bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A59: Targeting the glutamine dependent hexosamine biosynthesis pathway sensitizes pancreatic tumors to anti-PD1 therapy
Authors: Sharma Nikita S. , Gupta Vineet K. , Hadad Roey , Giri Bhuwan , Ferrantella Anthony , Dudeja Vikas , Saluja Ashok K. , Banerjee Sulagna
Published: June 2021

Background: Pancreatic cancer is notoriously difficult to treat owing to its late detection, metastasis and increased therapeutic resistance, leading to a 5-year survival rate of 9%. Immune evasive microenvironment further contributes to challenges in developing viable therapy options. As a result, anti-PD-1 therapy that has shown promising results in various cancers like melanoma, non-small cell lung cancer and kidney cancer is ineffective against pancreatic cancer patients. Hexosamine biosynthesis pathway (HBP), a glutamine-dependent metabolic shunt pathway responsible for producing UDP-GlcNAc and thus glycosylation, is overexpressed in pancreatic cancer. Our study shows that targeting HBP by a glutamine analog DON in pancreatic cancer can sensitize it to anti-PD1 immune therapy.

Methods: All experiments were done using primary cells isolated from spontaneous pancreatic cancer mice, KrasLSL.G12D/+; p53R172H/+; PdxCretg/+ (KPC), which are the epithelial cancer cells and the cancer-associated fibroblasts (CAFs) isolated from the same mice. 6-Diazo-5-oxo-L-norleucine (DON) was used as an inhibitor of GFAT, the rate-limiting enzyme of HBP. DON was used at a dose of 1mg/kg alone or 0.5mg/kg in combination with PD1 (100 microgm).

Results: Our study showed mice orthotopically implanted with KPC and CAF cells at a ratio of 1:9 showed a marked decrease in tumor volume when treated with DON. In addition to a decrease in tumor volume, these mice also had a profound decrease in their circulating tumor cells and a significant reduction in the collagen content in the tumor while the stromal content remained unaffected. Mice treated with low-dose DON in combination with low-dose anti-PD1 therapy showed a pronounced decrease in tumor volume as compared to either treatments alone. As expected, mice treated with the DON+PD1 combination had an increased infiltration of CD4 and CD8 cells.

Conclusion: Our study highlights for the first time an impressive response to PD1 therapy in pancreatic cancer when given in combination with DON, a glutamine analog. Since PDL1 depends on the HBP pathway for its glycosylation, inhibition by DON and the subsequent internalization of PDL1 leads to an increased immune response against pancreatic cancer that can be targeted therapeutically.

Note: This abstract was not presented at the conference.

Citation Format: Nikita S. Sharma, Vineet K. Gupta, Roey Hadad, Bhuwan Giri, Anthony Ferrantella, Vikas Dudeja, Ashok K. Saluja, Sulagna Banerjee. Targeting the glutamine dependent hexosamine biosynthesis pathway sensitizes pancreatic tumors to anti-PD1 therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A59.
Abstract A60: Targeted disruption of PI3K/Akt/mTOR signaling pathway induces cell cycle arrest, apoptosis, and autophagy, and inhibits inflammation, invasion and angiogenesis of OSCC cells
Authors: Aggarwal Sadhna , John Sarah , Sharma Suresh C. , Das Satya N.
Published: June 2021

Purpose: The phosphoinositide-3-kinase (PI3K) pathway is frequently altered in human cancer. This has led to the development and study of novel PI3K inhibitors for targeted therapy and also to overcome resistance to radiotherapy.

Method: The antitumor effects of PI3K inhibitors (PI-828, PI-103 and PX-866) in terms of cell proliferation, colony formation, induction of apoptosis, cell cycle arrest, invasion, autophagy, and pNF-κB/p65 translocation in SCC-4, SCC-9 and SCC-25 cells was studied by performing MTT, clonogenic, DAPI staining, propidium iodide staining, annexin-V binding, Matrigel invasion, acridine orange staining and immunofluorescence assay. Western blot assay was performed to assess the alteration in the expression of various proteins.

Results: PI-828 and PI-103 treatment exhibited dose-dependent inhibition of growth and proliferation of OSCC cells with a concomitant induction of apoptosis, altered cell cycle regulation and decreased invasiveness (p <0.01). PX-866 induced apoptosis, cell cycle arrest, autophagy and a significant decrease in the invasiveness of oral cancer cells as compared to untreated cells (p <0.01). These compounds significantly reduced expression of COX-2, Cyclin-D and VEGF in the treated cells besides cytoplasmic accumulation of pNF-κB/p65 protein. In addition to PI3Kα, inactivation of downstream components, i.e., Akt and mTOR, was seen.

Conclusion: PI3K inhibitors such as PI-103, PI-828 and PX-866 may be developed as potential chemotherapeutic agents for effective treatment of OSCC and other malignancies associated with activated PI3K/Akt pathway.

Note: This abstract was not presented at the conference.

