable disease. However, evidence of activity was observed in 4 of 15 evaluable patients. This activity included progression after 117 days treatment in 1 patient and reductions in CT tumour size or FDG-PET uptake and/or significant decreases in circulating CA19.9 levels in 3 others. Minnelide was generally well tolerated. Treatment related toxicities > 3 were: neutropenia 4 patients and cerebellar ataxia 1 patient.Exploratory analyses included ATAC-Seq on paired biopsies where gene ontology analyses showed downregulation of genes negatively regulating cell cycle and upregulation of genes controlling cell cycle checkpoints. Other exploratory analyses will be presented. Conclusions: The study identified a tolerable drug dose and evidence of activity in a highly chemotherapy-refractory population which provides encouraging proof for the principle of targeting TFIIH in human PDAC. Supported by SU2C, Cancer Research UK, Lustgarten Foundation Citation Format: David Propper, Erkut Borazanci, Peter J. O'Dwyer, Daniel Von Hoff, Joseph Hartlebury, Corina E. Antal, Gerard I. Evan, HaiYong Han, Michael Downes, Ronald Evans, Joshua Rabinowitz, Emmanuel Petricoin, Ashok Saluja, Mohana Velagapudi, Hitendra Patel. Phase II open label trial of Minnelide™ in patients with chemotherapy refractory metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT218.

Abstract 295: Fibroblast-specific endoglin deletion alters colonic immune infiltrate in premalignant colorectal lesions

Abstract Background: Endoglin expression on cancer-associated fibroblasts has been reported to promote tumor growth and metastasis, especially in colorectal cancer (CRC). However, little research has been done on the function of endoglin-expressing fibroblasts in the early stages of CRC and how it interacts with the host immune system. Here, we investigated the role of endoglin expression on fibroblasts during intestinal inflammation and tumorigenesis in a chemically induced colitis-associated CRC model. Methods: We generated two inducible fibroblast-specific endoglin (ENG) knockout mice Collagen1a1 CreERT2.ENGfl/fl (ENGCol1a1-/−) and Collagen1a2-CreERT.ENGfl/fl (ENGCol1a2-/−). Cre-mediated recombination in mice was induced by oral administration of tamoxifen for three consecutive days. Polyp formation was induced by a single injection of azoxymethane (AOM), followed by three cycles of dextran sodium sulphate (DSS). Analysis of the immune cell composition in the colons and polyps was done using flow cytometry. Hematoxylin and eosin (H&E) staining was performed to evaluate intestinal inflammation. Results: ENGCol1a1-/− mice showed a considerably higher number of colonic lesions than non-induced controls, whereas in the ENGCol1a2-/−, surprisingly no significant difference in the number of lesions was observed. Flow cytometry analysis revealed, compared to controls, the lesions of ENGCol1a1-/− mice had a significant increase in F4/80+ Ly6C− macrophages and Ly6G+ neutrophils, while those of ENGCol1a2-/− mice had a marked decrease in CD11C+ dendritic cells. Given the changes in immune cell infiltration, we also assessed acute inflammation, using a short-term DSS experiment. Interestingly, ENGCol1a1-/− mice lost significantly more weight compared to controls, indicating increased inflammation. Although weight loss did not differ in Col1a2 mice between the two groups, ENGCol1a2-/− mice had a much lower H&E staining score, indicating less colonic damage and inflammation. Furthermore, a decrease in F4/80+ Ly6C− macrophages was observed in the colons of both ENGCol1a1-/- and ENGCol1a2-/− mice, despite no change in the overall CD45+ immune cell population. Notably, the number of Ly6G+ neutrophils was decreased in the colons of ENGCol1a1-/− mice but increased in ENGCol1a2-/− mice. In addition, a decrease in CD11C+ dendritic cells was seen in the colons ENGCol1a2-/− mice. Conclusion: Our results suggest that AOM/DSS-induced polyp formation and infiltrating immune cells differ in ENGCol1a1-/− and ENGCol1a2-/− mice. Fibroblast-specific deletion of endoglin can reduce macrophage and dendritic cell infiltration, colonic damage, and protect mice from DSS colitis. Citation Format: Subinuer Abudukelimu, Mark J. Schoonderwoerd, Madelon Paauwe, Eveline S. de Jonge-Muller, Stef G. Janson, Nadine van Montfoort, Lukas JAC Hawinkels. Fibroblast-specific endoglin deletion alters colonic immune infiltrate in premalignant colorectal lesions [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 295.

Roles of pyroptosis and immune infiltration in aortic dissection.

