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Patient Demographics and Responses

Supplementary Table S2

Supplementary Figures S1-S7

Supplementary Figures 1-9

Despite the clinical success of T-cell checkpoint blockade, most patients with cancer still fail to have durable responses to immunotherapy. The molecular mechanisms driving checkpoint blockade resistance, whether preexisting or evolved, remain unclear. To address this critical knowledge gap, we treated B16 melanoma with the combination of CTLA-4, PD-1, and PD-L1 blockade and a Flt3 ligand vaccine (≥75% curative), isolated tumors resistant to therapy, and serially passaged them in vivo with the same treatment regimen until they developed complete resistance. Using gene expression analysis and immunogenomics, we determined the adaptations associated with this resistance phenotype. Checkpoint resistance coincided with acquisition of a “hypermetabolic” phenotype characterized by coordinated upregulation of the glycolytic, oxidoreductase, and mitochondrial oxidative phosphorylation pathways. These resistant tumors flourished under hypoxic conditions, whereas metabolically starved T cells lost glycolytic potential, effector function, and the ability to expand in response to immunotherapy. Furthermore, we found that checkpoint-resistant versus -sensitive tumors could be separated by noninvasive MRI imaging based solely on their metabolic state. In a cohort of patients with melanoma resistant to both CTLA-4 and PD-1 blockade, we observed upregulation of pathways indicative of a similar hypermetabolic state. Together, these data indicated that melanoma can evade T-cell checkpoint blockade immunotherapy by adapting a hypermetabolic phenotype.

Flow cytometry antibody catalog

Supplemental Contents and Legends

Coordinated manipulation of independent immune regulatory pathways in the tumor microenvironment—including blockade of T-cell checkpoint receptors and reversal of suppressive myeloid programs—can render aggressive cancers susceptible to immune rejection. Elevated toxicity associated with combination immunotherapy, however, prevents translation of the most efficacious regimens. We evaluated T-cell checkpoint–modulating antibodies targeting CTLA-4, PD-1, and 4-1BB together with myeloid agonists targeting either STING or Flt3 in the TRAMP-C2 model of prostate cancer to determine whether low-dose intratumoral delivery of these agents could elicit systemic control of multifocal disease. Intratumoral administration of the STING agonist cyclic di-GMP (CDG) or Flt3 Ligand (Flt3L) augmented the therapeutic effect of systemic triple checkpoint modulation and promoted the cure of 75% of mice with bilateral TRAMP-C2; however, when all agents were administered locally, only CDG mobilized abscopal immunity. Combination efficacy correlated with globally enhanced ratios of CD8+ T cells to regulatory T cells (Treg), macrophages, and myeloid-derived suppressor cells, and downregulation of the M2 marker CD206 on tumor-associated macrophages. Flt3L improved CD8+ T-cell and dendritic cell infiltration of tumors, but was diminished in efficacy by concomitant Treg expansion. Although intratumoral CDG/checkpoint therapy invokes substantial ulceration at the injection site, reduced CDG dosing can preserve tissue integrity without sacrificing therapeutic benefit. For high-order combinations of T-cell checkpoint antibodies and local myeloid agonists, systemic antibody administration provides the greatest efficacy; however, local administration of CDG and antibody provides substantial systemic benefit while minimizing the potential for immune-related adverse events. Cancer Immunol Res; 5(8); 676–84. ©2017 AACR.

Single-cell analysis clustering for gene profiling.

Purpose: Agonist antibodies targeting the T-cell costimulatory receptor 4-1BB (CD137) are among the most effective immunotherapeutic agents across preclinical cancer models. In the clinic, however, development of these agents has been hampered by dose-limiting liver toxicity. Lack of knowledge of the mechanisms underlying this toxicity has limited the potential to separate 4-1BB agonist–driven tumor immunity from hepatotoxicity.Experimental Design: The capacity of 4-1BB agonist antibodies to induce liver toxicity was investigated in immunocompetent mice, with or without coadministration of checkpoint blockade, via (i) measurement of serum transaminase levels, (ii) imaging of liver immune infiltrates, and (iii) qualitative and quantitative assessment of liver myeloid and T cells via flow cytometry. Knockout mice were used to clarify the contribution of specific cell subsets, cytokines, and chemokines.Results: We find that activation of 4-1BB on liver myeloid cells is essential to initiate hepatitis. Once activated, these cells produce interleukin-27 that is required for liver toxicity. CD8 T cells infiltrate the liver in response to this myeloid activation and mediate tissue damage, triggering transaminase elevation. FoxP3+ regulatory T cells limit liver damage, and their removal dramatically exacerbates 4-1BB agonist–induced hepatitis. Coadministration of CTLA-4 blockade ameliorates transaminase elevation, whereas PD-1 blockade exacerbates it. Loss of the chemokine receptor CCR2 blocks 4-1BB agonist hepatitis without diminishing tumor-specific immunity against B16 melanoma.Conclusions: 4-1BB agonist antibodies trigger hepatitis via activation and expansion of interleukin-27–producing liver Kupffer cells and monocytes. Coadministration of CTLA-4 and/or CCR2 blockade may minimize hepatitis, but yield equal or greater antitumor immunity. Clin Cancer Res; 24(5); 1138–51. ©2018 AACR.

Supplemental Figure 1: Peak of 4-1BB mediate liver transaminase level and gating strategy for flow cytometry analysis of liver immune infiltrates. Supplemental Figure 2: Representative flow cytometry analysis of liver immune infiltrates. Supplemental Figure 3: Administration of 4-1BB agonist antibodies initiates liver pathology through activation of liver-resident myeloid cells. Supplemental Figure 4: Effects of IL-27 pathway inactivation on CD4 T cells. Supplemental Figure 5: Representative flow cytometry analysis of liver immune infiltrates.