Citation Format: Sadhna Aggarwal, Sarah John, Suresh C. Sharma, Satya N. Das. Targeted disruption of PI3K/Akt/mTOR signaling pathway induces cell cycle arrest, apoptosis, and autophagy, and inhibits inflammation, invasion and angiogenesis of OSCC cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A60.
Abstract A63: Commensal microbiota promote lung tumorigenesis via γδ T cells
Authors: Jin Chengcheng , Zhao Chen , Lagoudas Georgia , Bhutkar Arjun , Hu Bo , Jacks Tyler
Published: June 2021

Lung cancer is closely associated with chronic inflammation, but the mechanisms underlying inflammation in this setting have not been clearly defined nor have the specific contributions of particular immune cell types been elucidated. The lung is a mucosal tissue colonized by a diverse bacterial community, and pulmonary infections commonly present in lung cancer patients are linked to clinical outcomes. Here we provide evidence that local microbiota provoke inflammation associated with lung adenocarcinoma by activating lung-resident γδT cells. Germ-free or antibiotic-treated mice were significantly protected from lung tumor initiation and progression induced by Kras mutation and p53 loss. Mechanistically, commensal bacteria stimulated Myd88-dependent IL-1β and IL-23 production from myeloid cells, inducing proliferation and activation of Vγ6+Vδ1+ γδ T cells that produced IL-17 and other effector molecules to promote inflammation and tumor cell proliferation. Our findings clearly link local microbiota-immune crosstalk to lung tumorigenesis, and thereby define key cellular and molecular mediators that may serve as effective targets in lung cancer treatment and prevention.

Citation Format: Chengcheng Jin, Chen Zhao, Georgia Lagoudas, Arjun Bhutkar, Bo Hu, Tyler Jacks. Commensal microbiota promote lung tumorigenesis via γδ T cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A63.
Abstract A67: Microbial polyamines and early detection of pancreatic cancer
Authors: Mendez Roberto , Kesh Kousik , Arora Nivedita , Di Martino Leá , McAllister Florencia , Merchant Nipun , Banerjee Sulagna , Banerjee Santanu
Published: June 2021

The lack of tools for early detection of pancreatic ductal adenocarcinoma (PDAC) is directly correlated to the abysmal survival rate in patients. In addition to several potential detection tools under active investigation, we present the gut microbiome and its metabolic complement as one of the earliest detection tools that could be useful in patients at high risk for PDAC. A combination of 16s pyrosequencing and whole-genome sequencing of gut microbiota in a spontaneous genetically engineered PDAC murine model (KRASG12DTP53R172HPdxCre or KPC) showed a progressive Proteobacterial and Firmicutes dominance in gut microbiota in early stages of PDAC development. Upon in silico reconstruction of active metabolic pathways within the altered microbial flora, polyamine and nucleotide biosynthetic pathways were found to be significantly elevated. These metabolic products are known to be actively assimilated by the host and eventually utilized by rapidly dividing cells for proliferation, validating their importance in the context of tumorigenesis. In KPC mice, as well as PDAC patients, we show significantly elevated serum polyamine concentration. Therefore, at the early stages of tumorigenesis, the gut microbial composition changes in a way to promote inflammation and release metabolites that foster host tumorigenesis, thereby fulfilling the “vicious cycle hypothesis” of the role of the microbiome in health and disease states. Our results provide a potential, precise, noninvasive tool for early detection of PDAC, which will result in improved outcomes.

Citation Format: Roberto Mendez, Kousik Kesh, Nivedita Arora, Leá Di Martino, Florencia McAllister, Nipun Merchant, Sulagna Banerjee, Santanu Banerjee. Microbial polyamines and early detection of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A67.
Abstract A72: IFNAR1 signaling regulates PMN-MDSCs immune suppressive activity in cancer
Authors: Fuchs Serge , Alicea-Torres Kevin M. , Gabrilovich Dmitry I.
Published: June 2021

Polymorphnuclear myeloid-derived suppressor cells (PMN-MDSCs) are a subset of MDSCs that are expanded and accumulated in several cancers. The main role of PMN-MDSCs in cancer is the suppression of antitumor immune responses, which leads to tumor progression, angiogenesis and metastasis. PMN-MDSCs within the tumor microenvironment (TME) have potent immune-suppressive effects compared to PMN-MDSCs in peripheral organs. However, the mechanism regulating the immune-suppressive activity of PMN-MDSCs within the TME remain poorly understood. Recently, it was shown that PMN-MDSCs from spleen of tumor-bearing mice treated with type 1 interferon (IFN1) have a decrease in their immune-suppressive activity. It has been established that tumor-associated PMN-MDSCs are more suppressive than those residing in the spleen; however, their response to IFN1 remains unclear. Therefore, our goal is to understand the role of IFN1 in the regulation of PMN-MDSCs’ function within the TME. We found that IFN1 receptor, IFNAR1, is downregulated in tumor-associated PMN-MDSCs compared to their counterparts in the spleen. In cancer patients IFNAR1 on PMN-MDSC was substantially lower than on neutrophils from healthy donors. IFNAR1 downregulation in PMN-MDSCs correlated with their immune-suppressive activity. To understand the importance of IFNAR1 signaling in PMN-MDSC biology, we utilized a knock-in mouse expressing a mutant form of IFNAR1 (Ifnar1SA) that is resistant to downregulation upon stimuli and stress. PMN-MDSCs from Ifnar1SA mice were defective in their immune-suppressive activity whereas PMN-MDSCs from Ifnar1-/- mice are as immunosuppressive as PMN-MDSCs from WT mice. In this study, we described the possible role of IFN1 in negative regulation of PMN-MDSCs function in cancer.