Introduction: Aortic dissection (AD) is often fatal, and its pathogenesis involves immune infiltration and pyroptosis, though the molecular pathways connecting these processes remain unclear. This study aimed to investigate the role of immune infiltration and pyroptosis in AD pathogenesis using bioinformatics analysis. Methods: Two Gene Expression Omnibus datasets and a Gene Cards dataset of pyroptosis-related genes (PRGs) were utilized. Immunological infiltration was assessed using CIBERSORT, and AD diagnostic markers were identified through univariate logistic regression and least absolute shrinkage and selection operator regression. Interaction networks were constructed using STRING, and weighted gene correlation network analysis (WGCNA) was employed to identify important modules and essential genes. Single-sample gene set enrichment analysis determined immune infiltration, and Pearson correlation analysis assessed the association of key genes with infiltrating immune cells. Results: Thirty-one PRGs associated with inflammatory response, vascular epidermal growth factor receptor, and Rap1 signaling pathways were identified. WGCNA revealed seven important genes within a critical module. CIBERSORT detected immune cell infiltration, indicating significant changes in immune cell infiltration and pyroptosis genes in AD and their connections. Discussion: Our findings suggest that key PRGs may serve as indicators for AD or high-risk individuals. Understanding the role of pyroptosis and immune cell infiltration in AD pathogenesis may lead to the development of novel molecular-targeted therapies for AD. Conclusion: This study provides insights into the molecular mechanisms underlying AD pathogenesis, highlighting the importance of immune infiltration and pyroptosis. Identification of diagnostic markers and potential therapeutic targets may improve the management of AD and reduce associated morbidity and mortality.

Genetic alterations in the neuronal development genes are associated with changes of the tumor immune microenvironment in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and is highly metastatic. Our prior studies have demonstrated the critical role of axon guidance pathway genes in PDAC and the connection between neuronal development and the tumor microenvironment. A recent study newly identified 20 neuronal development genes [disks large homolog 2 (DLG2), neuron-glial-related cell adhesion molecule (NRCAM), neurexin3 (NRXN3), mitogen-activated protein kinase 10 (MAPK10), platelet-derived growth factor D (PDGFD), protein kinase C epsilon (PRKCE), potassium calcium-activated channel subfamily M alpha 1 (KCNMA1), polycystic kidney and hepatic disease 1 (PKHD1), neural cell adhesion molecule 1 (NCAM1), neuregulin-1 (NRG1), zinc finger protein 667 (ZNF667), cystic fibrosis transmembrane conductance regulator (CFTR), acyl-CoA medium-chain synthetase-3 (ACSM3), complement 6 (C6), protein tyrosine phosphatase receptor type M (PTPRM), hypoxia-inducible factor 1 alpha (HIF1A), adenylyl cyclase 5 (ADCY5), adherens junctions-associated protein 1 (AJAP1), neurobeachin (NBEA), sodium voltage-gated channel alpha subunit 9 (SCN9A)] that are associated with perineural invasion and poor prognosis of PDAC. The relationship between genetic alterations in these 20 genes and tumor immune microenvironment (TME) has not previously been investigated. We hence applied the sequential multiplex immunohistochemistry results of biopsy specimens from 63 PDAC patients to investigate this relationship. We found that, except for PTPRM and NBEA, genetic alterations involving these 20 genes are associated with significant changes in the densities of major immune cell subtypes. Except for AJAP1, the copy number loss involving this panel of neuronal development genes is significantly associated with changes in immune cell infiltrates. In contrast, the copy number gain in fewer genes, including NRXN3, ZNF667, ACSM3, C6, ADCY5, SCN9A, and PRKCE, is significantly associated with changes in immune cell infiltrates. Our study suggested that neuronal development genes play a role in modulating TME in a pancreatic cancer setting.

Age-related genes USP2 and ARG2 are involved in the reduction of immune cell infiltration in elderly patients with rheumatoid arthritis.

There are large differences in clinical manifestations and biological markers between elderly patients with rheumatoid arthritis (EPRA, age >60) and younger patients with RA (YPRA, age ≤60), partly owing to variations in the immune system of different age groups. Here, we focused on the changes of immune cell infiltration in YPRA and EPRA. The R packages "ssGSEA" and "GSEA" were used to identify the changes in immune cell infiltration and immune-related pathways between the two groups. The R packages "WGCNA" and "DEseq2" were used to screen and verify age-related differentially expressed genes (DEGs). Hub genes were identified using Cytoscape and cytoHubba. Spearman correlation coefficient was conducted to evaluate correlations between hub age-related genes and immune cells. Compared with 54 established YPRA, several immune cells and immune-related pathways were markedly decreased in 29 EPRA synovial tissues. Moreover, 78 age-related DEGs related to amino acid and glycosphingolipid synthesis and metabolism were identified. USP2 and ARG2 were verified to be upregulated in EPRA, signifying that these two genes could effectively distinguish YPRA and EPRA and have potential as biomarkers. In addition, we found that USP2 was significantly negatively correlated with B cells and monocytes, while there was a significant negative association between ARG2 and T cells. In conclusion, this study is the first to systematically analyze changes in immune cell infiltration between YPRA and EPRA patients and obtain hub age-related genes, which may provide the basis for illuminating the pathogenesis of EPRA and informing treatment strategies.