Purpose:Intratumoral immunosuppression mediated by myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) represents a potential mechanism of immune checkpoint inhibitor (ICI) resistance in solid tumors. By promoting TAM and MDSC infiltration, IL1β may drive adaptive and innate immune resistance in renal cell carcinoma (RCC) and in other tumor types.Experimental Design:Using the RENCA model of RCC, we evaluated clinically relevant combinations of anti-IL1β plus either anti-PD-1 or the multitargeted tyrosine kinase inhibitor (TKI), cabozantinib. We performed comprehensive immune profiling of established RENCA tumors via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA sequencing (RNA-seq). Similar analyses were extended to the MC38 tumor model.Results:Analyses via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA-seq showed that anti-IL1β reduces infiltration of polymorphonuclear MDSCs and TAMs. Combination treatment with anti-IL1β plus anti-PD-1 or cabozantinib showed increased antitumor activity that was associated with decreases in immunosuppressive MDSCs and increases in M1-like TAMs.Conclusions:Single-cell RNA-seq analyses show that IL1β blockade and ICI or TKI remodel the myeloid compartment through nonredundant, relatively T-cell–independent mechanisms. IL1β is an upstream mediator of adaptive myeloid resistance and represents a potential target for kidney cancer immunotherapy.

Immune cell phenotyping of tumor draining lymph nodes from RENCA tumor bearing mice.

Visualization of top 5 upregulated RNA transcripts associated with each cluster.

Clusters from Intratumoral Immunocytes by Spectral Cytometry.

Transcript level assessment of angiogenic signatures in CD45+ intratumoral cells with anti-IL-1b and cabozantinib

Top 30 immune genes from Nanonstring I/O 360 gene list for clusters 1, 4, 8, and 12.

Comparison of combination anti-PD1/anti-IL1b and cabozantinib/anti-IL1b treatment.

Assessment of TME changes in MC38 tumors with combination cabozantinib and anti-interleukin-1b immunotherapy.

Evaluation of T cell depletion on the efficacy of combination cabozantinib and anti-interleukin-1b immunotherapy.

Current methods for biomarker discovery and target identification in immuno-oncology rely on static snapshots of tumor immunity. To thoroughly characterize the temporal nature of antitumor immune responses, we developed a 34-parameter spectral flow cytometry panel and performed high-throughput analyses in critical contexts. We leveraged two distinct preclinical models that recapitulate cancer immunoediting (NPK-C1) and immune checkpoint blockade (ICB) response (MC38), respectively, and profiled multiple relevant tissues at and around key inflection points of immune surveillance and escape and/or ICB response. Machine learning-driven data analysis revealed a pattern of KLRG1 expression that uniquely identified intratumoral effector CD4 T cell populations that constitutively associate with tumor burden across tumor models, and are lost in tumors undergoing regression in response to ICB. Similarly, a Helios-KLRG1+subset of tumor-infiltrating regulatory T cells (Tregs) was associated with tumor progression from immune equilibrium to escape, and were also lost in tumors responding to ICB. Validation studies confirmed KLRG1 signatures in human tumorinfiltrating CD4 T cells associate with disease progression in renal cancer. These findings nominate KLRG1+CD4 T cell populations as subsets for further investigation in cancer immunity and demonstrate the utility of longitudinal spectral flow profiling as an engine of dynamic biomarker and/or target discovery.

SUMMARYCompared to other malignancies, the tumor microenvironment (TME) of primary and castration-resistant prostate cancer (CRPC) is relatively devoid of immune infiltrates. While androgen deprivation therapy (ADT) induces a complex immune infiltrate in localized prostate cancer, both in animal models and humans, the TME composition of metastatic, castration-sensitive prostate cancer (mCSPC) is relatively unknown and the effects of ADT and other treatments are poorly characterized in this context. To address this challenge, we analyzed metastatic sites from patients enrolled on a phase 2 clinical trial (NCT03951831), in which men were treated with standard-of-care chemo-hormonal therapy with anti-PD-1 immunotherapy, at the single cell level. Longitudinal protein activity-based analysis of TME subpopulations identified immune subpopulations conserved across multiple metastatic sites, their dynamic, treatment-mediated evolution, and associated clinical response features. Our study revealed a therapy-resistant, transcriptionally distinct tumor subpopulation, which comprises an increasing number of cells in treatment-refractory patients, and identified several druggable targets in both tumor and immune cells as candidates to advance treatment and improve outcomes for patients with mCSPC.

The cell autonomous balance of immune-inhibitory and -stimulatory signals is a critical yet poorly understood process in cancer immune evasion. Using patient-derived co-culture models and humanized mouse models, we show that an intact CD58:CD2 interaction is necessary for anti-tumor immunity. Defects in this axis lead to multi-faceted immune evasion through impaired CD2-dependent T cell polyfunctionality, T cell exclusion, impaired intra-tumoral proliferation, and concurrent protein stabilization of PD-L1. We performed genome-scale CRISPR-Cas9 and CD58 coimmunoprecipitation mass spectrometry screens identifying CMTM6 as a key stabilizer of CD58, and show that CMTM6 is required for concurrent upregulation of PD-L1 in CD58 loss. Single-cell RNA-seq analysis of patient melanoma samples demonstrates that most TILs lack expression of primary costimulatory signals required for response to PD-1 blockade (e.g. CD28), but maintain strong CD2 expression, thus providing an opportunity to mobilize a so far therapeutically untapped pool of TILs for anti-tumor immunity. We identify two potential therapeutic avenues, including rescued activation of human CD2-expressing TILs using recombinant CD58 protein, and targeted disruption of PD-L1/CMTM6 interactions. Our work identifies an underappreciated yet critical axis at the nexus of cancer immunity and evasion, uncovers a fundamental mechanism of co-inhibitory and -stimulatory signal balancing, and provides new approaches to improving cancer immunotherapies.

