Interferon-gamma Signaling Research Articles

Prognostic Significance of Pretreatment Immune Cell Infiltration in Muscle Invasive Bladder Cancer Treated with Definitive Chemoradiation: Analysis of NRG RTOG 0524 and 0712

Chemoradiation therapy (CRT) is an organ conserving approach in the treatment of locally advanced bladder cancer. Chemoradiation is thought to potentially result in immunogenic stimulation, and bladder cancer is often a tumor with high immune cell infiltration. Thus, we aimed to profile the tumor immune microenvironment of bladder cancer and identify prognostic immune biomarkers for CRT response by profiling tumor samples from NRG/RTOG 0524 and 0712, two prospective trials of CRT in muscle invasive bladder cancer (MIBC). Pretreatment tissue samples from both trials were profiled using Cofactor Genomics ImmunoPrism, an RNA sequencing assay that uses gene expression profiles to quantify immune cell populations in the tumor microenvironment (TME). Differential gene expression was estimated for different immune cell type proportions across samples. Kaplan-Meier survival analysis and log rank tests were performed to evaluate differences in overall survival (OS) stratified by genes influenced by immune cell proportions or genes associated with immune response signatures. A total of 70 samples (43 from RTOG 0524 and 27 from RTOG 0712) underwent analysis using the ImmunoPrism assay. Immune cell proportions were as follows: CD8 T cells: median 1.2%, CD4 T cells: median 0.8%, Treg cells: median 9.2%, CD19 B cells: median 5.1%, M2 macrophages: median 0.8%, M1 macrophages: median 0%. Unbiased clustering based on gene expression profiles driven by immune cell proportions demonstrated two groups: cluster 1 with a low percentage of immune cells and shorter OS (median 31 months) and cluster 2 with a high percentage of immune cells and longer OS (median 101 months, p = 0.036). Higher expression of genes associated with T cell infiltration (CD8A and ICOS) was associated with improved OS (104 vs 35 months, p = 0.028, HR = 0.48 (0.25 - 0.94), p = 0.031) as was higher expression of IDO1, which is associated with the interferon gamma pathway (104 vs 35 months, p = 0.042, HR = 0.49 (0.24 - 0.99), p = 0.046). Bladder tumors have a wide range of immune cell infiltration in the TME. Increased immune cell proportions are prognostic for OS following CRT, as well as a higher expression of genes associated with T cell infiltration interferon gamma signaling. These findings have implications for the integration of immunotherapy in the definitive management of MIBC; and can be explored further in the ongoing NRG/SWOG 1806 trial.

Transcriptional Targets of TWIST1 in Human Mesenchymal Stem/Stromal Cells Mechanistically Link Stem/Progenitor and Paracrine Functions.

Despite extensive clinical testing, mesenchymal stem/stromal cell (MSC)-based therapies continue to underperform with respect to efficacy, which reflects the paucity of biomarkers that predict potency prior to patient administration. Previously, we reported that TWIST1 predicts inter-donor differences in MSC quality attributes that confer potency. To define the full spectrum of TWIST1 activity in MSCs, the present work employed integrated omics-based profiling to identify a high-confidence set of TWIST1 targets, which mapped to cellular processes related to ECM structure/organization, skeletal and circulatory system development, interferon gamma signaling, and inflammation. These targets are implicated in contributing to both stem/progenitor and paracrine activities of MSCs indicating these processes are linked mechanistically in a TWIST1-dependent manner. Targets implicated in extracellular matrix dynamics further implicate TWIST1 in modulating cellular responses to niche remodeling. Novel TWIST1-regulated genes identified herein may be prioritized for future mechanistic and functional studies.

Interspecies comparison of the early transcriptomic changes associated with hepatitis B virus exposure in human and macaque immune cell populations.

