BackgroundThe US is experiencing an epidemic of HPV+ oropharyngeal cancers (OPC), the rates and burden of which now exceed that for cervical cancer. Immunotherapy targeting programmed death 1 (PD-1) ...on tumor-infiltrating lymphocytes and/or its ligand PD-L1 on tumor cells, which was effective in several cancers has however, showed efficacy in only less than 15% of patients.MethodsWe used a preclinical HPV+ oral tumor model, mEER, consisting of mouse tonsil derived epithelial cells expressing HPV-16 E6 and E7 genes, along with the H-ras oncogene to test strategies for enhancing the efficacy of anti-PD-1 therapy.ResultsMonotherapy with PD-1 blocking antibody was ineffective against flank-implanted tumors, but induced regression in 54% of mice bearing orthotopic tongue tumors that correlated with higher CD8 T cell responses. Since the CD8+ T cells derived from tongue tumors also showed high levels of the immune checkpoint inhibitory receptor CTLA-4, we tested combination immunotherapy targeting both CTLA-4 and PD-1 together and observed 93.3% survival of mice bearing tumors in the tongue for the duration of our 100-day study. Protective immunity correlated with a significant decrease in immunosuppressive lymphoid and myeloid populations within the tumor microenvironment. Consistent with the reported capacity of interferon-driven PD-L1/PD-1 pathway induction to serve as a biomarker of response to PD-1 blockade, we observed elevated interferon signaling and significantly higher levels of PD-1/PD-L1 in tongue-implanted mEER tumors compared to those growing on the flank correlating with their preferential responsiveness to PD-1 blockade. More importantly, in a pseudometastasic mouse model bearing both flank and tongue tumors to represent metastatic disease, delivery of Stimulator of Interferon Induced Genes (STING) agonist into the flank tumors combined with systemic treatment with α-PD-1 and α-CTLA-4 antibodies resulted in sustained tumor regression in 71% of mice. In this case, productive abscopal anti-tumor immunity was associated with robust increases in the ratios of cytotoxic CD8+ T cells (CTL) versus regulatory T cells (Treg) and versus functional myeloid-derived suppressor cells (MDSC).ConclusionsThese results support combining α-PD-1 therapy with induction of IFN-α/β signaling via provision of STING agonist and/or through CTLA-4 blockade as potential treatment option for HNSCC patients, especially, those not responding to α-PD-1 monotherapy.
BackgroundIntratumoral injection of cyclic dinucleotide (CDN) agonists of the stimulator of interferon genes (STING) pathway engages innate immune activation and priming of adaptive immune effectors ...to foster local and distal tumor clearance. Despite proven therapeutic efficacy in preclinical models, a thorough understanding of how CDNs reprogram suppressive myeloid stroma in mouse and man is lacking.MethodsHere, we perform deep transcript-level and protein-level profiling of myeloid-derived suppressor cells and M2 macrophages following stimulation with CDNs of ascending potency. Additionally, we leverage orthotopic Kras+/G12DTP53+/R172HPdx1-Cre (KPC) derived models of pancreatic adenocarcinoma (PDAC) to determine the capacity for locally administered CDNs to sensitize PDAC to immune checkpoint blockade. We use bioluminescent in vivo imaging and 30-parameter flow cytometry to profile growth kinetics and remodeling of the tumor stroma post-therapy.ResultsHighly potent synthetic STING agonists repolarize suppressive myeloid populations of human and murine origin in part through inhibition of Myc signaling, metabolic modulation, and antagonism of cell cycle. Surprisingly, high-potency synthetic agonists engage qualitatively unique pathways as compared with natural CDNs. Consistent with our mechanistic observations, we find that intratumoral injection of the highest activity STING agonist, IACS-8803, into orthotopic pancreatic adenocarcinoma lesions unmasks sensitivity to checkpoint blockade immunotherapy. Dimensionality reduction analyses of high parameter flow cytometry data reveals substantial contributions of both myeloid repolarization and T cell activation underlying the in vivo therapeutic benefit of this approach.ConclusionsThis study defines the molecular basis of STING-mediated myeloid reprogramming, revealing previously unappreciated and qualitatively unique pathways engaged by CDNs of ascending potency during functional repolarization. Furthermore, we demonstrate the potential for high potency CDNs to overcome immunotherapy resistance in an orthotopic, multifocal model of PDAC.