Significance As we age, the innate immune system becomes dysregulated and is characterized by persistent inflammatory responses, and the chronic inflammation mediated by inflammatory receptors represents a key mechanism by which amyloid-beta (Aβ) drives the development of cognitive decline in Alzheimer’s disease (AD). A crucial aspect of this process is a failure to resolve inflammation, which involves the suppression of inflammatory cell influx and the endocytosis of inflammatory receptors. We found that ablation of the endosomal adaptor target of Myb1 (TOM1) worsens neuroinflammation, impairs microglial phagocytosis, and significantly exacerbates amyloid deposition. Conversely, restoration of TOM1 reverses these effects and reduces Aβ pathology. These results highlight the importance of endosomal adaptors and their interaction with inflammatory receptors in the pathogenesis of AD., Defects in interleukin-1β (IL-1β)–mediated cellular responses contribute to Alzheimer’s disease (AD). To decipher the mechanism associated with its pathogenesis, we investigated the molecular events associated with the termination of IL-1β inflammatory responses by focusing on the role played by the target of Myb1 (TOM1), a negative regulator of the interleukin-1β receptor-1 (IL-1R1). We first show that TOM1 steady-state levels are reduced in human AD hippocampi and in the brain of an AD mouse model versus respective controls. Experimentally reducing TOM1 affected microglia activity, substantially increased amyloid-beta levels, and impaired cognition, whereas enhancing its levels was therapeutic. These data show that reparation of the TOM1-signaling pathway represents a therapeutic target for brain inflammatory disorders such as AD. A better understanding of the age-related changes in the immune system will allow us to craft therapies to limit detrimental aspects of inflammation, with the broader purpose of sharply reducing the number of people afflicted by AD.

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.

Regulatory T cells (Tregs) are highly attractive targets for immunotherapy development, however, there remains a critical need for clinically actionable targeting strategies that specifically inhibit human tumor-infiltrating Tregs (TI-Tregs) while preserving function of cytotoxic effectors and peripheral non-tumor Tregs (P-Tregs). To this end, we sought to identify, validate, and target novel master regulators of TI-Tregs by leveraging a suite of next-generation bioinformatic tools and rigorous ex vivo and in vivo screening and validation methodologies. Specifically, we performed VIPER (Virtual Inference of Protein Activity) analysis on a large dataset of T cell transcriptional profiles from matched peripheral blood and tumors of 36 human patients, yielding 17 master regulator (MR) proteins predicted to uniquely drive the TI-Treg phenotype across cancers. To identify putative therapeutics that modulate TI-Treg MRs, we performed a systematic ex vivo drug screen with an unbiased panel of 1,554 FDA-approved and experimental compounds coupled to RNA sequencing (PLATE-Seq) on human TI-Tregs and P-Tregs. Drug candidates with preferential cytotoxic activity on TI-Tregs versus P-Tregs that also reversed MR transcriptional activity were thoroughly validated in vivo in the MC38 tumor model. In parallel, we performed a pooled in vivo CRISPR/Cas9 screen via the CHIME (CHimeric IMmune Editing) system to identify candidate MRs that regulate TI-Treg recruitment to and/or retention within MC38 tumors. By these approaches, we successfully validated an as-yet poorly described TI-Treg MR, TRPS1 (Transcriptional Repressor GATA Binding 1), which after genetic deletion across the hematopoietic compartment diminished TI-Tregs while preserving P-Tregs in MC38-bearing mice, and led to enhanced spontaneous control of MCA205 sarcomas. In addition, we found the widely used nucleoside analog chemotherapeutic Gemcitabine exhibits preferential inhibitory activity against human TI-Tregs versus P-Tregs, and validated in mice that at sub-clinical dose levels Gemcitabine exhibits immune-dependent therapeutic activity that significantly potentiates checkpoint blockade control of late-stage MC38 tumors. By single cell RNA sequencing of TI- and P-Tregs flow-sorted from Gemcitabine-treated mice, we observe specific depletion of a TI-Treg subset enriched for expression of TI-Treg MRs, including TRPS1. Together, these studies reveal new putative regulators of human TI-Tregs and identify a readily available clinical therapeutic that exhibits inhibitory activity against them. These findings specifically warrant future investigation into the role of TRPS1 in TI-Treg activity and clinical evaluation of low-dose Gemcitabine as a Treg-targeting strategy capable of enhancing checkpoint blockade immunotherapy. Citation Format: Casey R. Ager, Aleksandar Obradovic, Mikko Turunen, Mohsen Khosravi-Maharlooei, Charles Karan, Andrea Califano, Charles G. Drake. Elucidating and targeting master regulators of tumor infiltrating regulatory T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3612.

Despite the clinical success of T-cell checkpoint blockade, most patients with cancer still fail to have durable responses to immunotherapy. The molecular mechanisms driving checkpoint blockade resistance, whether preexisting or evolved, remain unclear. To address this critical knowledge gap, we treated B16 melanoma with the combination of CTLA-4, PD-1, and PD-L1 blockade and a Flt3 ligand vaccine (≥75% curative), isolated tumors resistant to therapy, and serially passaged them in vivo with the same treatment regimen until they developed complete resistance. Using gene expression analysis and immunogenomics, we determined the adaptations associated with this resistance phenotype. Checkpoint resistance coincided with acquisition of a “hypermetabolic” phenotype characterized by coordinated upregulation of the glycolytic, oxidoreductase, and mitochondrial oxidative phosphorylation pathways. These resistant tumors flourished under hypoxic conditions, whereas metabolically starved T cells lost glycolytic potential, effector function, and the ability to expand in response to immunotherapy. Furthermore, we found that checkpoint-resistant versus -sensitive tumors could be separated by noninvasive MRI imaging based solely on their metabolic state. In a cohort of patients with melanoma resistant to both CTLA-4 and PD-1 blockade, we observed upregulation of pathways indicative of a similar hypermetabolic state. Together, these data indicated that melanoma can evade T-cell checkpoint blockade immunotherapy by adapting a hypermetabolic phenotype.

Pancreatic ductal adenocarcinoma (PDAC) is clinically unresponsive to immune checkpoint blockade (ICB) immunotherapy. Dense immunosuppressive myeloid stroma (MS) and consequent T cell exclusion from the tumour microenvironment renders PDAC resistant to immune-based therapies. Innate immune activation of the MS via cyclic dinucleotide (CDN) agonists of the STING (Stimulator of Interferon Genes) pathway can trigger T cell infiltration into cold tumours leading to robust anti-tumour immunity. Despite proven therapeutic efficacy in preclinical models, the cellular mechanisms of how CDNs reprogram the suppressive MS to sensitise tumours to ICB is poorly understood. Using multi-omic profiling of MDSCs and M2 Macrophages of human and murine origin, we show that the synthetic STING agonist, IACS-8803, rewires these populations from immunosuppressive to immune-permissive phenotypes in part through inhibition of c-Myc signaling, energy metabolism modulation, and antagonism of cell cycle. Furthermore, dimensionality reduction analyses of multiparameter flow cytometry data and survival studies in a KPC-derived model of PDAC show a proinflammatory remodeling of the MS and an increased T/NK cell infiltration and function by synthetic CDN STING agonists into the tumour that underlies an amplified therapeutic response to ICB. We are the first to report that synthetic CDN STING agonists affect MDSC and M2 macrophage repolarization through altering energy metabolism, cell cycle dynamics and c-Myc signalling. This study uncovers molecular and cellular mechanisms by which high-potency STING agonists drive a proinflammatory conversion of the tumour MS to overcome resistance to ICB in an aggressive orthotopic tumour model of PDAC. Supported by grants from PanCan (206100-90-100300-54-00006617-11111)