Citation Format: Serge Fuchs, Kevin M. Alicea-Torres, Dmitry I. Gabrilovich. IFNAR1 signaling regulates PMN-MDSCs immune suppressive activity in cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A72.
Abstract A80: The effects of CCL3 on dendritic cell migration and immune cell activation
Authors: Schaller Teilo H. , Batich Kristen A. , Hotchkiss Kelly , Cui Xiuyu , Sanchez-Perez Luis , Sampson John H.
Published: June 2021

Glioblastoma (GBM) remains uniformly lethal and current therapy is ineffective and incapacitating. Standard therapies are limited by nonspecific toxicity, and the median overall survival for patients with GBM remains <15 months. Immunotherapy for GBM holds significant promise, as substantial evidence suggests that T cells can eradicate large, well-established tumors in mice and humans. Dendritic cell (DC) vaccines can, in some cases, effectively stimulate such T-cell responses as shown by several remarkable cases of individual patient responders. However, overall objective responses in early-phase clinical trials with tumor-targeted DC vaccination have remained under 15%. In a recent study in patients with newly diagnosed GBM published in Nature, we demonstrated that unilaterally preconditioning one vaccine site with the inflammatory recall antigens Tetanus/diphtheria toxoid (Td) resulted in increased bilateral DC migration to the draining lymph nodes and a significant increase in progression-free survival and overall survival—with half of the Td-treated patients living past 4.5 years. The benefit of vaccine site preconditioning could be recapitulated in a mouse model, as we found that Td increased DC migration to the draining lymph nodes and suppressed tumor growth in an antigen-dependent manner. In a search for potential mediators of these effects, we found that the only cytokine or chemokine significantly upregulated after Td preconditioning in both patients and mice was the chemokine (C-C motif) ligand 3 (CCL3). Furthermore, our data show that a source for CCL3 appears to be Td-specific CD4+ memory effector T cells (CD4Td-mem). In experiments where wild-type mice received either CCL3(-/-) or wild-type CD4Td-mem, DC migration was increased only in the wild-type CD4Td-mem setting, signifying that CCL3 from these cells is necessary for DC migration. Finally, we have found that exogenous CCL3 alone can enhance bilateral DC migration to the draining lymph nodes and reduce tumor growth similar to Td preconditioning, suggesting that exogenous CCL3 may be used to treat patients and increase objective responses associated with DC vaccines.

Citation Format: Teilo H. Schaller, Kristen A. Batich, Kelly Hotchkiss, Xiuyu Cui, Luis Sanchez-Perez, John H. Sampson. The effects of CCL3 on dendritic cell migration and immune cell activation [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A80.
Abstract A84: B7-H3 promotes antitumor T-cell suppression via MDSCs in colorectal cancer
Authors: Jayakumar Asha , Bothwell Alfred
Published: June 2021

One of the mechanisms causing resistance to checkpoint inhibitor treatment is accumulation of suppressive immune cells including myeloid-derived suppressor cells (MDSCs). In CRC patients, accumulation of MDSCs correlates with poor overall survival. Among a subset of CRC patients with high mutation burden, 31.1% respond to checkpoint inhibitor treatment. This implies that targeting resistance mechanisms such as MDSCs in these CRC patients could boost the efficacy of this treatment. B7-H3 (CD279), a member of the B7 family, is expressed on myeloid cells and suppresses T-cell proliferation. Therefore, we hypothesize that B7-H3 on MDSCs could promote CRC tumor growth by suppressing cytotoxic CD8 T cells. We used polyps from ApcMin/+ mice, an intestinal tumor model and a transplantable CRC tumor model such as MC38 colon carcinoma to assess the role of B7-H3. Our findings show that B7-H3 is upregulated in ApcMin/+ polyps and is expressed on in vitro derived monocytic MDSCs (M-MDSCs). The frequency of MDSCs increased with tumor growth, indicating the close correlation between MDSCs and disease burden. Blocking B7-H3 with an antibody in MC38 tumor model significantly reduced tumor volume (control group receiving isotype antibody: 1672.2 ± 121 mm3; B7-H3 antibody treated group: 892.7 ± 36.4 mm3; p<0.05). The frequency of CD4 and CD8 T cells in spleens and tumors of B7-H3 antibody-treated mice was increased, but this was not statistically significant. However, the frequency of CD8 cells producing IFNγ or granzyme B was significantly increased in tumors of B7-H3 antibody-treated mice. This shows that inhibition of B7-H3 in CRC regresses tumor growth and expands cytotoxic CD8 T cells in MC38 tumors. Previously we showed that reduction of intestinal tumors in ApcMin/+Stat6-/- mice correlated with reduced MDSCs and increased CD8 cytotoxic activity, indicating that MDSCs suppressed CD8 cells. Therefore, B7-H3 suppresses cytotoxic CD8 response in colon carcinoma, possibly by mediating the suppressive function of MDSCs. Clinical trials using dual targeting antibodies to B7-H3 and CD3 recruit T cells to kill tumor cells expressing B7-H3. However, impaired T-cell effector function by MDSCs could limit the efficacy of this therapy. Targeting B7-H3 on MDSCs could overcome resistance and boost the efficacy of combination treatments.