BackgroundHuman papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HPV+ HNSCC) is a disease that has moderate response to anti-PD-1/L1 immune checkpoint blockade, with the response ...rates less than 20% and median progression-free survival less than 3 months. A greater understanding of tumor intrinsic and extrinsic factors that restrict anti-tumor immunity in the tumor immune microenvironment (TIME) is needed to identify other immune checkpoints to enhance therapeutic efficacy.MethodsTwo cohorts (TCGA n=72 and a separate cohort n=84) of surgically resected, treatment-naïve HPV+ HNSCC with RNA-seq were analyzed to understand the immune features. In addition, single-cell RNA-seq and TCR-seq were performed on 18 cases to further delineate the immune molecules' interactions. An immune-competent murine HPV+ HNSCC model was used to preliminarily evaluate the therapeutic efficacy.ResultsIn two bulk-sequenced HPV+ HNSCC cohorts, TIGIT ligands PVR and NECTIN2 were found to associate with an epithelial-to-mesenchymal gene expression signature, suppression of IFNα and IFNγ signaling, a stromal-enriched or immune-excluded TIME, and poor survival. Single-cell RNA-seq of over 72,000 cells of HPV+ HNSCC revealed that the PVR/NECTIN ligand TIGIT was highly prevalent in T-cells (34%), significantly higher than PD1- (20%, p<0.01). There is an enrichment of cell-cell interactions mediated by TIGIT-PVR/NECTIN2 in the TIME of HPV+HNSCC versus normal tonsil. TIGIT was the most differentially upregulated immune checkpoint on clonally expanded CD8+T-cells and was abundant on antigen-experienced, tissue-resident memory CD8+T-cell and T-regulatory subsets. TIGIT ligands PVR, NECTIN1, and NECTIN2 were abundant on mature regulatory dendritic cells (DCs), immunosuppressive plasmacytoid (p)DCs, and macrophages, respectively. TIGIT and PD-1 co-blockade in the mEER syngeneic murine model significantly reduced tumor growth, improved survival, restored effector function of HPV16E7-specific CD8+T cells, natural killer cells, and DCs, and conferred tumor re-challenge protection.ConclusionsTIGIT-PVR/NECTIN receptors/ligands are more abundant than PD-1/L1 in the TIME of HPV+ HNSCC. Co-blockade of TIGIT and PD-1 immune checkpoints enhanced anti-tumor efficacy in a CD8+ T-cell-dependent manner and conferred long-term immune protection in a murine model. Our study nominates TIGIT as a therapeutic target for HPV+ HNSCC.
Abstract
Despite recently approved immunotherapy combinations, Hepatocellular carcinoma (HCC) remains among the most therapeutically intractable cancers with a 5-year survival rate of only 18%. ...Underlying chronic liver disease due to hepatitis B/C infection, alcohol abuse, hemochromatosis, or nonalcoholic steatohepatitis promotes HCC development. Current therapeutic strategies include surgery, radiotherapy and multikinase inhibitors alone or with immune-checkpoint blockade (ICB). In particular, the receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF) are known drivers of HCC through promotion of tumor growth, survival, tissue invasion, and angiogenesis. Previously, our collaborators showed that the limited clinical benefit of MET kinase inhibition was partially a result of induced upregulation PD-L1 leading to local T cell suppression. We hypothesized that combining MET inhibition with blockade of the PD-1 immune checkpoint would overcome the limitations of each individual approach and act synergistically to promote HCC tumor regression. We first validated that both MET selective (Capmatinib and Tivantinib) and non-selective (Cabozantinib) inhibitors all induced PD-L1 on HCC cell lines in vitro. Next, we compared Capmatinib and Cabozantinib alone and with PD-1 ICB in vivo in the ICB-sensitive Hepa1-6 and HCA-1 models of HCC. In HCA-1, statistically significant benefit of the MET inhibitor and αPD-1 combination was evident in limitation of tumor growth and extension of survival. In each case, the Capmatinib combination trended toward better outcomes with PD-1 blockade. Mechanistically, the Capmatinib and αPD-1 combination decreased tumor Treg cells while promoting increased accumulation of activated, non-exhausted, proliferating CD8 T cells. Central memory CD8 T cell frequencies were increased by the combination during the effector phase, and combination treated animals were better protected against subsequent rechallenge than those cured by αPD-1 alone. In the myeloid compartment, M1 macrophage frequencies were increased while PMN-MDSC both decreased in frequency and in Arginase 1 levels. Finally, we studied a serially-passaged, αPD-1 refractory DEN HCC model. Despite lack of efficacy of either component therapy, the MET inhibitor and αPD-1 combination significantly extended survival and inhibited tumor growth. As in the αPD-1 sensitive setting, Capmatinib appeared to offer the most therapeutic benefit with αPD-1. Ongoing studies are focused on characterizing changes mediated by the combination therapy which confer aPD-1 sensitivity to the otherwise refractory DEN HCC tumor microenvironment. Molecular studies are focused on understanding the superior ICB potentiating capacity of Type I MET inhibitors versus the other classes. Given the clinical availability of numerous MET and PDL-1 inhibitors, we hope to inform near-term optimization of MET and αPD-1 clinical trial design for HCC.