Despite the success of immune checkpoint blockade against melanoma, many "cold" tumors like prostate cancer remain unresponsive. We found that hypoxic zones were prevalent across preclinical prostate cancer and resisted T cell infiltration even in the context of CTLA-4 and PD-1 blockade. We demonstrated that the hypoxia-activated prodrug TH-302 reduces or eliminates hypoxia in these tumors. Combination therapy with this hypoxia-prodrug and checkpoint blockade cooperated to cure more than 80% of tumors in the transgenic adenocarcinoma of the mouse prostate-derived (TRAMP-derived) TRAMP-C2 model. Immunofluorescence imaging showed that TH-302 drives an influx of T cells into hypoxic zones, which were expanded by checkpoint blockade. Further, combination therapy reduced myeloid-derived suppressor cell density by more than 50%, and durably reduced the capacity of the tumor to replenish the granulocytic subset. Spontaneous prostate tumors in TRAMP transgenic mice, which completely resist checkpoint blockade, showed minimal adenocarcinoma tumor burden at 36 weeks of age and no evidence of neuroendocrine tumors with combination therapy. Survival of Pb-Cre4, Ptenpc-/-Smad4pc-/- mice with aggressive prostate adenocarcinoma was also significantly extended by this combination of hypoxia-prodrug and checkpoint blockade. Hypoxia disruption and T cell checkpoint blockade may sensitize some of the most therapeutically resistant cancers to immunotherapy.

Purpose: Agonist antibodies targeting the T-cell costimulatory receptor 4-1BB (CD137) are among the most effective immunotherapeutic agents across preclinical cancer models. In the clinic, however, development of these agents has been hampered by dose-limiting liver toxicity. Lack of knowledge of the mechanisms underlying this toxicity has limited the potential to separate 4-1BB agonist–driven tumor immunity from hepatotoxicity. Experimental Design: The capacity of 4-1BB agonist antibodies to induce liver toxicity was investigated in immunocompetent mice, with or without coadministration of checkpoint blockade, via (i) measurement of serum transaminase levels, (ii) imaging of liver immune infiltrates, and (iii) qualitative and quantitative assessment of liver myeloid and T cells via flow cytometry. Knockout mice were used to clarify the contribution of specific cell subsets, cytokines, and chemokines. Results: We find that activation of 4-1BB on liver myeloid cells is essential to initiate hepatitis. Once activated, these cells produce interleukin-27 that is required for liver toxicity. CD8 T cells infiltrate the liver in response to this myeloid activation and mediate tissue damage, triggering transaminase elevation. FoxP3+ regulatory T cells limit liver damage, and their removal dramatically exacerbates 4-1BB agonist–induced hepatitis. Coadministration of CTLA-4 blockade ameliorates transaminase elevation, whereas PD-1 blockade exacerbates it. Loss of the chemokine receptor CCR2 blocks 4-1BB agonist hepatitis without diminishing tumor-specific immunity against B16 melanoma. Conclusions: 4-1BB agonist antibodies trigger hepatitis via activation and expansion of interleukin-27–producing liver Kupffer cells and monocytes. Coadministration of CTLA-4 and/or CCR2 blockade may minimize hepatitis, but yield equal or greater antitumor immunity. Clin Cancer Res; 24(5); 1138–51. ©2018 AACR.

Coordinated manipulation of independent immune regulatory pathways in the tumor microenvironment—including blockade of T-cell checkpoint receptors and reversal of suppressive myeloid programs—can render aggressive cancers susceptible to immune rejection. Elevated toxicity associated with combination immunotherapy, however, prevents translation of the most efficacious regimens. We evaluated T-cell checkpoint–modulating antibodies targeting CTLA-4, PD-1, and 4-1BB together with myeloid agonists targeting either STING or Flt3 in the TRAMP-C2 model of prostate cancer to determine whether low-dose intratumoral delivery of these agents could elicit systemic control of multifocal disease. Intratumoral administration of the STING agonist cyclic di-GMP (CDG) or Flt3 Ligand (Flt3L) augmented the therapeutic effect of systemic triple checkpoint modulation and promoted the cure of 75% of mice with bilateral TRAMP-C2; however, when all agents were administered locally, only CDG mobilized abscopal immunity. Combination efficacy correlated with globally enhanced ratios of CD8+ T cells to regulatory T cells (Treg), macrophages, and myeloid-derived suppressor cells, and downregulation of the M2 marker CD206 on tumor-associated macrophages. Flt3L improved CD8+ T-cell and dendritic cell infiltration of tumors, but was diminished in efficacy by concomitant Treg expansion. Although intratumoral CDG/checkpoint therapy invokes substantial ulceration at the injection site, reduced CDG dosing can preserve tissue integrity without sacrificing therapeutic benefit. For high-order combinations of T-cell checkpoint antibodies and local myeloid agonists, systemic antibody administration provides the greatest efficacy; however, local administration of CDG and antibody provides substantial systemic benefit while minimizing the potential for immune-related adverse events. Cancer Immunol Res; 5(8); 676–84. ©2017 AACR.

Alzheimer’s disease (AD), the most common age-related neurodegenerative disorder, is currently conceptualized as a disease of synaptic failure. Synaptic impairments are robust within the AD brain and better correlate with dementia severity when compared with other pathological features of the disease. Nevertheless, the series of events that promote synaptic failure still remain under debate, as potential triggers such as β-amyloid (Aβ) can vary in size, configuration and cellular location, challenging data interpretation in causation studies. Here we present data obtained using adeno-associated viral (AAV) constructs that drive the expression of oligomeric Aβ either intra or extracellularly. We observed that expression of Aβ in both cellular compartments affect learning and memory, reduce the number of synapses and the expression of synaptic-related proteins, and disrupt chemical long-term potentiation (cLTP). Together, these findings indicate that during the progression AD the early accumulation of Aβ inside neurons is sufficient to promote morphological and functional cellular toxicity, a phenomenon that can be exacerbated by the buildup of Aβ in the brain parenchyma. Moreover, our AAV constructs represent a valuable tool in the investigation of the pathological properties of Aβ oligomers both in vivo and in vitro.

