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BackgroundImmune checkpoint inhibitors (ICI) have improved patient survival in some cancer types but yielded limited success in breast cancer. Combinations of ICI (αPD-L1/PD-1) and chemotherapy have been FDA-approved for metastatic TNBC patients, and potentially in the early breast cancer setting, but many patients remain non-responsive to ICI. B7-H4 is a B7 family ligand with proposed immunosuppressive functions being explored as a cancer immunotherapy target and may be associated with resistance to αPD-L1. We confirmed an inverse expression pattern between B7-H4 and PD-L1 in breast tumor cells, which has previously been noted by others. B7-H4 was expressed in immune-excluded tumors, while PD-L1 was expressed in immune-infiltrated tumors. Based on these findings, we hypothesized ectopic B7-H4 expression would induce αPD-L1 resistance through immune cell suppression in vivo.MethodsUsing an immunocompetent and αPD-L1-sensitive EMT6 orthotopic mammary cancer model, we induced ectopic expression of B7-H4 and performed animal survival studies to assess therapy response, and RNA analysis to assess changes to cell signaling among tumor infiltrating immune cells. Finally, we performed transcriptomic correlation analyses from the cancer cell line encyclopedia dataset to identify potential regulators of B7-H4 in breast cancer.ResultsIn the αPD-L1-sensitive EMT6 mammary cancer model, tumors with cell-surface B7-H4 expression were more resistant to immunotherapy. Additionally, tumor infiltrating immune cells had reduced immune activation signaling based on transcriptomic analysis. We also observed strong correlation with B7-H4 mRNA and epithelial cell markers, in contrast to gene expression markers of mesenchymal cells.ConclusionsOur data support the hypothesis that B7-H4 induces tumor resistance to αPD-L1 ICI through an immunosuppressive function. Additionally, the strong correlation of B7-H4 to epithelial cell markers suggests a potential regulatory mechanism of B7-H4 expression independent of PD-L1 regulation.

BackgroundDespite the broad success of immune checkpoint inhibition (ICI e.g. anti-Programmed Death Ligand-1 [PD-L1]) in cancer treatment, tumor-intrinsic factors leading to intrinsic and acquired resistance are poorly understood. Tumor specific MHC-I expression is indispensable for anti-PD-1/L1 response as complete loss of MHC-I via B2M deletion results in inability of CD8+ T cells to recognize tumor-associated antigens. However, MHC-I is heterogeneously downregulated or lost in many tumor types. Tumor cell destruction can also occur through non-synaptic mechanisms, in a so-called ‘field effect’. Therefore, we modeled heterogeneous loss of MHC-I expression in breast cancer and experimentally evaluated how heterogeneous MHC-I loss affects response to anti-PD-L1 therapy.MethodsWe performed quantitative immunofluorescence for MHC-I and Pan-CK on breast cancer tumors (n=410). To determine the functional effect of MHC-I heterogeneity on anti-PD-L1 response, we used an immunocompetent EMT6 orthotopic mammary tumor model which ubiquitously expresses MHC-I at baseline. Using CRISPR/Cas9, we engineered EMT6 cells with B2m loci excision resulting in complete knockout of MHC-I on the cell surface. We then orthotopically implanted B2m-comtetent and B2m-KO cells at varying inoculum ratios (100:0, 90:10, 50:50, 10:90, 0:100) into syngeneic Balb/C mice and assessed immune responsiveness and efficacy of checkpoint inhibition. Additionally, to look at how loss of MHC-I affects the tumor microenvironment we will use the PanCancer Immune NanoString panel (n=770 genes) to evaluate gene expression patterns in tumor cells and infiltrating immune cells.ResultsIn patient samples, we identified high diversity in MHC-I expression across all clinical subtypes, with triple negative breast cancer (TNBC) having the highest MHC-I expression. Chemotherapy-treated tumors had higher MHC-I levels than untreated tumors. In mice when 10% of cells were B2m-KO, we observed a 50% reduction in complete eradication of EMT6 tumors with aPD-L1 treatment and reduced disease-stabilization and no complete responses when a 50% mixture of MHC-I deficient cells. An increasing percentage of B2m KO leads to worse outcomes overall and a decrease in infiltrating T cells.ConclusionsOur work suggests that there is an ICI-responsive phenotype that is driven by heterogeneity in MHC-I expression levels. As little as 10% of tumor specific MHC-I loss can lead to therapeutic resistance and a decrease in complete responders. This represents that early TNBC may be less responsive to single-agent PD-L1 due to specific percentages of MHC-I loss. MHC-I expression can influence therapy outcomes and potentially lead to novel observations of how to overcome lack of, or limited, MHC-I expression.