Effect of tumor CD276 expression on infiltrating immune cells and clinicopathological features of prostate cancer.

Advanced prostate cancer (PCa) is often resistant to immunotherapy. In this study, we examined the role of CD276 in mediating immunotherapeutic effects through changes in immune cell infiltration. Using transcriptomic and proteomic analyses, CD276 was identified as a potential target for immunotherapy. Subsequent in vivo and in vitro experiments confirmed its role as a potential mediator of immunotherapeutic effects. Multi-omic analysis suggested that CD276 was identified as a key molecule regulating the immune microenvironment (IM). In vivo experiments revealed that CD276 knockdown was found to enhance CD8+ T cell infiltration into the IM. Immunohistochemical analysis of PCa samples further confirmed the same findings. CD276 was found to inhibit the enrichment of CD8+ T cells in PCa. Thus, CD276 inhibitors may be potential targets for immunotherapy.

Abstract 4892: MMAE drives immunomodulatory changes in a preclinical xenograft model that are distinct from other clinical-stage ADC payloads

Abstract Monomethyl auristatin E (MMAE), the payload delivered by vedotin ADCs, exerts its cytotoxic effects via microtubule disruption and induction of ER stress, leading to apoptosis and tumor cell death. In addition, MMAE also has the ability to induce immunogenic cell death (ICD) and immune modulation in the tumor microenvironment (TME) (Liu, 2020; Gray, 2020). Other clinical-stage and approved ADCs incorporate payloads that also cause microtubule disruption (DM1, DM4), or that drive DNA damage through topoisomerase I inhibition (exatecan) as the primary mechanism of action (MOA). While ADCs with different linker payloads produce clinical benefit, their long-term impact on survival and how they pair with PD(L)1 agents may differ based on their ability to elicit immune modulation. In this study we have investigated how these payloads compare to MMAE in their ability to drive immune changes in the TME and describe how these changes could support anti-tumor immunity and improve outcomes in the clinic.Building upon an initial in vitro assessment in which we observed superior induction of ICD markers and underlying ER stress with an MMAE ADC, we utilized a B7H4-expressing MDA-MB-468 xenograft model of triple-negative breast cancer to further characterize the ICD properties of ADC payloads. Tumor-bearing mice were treated with a single dose of B7H4-targeted antibody conjugated to either MMAE, DM1, DM4 or exatecan. Tumors were harvested seven days after treatment and processed for immunohistochemistry (IHC) to assess changes in immune cell infiltration and analyzed by RNA-seq to determine alterations in gene expression. Payload-driven anti-tumor activity was observed with all ADC treatments, each leading to a similar reduction in tumor volume compared to vehicle or unconjugated antibody. IHC staining of tumor sections for F4/80, a mouse macrophage marker, revealed that of the microtubule-disrupting agents, only MMAE significantly increased F4/80+ macrophage infiltration in both the tumor nests and stroma. Increased F4/80+ macrophages in tumor nests and stroma was also observed as a result of B7H4-exatecan ADC treatment. RNA-seq analysis revealed a unique profile for the MMAE conjugate, with an increase in human transcripts encoding cytokine and type I interferon response genes. In summary, the anti-tumor activity of the B7H4-MMAE ADC resulted in recruitment of mouse innate immune cells to the xenograft tumors. Importantly, immune response genes related to ICD were increased following B7H4-MMAE treatment compared to DM1, DM4, and exatecan conjugates. Together, these data suggest that treatment with vedotin ADCs results in robust immunomodulatory changes in vivo that are distinct from other clinical ADC payloads. Citation Format: Michelle Ulrich, Kerry Klussman, John J. Gosink, Sean Allred, Kelly Hensley, Piper M. Treuting, Nikhil J. Parekh, Elizabeth E. Gray, Shyra J. Gardai. MMAE drives immunomodulatory changes in a preclinical xenograft model that are distinct from other clinical-stage ADC payloads. [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 4892.

A Study on Immune Cell Infiltration in Lung Adenocarcinoma.