Diabetes mellitus (DM) is one of the most devastating diseases that currently affects the aging population. Recent evidence indicates that DM is a risk factor for many brain disorders, due to its direct effects on cognition. New findings have shown that the microtubule‐associated protein tau is pathologically processed in DM; however, it remains unknown whether pathological tau modifications play a central role in the cognitive deficits associated with DM. To address this question, we used a gain‐of‐function and loss‐of‐function approach to modulate tau levels in type 1 diabetes (T1DM) and type 2 diabetes (T2DM) mouse models. Our study demonstrates that tau differentially contributes to cognitive and synaptic deficits induced by DM. On one hand, overexpressing wild‐type human tau further exacerbates cognitive and synaptic impairments induced by T1DM, as human tau mice treated under T1DM conditions show robust deficits in learning and memory processes. On the other hand, neither a reduction nor increase in tau levels affects cognition in T2DM mice. Together, these results shine new light onto the different molecular mechanisms that underlie the cognitive and synaptic impairments associated with T1DM and T2DM.

Despite intensive research efforts over the past few decades, the mechanisms underlying the etiology of sporadic Alzheimer's disease (AD) remain unknown. This fact is of major concern because the number of patients affected by this medical condition is increasing exponentially and the existing treatments are only palliative in nature and offer no disease modifying affects. Interestingly, recent epidemiological studies indicate that diabetes significantly increases the risk of developing AD, suggesting that diabetes may play a causative role in the development of AD pathogenesis. Therefore, elucidating the molecular interactions between diabetes and AD is of critical significance because it might offer a novel approach to identifying mechanisms that may modulate the onset and progression of sporadic AD cases. This review highlights the involvement of several novels pathological molecular mechanisms induced by diabetes that increase AD pathogenesis. Furthermore, we discuss novel findings in animal model and clinical studies involving the use of anti-diabetic compounds as promising therapeutics for AD.

Advances in our understanding of tumor immune biology and development of cancer immunotherapies have led to improved outcomes for patients who suffer from aggressive cancers such as melanoma. Despite the clinical success of immune checkpoint blockade, a majority of patients still fail to respond, and the underlying mechanisms that drive resistance remain unclear. To understand why a subset of tumors fail to respond to immunotherapy, we established a novel murine model of melanoma that is fully resistant to immune checkpoint blockade. By in vivo passaging nonresponding B16 melanoma tumor cells, we selected for a resistant variant that fails to respond to the combination of CTLA-4, PD-1, and PD-L1 blockade. In comparing gene expression of parental versus resistant tumor cells and analyzing the corresponding immune infiltrate, we determined the adaptations associated with resistance to therapy. We found that evasion of immunotherapy was associated with a “hypermetabolic” phenotype, characterized by an upregulation of glycolytic, oxidoreductase, and mitochondrial oxidative phosphorylation pathways to establish a hypoxic, metabolically hostile microenvironment. Enforced expression of two key genes associated with these pathways in parental tumor cells was sufficient to mediate resistance to triple checkpoint blockade. Flow cytometry assays determined that T cells infiltrating resistant tumors had diminished glycolytic capacity and effector function, indicating a metabolic disadvantage. Consistent with our findings, melanoma patients who failed dual checkpoint blockade exhibited similar metabolic alterations as seen in our resistant variant. Using a novel MRI-based imaging approach, we observed distinct metabolic changes that stratified responding versus nonresponding tumors in live mice. Applying this method to patients could provide insight into predicting response rates to checkpoint modulation. Overall, our data indicate that resistant melanoma tumor cells acquire a “hypermetabolic” phenotype to establish a hostile microenvironment that is capable of inhibiting the antitumor immune response. This abstract is also being presented as Poster A24. Citation Format: Ashvin R. Jaiswal, Arthur J. Liu, Shivanand Pudakalakatti, Prasanta Dutta, Priyamvada Jayaprakash, Todd Bartkowiak, Casey Ager, Zhiqiang Wang, Alex Reuben, Zachary Cooper, Cristina Ivan, Zhenlin Ju, Felix Nwajei, Jing Wang, Michael A. Davies, R. Eric Davis, Jennifer A. Wargo, Pratip K. Bhattacharya, David S. Hong, Michael A. Curran. Melanoma evolves complete immunotherapy resistance through acquisition of a hypermetabolic phenotype [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR02.

The majority of Alzheimer’s disease (AD) cases are late-onset and occur sporadically, however most mouse models of the disease harbor pathogenic mutations, rendering them better representations of familial autosomal-dominant forms of the disease. Here, we generated knock-in mice that express wildtype human Aβ under control of the mouse App locus. Remarkably, changing 3 amino acids in the mouse Aβ sequence to its wild-type human counterpart leads to age-dependent impairments in cognition and synaptic plasticity, brain volumetric changes, inflammatory alterations, the appearance of Periodic Acid-Schiff (PAS) granules and changes in gene expression. In addition, when exon 14 encoding the Aβ sequence was flanked by loxP sites we show that Cre-mediated excision of exon 14 ablates hAβ expression, rescues cognition and reduces the formation of PAS granules., Most instances of Alzheimer’s disease (AD) are sporadic or not associated with a particular mutation. Here, the authors develop knock-in mice that express wildtype human Aβ under control of the mouse App locus, which may have potential for modelling some aspects of sporadic late onset AD.

Activation of the stimulator of interferon genes (STING) pathway by both exogenous and endogenous cytosolic DNA results in the production of interferon beta (IFN-β) and is required for the generation of cytotoxic T-cell priming against tumor antigens. In the clinical setting, pharmacological stimulation of the STING pathway has the potential to synergize with immunotherapy antibodies by boosting anti-tumor immune responses. We report the discovery of two highly potent cyclic dinucleotide STING agonists, IACS-8803 and IACS-8779, which show robust activation of the STING pathway in vitro and a superior systemic anti-tumor response in the B16 murine model of melanoma when compared to one of the clinical benchmark compounds.