BackgroundImmune checkpoint inhibitors (ICI) improve patient survival in some cancer types but yield limited success in breast cancer. Phase-III clinical trials in triple-negative breast cancer (TNBC) patients, who harbor extensive tumor-infiltrating lymphocytes, demonstrate increased progression-free survival (IMpassion130) and pathologic complete response (KEYNOTE-522). Consequently, combinations of ICI and chemotherapy have been FDA-approved for metastatic TNBC patients. However, the therapeutic benefit of ICI alone and the most efficacious chemotherapy combinations are poorly characterized. We sought to model ICI response in vivo to elucidate the mechanisms of immunotherapy efficacy in breast cancer and ascertain the therapeutic benefits of different chemotherapeutic combinations with ICI.MethodsUsing an immunocompetent EMT6 orthotopic mammary tumor model, we investigated the efficacy of single-agent immunotherapy and in combination with standard-of-care chemotherapy (paclitaxel [PAC] or doxorubicin [DOX]). We used single-cell RNA sequencing and bulk RNA and T-cell receptor (TCR) sequencingto assess the cellular landscape of the primary tumor in response to combinatorial therapeutic strategies and identify systemic genetic alterations and T-cell expansion, respectively.ResultsSingle-agent anti-PD-L1 robustly suppressed primary tumor growth (p =0.0046) and extended survival (pConclusionsWe identify a heterogeneously ICI-responsive in vivo model that emulates TNBC patient response to combinatorial ICI approaches. We describe single-agent ICI efficacy in upregulating cytotoxic immune cell infiltration and expansion within the primary tumor that diminishes tumor growth and enhances survival. Moreover, this study describes differential responses in a genetically similar host, which reflects heterogeneous patient response to ICI. Further characterization may identify systemic biomarkers and tumor antigen-specific T cell clones to accurately predict immunotherapy response in patients and uncover mechanisms for sensitizing refractory tumors to ICI

BackgroundGlucose fixation is a hallmark clear cell renal carcinoma (ccRCC).1 2 Our group has shown unique metabolic enzyme utilization between malignant cells and infiltrating cells. Additionally, we uncovered the glycolytic nature of tumor infiltrating myeloid cells.3 Therefore, we decided to investigate the role of the hexokinase isoforms (HK1,2/3, GCK, and HKDC1) in the ccRCC tumor microenvironment (TME).MethodsFor this study, we performed immunogenomic analyses across ccRCC samples available via The Cancer Genome Atlas (TCGA).4 5 Additionally, we examined the expression of hexokinases in the neoadjuvant VEGF inhibitor setting6 as well as correlation to a poor prognostic macrophage subset.7 Our group also performed single cell-ATAC seq on methocult cultures to further characterize the metabolic features of hematopoiesis. We additionally implemented qPCR on magnetically sorted bone marrow as well as myeloid cell culture to further interrogate the role of HK3 in macrophage biology and in-situ RNA hybridization (RNA-ISH) to describe the subpopulation of HK3+ cells in the ccRCC TME.ResultsGene set enrichment analysis confirmed HK1/2’s role in anabolic metabolism. GCK was barely detectable in these samples while HKDC1 expression decreased in ccRCC tumors. Intriguingly, patients with elevated expression of HK3 had an enrichment of interferon gamma response signature. In our evaluation of the TCGA, only HK3 expression correlated with poor outcome in ccRCC. CiberSortX demonstrated that HK3 expressing tumors correlated with the presence M2 macrophages while other HK family enzymes had marginal association with immune infiltrate. HK3 was the only hexokinase found to be significantly elevated with neoadjuvant pazopanib treatment in addition to being enriched in ccRCC patients with high levels of poor prognostic macrophages. RNA-ISH confirms HK3 expression is limited to myeloid cells in ccRCC tumors. The myeloid specific nature of HK3 is supported by transcript analysis from MC38 tumors, and qPCR on mouse bone marrow. Myeloid specificity for HK3 isoform expression is not restricted to malignancy; HK3 is one of a handful of genes that define myeloid