As a vital part of the tumor environment, immune cells affect the progression of tumors, and their composition and role vary in different types of tumors and influence prognosis. These immune cells have the potential to be beneficially targeted for immunotherapy, or, conversely, they may react negatively, even leading to drug resistance. For these reasons, probing into the composition and possible effects of immune cells in lung cancer is conducive to discovering valuable therapeutic targets. The lung adenocarcinoma gene expression data were downloaded from the TCGA database (https://cancergenome.nih.gov/; https://portal.gdc.cancer.gov/), and the lung adenocarcinoma gene expression matrix was converted into an immune cell-matrix using CIBERSORT software (https://cibersort.stanford.edu/), followed by an analysis of immune cells in lung adenocarcinoma tissues. The results showed that among all immune cells in lung adenocarcinoma tissues, macrophages (Mφ) had the highest number, followed by T cells. The number of plasma cells in lung adenocarcinoma tissues was higher than in adjacent normal tissues. Compared with those in adjacent normal tissues, the number of resting memory clusters of differentiation 4 (CD4)+ T cells was lower, whereas active memory CD4+ T cells were higher in lung adenocarcinoma tissues. In addition, the number of CD8+ T cells was negatively related to that of resting memory CD4+ T cells, with a correlation coefficient of -0.44, whereas it showed a positive association with the number of active memory CD4+ T cells, with a correlation coefficient of 0.47. It was found that among various immune cells infiltrating lung adenocarcinoma tissues, unstimulated Mφ (M0), alternatively activated Mφ (M2), and resting memory CD4+ T cells accounted for the largest proportions. However, these three types of immune cells were found to be lower in lung adenocarcinoma tissues than in adjacent normal tissues. Immune cells infiltrating lung adenocarcinoma tissues are complex, which affect the development and progression of the tumor and may also be a significant cause of drug resistance. Studying the changes in immune cell infiltration during the development of specific types of tumors contributes to disease progression interpretation, prognosis assessment, and potential solutions to the existing drug resistance issue. In this paper, the status of immune cells in lung adenocarcinoma tissues was preliminarily discussed based on the database mining, but more experimental studies and in-depth discussions are needed in the future.

Fusobacterium is enriched in oral cancer and promotes induction of programmed death-ligand 1 (PD-L1)

Recently, increased number of studies have demonstrated a relationship between the oral microbiome and development of head and neck cancer, however, there are few studies to investigate the role of oral bacteria in the context of the tumor microenvironment in a single head and neck subsite. Here, paired tumor and adjacent normal tissues from thirty-seven oral tongue squamous cell carcinoma (SCC) patients were subjected to 16S rRNA gene sequencing and whole exome sequencing (WES), in addition to RNA sequencing for tumor samples. We observed that Fusobacterium was significantly enriched in oral tongue cancer and that Rothia and Streptococcus were enriched in adjacent normal tissues. A decrease in alpha diversity was found in tumor when compared to adjacent normal tissues. While increased Fusobacterium in tumor samples was not associated with changes in immune cell infiltration, it was associated with increased PD-L1 mRNA expression. Therefore, we examined the effects of Fusobacterium on PD-L1 expression in head and neck SCC cell lines. We demonstrated that infection with Fusobacterium species can increase both PD-L1 mRNA and surface PD-L1 protein expression on head and neck cancer cell lines. The correlation between Fusobacterium and PD-L1 expression in oral tongue SCC, in conjunction with the ability of the bacterium to induce PD-L1 expression in vitro suggests a potential role for Fusobacterium on modulation of the tumor immune microenvironment in head and neck cancer.

ARL4C Regulates the Progression of Clear Cell Renal Cell Carcinoma by Affecting the Wnt/β-Catenin Signaling Pathway.

Purpose To investigate the expression of the ADP-ribosylation factor (ARF)-like proteins (ARLs) and ARL4C in clear cell renal cell carcinoma (ccRCC) based on bioinformatics analysis and experimentally determine the effect and mechanism of ARL4C on cellular properties involved in ccRCC progression. Methods After downloading the data of cancer patients from the TCGA database, we used various bioinformatics analysis websites and methods to analyze the expression and function of ARLs and ARL4C. The differential expression of ARL4C in clinical renal cancer tissues versus adjacent normal tissues was further verified using immunohistochemistry and real-time quantitative reverse-transcription (qRT-PCR). qRT-PCR was used to explore the expression of ARL4C mRNA in normal renal cells versus different ccRCC cell lines, and the protein expression of ARL4C was further verified using western blotting. CCK-8, colony formation, and EdU assays were used to determine the effect of ARL4C knockdown on ccRCC cell proliferation. We also used wound healing and Transwell assays to analyze the changes in ccRCC cell migration and invasion following ARL4C knockdown. Finally, we used western blotting to probe the molecular mode of action of ARL4C in ccRCC cells after exposure to Wnt signaling pathway agonists. Results Biological function analysis showed that methylation of ARL4C and changes in immune cell infiltration and targeted drug sensitivity caused by altered ARL4C expression affected the prognosis of ccRCC. Further bioinformatics analysis suggested that the expression of ARL4C mRNA was increased in ccRCC, and this was associated with a poor prognosis in ccRCC patients. Increased expression of ARL4C was further verified using qRT-PCR and western blotting of human ccRCC tissue samples. Downregulation of ARL4C significantly inhibited the proliferation, migration, and invasion of ccRCC cells, and activation of the Wnt/β-catenin pathway promoted the expression of ARL4C. As an essential downstream effector of the Wnt signaling pathway, ARL4C increased the expression of cyclin D1 and c-myc, thereby increasing the ability of the cells to undergo epithelial-mesenchymal transition (EMT) and ccRCC progression. Conclusions As a critical factor in the Wnt/β-catenin pathway, ARL4C regulates EMT and progression in ccRCC.