Citation Format: Asha Jayakumar, Alfred Bothwell. B7-H3 promotes antitumor T-cell suppression via MDSCs in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A84.
Abstract A85: The role of LAP positive immune cells in cancer
Authors: Gabriely Galina , da Cunha Andre , Rezende Rafael , Skillin Nathaniel , Kenyon Brendan , Walton Lena , Gopal Murugaiyan , Weiner Howard
Published: June 2021

Immunotherapy that acts by counteracting the suppressive environment established by the tumor became a very promising approach for the treatment of cancer. Regulatory immune cells promote cancer by suppressing antitumor immune responses, but there are few cell surface molecules that can specifically be targeted to neutralize their function. We characterized a regulatory T-cell population that expresses surface latency-associated peptide (LAP). LAP is an adaptor and regulator of TGF-beta, a potent immunosuppressive cytokine. An increase in LAP+ CD4 T cells has been reported in human cancer, including head and neck and colorectal cancer. To study the regulatory role of LAP+ cells in cancer, we developed a murine monoclonal anti-LAP antibody. We found that treatment with anti-LAP antibody reduces tumor growth and increases survival in various cancer models, including colon carcinoma, glioblastoma and melanoma. Anti-LAP antibody is able to block TGF-beta release from cells expressing LAP and also reduces both the number and suppressive abilities of tumor-associated LAP+ regulatory cells. Anti-LAP antibody treatment triggers a profound peripheral immune response by acting on both innate and adaptive arms of the immune system. A synergistic antitumor effect was observed by combining anti-LAP antibody with dendritic cell vaccination. To study different LAP+ immune cells on a single-cell level, we developed a 19-panel flow cytometry approach. Based on the TCGA data analysis, the expression of LAP-associated genes correlates inversely with patient survival in a number of cancers. In summary, LAP+ immune cells contribute to cancer progression, and targeting these cells for cancer treatment represents a promising immunotherapeutic approach against cancer.

Citation Format: Galina Gabriely, Andre da Cunha, Rafael Rezende, Nathaniel Skillin, Brendan Kenyon, Lena Walton, Murugaiyan Gopal, Howard Weiner. The role of LAP positive immune cells in cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A85.
Abstract A86: PI3Kγ inhibition activates T cell memory and relieves T cell exhaustion
Authors: Takahashi Hideyuki , Pathria Paulina , Shepard Ryan , Shih Ann , Louis Tiani L. , Varner Judith A.
Published: June 2021

Introduction: Tumor-associated macrophages promote immunosuppressive microenvironment in head and neck squamous cell carcinoma (HNSCC). We previously reported that macrophage PI3-kinase γ (PI3Kγ) controls a critical switch between immune stimulation and suppression during inflammation and cancer. The aim of the present study was to investigate the effect of PI3Kγ inhibition on T-cell immune response, especially on T-cell memory and exhaustion status using mouse models of HNSCC.

Materials and Methods: Wild-type (WT) or Pik3cg-/- 6- to 8-week-old male syngeneic C57Bl/6J mice were implanted with HPV+ MEER tumor cells (mouse HPV+ HNSCC cell line) by subcutaneous injection. Tumors, draining lymph nodes and spleens were isolated, then analyzed using flow cytometry or mass cytometry. Mice that completely cleared tumors were reinjected with tumor cells and tumor growth was monitored. CD90.2+ T cells or CD19+ B cells that were harvested from spleens of WT or Pik3cg-/- tumor-inoculated mice were mixed 1:1 with viable tumor cells and injected into the flanks of naive WT mice.

Results: Mice lacking PI3Kγ exhibited suppressed growth of implanted HPV+ MEER tumors. The proportion of T cells, especially CD8+ T cells, significantly increased in tumors from Pik3c-/- mice. T cells from Pik3cg-/- tumors expressed significantly more granzyme B and less T-cell exhaustion markers. The proportion of CD8+ effector memory T cells significantly increased in spleens from Pik3cg-/- mice. Mice that were implanted with both tumor cells and T cells from spleens of tumor-bearing Pik3cg-/- mice exhibited significant suppression of tumor growth. All mice that had previously cleared tumors dramatically suppressed tumor growth when rechallenged with tumor cells and remained cancer-free.

Conclusion: Pik3cg-/- mice showed more activated T-cell immune response and T-cell memory than WT, resulting in significant suppression of tumor growth. These results suggest that PI3Kγ-targeted therapy might enhance the activity of checkpoint inhibitors through the activation of T-cell immune response in patients with HNSCC.

Citation Format: Hideyuki Takahashi, Paulina Pathria, Ryan Shepard, Ann Shih, Tiani L. Louis, Judith A. Varner. PI3Kγ inhibition activates T cell memory and relieves T cell exhaustion [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A86.
Abstract A89: Identification of novel costimulatory pathways in T cells with functional assays on the Membrane Proteome Array (MPA)
Authors: Sullivan Jonathan T. , Huston-Paterson Duncan , Azuelos Charles , Doranz Benjamin J.
Published: June 2021

To identify membrane proteins involved in cytotoxic T-cell (CTL) stimulation, we screened our proprietary Membrane Proteome Array (MPA) for activation of CTLs. The MPA comprises 5,300 different human membrane proteins, each overexpressed in live HEK-293 cells. CTLs were layered on the MPA, incubated overnight, stained for an activation marker, and quantified for expression by high-throughput flow cytometry. We identified 38 putative membrane proteins involved in CTL activation. Eight of these “hits” are known activators of CTLs, such as CD86. Other hits include membrane proteins that are involved in multiple functional activities. Here, we describe the functional validation of two of these previously unreported stimulatory proteins. For Hit A, we converted the extracellular domain to an Fc-fusion protein and used the construct to probe the MPA for binding partners. We identified two proteins that interact with Hit A: one protein found on neurons and one protein found on T cells. Hit B was a commercially available molecular agonist. Using the agonist in our assay, we were able to modulate the extent to which this target stimulated CTLs. In addition to identifying factors of CTL activation, we are currently using the MPA to identify novel viral receptors and therapeutic targets for neurodegenerative diseases, as well as deorphaning and testing the specificity of antibodies.