identity from scATAC sequencing of in vitro differentiated CD34+ hematopoietic stem cells. Our ongoing in vitro studies indicate that M1 polarization (+LPS/IFNg) increases expression of HK1/2/3, consistent with the anabolic phenotype of activate macrophages. However, stimulation with IFNg alone only elevates the expression of HK3.ConclusionsHK3 is a myeloid specific interferon gamma responsive gene, whose expression imparts poor prognosis for ccRCC cancer patients, while HK1/HK2 contribute significantly to the glucose uptake/pseudohypoxic phenotype seen throughout the ccRCC TME.AcknowledgementsN/aTrial RegistrationN/AReferencesCourtney KD, et al. Isotope tracing of human clear cell renal cell carcinomas demonstrates suppressed glucose oxidation in vivo. Cell metabolism 2018;28(5):793–800.e2.Linehan WM, et al. The metabolic basis of kidney cancer. Cancer Discov 2019;9(8):1006–1021.Reinfeld BI, et al. Cell-programmed nutrient partitioning in the tumour microenvironment. Nature 2021.Ricketts CJ, et al. The cancer genome atlas comprehensive molecular characterization of renal cell carcinoma. Cell reports 2018;23(1):313–326.e5.Creighton CJ, et al. Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature 2013;499(7456):43–49.Wood CG, et al. Neoadjuvant pazopanib and molecular analysis of tissue response in renal cell carcinoma. JCI Insight 2020;5(22).Obradovic A, et al. Single-cell protein activity analysis identifies recurrence-associated renal tumor macrophages. Cell 2021;184(11):2988–3005.e16.Ethics ApprovalThis clinical trial [in Reference 6] was approved by the IRBs at the University of Carolina at Chapel Hill (Office of Human Research Ethics) and MD Anderson (Office of Human Subjects Protection), and the research was conducted according to the Declaration of Helsinki principles. All participants provided written informed consent before the initiation of any research procedures.The studies were conducted in accordance with the guidelines approved by the Institutional Review Board (IRB) protocols, AAAO5706 and AAAA9967, respectively. Patients provided consent prior to taking part in the study. This is the clinical data take from the study in reference 7.All other studies referenced in the above abstract were conducted in accordance with the Declaration of Helsinki principles under a protocol approved by the Vanderbilt University Medical Center (VUMC) Institutional Review Board (protocol no. 151549). Informed consent was received from all patients before inclusion in the study by the Cooperative Human Tissue Network at VUMC. This is the clinical data take from the study in reference 3 and 7All mouse procedures were performed under Institutional Animal Care and Use Committee (IACUC)-approved protocols from VUMC and conformed to all relevant regulatory standards. The mouse protocol ID is 19000125

BackgroundNearly half of all U.S. oncology patients meet FDA eligibility criteria to receive treatment with an immune checkpoint inhibitor (ICI). With increasing use of ICIs, preventing, diagnosing and treating immune-related adverse events (irAEs) are urgent clinical challenges. Myocarditis is an uncommon irAE, affecting < 1% of ICI-treated patients, but is highly fatal, with a mortality rate of nearly 50%. Genetically altered Pdcd1-/-Ctla4± mice die prematurely and specifically due to myocarditis. This model recapitulates the clinical and pathological features of ICI-myocarditis, including abundant cardiac infiltrating CD8+ T cells. The potential autoantigen(s) involved in ICI-myocarditis are unknown for both human disease and our murine model.MethodsWe used Pdcd1-/-Ctla4± mice on the C57BL6 background as a model of ICI-myocarditis. Single cell RNA and T cell receptor (TCR) sequencing was performed on sorted CD45+ cardiac immune cells from four affected Pdcd1-/-Ctla4± mice compared to six healthy wild type mice. The most three clonal TCRs (TCR-A, B, C), derived from two independent Pdcd1-/-Ctla4± mice, were reconstructed using stiTChR and transduced into reporter T cell lines for antigen discovery. Alpha-myosin was selected as a candidate autoantigen due to lack of presentation in the thymus. Reporter TCR-A, B, and C cells were screened using a library of overlapping 20 amino acid peptides