Characteristics of Prognostic Programmed Cell Death-Related Long Noncoding RNAs Associated With Immune Infiltration and Therapeutic Responses to Colon Cancer.

Programmed cell death (PCD) plays an important role in the onset and progression of various cancers. The molecular events surrounding the occurrence of abnormally expressed long noncoding RNAs (lncRNAs) leading to colon cancer (CC) have become a focus. We comprehensively evaluated the roles of PCD-related lncRNAs in the clinical management of CC and their immune responses. Therefore, we screened 41 prognostic PCD-related lncRNAs in The Cancer Genome Atlas database using co-expression analysis and assigned patients to groups according to the results of cluster analysis. The immune response and functions of cluster 2 were substantially suppressed, which might explain the poor prognosis in this group. A prognostic model comprising eight PCD-related lncRNAs was developed, and its effectiveness was verified using an external database. High-and low-risk groups had different epigenetic modifications and changes in immune cell infiltration. Patients in the high-risk group were resistant to immunotherapy and various chemotherapeutic drugs. Studies in vitro and in vivo further confirmed a carcinogenic role of the lncRNA U62317.4. Our findings of the prognostic value of PCD-related lncRNAs revealed their important roles in immune response disorders, thus providing valuable insights into the clinical management and molecular mechanisms of CC.

Endothelial-Specific Loss of Sphingosine-1-Phosphate Receptor 1 Increases Vascular Permeability and Exacerbates Bleomycin-induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease which leads to significant morbidity and mortality from respiratory failure. The two drugs currently approved for clinical use slow the rate of decline in lung function but have not been shown to halt disease progression or reverse established fibrosis. Thus, new therapeutic targets are needed. Endothelial injury and the resultant vascular permeability are critical components in the response to tissue injury and are present in patients with IPF. However, it remains unclear how vascular permeability affects lung repair and fibrosis following injury. Lipid mediators such as sphingosine-1-phosphate (S1P) are known to regulate multiple homeostatic processes in the lung including vascular permeability. We demonstrate that endothelial cell-(EC) specific deletion of the S1P receptor 1 (S1PR1) in mice (EC-S1pr1-/-) results in increased lung vascular permeability at baseline. Following a low-dose intratracheal bleomycin challenge, EC-S1pr1-/- mice had increased and persistent vascular permeability compared with wild-type mice, which was strongly correlated with the amount and localization of resulting pulmonary fibrosis. EC-S1pr1-/- mice also had increased immune cell infiltration and activation of the coagulation cascade within the lung. However, increased circulating S1P ligand in ApoM-overexpressing mice was insufficient to protect against bleomycin-induced pulmonary fibrosis. Overall, these data demonstrate that endothelial cell S1PR1 controls vascular permeability in the lung, is associated with changes in immune cell infiltration and extravascular coagulation, and modulates the fibrotic response to lung injury.

536MO A phase I, first-in-human, safety, pharmacokinetic, and pharmacodynamic study of oral dubermatinib (TP-0903) in patients with advanced solid tumours

AXL is upregulated in many solid tumors and may drive resistance to targeted cancer therapeutics, traditional chemotherapies, and immuno-oncology agents. Dubermatinib is a novel, orally administered small molecule AXL kinase inhibitor affecting mesenchymal-epithelial transition while targeting oncogenesis and chemoresistance. Ph I enrolled 45 pts with advanced solid tumors across 9 dose levels (1.5-37mg/m2) and 50mg flat dose. The RPh2D was determined as 50mg dubermatinib QD 21/28 days based on dose-proportionate drug accumulation including metabolites, and thrombocytopenia. Five expansion cohorts enrolled 1) dubermatinib plus IO with progression after response (n=19); 2) dubermatinib plus EGFR inhibitor in EGFR+ NSCLC with progression after response (n=18); 3) KRAS+ CRC (n=47); 4) platinum refractory/resistant ovarian cancer (n=22); 5) BRAFmut/wt melanoma (n=13). In 125 patients receiving at least one 50mg dose the most frequently observed Gr3/4 AEs were anemia, diarrhea (7/5.6%); hyponatremia, dyspnea (5/4.0%); vomiting, ascites, fatigue, incr alkphos, decr potassium, PE (3/2.4%). PK values for plasma dubermatinib and active metabolites rose incrementally with dose (t½ 12-20hrs). Day 1 Cmax and AUC0-24 (50mg) were 14.8 ng/mL and 160.9 ng*hr/mL, respectively. Increases were observed at Day 21. Clinical activity included 4 PRs: 2 NSCLC (1 pt 37mg/m2 dubermatinib+TKI, 1 pt 50mg dubermatinib+nivolumab), 1 melanoma pt. 9mg/m2 and 1 cholangiocarcinoma pt. 50mg, both single agent dubermatinib. Dose reduction rate at RP2D was <10% with overall CBR observed in 18.4% (23/125) of pts including 4 PRs and SD>4 months in heavily pretreated patients. PD evaluation of pre, post-treatment tumor biopsies and PBMCs correlated with drug activity (dose dependent sAXL reduction and GAS6 increase on treatment Day 8). Changes in immune cell infiltration, activity of antigen presenting cells and polarization of macrophages will be presented. Dubermatinib at 50mg orally once daily was tolerated, with evidence of AXL engagement in surrogate tissues and preliminary evidence of clinical activity. Combination studies with TKIs and IO agents are being analyzed.