Hepatitis B virus (HBV) infection affects 300 million individuals worldwide, representing a major factor for the development of hepatic complications. Although existing antivirals are effective in suppressing replication, eradication of HBV is not achieved. Therefore, a multi-faceted approach involving antivirals and immunomodulatory agents is required. Non-human primates are widely used in pre-clinical studies due to their close evolutionary relationship to humans. Nonetheless, it is fundamental to identify the differences in immune response between humans and these models. Thus, we performed a transcriptomic characterization and interspecies comparison of the early immune responses to HBV in human and cynomolgus macaques. We characterized early transcriptomic changes in human and cynomolgus B cells, T cells, myeloid and plasmacytoid dendritic cells (pDC) exposed to HBV ex vivo for 2 hours. Differentially-expressed genes were further compared to the profiles of HBV-infected patients using publicly-available single-cell data. HBV induced a wide variety of transcriptional changes in all cell types, with common genes between species representing only a small proportion. In particular, interferon gamma signaling was repressed in human pDCs. At the gene level, interferon gamma inducible protein 16 (IFI16) was upregulated in macaque pDCs, while downregulated in humans. Moreover, IFI16 expression in pDCs from chronic HBV-infected patients anti-paralleled serum HBsAg levels. Our characterization of early transcriptomic changes induced by HBV in humans and cynomolgus macaques represents a useful resource for the identification of shared and divergent host responses, as well as potential immune targets against HBV.

Investigation of molecular mechanisms of Ulmus davidiana against gastric cancer: A network pharmacology study incorporating gene expression data and molecular docking analysis

Ulmus davidiana (UD), has long been utilized in traditional medicine to treat various diseases, particularly gastric cancer (GC). However, the molecular mechanism behind its health-protective effect has yet to be clarified. An extensive literature search was conducted to collect all reported phytochemicals of the plant, which were then filtered for druglike properties. SwissTargetPrediction was utilized to predict targets for phytochemicals and functional enrichment of targets was performed for GO, pathways, and diseases. A network pharmacology approach was used to investigate all possible targets and targets specifically associated with GC. The expression of target genes was also studied for their expression in the TCGA patient GC dataset from TCGA through GEPIA 2.0. Cytoscape and STRING were used for network construction and analysis, while core targets and filtered phytochemicals were employed for molecular docking. In total, 396 targets for screened compounds, of which 242 targets were associated with GC. Among these, 87 targets were differentially expressed in GC. Several highly differentially expressed genes (MMP7, TOP2A, CCNB2, MMP12, and CCNB2) were found to be strongly associated with gastric cancer. Target enrichment categories were correlated with the known bioactivities of UD such as antioxidant, anti-inflammatory, and anticancer properties. Cancer and neurological complications-related diseases were prevalent in the associated disease network. The interferon-gamma signaling pathway was identified as the most enriched pathway for gastric cancer targets (GT). CCKR signaling was found to be the most connected pathway in the GC network. Core targets (HSP90AA1, STAT3, SRC, ESR1, and EGFR) were determined through protein-protein-interaction network analysis. Molecular docking revealed phytochemicals (torilin, 1-hydroxytorilin, and butyl alpha-D-fructofuranoside) with high docking scores and strong interactions with core targets. The key findings of the study, including phytochemicals, targets, and pathways, are suggested for further validation.

IGF2BP family of RNA-binding proteins regulate innate and adaptive immune responses in cancer cells and tumor microenvironment.

Insulin-like growth factor 2 mRNA-binding proteins (IGF2BP1, IGF2BP2, and IGF2BP3) are a family of RNA-binding proteins that play an essential role in the development and disease by regulating mRNA stability and translation of critical regulators of cell division and metabolism. Genetic and chemical inhibition of these proteins slows down cancer cell proliferation, decreases invasiveness, and prolongs life span in a variety of animal models. The role of RNA-binding proteins in the induction of tissues' immunogenicity is increasingly recognized, but, the impact of the IGF2BPs family of proteins on the induction of innate and adaptive immune responses in cancer is not fully understood. Here we report that downregulation of IGF2BP1, 2, and 3 expression facilitates the expression of interferon beta-stimulated genes. IGF2BP1 has a greater effect on interferon beta and gamma signaling compared to IGF2BP2 and IGF2BP3 paralogs. We demonstrate that knockdown or knockout of IGF2BP1, 2, and 3 significantly potentiates inhibition of cell growth induced by IFNβ and IFNγ. Mouse melanoma cells with Igf2bp knockouts demonstrate increased expression of MHC I (H-2) and induce intracellular Ifn-γ expression in syngeneic T-lymphocytes in vitro. Increased immunogenicity, associated with Igf2bp1 inhibition, "inflames" mouse melanoma tumors microenvironment in SM1/C57BL/6 and SW1/C3H mouse models measured by a two-fold increase of NK cells and tumor-associated myeloid cells. Finally, we demonstrate that the efficiency of anti-PD1 immunotherapy in the mouse melanoma model is significantly more efficient in tumors that lack Igf2bp1 expression. Our retrospective data analysis of immunotherapies in human melanoma patients indicates that high levels of IGF2BP1 and IGF2BP3 are associated with resistance to immunotherapies and poor prognosis. In summary, our study provides evidence of the role of IGF2BP proteins in regulating tumor immunogenicity and establishes those RBPs as immunotherapeutic targets in cancer.