Immune checkpoint blockade is effective in “hot” tumors like melanoma with pre-existing immune infiltrates; however, “cold” tumors like prostate cancer fail to respond. We found that prostate cancers harbor regions of hypoxia that resist T cell infiltration even in the context of anti-CTLA-4 (cytotoxic T lymphocyte associated protein-4) and anti-PD-1 (programmed cell death protein 1) blockade. These hypoxic zones serve as islands of immune privilege through the recruitment and suppressive polarization of immature myeloid cells into myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM). We found that targeted hypoxia ablation using TH-302, a hypoxia-activated prodrug, sensitized both transplantable and spontaneous models of prostate cancer to checkpoint blockade, coincident with enhanced T cell infiltration and effector function and loss of MDSC recruitment and suppressive function. Tumors treated with the combination of TH-302 and checkpoint blockade showed a reduced capacity to suppressively polarize new myeloid immigrants, implying a durable reconditioning of the tumor microenvironment (TME) into an immune-infiltrated, pro-inflammatory milieu. T cells infiltrating combination-treated tumors exhibited increased mitochondrial respiration, consistent with creation of a metabolically favorable milieu for T cell function. Based on these findings, we hypothesized that other approaches capable of metabolically rewiring the TME should promote anti-tumor immunity and sensitize checkpoint blockade-resistant tumors to immunotherapy. With this in mind, we performed a longitudinal study comparing a panel of different mitochondrial respiration inhibitors and a glutaminase inhibitor for their efficacy in reducing hypoxia, improving T cell infiltration and decreasing myeloid cell recruitment and suppressive polarization using immunofluorescence staining and confocal microscopy. Our preliminary data suggests that inhibitors targeting mitochondrial respiration, rather than those targeting glutamine metabolism synergize with checkpoint blockade and exhibit the highest efficacy in increasing T cell recruitment. We continue to characterize the dynamics of hypoxia reduction, duration of normalization following drug withdrawal, and impact on the immune microenvironment of these diverse approaches to metabolic reconditioning. Citation Format: Priyamvada Jayaprakash, Midan Ai, Arthur Liu, Pratha Budhani, Todd Bartkowiak, Jie Sheng, Casey Ager, Courtney Nicholas, Ashvin Jaiswal, Yanqiu Sun, Krishna Shah, Sadhana Balasubramanyam, Nan Li, Guocan Wang, Jing Ning, Anna Zal, Tomasz Zal, Michael Curran. Targeting hypoxia-induced immune suppression to overcome immunotherapy resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5011.

TPS2669 Background: Drugs targeting pathogen associated molecular patterns are an attractive strategy to stimulate the immune system. Toll-like receptors (TLR) have generated significant interest as an effective means of stimulating the immune system that result in the killing of tumor cells. TLR-9 agonists can function to promote an early immune response and are an appealing partner for combination with checkpoint blockade to improve immune activation. Lefitolimod (MGN1703) is a covalently closed dumbbell-shaped DNA molecule that functions as a TLR-9 agonist. We developed a clinical trial combining lefitolimod with ipilimumab (anti-CTLA4) in patients with advanced malignancies. In the dose-escalation phase 19 patients were enrolled and no DLTs were encountered. Safety data and maximum planned dose level of lefitolimod at 120mg weekly and ipilimumab 3mg/kg every 3 weeks was previously presented. Adverse events related to the combination included fatigue, appetite loss, rash, and anemia. Methods: This trial (NCT02668770) was designed to evaluate the safety profile and maximum tolerated dose of lefitolimod with ipilimumab. A 3+3 trial design was used to establish safety of the combination at each dose level and guide the decision to escalate dose. Lefitolimod is administered via subcutaneous (SC) injection weekly while ipilimumab is given at 3mg/kg intravenous on day 8 of each 3 week cycle. Lefitolimod starting dose was 15mg SC weekly with 3 dose level escalations up to 120mg SC weekly. Patients receive treatment for 4 cycles (total 12 weeks) with the combination, and those with stable disease or response were eligible to remain on lefitolimod therapy for up to 1 year. Eligible patients have a metastatic or unresectable solid tumor refractory to standard therapies, ECOG ≤ 2, and normal organ and bone marrow function. Patients are allowed to have received prior checkpoint blockade agents. Enrollment in expansion cohorts in ongoing. To better understand relevant immunologic changes associated with treatment, paired pre- and post-treatment biopsies of target lesions and peripheral blood collection during treatment is required for target expansion cohort patient populations. In addition to evaluating target patient populations at the combination dose established during escalation, an expansion cohort for patients with cutaneous metastases involves combination treatment with intratumoral delivery of lefitolimod. Clinical trial information: NCT02668770.

The incidence and prevalence of neurodegenerative disorders (e.g., Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD), etc.) are growing rapidly due to increasing life expectancy. Researchers over the past two decades have focused their efforts on the development of animal models to dissect the molecular mechanisms underlying neurodegenerative disorders. Existing models, however, do not fully replicate the symptomatic and pathological features of human diseases. This chapter focuses on animal models of AD, as this disorder is the most prevalent of the brain degenerative conditions afflicting society. In particular, it briefly discusses the current leading animal models, the translational relevance of the preclinical studies using such models, and the limitations and shortcomings of using animals to model human disease. It concludes with a discussion of potential means to improve future models to better recapitulate human conditions.