A phase Ib/IIa study of rucaparib (PARP inhibitor) combined with nivolumab in metastatic castrate-resistant prostate cancer.

TPS270 Background: Immune checkpoint blockade (ICB) therapies have had a major impact across a wide range of cancers. However, only subsets of patients across all malignancies benefit from ICB. In particular, metastatic castrate-resistant prostate cancers (mCRPC) have shown very limited responses to ICB. While there is ongoing work to identify predictive biomarkers to ICB responsiveness, early preclinical data from our group suggests that targeting fundamental DNA repair pathways could markedly increase the fraction of patients responsive to immunotherapeutic interventions. Based on these preclinical studies, we are conducting an investigator-initiated Phase Ib/IIa co-clinical trial of rucaparib and nivolumab singly and in combination, in mCRPC patients. Methods: Patients are randomized to one of three arms – rucaparib, nivolumab, or both drugs in combination for 4 weeks. Metastatic biopsy samples are collected at baseline and after 4 weeks on treatment, after which all arms switch to combination therapy. The primary objective is to assess feasibility of the combination, and to elucidate changes in T cell infiltration by RNA-seq analysis using established T-cell non-inflamed and inflamed gene signatures. Secondary objectives are to assess changes in immune cell infiltration via flow cytometry, multiplex IHC, transparent tissue tomography (3D mapping) and single-cell RNA-seq. We will correlate changes in the metastatic tumor microenvironment (TME) at baseline and following 4 weeks of treatment, with genomic alterations and clinical responses. We have currently enrolled 12 patients to the study, and collected pre- and 4 week on-treatment biopsies. This study utilizes novel emerging technologies for in-depth TME analysis that will unravel the impact of PARP inhibition, singly and in combination with PD-1 blockade, on specific immune subsets within the TME. The correlative analyses will also lead to the discovery of novel biomarkers of response/resistance, and suggest additional immuno-oncology combinations for specific genomic subsets of mCRPC. Clinical trial information: NCT03572478.

Abstract A31: Investigating the effect of myeloid Arg1 deletion on tumor growth and CD8+ T-cell infiltration and activation in pancreatic cancer

Abstract PDA is characterized by an extensive fibroinflammatory stroma. This fibroinflammatory stroma is mainly composed of fibroblasts and tumor-infiltrating immune cells. The most abundant infiltrating immune cells are myeloid cells. Myeloid cells including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells, and granulocytes are required for PDA tumor growth and maintenance. Myeloid cells have the ability to suppress antitumor T-cell responses in PDA, as depletion of myeloid cells restores CD8+ T-cell immunity. Our previous characterization of myeloid cells infiltrating the neoplastic pancreas has revealed that myeloid cells express high levels of Arginase 1 (Arg1), an enzyme that depletes the amino acid L-arginine from the microenvironment and a signature marker of immunosuppressive macrophages and TAMs. In turn, L-arginine is required for CD8+ T-cell activation. An increase in Arginase levels has been reported also in other cancers, including lung, gastrointestinal, and bladder cancer. Thus, Arg1 expression in myeloid cells might be a key mediator of immune suppression, although this possibility has not been investigated directly in pancreatic cancer. Based on these observations, we test the hypothesis that myeloid cell polarization in the tumor microenvironment mediates immune suppression in PDA through expression of Arginase 1. The objective of this study is to provide novel insights into the role of Arg1 in pancreatic cancer, and the overall goal is to identify new therapeutic targets for combination therapy in pancreatic cancer. We used a genetically engineered mouse model (LysM-Cre;Arg1f/f) to delete the Arg1 gene, specifically from the myeloid cell compartment (LysM+ cells), including macrophages and neutrophils. We orthotopically implanted primary mouse pancreatic cancer cell lines into C57BL/6 LysM-Cre;Arg1f/f and wild-type (WT) mice. We evaluated tumor growth and CD8+ T-cell infiltration and activation between LysM-Cre;Arg1f/f and WT control mice. We used MRI imaging to determine tumor volume, and we used immunofluorescence and mass cytometry analysis to investigate changes in immune cell infiltration. We confirmed Arg1 depletion in LysM-Cre;Arg1f/f mice by Western blotting, co-immunofluorescence, and mass cytometry. We observed a decreasing trend in tumor growth and volume in LysM-Cre;Arg1f/f mice, an increase in iNOS expression (a marker of inflammatory macrophages), and an increase in CD8+ T-cell number and activity compared to the WT. These results support the notion that Arg1 might be a moderator of immune suppression in pancreatic cancer. Citation Format: Rosa E. Menjivar, Christopher Halbrook, Ashley Velez, Fatima Lima, Carlos Espinoza, Stefanie Galban, Yaqing Zhang, Costas Lyssiotis, Marina Pasca di Magliano. Investigating the effect of myeloid Arg1 deletion on tumor growth and CD8+ T-cell infiltration and activation in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A31.