Sex and interferon gamma signaling regulate microglia migration in the adult mouse cortex in vivo

Although microglia possess the unique ability to migrate, whether mobility is evident in all microglia, is sex dependent, and what molecular mechanisms drive this, is not well understood in the adult brain. Using longitudinal in vivo two-photon imaging of sparsely labeled microglia, we find a relatively small population of microglia (~5%) are mobile under normal conditions. Following injury (microbleed), the fraction of mobile microglia increased in a sex-dependent manner, with male microglia migrating significantly greater distances toward the microbleed relative to their female counterparts. To understand the signaling pathways involved, we interrogated the role of interferon gamma (IFNγ). Our data show that in male mice, stimulating microglia with IFNγ promotes migration whereas inhibiting IFNγ receptor 1 signaling inhibits them. By contrast, female microglia were generally unaffected by these manipulations. These findings highlight the diversity of microglia migratory responses to injury, its dependence on sex and the signaling mechanisms that modulate this behavior.

Prognostic significance of pretreatment immune cell infiltration in muscle invasive bladder cancer treated with definitive chemoradiation: Analysis of NRG RTOG 0524 and 0712.

4523 Background: Chemoradiation therapy (CRT) is an organ conserving approach in the treatment of locally advanced bladder cancer. Chemoradiation is thought to potentially result in immunogenic stimulation, and bladder cancer is often a tumor with high immune cell infiltration. Thus, we aimed to profile the tumor immune microenvironment of bladder cancer and identify prognostic immune biomarkers for CRT response by profiling tumor samples from NRG/RTOG 0524 and 0712, two prospective trials of CRT in muscle invasive bladder cancer (MIBC). Methods: Pretreatment tissue samples from both trials were profiled using Cofactor Genomics ImmunoPrism, an RNA sequencing assay that uses gene expression profiles to quantify immune cell populations in the tumor microenvironment (TME). Differential gene expression was estimated for different immune cell type proportions across samples. Kaplan-Meier survival analysis and log rank tests were performed to evaluate differences in overall survival (OS) stratified by genes influenced by immune cell proportions or genes associated with immune response signatures. Results: A total of 70 samples (43 from RTOG 0524 and 27 from RTOG 0712) underwent analysis using the ImmunoPrism assay. Immune cell proportions were as follows: CD8 T cells: median 1.2%, CD4 T cells: median 0.8%, Treg cells: median 9.2%, CD19 B cells: median 5.1%, M2 macrophages: median 0.8%, M1 macrophages: median 0%. Unbiased clustering based on gene expression profiles driven by immune cell proportions demonstrated two groups: cluster 1 with a low percentage of immune cells and shorter OS (median 31 months) and cluster 2 with a high percentage of immune cells and longer OS (median 101 months, p = 0.036). Higher expression of genes associated with T cell infiltration ( CD8A and ICOS) was associated with improved OS (104 vs 35 months, p = 0.028, HR = 0.48 (0.25 – 0.94), p = 0.031) as was higher expression of IDO1, which is associated with the interferon gamma pathway (104 vs 35 months, p = 0.042, HR = 0.49 (0.24 – 0.99), p = 0.046). Conclusions: Bladder tumors have a wide range of immune cell infiltration in the TME. Increased immune cell proportions are prognostic for OS following CRT, as well as a higher expression of genes associated with T cell infiltration interferon gamma signaling. These findings have implications for the integration of immunotherapy in the definitive management of MIBC; and can be explored further in the ongoing NRG/SWOG 1806 trial.

Immunogenomic Landscape of Neuroendocrine Prostate Cancer.