Immunosuppressive myeloid populations including tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) are abundant within pancreatic adenocarcinoma (PDAC) tumors and play critical roles in constraining cytotoxic T-cell function in the tumor microenvironment. We hypothesized that intratumoral engagement of innate pathogen recognition receptors such as Stimulator of Interferon Genes (STING) could induce proinflammatory polarization of the myeloid stroma and liberate the antitumor T-cell response to regress refractory PDAC tumors in the presence of checkpoint blockade.We developed and characterized a novel cyclic dinucleotide (CDN) STING agonist IACS-8803, and found that 8803 activates downstream STING signaling in both human (THP-1) and murine (J774) myeloid reporter cells with over 10-fold greater potency than ML-RR-S2-CDA, the first-in-class CDN currently undergoing clinical evaluation (NCT02675439, NCT03172936). Intratumoral delivery of 8803 into subcutaneous B16 melanoma and PDAC tumors additionally revealed a greater capacity to induce tumor regression relative to ML-RR-S2-CDA. In order to evaluate the specific effects of 8803 on the phenotype and function of suppressive myeloid populations, we generated in vitro polarized human M2 macrophages and murine bone marrow-derived MDSC. Upon exposure to 8803, we observe downregulation of M2 markers CD163, LAP/TGF-β, and Arginase on human M2 macrophages, concomitant with upregulation of M1 markers CD86, CD80, and IL-6. Additionally, 8803-stimulated murine MDSC exhibit reduced T-cell suppressive capacity compared to unstimulated MDSC. In these studies, we consistently observe that the magnitude of phenotypic and functional repolarization by 8803 is superior to that of ML-RR-S2-CDA as well as other known CDN, 2’3’-cGAMP and c-di-GMP. Therefore, we describe IACS-8803 as a novel, highly potent STING agonist with the capacity to induce inflammatory repolarization in suppressive myeloid cells of both human and murine origin. We next investigated the capacity for intratumoral delivery of IACS-8803 to sensitize murine pancreatic cancer to checkpoint blockade and to mobilize systemic immunity against disseminated lesions. We utilized mT4-2D, a novel pancreatic cancer cell line from Kras+/LSL-G12D Tp53+/LSL-R172H Pdx1-Cre tumor organoids. We isolateda single cell clone of mT4-2D with reduced in vivo growth kinetics (termed mT4-LS), as well as a clone that maintains the aggressive nature of the parental line (termed mT4-LA). Mice bearing 10-day established orthotopic and subcutaneous mT4-LS tumors received standard regimens of αCTLA-4, αPD-1, or combined αCTLA-4/αPD-1 in the presence or absence of 8803 CDN injected into the orthotopic pancreatic tumor. We find single-agent treatment with 8803, αCTLA-4, αPD-1, or αCTLA-4/αPD-1 can cure 40-60% of mice of both orthotopic and subcutaneous tumors in this system; however, combining 8803 with checkpoint blockade completely eradicates both injected and distal mT4-LS tumors in all mice. We replicated these studies using the highly aggressive and refractory mT4-LA model, and found that combination therapy with intra-pancreatic 8803 and systemic αCTLA-4/αPD-1 significantly extends survival compared to 8803 or αCTLA-4/αPD-1 alone (p=0.001, p=0.0086, respectively). Analysis of treated mT4-LA tumors by flow cytometry reveals that combination therapy enhances the cytotoxic potential of CD8 T-cells at both injected and uninjected lesions, and promotes dendritic cell proliferation within the pancreatic milieu. These studies provide a preclinical rationale for pursuing the use of STING-activating CDN as a localized approach to sensitize refractory PDAC tumors to checkpoint blockade immunotherapy. Citation Format: Casey R. Ager, Maria E. Di Francesco, Philip Jones, Michael A. Curran. Intratumoral delivery of a novel STING agonist synergizes with checkpoint blockade to regress multifocal pancreatic cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A050.

Background: Resistance of pancreatic ductal adenocarcinoma (PDAC) to checkpoint blockade therapy is commonly attributed to its status as an immunologically cold tumor. However, it remains to be determined whether the lack of endogenous immunity to PDAC is predominantly due to a paucity of targetable neoantigens or to the unique tolerogenic environment of malignant pancreatic tissue. Using an implantable model derived from the Kras+/LSL-G12DTrp53+/LSL-R172HPdx1-Cre (KPC) mouse, we investigated how tissue implantation site and exogenous antigen presentation influenced immune surveillance of KPC tumors and response to combination immunotherapy. Methods: The KPC organoid-derived cell line mT4-2D was stably transduced with various expression constructs including luciferase (mT4-Luc), ovalbumin (mT4-OVA), or mCherry (mT4-Cherry). Each line was implanted subcutaneously (2.5x105 cells) or orthotopically at indicated doses to assess effects of exogenous antigen expression on engraftment efficiency. To evaluate how tissue environment governs response to therapy, mice were co-implanted with mT4-Luc at both orthotopic and subcutaneous sites and were exposed to combination therapy consisting of intratumoral STING agonist ML-RR-CDA (10ug/mouse) and checkpoint antagonists αCTLA-4 and αPD-1 beginning on day 14 post-implantation. Tumor growth at both sites was monitored weekly by IVIS bioluminescent imaging and mice were followed for survival, or tumors were harvested on day 25 for analysis of immune infiltrates by flow cytometry. Results: mT4 tumors grew aggressively at both subcutaneous and orthotopic sites, with doses as low as 3.5x104 causing mortality within 3 weeks post-implantation. However, expression of either OVA or mCherry resulted in spontaneous clearance in 100% of mice, regardless of implantation site. Luciferase expression yielded progressing tumors, and ex vivo peptide restimulation failed to identify Luciferase-specific T cells within mT4-Luc challenged mice; suggesting that Luciferase is a minimally immunogenic protein in C57BL/6 mice. Despite the poor immunogenicity of the mT4 model, we found that intratumoral injections of ML-RR-CDA delayed growth of subcutaneously-implanted mT4, and that concomitant administration of αCTLA-4/αPD-1 induced complete clearance in ~40% of mice. In contrast, intratumoral delivery of ML-RR-CDA was ineffective against orthotopic mT4 tumors, and combination therapy with αCTLA-4/αPD-1 achieved only modest delays in tumor progression. Flow cytometric analysis of tumor infiltrating immune populations in mice with co-implanted orthotopic and subcutaneous lesions revealed dichotomies in the density of CD8 T cells, CD4 effector T cells, and dendritic cell populations between tissue sites in response to local therapy. Conclusions: Although implantable cell lines isolated from KPC tumors are highly aggressive and resistant models of PDAC, we found that engraftment and growth of mT4-2D was completely negated upon forced expression of a single foreign protein antigen. This suggests that the lack of targetable mutanome products may explain the resistance of KPC and potentially human PDAC tumors to immunotherapy. However, when implanted subcutaneously, mT4-2D was sensitive to in situ STING activation in combination with checkpoint blockade, which indicates that immune responses against tissue-specific antigens are potentially sufficient to control growth of KPC tumors when removed from the suppressive pancreatic environment. Flow analysis revealed a greater capacity of CD8 T cells, CD4 T cells, and dendritic cells to infiltrate subcutaneous vs orthotopic tumors following intratumoral therapy, reinforcing the theory that therapies which enhance infiltration of select immune populations are needed in order to sensitize PDAC tumors to therapy. Citation Format: Casey R. Ager, Michael A. Curran. Effects of tissue site and antigenicity on KPC-derived pancreatic tumor growth and response to combination immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B52.