Physical Activity Plus Energy Restriction Prevents 4T1.2 Mammary Tumor Progression, MDSC Accumulation, and an Immunosuppressive Tumor Microenvironment.

Physical activity and the prevention of weight gain decrease breast cancer incidence and improve survival. Unraveling the biological mechanisms underlying these cancer prevention effects is difficult because activity and dietary restriction are often linked. The goal of this study was to determine whether physical activity (PA), preventing weight gain via energy restriction (ER), or the combination was most effective in delaying tumor growth, reducing metastatic progression, and improving survival in the 4T1.2 mammary tumor model. Furthermore, we determined whether any of these interventions prevented the expansion of protumor immunosuppressive cells and altered the tumor microenvironment (TME). Female BALB/c mice (n = 7-20/group) were randomized to sedentary (SED) or PA wheel cages and fed ad libitum (AL) or 90% of control food intake (ER). After 8 weeks on the interventions, mice were inoculated with 5 × 104 4T1.2luc cells into the 4th mammary fat pad and continued on their respective intervention. PA+ER significantly delayed primary tumor growth (final tumor volume, 0.193 ± 0.042 vs. 0.369 ± 0.049 cm3, P < 0.001), reduced metastatic burden in the lungs (0.72 ± 0.36 vs. 16.27 ± 6.98, P = 0.054) and increased survival (median survival, 68 vs 40 days, P = 0.043) compared with SED+AL mice. PA+ER also reduced the expression level of metastatic and immunosuppressive genes and resulted in favorable changes in immune cell infiltrates in the tumor. These data suggest that both PA and ER are needed to reduce tumor growth, delay metastatic progression, and improve survival, and that this protection is associated with changes in immune-mediated mechanisms.

Abstract 3079: Pharmacodynamic evaluation of the novel SUMOylation inhibitor TAK-981 in a mouse tumor model

Abstract TAK-981 is a small molecule inhibitor of the SUMOylation enzyme cascade which is currently in a Phase I clinical trial (NCT03648372). TAK-981 selectively inhibits the activity of the SUMO activating enzyme, forming a covalent adduct between TAK-981 and SUMO, thereby decreasing levels of SUMOylated proteins. A central feature of pharmacological inhibition of SUMOylation is production of type I IFNs in immune cells, promoting both innate and adaptive immune responses. TAK-981 demonstrates antitumor activity, including complete regression (CR) of some tumors, in immune-competent BALB/c mice bearing syngeneic A20 lymphoma tumors. This model was utilized to investigate 1) target engagement and pathway inhibition, 2) modulation of IFN-regulated genes in tumor and blood, 3) dependence of antitumor activity on Type I IFN signaling and adaptive immunity, and 4) changes in immune cell infiltrates in tumors. IHC analysis of tumors showed TAK-981-SUMO adduct formation and decrease of SUMOylated protein levels after a single IV dose of 7.5 mg/kg TAK-981, as measured by assays for TAK-981-SUMO adduct and SUMO2/3 conjugates, respectively. IFN-regulated genes including CXCL10, IFIT1, and ISG15 are coordinately upregulated 4-8 hrs post-dose in tumor and in blood, as demonstrated by Nanostring, and plasma levels of IP-10 (CXCL10) increase. Single cell RNAseq experiments on dissociated tumors demonstrated upregulation of IFN-regulated genes in T cells and monocytes in the tumor microenvironment. TAK-981-mediated antitumor activity is fully dependent on Type I IFN signaling; administration of a neutralizing antibody against the IFN alpha/beta receptor 1 (IFNAR) before dosing TAK-981 results in the loss of antitumor activity against A20. TAK-981-mediated antitumor activity is also dependent on the adaptive immune system, as demonstrated by two experiments in mice lacking T cells (BALB/c Rag2 KO mice, and mice treated with an anti-CD8 antibody to deplete T cells). Antitumor response was diminished in both settings, with no CRs achieved in either model. Flow cytometric analysis of immune infiltrates in A20 tumors grown in BALB/c mice indicated that TAK-981 response is accompanied by an increase in activated T cells in the tumor microenvironment as well as increased NK cells, potentially supporting a role for both innate and adaptive immunity in the MOA. Evaluation of pharmacodynamic biomarkers in Phase I clinical development will be informed by these preclinical studies. Citation Format: Allison J. Berger, Sharon Friedlander, Omid Ghasemi, Stephen Grossman, Erik Koenig, Eric Lightcap, Michael Milhollen, Pooja Shah, Gary Shapiro, Vaishali Shinde, Keli Song, Kristina Xega, Agatha Zawadzka, Dennis Huszar. Pharmacodynamic evaluation of the novel SUMOylation inhibitor TAK-981 in a mouse tumor model [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 3079.