Patients with neuroendocrine prostate cancer (NEPC) are often managed with immunotherapy regimens extrapolated from small-cell lung cancer (SCLC). We sought to evaluate the tumor immune landscape of NEPC compared with other prostate cancer types and SCLC. In this retrospective study, a cohort of 170 patients with 230 RNA-sequencing and 104 matched whole-exome sequencing data were analyzed. Differences in immune and stromal constituents, frequency of genomic alterations, and associations with outcomes were evaluated. In our cohort, 36% of the prostate tumors were identified as CD8+ T-cell inflamed, whereas the remaining 64% were T-cell depleted. T-cell-inflamed tumors were enriched in anti-inflammatory M2 macrophages and exhausted T cells and associated with shorter overall survival relative to T-cell-depleted tumors (HR, 2.62; P < 0.05). Among all prostate cancer types in the cohort, NEPC was identified to be the most immune depleted, wherein only 9 out of the 36 total NEPC tumors were classified as T-cell inflamed. These inflamed NEPC cases were enriched in IFN gamma signaling and PD-1 signaling compared with other NEPC tumors. Comparison of NEPC with SCLC revealed that NEPC had poor immune content and less mutations compared with SCLC, but expression of checkpoint genes PD-L1 and CTLA-4 was comparable between NEPC and SCLC. NEPC is characterized by a relatively immune-depleted tumor immune microenvironment compared with other primary and metastatic prostate adenocarcinoma except in a minority of cases. These findings may inform development of immunotherapy strategies for patients with advanced prostate cancer.

Class A capsid assembly modulator RG7907 clears HBV-infected hepatocytes through core-dependent hepatocyte death and proliferation.

Effective therapies leading to a functional cure for chronic hepatitis B are still lacking. Class A capsid assembly modulators (CAM-As) are an attractive modality to address this unmet medical need. CAM-As induce aggregation of the HBV core protein (HBc) and lead to sustained HBsAg reductions in a chronic hepatitis B mouse model. Here, we investigate the underlying mechanism of action for CAM-A compound RG7907. RG7907 induced extensive HBc aggregation in vitro , in hepatoma cells, and in primary hepatocytes. In the adeno-associated virus (AAV)-HBV mouse model, the RG7907 treatment led to a pronounced reduction in serum HBsAg and HBeAg, concomitant with clearance of HBsAg, HBc, and AAV-HBV episome from the liver. Transient increases in alanine transaminase, hepatocyte apoptosis, and proliferation markers were observed. These processes were confirmed by RNA sequencing, which also uncovered a role for interferon alpha and gamma signaling, including the interferon-stimulated gene 15 (ISG15) pathway. Finally, the in vitro observation of CAM-A-induced HBc-dependent cell death through apoptosis established the link of HBc aggregation to in vivo loss of infected hepatocytes. Our study unravels a previously unknown mechanism of action for CAM-As such as RG7907 in which HBc aggregation induces cell death, resulting in hepatocyte proliferation and loss of covalently closed circular DNA or its equivalent, possibly assisted by an induced innate immune response. This represents a promising approach to attain a functional cure for chronic hepatitis B.

High expression of RNF31 is associated with tumor immune cell infiltration and leads to poor prognosis in liver hepatocellular carcinoma

Ring finger protein 31 (RNF31) has been found to play an important role in tumor immunity. However, the role of RNF31 in liver hepatocellular carcinoma (LIHC) has not been reported. Therefore, we investigated the expression and prognostic value of RNF31 in patients with LIHC and explored its relationship with immune cell infiltration. The Cancer Genome Atlas liver hepatocellular carcinoma (TCGA-LIHC) dataset was downloaded to analyse the impact of RNF31 on the prognosis and immune cell infiltration of LIHC. The Tumor Immune Estimation Resource (TIMER) database was used to analyse the correlation between RNF31 and tumor immune cell infiltration in LIHC. Additionally, we analysed the relationship between RNF31 and tumor necrosis factor (TNF) as well as the interferon-gamma (IFN-γ) signaling pathway. The expression of RNF31 in LIHC was significantly higher than that in normal tissues. Increased RNF31 expression was associated with decreased overall survival (OS) and relapse-free survival (RFS). An increase in RNF31 expression was closely related to the infiltration levels of immune cells (e.g., natural killer (NK) cells, CD8 + T cells, and B cells). RNF31 was also positively correlated with the expression of immune checkpoint genes in LIHC. Moreover, RNF31 may participate in TNF and IFN-γ signaling pathways. In conclusion, RNF31 is a potentially valuable prognostic biomarker in LIHC. RNF31 is also associated with immune cell infiltration in LIHC. RNF31 may be a potential target for immunotherapy of LIHC.