Citation Format: Jonathan T. Sullivan, Duncan Huston-Paterson, Charles Azuelos, Benjamin J. Doranz. Identification of novel costimulatory pathways in T cells with functional assays on the Membrane Proteome Array (MPA) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A89.
Abstract A92: Assessing the T-cell exhaustion status of TCR discovery outputs
Authors: Mallini Paraskevi , Bravo-Lopes Filipa , Lech Karolina , Malla Sunir , Busz Maria , Davies Nathaniel , Hurst Jacob , Williams Luke , Davis Sterenn , Teng Michelle
Published: June 2021

Identification of antigen-specific T cell receptors (TCRs) is an essential component of the ImmTAC (Immune mobilizing monoclonal TCRs Against Cancer) platform at Immunocore. ImmTAC molecules are formed of a soluble TCR fused to an anti-CD3 effector function that engages, redirects and activates T cells to kill cancerous cells. TCRs recognize peptide antigens presented on the surface of the cancer cell by human leukocyte antigen (HLA). Thus, a fundamental step in our pipeline is to isolate T-cell receptors specific for validated cancer antigens for downstream development. T cells are isolated from whole blood of healthy donors to identify antigen-specific T cell clones using an in vitro model that consists of several stimulation steps. Activated CD8+ T cells are sorted into single cells for subsequent analysis and characterization. Despite the relative success of this approach, often analysis of T-cell clones is limited by low or absent mRNA expression for either or both TCR alpha (TRAV) and beta (TRBV) chains. We predict that TCR downregulation may result from overstimulation of T cells, leading to exhaustion. Here, we aimed to establish an in vitro model of T-cell exhaustion that would enable us to better understand the molecular mechanisms behind this process and thus facilitate method development. We induced extreme conditions of stimulation using CD3/CD28 beads and recombinant human IL-2 (rhIL-2) for 2-3 weeks. Whole-transcriptome analysis of the resulting activated T cells was compared to rested cells using a 5′ single-cell RNA-seq method from 10X Genomics (California, USA). Our preliminary analysis indicates that the activated T cells express several checkpoint inhibitors at higher levels. Our findings will be further investigated by performing full-length RNA sequencing of previously isolated single T-cell clones with low TRAV and/or TRBV detection rates. By identifying a molecular signature that represents the phenotype of exhausted T cells, we can potentially assess the state of T cells in TCR discovery outputs. Most importantly, determination of the expression levels of inhibitory receptors that are responsible for the decrease or loss of TCR expression is expected to enhance our understanding of the potential impact of T-cell exhaustion.

Citation Format: Paraskevi Mallini, Filipa Bravo-Lopes, Karolina Lech, Sunir Malla, Maria Busz, Nathaniel Davies, Jacob Hurst, Luke Williams, Sterenn Davis, Michelle Teng. Assessing the T-cell exhaustion status of TCR discovery outputs [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A92.
Abstract A98: Molecular dynamics study of pH-dependent interactions between immune checkpoint receptor PD-1 and PD-L1
Authors: Alexandrov Vitaly , Klyukin Konstantin
Published: June 2021

Understanding of how small variations in checkpoint protein chemistry (e.g., mutations) and tumor immune microenvironment (e.g., pH) influence receptor-ligand binding and define both the mechanisms and magnitude of immune response is of great importance, but the molecular details are still missing. Here, we employ enhanced free-energy sampling molecular dynamics simulations to unveil pH-dependent binding/unbinding mechanisms and kinetics of PD-1/PD-L1 interactions. By identifying the binding pocket in the PD-1/PD-L1 complex, we show a key role of the His68 protonation state in PD-1 in the complex stabilization at low pH. Specifically, we estimate the unbinding free-energy barrier to be around 15 kJ/mol higher at pH 5.5 than at pH 7.4. The estimated ratio of binding (kon) and unbinding (koff) rate constants is found to agree well with recent experimental data on KD values at different pH. Also, simulations show an important role of solvation state of the binding pocket in mediating polar interactions at the PD-1/PD-L1 binding interface. These initial molecular insights are important for further engineering of binding/unbinding kinetics to formulate more efficient immune checkpoint blockade strategies.