Abstract CT165: Phase II open label trial of minnelide™ in patients with chemotherapy refractory metastatic pancreatic cancer

Abstract BackgroundTriptolide and its water-soluble prodrug, Minnelide™, have shown promising activity against pancreatic ductal adenocarcinoma (PDAC) in both in vitro and in vivo experiments. Triptolide induces apoptosis in pancreatic cancer cells by different pathways, including caspase-dependent apoptotic death and caspase -independent autophagic death. It inhibits the activity of the TFIIH general transcription factor complex and modulates the transcriptional landscape of both the stroma and cancer cell compartments. In the epithelial tumour compartment, triptolide down-regulates key transcription factors such as c-MYC. In the phase I clinical trial of Minnelide™ significant activity in highly refractory metastatic pancreatic carcinoma was observed. Hence we undertook this study in patients with chemotherapy refractory metastatic PDAC. MethodsEligibility criteria include stage IV PDAC that has progressed on standard chemotherapy, Karnofsky performance status ≥ 70%, and measurable disease. Planned sample size is up to 35 patients with interim analysis after the first 18 evaluable patients, aiming for at least a 20% difference in DCR (10% v 30%). Minnelide™ 0.67 mg/m2 is administered intravenously over 30 minutes on days 1-21, followed by a 7 day rest period in a 28 day cycle. Exploratory objectives include: analysis of tumor texture on radiologic scans as a non-invasive biomarker for response, as well as analyses of paired tumour biopsies for changes in immune cell infiltration, stromal activation, chromatin accessibility, phopshoproteomics and metabolism. # NCT03117920The trial opened 4/2017 and as of 12/2018, 18 patients have been treated. Supported by SU2C, Cancer Research UK, Lustgarten Foundation Citation Format: David Propper, Haiyong Han, Daniel Von Hoff, Erkut Borazanci, Tannishtha Reya, Jonathan Ghergurovich, Irina Pshenichnaya, Corina Antal, Rachel Condjella, Shreya Sharma, Peter O'Dwyer, Trevor Littlewood, Hitendra Patel, Ashok Saluja, Mohana Velagapudi, Lifeng Yang, Michael Downes, Ronald Evans, Gerard Evan. Phase II open label trial of minnelide™ in patients with chemotherapy refractory metastatic pancreatic cancer [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 CT165.

Abstract 1207: Reversing lactate-driven immunosuppression using the novel, potent and selective MCT4 inhibitor AZD0095

Abstract Metabolic adaptation to a Warburg phenotype is a hallmark of cancer, where tumors rely on glucose to produce ATP and building blocks required to support rapid tumor growth. Glycolytic metabolism requires continuous export of lactate from cells by monocarboxylate transporters (MCTs) resulting in accumulation of lactic acid in the tumor microenvironment. Tumor-derived lactate is immune-suppressive and has been shown to modify immune cell function. Inhibition of the MCT4 lactate transporter has the potential to reduce lactate in the tumor microenvironment and reverse immune suppression. AZD0095 is a highly selective, potent inhibitor of MCT4 with a cellular activity of 1-3 nM with a &amp;gt;1000-fold selectivity over MCT1. We confirm that lactic acid inhibits T cell proliferation and viability. AZD0095 does not inhibit T cell proliferation or survival (IC50 &amp;gt; 10 μM). Oral administration of AZD0095 (10-100 mg/kg BID) modulates lactate transport in mouse syngeneic models which do not express the MCT1 lactate transporter. AZD0095 treatment causes an increase in tumor-infiltrating lymphocytes and an increase in cytotoxic T cells in combination with α-PD1. AZD0095 treatment suppresses myeloid infiltration as a monotherapy and in combination with checkpoint inhibitor. Consistent with the observed changes in immune cell infiltration, the combination of AZD0095 with checkpoint inhibitors α-PD1 or α-CTLA4 (10 mg/kg 3x week) enhances the anti-tumor activity of checkpoint inhibitor monotherapy in MC-38 and EMT6 (MCT1 knock-out) mouse syngeneic models. These data demonstrate that AZD0095 can reverse lactate-driven immunosuppression and enhance response to checkpoint inhibition in pre-clinical models. Citation Format: Susan E. Critchlow, Gareth Hughes, Anna Staniszewska, Matt King, Filippos Michopoulos, Lorna Hopcroft, Martin R. Brown, Jenna Bradley, Beverley Hammond, Pablo MorentinGutierrez, Larissa Carnevalli, Elizabeth Hardaker, Frederick W. Goldberg. Reversing lactate-driven immunosuppression using the novel, potent and selective MCT4 inhibitor AZD0095 [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 1207.