Genetic Differences between 129S Substrains Affect Antiretroviral Immune Responses.

Inbred mouse lines vary in their ability to mount protective antiretroviral immune responses, and even closely related strains can exhibit opposing phenotypes upon retroviral infection. Here, we found that 129S mice inherit a previously unknown mechanism for the production of anti-murine leukemia virus (MLV) antibodies and control of infection. The resistant phenotype in 129S1 mice is controlled by two dominant loci that are independent from known MLV resistance genes. We also show that production of anti-MLV antibodies in 129S7 mice, but not 129S1 mice, is independent of interferon gamma signaling. Thus, our data indicate that 129S mice inherit an unknown mechanism for control of MLV infection and demonstrate that there is genetic variability in 129S substrains that affects their ability to mount antiviral immune responses. IMPORTANCE Understanding the genetic basis for production of protective antiviral immune responses is crucial for the development of novel vaccines and adjuvants. Additionally, characterizing the genetic and phenotypic variability in inbred mice has implications for the selection of strains for targeted mutagenesis, choice of controls, and for broader understanding of the requirements for protective immunity.

Abstract LB285: Single cell characterization of the adjacent primed tissues and metastatic microenvironment of adrenocortical carcinoma reveals profound molecular and cellular reprograms that dictate metastatic progression and disease outcome

Abstract Metastasis is the strongest predictor of outcome in cancer; and early interventions are needed to alter disease course and patient outcome. Adrenocortical carcinoma (ACC) is a rare adrenal cancer that has high mortality due to high rate of metastasis. The therapeutic options of metastatic-ACC include surgery and combination of chemotherapeutic regimens, but with very poor outcomes. The heterogeneous composition of immune and stromal cells in the metastatic microenvironment (MME) and adjacent primed tissues, and their crosstalk with malignant cells are critical determinants of cancer metastatic progression and response to therapies, which remain poorly understood for ACC. Therefore, to comprehensively characterize ACC-MME and adjacent primed tissues, and to identify global and tissue-specific reprogramming, we performed single cell RNA sequencing of metastatic-ACC and adjacent primed tissues from the liver and lung. Our studies revealed that ACC-MME undergoes aberrant vascularization with global depletion of capillary endothelial cells (ECs) and enrichment for Tip/Stalk-like ECs, which are significantly associated with poor disease outcome. Moreover, ECs in ACC-MME are highly pro-tumorigenic, showing global upregulation of tumor-promoting gene signatures, and key signaling pathways responsible for angiogenesis, proliferation, migration, and immune suppression, as well depletion of effective anti-tumor immune response programs such as antigen presentation and interferon gamma signaling. Furthermore, there is a major shift in tissue-specific and universal myeloid populations in ACC-MME and adjacent primed tissues, exhibiting global enrichment of dysfunctional DCs and immunosuppressive macrophages, and liver specific pDCs in MME, whereas, MDSCs are broadly enriched in both MME and adjacent primed tissues suggesting these primed for metastasis environments are immune suppressive. CD8 T-cells in ACC-MME express a dysfunctional gene signature, showing downregulation of key effector function genes. We also found an enrichment of immunosuppressive CD4-T-regs in ACC-MME that represent dysregulation of immune checkpoint molecules including CTLA4 and LAG3. Finally, immune and stroma cell populations in ACC MME and adjacent primed tissues show a commonality in reprogramming, favorable for tumor growth and metastasis, across diverse cell and tissue types in comparison to normal tissue samples. Taken together, these findings suggest that ACC-MME and its adjacent primed tissue microenvironments are reprogrammed to immunosuppressive and tumor-promoting states that orchestrate metastasis of ACC. Citation Format: Mohd Omar Faruk Sikder, Vishaka D. Gopalan, Sridhar Hannenhalli, Rosandra N. Kaplan. Single cell characterization of the adjacent primed tissues and metastatic microenvironment of adrenocortical carcinoma reveals profound molecular and cellular reprograms that dictate metastatic progression and disease outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB285.