Citation Format: Vitaly Alexandrov, Konstantin Klyukin. Molecular dynamics study of pH-dependent interactions between immune checkpoint receptor PD-1 and PD-L1 [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A98.
Abstract B06: Immune inhibitory receptors restrain hyperactivated effector T cells in the tumor microenvironment
Authors: Lu Binfeng , Yang Min , Du Wenwen , Zhai Wensi , Sun Runzi , Yue Cuihua , Jiang Jingting
Published: June 2021

The immune checkpoint blockade (ICB) immunotherapy of cancer has prolonged overall survival for some patients; however, the response rates are still low. Rational design of combination therapy targeting multiple immune checkpoint inhibitors promises to significantly improve the current ICB therapy. TIM-3 and LAG3 are highly expressed in the tumoral-infiltrating lymphocytes (TIL) and monoclonal antibodies targeting these immune inhibitors have been subjected to intensive clinical development. However, the underlying mechanisms are not fully understood. In order to dissect the mechanisms, we first systematically examined Tim-3 expression in immune cells in mouse tumors. We found Tim-3 was expressed in large portions of Treg, CD4, CD8 T cells, DC1, M1, and MDSC. Administration of a none-depleting Tim-3 antibody alone did not affect tumor progress, but combination treatment with Tim-3 and PD-1 mAbs resulted in a synergistic antitumor efficacy. Mechanistically, treatment with either Tim-3 mAbs or PD-1/Tim-3 mAbs led to a decrease of Treg in the tumor microenvironment (TME). In addition, Tim-3 mAbs and PD-1 mAbs either alone or synergistically increased IFN-γ and granzymes production by CD4+ and CD8+ TIL. Interestingly, combined treatment with Tim-3 and PD-1 mAbs greatly increased the expression of Lag3 in TIL, suggesting induction of hyperactivated effector T cells is associated with increased level of checkpoint inhibitory molecules. We further found that the antitumor efficacy with triple combination of PD-1, Tim-3, and Lag3 mAbs was much greater than double combination. Our data demonstrated that checkpoint inhibitors are genetically encoded to control the graded activation of effector T cells and simultaneous blockade of several inhibitors is necessary to fully unleash the power of immunotherapy of cancer.

Citation Format: Binfeng Lu, Min Yang, Wenwen Du, Wensi Zhai, Runzi Sun, Cuihua Yue, Jingting Jiang. Immune inhibitory receptors restrain hyperactivated effector T cells in the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B06.
Abstract B32: Cxcr3-expressing leukocytes are necessary for neurofibroma formation in mice
Authors: Fletcher Jonathan S. , Wu Jianqiang , Jessen Walter J. , Pundavela Jay , Dombi Eva , Kim Mi-Ok , Rizvi Tilat A. , Chetal Kashish , Salomonis Nathan , Ratner Nancy
Published: June 2021

Plexiform neurofibroma is a major contributor to morbidity in neurofibromatosis type I (NF1) patients. Macrophages and mast cells infiltrate neurofibroma, and data from mouse models implicate these leukocytes in neurofibroma development. Anti-inflammatory therapy targeting these cell populations has been suggested as a means to prevent neurofibroma development. Here, we compare gene expression in inflamed nerves from NF1 models that invariably form neurofibroma to those with inflammation driven by EGFR overexpression, which rarely progresses to neurofibroma. We find that the chemokine Cxcl10 is uniquely upregulated in NF1 mice that invariably develop neurofibroma. Global deletion of the Cxcl10 receptor Cxcr3 prevented neurofibroma development in these neurofibroma-prone mice. Cxcr3 expression localized to T cells and dendritic cells (DCs) in both inflamed nerves and neurofibromas. These data support a heretofore unappreciated role for T cells/DCs in neurofibroma initiation.

Citation Format: Jonathan S. Fletcher, Jianqiang Wu, Walter J. Jessen, Jay Pundavela, Eva Dombi, Mi-Ok Kim, Tilat A. Rizvi, Kashish Chetal, Nathan Salomonis, Nancy Ratner. Cxcr3-expressing leukocytes are necessary for neurofibroma formation in mice [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B32.
Abstract B40: Translational control of tumor immune escape via the eIF4F-STAT1-PDL1 axis in melanoma
Authors: Cerezo Michael , Guemiri Ramdane , Girault Isabelle , Robert Caroline , Vagner Stephan
Published: June 2021

Preventing the immune escape of tumor cells by blocking inhibitory checkpoints, such as the interaction between Programmed Death Ligand 1 (PD-L1) and Programmed Death-1 (PD-1) receptor, is a powerful anticancer approach. However, many patients do not respond to checkpoint blockade. Tumor PD-L1 expression is a potential efficacy biomarker, but the complex regulatory mechanisms underlying its regulation are not completely understood. Here, we show that the eIF4F eukaryotic translation initiation complex, which binds the 5’ cap of mRNAs, regulates the surface expression of interferon-induced PD-L1 on cancer cells by regulating translation of the mRNA encoding the STAT1 transcription factor. eIF4F complex formation correlates with response to immunotherapy in human melanoma. Pharmacologic inhibition of eIF4A, the RNA helicase component of eIF4F, elicits powerful antitumor immune-mediated effects via PD-L1 downregulation. Thus, eIF4A inhibitors, in development as anti-ancer drugs, may also act as cancer immunotherapies.