derived from alpha-myosin in co-culture with bone marrow derived dendritic cells.ResultsTreatment with anti-CD8, but not anti-CD4, depleting antibodies rescues survival of Pdcd1-/-Ctla4± mice. Furthermore, adoptive transfer of splenocytes from Pdcd1-/-Ctla4± mice with myocarditis to Rag1-/- recipient mice was sufficient to induce fatal myocarditis. Single cell RNA/TCR sequencing on the cardiac immune infiltrate of Pdcd1-/-Ctla4± mice identified highly activated, clonal CD8+ T cells as the dominant cell population. The TCR-A cell line, the most clonal TCR identified in single cell TCR sequencing, activates NFAT, NFkB, and AP-1 reporters in response to the alpha-myosin epitope VIQYFASI. The TCR-B and TCR-C cell lines activate their reporters in response to the alpha myosin peptide DALLVIQWNIRAFMGVKNWP, indicating that alpha-myosin is an autoantigen in this mouse model of ICI-myocarditis.ConclusionsClonal, activated CD8+ T cells are critical for the development of ICI-myocarditis. Alpha-myosin is an autoantigen recognized by the most clonal cardiac CD8+ T cells. Efforts are currently underway to determine whether human TCRs derived from ICI-myocarditis samples recognize similar antigens. These studies are the first to identify a candidate autoantigen in ICI-myocarditis and may yield new insights into irAE pathogenesis.Ethics ApprovalAll animal experiments were in accordance with the VUMC Institutional Animal Care and Use Committee (IACUC), protocol # M2000067

BackgroundNeuroblastoma (NB) is a childhood cancer for which new treatment options are needed. The success of immune checkpoint blockade in the treatment of adult solid tumors has prompted the exploration of immunotherapy in NB; however, clinical evidence indicates that the vast majority of NB patients do not respond to single-agent checkpoint inhibitors. This motivates a need for therapeutic strategies to increase NB tumor immunogenicity. The goal of this study was to evaluate a new immunotherapeutic strategy for NB based on potent activation of the stimulator of interferon genes (STING) pathway.MethodsTo promote STING activation in NB cells and tumors, we utilized STING-activating nanoparticles (STING-NPs) that are designed to mediate efficient cytosolic delivery of the endogenous STING ligand, 2’3’-cGAMP. We investigated tumor-intrinsic responses to STING activation in both MYCN-amplified and non-amplified NB cell lines, evaluating effects on STING signaling, apoptosis, and the induction of immunogenic cell death. The effects of intratumoral administration of STING-NPs on CD8+T cell infiltration, tumor growth, and response to response to PD-L1 checkpoint blockade were evaluated in syngeneic models of MYCN-amplified and non-amplified NB.ResultsThe efficient cytosolic delivery of 2’3’-cGAMP enabled by STING-NPs triggered tumor-intrinsic STING signaling effects in both MYCN-amplified and non-amplified NB cell lines, resulting in increased expression of interferon-stimulated genes and pro-inflammatory cytokines as well as NB cell death at concentrations 2000-fold to 10000-fold lower than free 2’3’-cGAMP. STING-mediated cell death in NB was associated with release or expression of several danger associated molecular patterns that are hallmarks of immunogenic cell death, which was further validated via cell-based vaccination and tumor challenge studies. Intratumoral administration of STING-NPs enhanced STING activation relative to free 2’3’-cGAMP in NB tumor models, converting poorly immunogenic tumors into tumoricidal and T cell-inflamed microenvironments and resulting in inhibition of tumor growth, increased survival, and induction of immunological memory that protected against tumor re-challenge. In a model of MYCN-amplified NB, STING-NPs generated an abscopal response that inhibited distal tumor growth and improved response to PD-L1 immune checkpoint blockade.ConclusionsWe have demonstrated that activation of the STING pathway, here enabled by a nanomedicine approach, stimulates immunogenic cell death and remodels the tumor immune microenvironment to inhibit NB tumor growth and improve responses to immune checkpoint blockade, providing a multifaceted immunotherapeutic approach with potential to enhance immunotherapy outcomes in NB.