CD74: a prospective marker for reactive microglia?

CD74: a prospective marker for reactive microglia?

Abstract 667: Single-cell functional genomics of natural killer cell evasion by tumor cells

Abstract Background: Natural killer (NK) cells are emerging as a promising therapeutic option in cancer. To better understand how cancer cells evade NK cells, we studied interacting NK and blood cancer cells using single-cell and genome-scale functional genomics screens. Methods: We performed multiplexed single-cell RNA-seq (scRNA-seq) on co-cultures of NK cells and 26 cell lines representing diverse blood cancers. Using screens of pooled DNA-barcoded cell lines (PRISM), we quantified the sensitivity of over 60 blood cancer cell lines and integrated the results with CCLE multi-omics to uncover molecular correlates of tumor cell susceptibility to NK cells. We performed 12 genome-scale CRISPR loss-of-function and gain-of-function screens of cancer-cell intrinsic NK cell resistance mechanisms across 7 blood cancer cell lines. Finally, we investigated the mechanisms-of-action of 65 genome-scale screen hits using CRISPR screens with scRNA-seq readout in both tumor and NK cells. Results: At single-cell resolution, interaction of NK and cancer cells induced distinct activation states in both cell types depending on the cancer cell lineage and molecular phenotype. NK cells transitioned either into an activated state characterized by 4-1BB, GITR, TIM-3, and TIGIT or a state marked by type I interferon signature. Tumor cells responded to NK cell attack by activating interferon gamma (IFNy) signaling, inducing MHC class I. The activation states correlated with sensitivity to NK cells, ranging from more sensitive myeloid to more resistant B-lymphoid cancers. Molecular correlates of increased sensitivity included expression of activating receptor ligands NCR3LG1, PVR, and ULBP1, whereas TRAF3 mutations correlated with resistance. CRISPR screens uncovered cancer cell-intrinsic genes driving sensitivity and resistance, including antigen presentation and death receptor signaling mediators and adhesion molecules. The screens identified new ​​blood cancer-specific NK cell inhibitory regulators (SELPLG, SPN, and MYB ) and genes previously underappreciated in NK cell evasion, including protein fucosylation and transcriptional regulators (e.g. GFI1B). CRISPR screens with scRNA-seq readout identified MHC-I, IFNy, and NF-κB regulation as underlying mechanisms. Cancer cell knockout of positive regulators of NK cell response (CD58, NCR3LG1) induced an inactive NK cell state, providing experimental evidence how cancer cell-intrinsic genetic alterations can shape the molecular profile of attacking immune cells to promote immune evasion. Conclusions: By integrating diverse functional genomics screens and patient genomic profiles, we provide a comprehensive landscape of potential biomarkers and functionally validated genetic mechanisms which influence how NK cells recognize and kill malignant cells. The results offer a roadmap to facilitate development of NK-cell based immunotherapy for blood cancers and beyond. Citation Format: Olli Dufva, Sara Gandolfi, Jani Huuhtanen, Olga Dashevsky, Khalid Saeed, Jay Klievink, Petra Nygren, Jonas Bouhlal, Jenni Lahtela, Anna Näätänen, Bishwa Ghimire, Tiina Hannunen, Pekka Ellonen, Hanna Duàn, Jason Theodoropoulos, Essi Laajala, Jouni Härkönen, Petri Pölönen, Merja Heinäniemi, Shizuka Yamano, Ryosuke Shirasaki, David Barbie, Jennifer Roth, Rizwan Romee, Michal Sheffer, Harri Lähdesmäki, Dean A. Lee, Ricardo De Matos Simoes, Matti Kankainen, Constantine S. Mitsiades, Satu Mustjoki. Single-cell functional genomics of natural killer cell evasion by tumor cells [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 667.