Citation Format: Michael Cerezo, Ramdane Guemiri, Isabelle Girault, Caroline Robert, Stephan Vagner. Translational control of tumor immune escape via the eIF4F-STAT1-PDL1 axis in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B40.
Abstract B41: BRAF targeting sensitizes resistant melanoma to cytotoxic T cells
Authors: Atay Cigdem , Kwak Taekyoung , Lavilla-Alonso Sergio , Richards Allison , Moberg Valerie , Pilon-Thomas Shari , Schell Michael , Messina Jane L. , Rebecca Vito W. , Zhang Gao , Weber Jeffrey S. , Herlyn Meenhard , Sarnaik Amod A. , Gabrilovich Dmitry I.
Published: June 2021

Although treatment of combination therapy with BRAF and MEK inhibitors in patient with melanoma has shown longer duration of response and higher rate of tumor responses, recurrence of tumor from dual targeted therapy in patients with metastatic melanoma has still remained a challenge to overcome. Here, we provide new therapeutic approaches for BRAF inhibitors-resistant melanoma model that upregulation of mannose-6-phosphate receptor (M6PR) on melanoma by treatment of BRAF inhibitors enhances tumor cytotoxicity level via Granzyme B uptake, which is major component of cytotoxic activity of cytotoxic T lymphocytes (CTLs). Our results demonstrate that treatment of BRAF inhibitor increases M6PR expression temporally in both sensitive and resistant BRAFi melanoma cell lines as well as tumor-bearing mouse models (NOD/SCID). Cytotoxic activity of human melanoma cells by co-culture with patient-derived tumor-infiltrating lymphocytes (TILs) shows correlation with M6PR expression level. Furthermore, treatment of mice bearing resistant tumor with BRAFi enhances the antitumor effect of adaptive TILs transfer. In pilot clinical trials of 16 patients with metastatic melanoma, our results support that administration of BRAF inhibitor to patient shows high level of M6PR expression on tumor tissues. Also, combination treatment of BRAF inhibitor and adaptive cell transfer with patient-derived TILs shows promising clinical results in terms of longer response and tumor-free survival. Taken together, BRAF targeted therapy upregulates M6PR level, which enhances activity of TILs to kill resistant melanoma, providing new therapeutic approaches for patients who have recurrence melanoma.

Note: This abstract was not presented at the conference.

Citation Format: Cigdem Atay, Taekyoung Kwak, Sergio Lavilla-Alonso, Allison Richards, Valerie Moberg, Shari Pilon-Thomas, Michael Schell, Jane L. Messina, Vito W. Rebecca, Gao Zhang, Jeffrey S. Weber, Meenhard Herlyn, Amod A. Sarnaik, Dmitry I. Gabrilovich. BRAF targeting sensitizes resistant melanoma to cytotoxic T cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B41.
Abstract B45: NFAT5/TonEBP mediated anti-tumor efficiency of high salt activated CD4+T lymphocytes
Authors: Amara Suneetha , Yenuga Pooja , Zent Roy , Tiriveedhi Venkataswarup
Published: June 2021

Cell-based immunotherapy is rapidly emerging as a promising alternative to conventional chemotherapy-based treatment for cancer. Previous studies have shown that high salt treatment induces naive CD4+T lymphocytes to differentiate into a highly proinflammatory Th17 phenotype. In our current study, we analyzed the ability of salt treatment to induce cancer-specific Th17 lymphocytes. Naive CD4+T cells isolated by immunomagnetic bead isolation from splenocytes of 10-week-old BALB/c mice were treated in vitro with high salt (Δ0.05 M NaCl) along with CD3 monoclonal antibodies (mAbs), CD28 mAbs and IL-2 for 4 days along with heat killed 4T1 breast cancer cells. The isolated CD4+CCR6+CD45RA-CXCR3- cells were tested for IL-17 expression by flow cytometry and determined to be >95% pure. Orthotopic murine breast tumor models established by injection of 4T1 cells into the mammary fat pad of Nu/J mice were administered two doses of (1 × 106) salt induced Th17 cells on day 7 and day 12, post-tumor implantation. By day 28, the high salt induced Th17 cells cohort demonstrated significantly reduced tumor growth (97 ± 34 mm3 vs 512 ± 91 mm3, untreated control, p<0.05) and lung metastasis. However, no effect on tumor growth was noted when these 4T1 sensitized high salt treated Th17 cells were injected into Nu/J mice with prostrate (RM1) and lung tumors (KLN205), thus suggesting that the specificity of salt induced Th17 cells to breast tumors. Similarly, in vitro high salt mediated differentiation of Th17 cells when co-treated with heat killed RM1 and KLN205 cancer cells were able to induce respective murine tumor regression. Mechanistic studies demonstrated that the high salt induction to the Th17 phenotype was mediated by the tonicity-dependent transcription factor, TonEBP/NFAT5. The shRNA-based knockdown of TonEBP abrogated the induction of the Th17 phenotype and expression of proinflammatory cytokines IL-17, IL-1β and IL-6. Taken together, our data suggests that high salt mediated in vitro differentiation of Th17 cells could induce tumor specific anticancer CD4+T lymphocytes, which may give rise to a cell-based anticancer immunotherapy application.

Citation Format: Suneetha Amara, Pooja Yenuga, Roy Zent, Venkataswarup Tiriveedhi. NFAT5/TonEBP mediated anti-tumor efficiency of high salt activated CD4+T lymphocytes [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B45.
Abstract B48: PBRM1 loss promotes resistance to immunotherapy in RCC
Authors: Liu Xian-De , Kong Wen , Hoang Anh , Zhang Xuesong , Zhou Lijun , Pilie Patrick G. , Isgandrova Sevinj , Moczygemba Margie M. , Jonasch Eric
Published: June 2021

PBRM1 is a subunit of a SWI/SNF chromatin remodeling complex, and more than 30% of clear cell RCCs show PBRM1 mutations. The function of PBRM1 in tumor immune microenvironment, tumor prognosis and response to immune checkpoint therapies still remain unclear. Here we found that Pbrm1 knockout in mouse Renca RCC cells decreased the activity of IFNγ-STAT1 signaling pathway and consequently the expression of chemokines that recruit effector CD8+ T cells. Pbrm1 deficient murine RCC tumors showed reduced infiltration of CD8+ T cells, which was associated with less PD-1 expression. Pbrm1 deficient tumors demonstrated longer latency but they were more resistant to anti-PD1 treatment. This study provided mechanistic insights and therapeutic guideline to manage ccRCC associated with PBRM1 inactivation.