Citation Format: Ricardo A. de Azevedo, Broderick Turner, Priyamvada Jayaprakash, Krithikaa Rajkumar Bhanu, Anupallavi Srinivasamani, Brittany Morrow, Michelle Winkler, Shweta Mahendra Hedge, Arthur Liu, Ravaen Slay, Michael A. Curran. Proposing the best MET inhibitor to improve anti-PD-1 efficacy in HCC. 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 5745.
BackgroundBackground: Radiotherapy of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)-expressing CRC cells systemically; however, incidences of abscopal ...tumor remission are extremely rare. We sought to unravel the post-irradiation immune escape mechanisms in CRC.MethodsMethodsFlow cytometry, gene knockdown, RNA and T cell receptor sequencing, and multiple murine syngeneic CRC models were used to interrogate mechanisms of CRC immune evasion following radiotherapy. Comparison of immunohistochemistry staining between pretreatment biopsy and post-irradiation surgical specimens was performed in rectal patients who underwent neoadjuvant radiotherapy with 5 Gy for 5 fractions.ResultsResultsWe find that CRC cells utilize a common DNA repair signaling pathway — ATR/Chk1/STAT3 — to upregulate both CD47 and PD-L1 in response to radiotherapy, which through engagement of SIRPα and PD-1 suppresses the capacity of antigen-presenting cells (APCs) to phagocytose them thereby preventing TAA cross-presentation. This post-irradiation CD47 and PD-L1 upregulation can be observed in CRC cells treated with either photon or proton radiotherapy and across a wide variety of human solid tumor cells. Concordantly, rectal cancer patients who responded poorly (tumor regression grade 4–5, n = 10) to neoadjuvant radiotherapy exhibited significantly elevated post-irradiation CD47 levels (P = 0.005). In murine CRC models, the combination of radiotherapy, αSIRPα, and αPD-1 (RSP) profoundly enhances TAA uptake, activation of innate immune sensors, and TAA cross-priming across various antigen-presenting myeloid populations in the irradiated tumor microenvironment and facilitates TAA-presenting APC migration to secondary lymphoid organs. Furthermore, we observed robust production of TAA-specific CD8 T cells, functional activation of effector T cells, and increased tumor-infiltrating T cell clonality and clonal diversity in mice treated with RSP. Importantly, radiotherapy coupled with phagocytosis checkpoint blockade significantly improves complete response rates in both irradiated and abscopal tumors and prolongs survival in three distinct murine CRC models, including a cecal orthotopic model. In addition, αSIRPα exerts superior tumoricidal efficacy than αCD47 in combination with RT and αPD-1. We find RSP efficacy to be STING dependent as knockout animals lose most benefit of phagocytosis checkpoint blockade.ConclusionATR-mediated CD47 and PD-L1 upregulation restrains radiation-induced immune priming in CRC. Blockade of the phagocytosis checkpoints SIRPα and PD-1 during radiotherapy promotes vigorous anti-CRC immune priming leading to systemic tumor regression.AcknowledgementsThis study is supported in part by NIH grant P30 CA16672, the MD Anderson Andrew Sabin Family Fellowship, and Chang Gung Memorial Hospital grant CMRPG3K1751. RCH was supported by the CPRIT Research Training Grant (RP170067) and Ralph B. Arlinghaus Ph.D. Scholarship. The authors are grateful to the members of the Advanced Cytometry & Sorting Facility at South Campus, Tissue Bank of Chang Gung Memorial Hospital at Linkou, and MHC Tetramer Core Facility at Baylor College of Medicine for their invaluable help.Ethics ApprovalThis study was approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taiwan; approval number: 202001191B0C601.
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