Abstract 707: High major histocompatibility complex class I expression overcomes cancer immunotherapy resistance due to interferon gamma signaling pathway defects

Abstract IFN-γ signaling pathway defects such as JAK mutations are well-known mechanisms of resistance to immune checkpoint inhibitors (ICIs). However, some patients respond to ICIs despite IFN-γ signaling pathway defects, and the detailed mechanisms remain unclear. Here, we created JAK-deleted cancer cell lines and evaluated antitumor immunity. As a result, several tumors responded to PD-1 blockade despite JAK deletion. Such sensitive tumors had high major histocompatibility complex class I (MHC-I) expression despite IFN-γ signaling pathway defects, whereas MHC-I expression was reduced by JAK deletion in resistant tumors. In particular, if cancer cells had high MHC-I expression despite defects in IFN-γ signaling pathways, chemokines that recruit effector T cells were mainly produced by immune cells rather than cancer cells in the tumor microenvironment. Accordingly, we found a response to ICIs in a patient with JAK-negative head and neck squamous cell carcinoma, whose human leukocyte antigen class I (HLA-I) expression level was maintained. In addition, clustered regularly interspaced short palindromic repeats (CRISPR) screening to identify molecules with elevated MHC-I expression independent of IFN-γ signaling pathways demonstrated that guanine nucleotide-binding protein subunit gamma 4 (GNG4) maintained MHC-I expression via the NF-κB signaling pathway. Our results indicate that patients with IFN-ɤ signaling pathway defects are not always resistant to ICIs, and highlight the importance of MHC-I expression among the pathways, including inhibitory effects on cellular proliferation or chemokine production, and the possibility of NF-κB-targeted therapies to overcome such resistance. Citation Format: Katsushige Kawase, Shusuke Kawashima, Joji Nagasaki, Takashi Inozume, Hiromasa Yamamoto, Masahito Kawazu, Toyoyuki Hanazawa, Yosuke Togashi. High major histocompatibility complex class I expression overcomes cancer immunotherapy resistance due to interferon gamma signaling pathway defects [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 707.

Promoting immunotherapy of cancer with focused ultrasound

Immunotherapy (ITx) has revolutionized cancer treatment. Nonetheless, for several indications, many patients do not respond to ITx due to poor immune cell infiltration and/or limited ITx penetration into solid tumors. Both thermally ablative (T) and mechanical (M) forms of focused ultrasound (FUS) may be capable of overcoming these challenges. For example, the pre-clinical application of T-FUS in combination with myeloreductive gemcitabine elicits immunological control of 4T1 breast tumors and metastases, results that inspired the Br54 clinical trial for early stage breast cancer patients at UVA. Meanwhile, our Br48 clinical trial, which combines αPD-1 ITx with T-FUS in Stage III/IV breast cancer patients, shows that the well-tolerated treatment yields reduced regulatory T cells and transcriptional alterations consistent with inflammasome activation, interferon gamma signaling and αβ T cell activation. Regarding M-FUS, in pre-clinical brain tumor studies, we have used ImmunoPET imaging to optimize the timing of ITx antibodies (i.e., αCD47) administration with M-FUS-mediated blood-tumor barrier opening. This readily translatable treatment controlled GL261 tumors and improved survival markedly over previous studies that required multi-fold more αCD47 administration to achieve an effect. Overall, our pre-clinical and clinical results support the use of both T-FUS and M-FUS in combination with ITx for multiple cancer indications.

Abstract PD4-03: PD4-03 Pelareorep primes the tumor for checkpoint inhibition therapy by activating the interferon-gamma signaling pathway and tumor inflammation signature in early breast cancer patients - results of the AWARE-1 trial