Citation Format: Xian-De Liu, Wen Kong, Anh Hoang, Xuesong Zhang, Lijun Zhou, Patrick G. Pilie, Sevinj Isgandrova, Margie M. Moczygemba, Eric Jonasch. PBRM1 loss promotes resistance to immunotherapy in RCC [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B48.
Abstract B53: Immunomodulatory effect of prostaglandin dehydrogenase in metastatic colon cancer
Authors: Satapathy Shakti R. , Sjolander Anita
Published: June 2021

Cyclooxygenase-2, the key player in the biosynthesis of prostaglandin-E2 (PGE2) is often upregulated in colon cancer (CC). TNF-α, the main regulator of COX-2 and mPGES-1, contributes to the increased synthesis of PGE2, which is inhibited by overexpressed prostaglandin dehydrogenase (15-PGDH), a tumor suppressor playing major role in PGE2 catabolism but often downregulated in CC. Interestingly, CC patients expressing high levels of cysteinyl leukotriene receptor-2 (CysLT2R) have good prognosis and therefore, we investigated a potential link between CysLT2R-signaling and 15-PGDH in metastatic CC. Elevation of 15-PGDH expression by LTC4, a CysLT2R ligand, exhibited anti-tumor activity in colon cancer cells with significant phosphorylation of β-catenin with concurrent activation of CASPASE-3 and dramatic down-regulation of TNF-α mRNA expression in two different colon cancer cells. Colony-stimulating factor 1 (CSF-1) is a key regulator of macrophage differentiation that supports the protumorigenic role of tumor-associated macrophages (TAMs). We observed high CSF-1 in metastatic colon cancer with low CysLT2R expression, which is correlated with high level of CD-8+ T-cells and CD-163+ TAMs. CC cell lines produced high amount of CSF-1 when co-cultured with CC-specific CD-8+ T-cell or exposed to T-cell derived cytokines in vitro and exhibited significant reduction with LTC4 stimulation or 15-PGDH overexpression. Furthermore, longer use of 15-PGDH led to downregulation in the PD-L1 in a time-dependent manner. Combination of anti-CSF-1, anti-PD-1 and LTC4 exhibited significant reduction in CC tumor rich in macrophages. Blockade of PD-1/PD-L1 axis also facilitated upregulation of IFN-γ in γδ-T cells. Hence restoration of 15-PGDH expression through CysLT2R-signaling promotes immunomodulatory effect, indicating a promising therapeutic alternative for metastatic CC with high TAM infiltration.

Citation Format: Shakti R. Satapathy, Anita Sjolander. Immunomodulatory effect of prostaglandin dehydrogenase in metastatic colon cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B53.
Abstract B72: Lineage specifiers SOX2 and NKX2-1 inversely regulate tumor cell fate and neutrophil recruitment in lung cancer
Authors: Mollaoglu Gurkan , Jones Alex , Wait Sarah , Mukhopadhyay Anandaroop , Jeong Sangmin , Arya Rahul , Camolotto Soledad , Mosbruger Timothy , Stubben Chris , Conley Christopher , Bhutkar Arjun , Vahrenkamp Jeffery , Berrett Kristofer , Cessna Melissa , Lane Thomas , Witt Benjamin , Salama Mohamed , Gertz Jason , Jones Kevin , Snyder Eric , Oliver Trudy
Published: June 2021

The major types of non-small cell lung cancer, squamous cell carcinoma and adenocarcinoma, have distinct tumor immune microenvironments. Understanding the mechanisms underlying these differences is of particular importance given the success and current limitations of immunotherapy. We developed multiple mouse models of lung cancer to demonstrate that NKX2-1 potently suppresses SOX2-driven squamous tumorigenesis by repressing adeno-to-squamous transdifferentiation. Furthermore, SOX2 recruits, whereas NKX2-1 suppresses, tumor-associated neutrophils (TANs) at least partly through inverse regulation of the chemoattractant CXCL5/6. Single-cell RNA sequencing revealed that TANs exhibit tumor-promoting features and distinct gene expression profiles compared to blood neutrophils. Finally, TANs cooperate with squamous-associated genetic alterations to promote squamous tumors. These data reveal how transcription factors with key functions in normal development dictate not only cancer cell identity but also distinct tumor immune microenvironments.

Citation Format: Gurkan Mollaoglu, Alex Jones, Sarah Wait, Anandaroop Mukhopadhyay, Sangmin Jeong, Rahul Arya, Soledad Camolotto, Timothy Mosbruger, Chris Stubben, Christopher Conley, Arjun Bhutkar, Jeffery Vahrenkamp, Kristofer Berrett, Melissa Cessna, Thomas Lane, Benjamin Witt, Mohamed Salama, Jason Gertz, Kevin Jones, Eric Snyder, Trudy Oliver. Lineage specifiers SOX2 and NKX2-1 inversely regulate tumor cell fate and neutrophil recruitment in lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B72.