Abstract Background The status of the tumor microenvironment (TME) can profoundly affect the response to immune-based therapies for the treatment of cancer. Results previously reported from the AWARE-1 study in early breast cancer patients demonstrated that pelareorep (pela, an oncolytic reovirus), alone or in combination with checkpoint inhibitor (CPI) therapy, modified the inflammatory state of the TME. We also showed that many of pela’s effects on the TME were enhanced by the addition of checkpoint blockade. Here, we report the effect of treatment with pela on selected molecular markers associated with enhanced anti-tumor immunity. Methods Newly diagnosed HR+/HER2- early BC patients were enrolled into two cohorts: Cohort 1 (C1): pela + letrozole (n=10); and Cohort 2 (C2): pela + letrozole + atezolizumab (n=10). Pela was intravenously administered on days 1, 2 and 8, 9, and atezolizumab was given on day 3. For this analysis, tumor biopsies (FFPE samples) collected pre-treatment (D1) and on days 3 (D3, prior to the atezolizumab administration) were examined by GeoMx digital spatial profiling (DSP, using Nanostring’s Cancer Transcriptome Atlas [CTA]). Moreover, the expression of 770 immune-related genes was analyzed using a specific immune panel (n=20). Gene Set Enrichment Analysis (GSEA) (version 4.1.0) was used to assess pela-induced activation pathways. Results GeoMx DSP showed that pela therapy significantly activated IFN-gamma signaling and associated interferon response genes from D1 to D3 (Normalized Enrichment Score [NES] = 3.3, p-values &amp;lt; 0.02) in the cytokeratin-positive subset of the tumor samples. GSEA of the immune dataset (730 immune genes + 30 housekeeping genes) from the whole tissue also showed a significant upregulation of IFN-gamma signaling pathway genes (FDR &amp;lt; 25%, p-value&amp;lt; 0.001). Increases were also observed in genes reported to be associated with an enhanced tumor inflammatory signal (TIS) including PD-L1, IDO1, HLA-E and STAT1 (p-values &amp;lt; 0.005). Conclusions These results demonstrate that treatment with pela alters the TME to induce and enhance anti-tumor immunity. This enhancement of anti-tumor immunity may potentiate the TME for CPI therapy. Citation Format: Houra Loghmani, Joaquín Gavilá, Luis Manso, Matt Coffey, Richard Trauger, Fernando Salvador, Tomás Pascual, Aleix Prat, Thomas Heineman. PD4-03 Pelareorep primes the tumor for checkpoint inhibition therapy by activating the interferon-gamma signaling pathway and tumor inflammation signature in early breast cancer patients - results of the AWARE-1 trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD4-03.

Spatially restricted tumour-associated and host-associated immune drivers correlate with the recurrence sites of pancreatic cancer

ObjectiveMost patients with pancreatic ductal adenocarcinoma (PDAC) will experience recurrence after resection. Here, we investigate spatially organised immune determinants of PDAC recurrence.DesignPDACs (n=284; discovery cohort) were classified according to recurrence...

Machine learning identifies T cell receptor repertoire signatures associated with COVID-19 severity

T cell receptor (TCR) repertoires are critical for antiviral immunity. Determining the TCR repertoire composition, diversity, and dynamics and how they change during viral infection can inform the molecular specificity of host responses to viruses such as SARS-CoV-2. To determine signatures associated with COVID-19 disease severity, here we perform a large-scale analysis of over 4.7 billion sequences across 2130 TCR repertoires from COVID-19 patients and healthy donors. TCR repertoire analyses from these data identify and characterize convergent COVID-19-associated CDR3 gene usages, specificity groups, and sequence patterns. Here we show that T cell clonal expansion is associated with the upregulation of T cell effector function, TCR signaling, NF-kB signaling, and interferon-gamma signaling pathways. We also demonstrate that machine learning approaches accurately predict COVID-19 infection based on TCR sequence features, with certain high-power models reaching near-perfect AUROC scores. These analyses provide a systems immunology view of T cell adaptive immune responses to COVID-19.

Unrevealing of dysregulated hub genes linked with immune system and inflammatory signaling pathways in the pathogenesis of irritable bowel syndrome by system biology approaches

Irritable bowel syndrome (IBS) is a chronic functional gastrointestinal disorder characterized by abdominal pain and altered bowel movements. This disorder affects up to 10–23% of people globally and can affect both children and adults. Therefore, it is important to uncover early and novel therapeutic biomarkers and also reveal the pathobiology mechanisms of IBS. The main goal of this study is to investigate the hub genes and putative molecular mechanisms in IBS. Firstly, we identified 73 mutual differentially expressed genes (DEGs) from three RNA-seq databases. Then, functional gene annotation and pathway analysis of these common DEGs were enriched with interferon signaling, interferon gamma signaling, antigen processing-cross presentation, adaptive immune system, cytokine signaling in immune system, type II interferon signaling (IFNG), notch signaling pathway, MAPK signaling pathway, and IL-4 signaling pathway. In addition, a total number of 9 hub genes discovered as candidates of potential therapeutic biomarkers of IBS pathogenesis. Furthermore, transcription factors and regulatory protein kinases were identified as key signature molecules linked with IBS pathobiology. Taken together, this current study provided a novel understanding of biological mechanisms of IBS and may provide promising therapeutic targets.