Background: Despite the success of T-cell checkpoint blockade antibodies in treating an array of cancers, the majority of patients still fail to respond to these therapies, or respond transiently and then relapse. The molecular mechanisms that drive lack of response to checkpoint blockade, whether pre-existing or evolved on therapy, remain unclear. Materials and Methods: To address this critical gap in clinical knowledge, we established a mouse model of melanoma designed to elucidate the molecular mechanisms underlying immunotherapy resistance. Through multiple in vivo passages, we selected a B16 melanoma tumor line that evolved complete resistance to combination blockade of CTLA-4, PD-1, and PD-L1, which cures ~80% of mice of the parental tumor. Using gene expression analysis, proteomics, and immunogenomics, we determined the adaptations engaged by this melanoma to become completely immunotherapy resistant. NMR spectroscopy, Seahorse XF analysis, flow cytometry, confocal microscopy, and Western blot analysis provided further insight into the mechanisms driving checkpoint blockade resistance. Results: Acquisition of immunotherapy resistance by these melanomas was driven by coordinate upregulation of the glycolytic and aldose reductase pathways to create a metabolically hostile microenvironment in which T-cell function is profoundly suppressed. When reintroduced into the parental tumor, the genes most closely associated with these metabolic adaptations confer enhanced immunotherapy resistance. We have validated upregulation of these pathways in a unique cohort of melanoma patients who failed dual checkpoint blockade. Additionally, we employed MRI imaging to visualize metabolic changes acquired by resistant tumors in live mice. Clinical application of this technique could provide a much-needed noninvasive tool to predict immunotherapeutic sensitivity of patients. Conclusion: Upregulation of glycolytic metabolism and the aldose reductase pathway by melanoma tumor cells cripples T cells in the microenvironment and confers resistance to checkpoint blockade. This abstract is also being presented as Poster A71. Citation Format: Ashvin R. Jaiswal, Shivanand Pudakalakatti, Prasanta Dutta, Arthur Liu, Todd Bartkowiak, Casey Ager, Cristina Ivan, Richard Eric Davis, Michael A. Davies, Jennifer Wargo, James P. Allison, Pratip K. Bhattacharya, David Hong, Michael A. Curran. Metabolic adaptations establish immunotherapy resistance in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr PR08.

Background: Despite the success of T-cell checkpoint blockade antibodies in treating an array of cancers, the majority of patients still fail to respond to these therapies, or respond transiently and then relapse. The molecular mechanisms that drive lack of response to checkpoint blockade, whether pre-existing or evolved on therapy, remain unclear. Materials and Methods: To address this critical gap in clinical knowledge, we established a mouse model of melanoma designed to elucidate the molecular mechanisms underlying immunotherapy resistance. Through multiple in vivo passages, we selected a B16 melanoma tumor line that evolved complete resistance to combination blockade of CTLA-4, PD-1, and PD-L1, which cures ~75% of mice of the parental tumor. Using gene expression analysis, proteomics, and immunogenomics, we determined the adaptations engaged by this melanoma to become completely immunotherapy resistant. NMR spectroscopy, Seahorse XF Analysis, flow cytometry, confocal microscopy and Western blot analysis provided further insight into the mechanisms driving checkpoint blockade resistance. Results: Acquisition of immunotherapy resistance by these melanomas was driven by acquisition of a hypermetabolic state through coordinate upregulation of both glycolysis and oxidative phosphorylation (OxPhos). Depletion of essential nutrients from the tumor microenvironment by these resistant tumors functionally compromises infiltrating T cells to an extent that cannot be rescued by even triple checkpoint blockade. The glycolysis and OxPhos-associated genes most highly induced by these resistant melanomas were capable of conferring enhanced resistance to checkpoint blockade when reintroduced monogenically into the parental tumor. We have validated upregulation of these pathways in a unique cohort of melanoma patients who failed dual checkpoint blockade. Additionally, we employed a novel MRI imaging technique to visualize metabolic changes acquired by resistant tumors in live mice. Clinical application of this technique could provide a much-needed noninvasive tool to predict immunotherapeutic sensitivity of patient cancers. Conclusion: Acquisition of a hypermetabolic state by melanoma tumor cells cripples T cells in the microenvironment and confers complete resistance to checkpoint blockade. Citation Format: Ashvin R. Jaiswal, Shivanand Pudakalakatti, Dutta Prasanta, Arthur Liu, Todd Bartkowiak, Casey Ager, Michael A. Davies, Richard E. Davis, Jennifer Wargo, Pratip K. Bhattacharya, David S. Hong, Michael A. Curran. Metabolic adaptations confer immunotherapy resistance in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1700.

Summary Alzheimer's disease (AD) is a devastating neurodegenerative disorder that impairs memory and causes cognitive and psychiatric deficits. New evidences indicate that AD is conceptualized as a disease of synaptic failure, although the molecular and cellular mechanisms underlying these defects remain to be elucidated. Determining the timing and nature of the early synaptic deficits is critical for understanding the progression of the disease and for identifying effective targets for therapeutic intervention. Using single‐synapse functional and morphological analyses, we find that AMPA signaling, which mediates fast glutamatergic synaptic transmission in the central nervous system (CNS), is compromised early in the disease course in an AD mouse model. The decline in AMPA signaling is associated with changes in actin cytoskeleton integrity, which alters the number and the structure of dendritic spines. AMPA dysfunction and spine alteration correlate with the presence of soluble but not insoluble Aβ and tau species. In particular, we demonstrate that these synaptic impairments can be mitigated by Aβ immunotherapy. Together, our data suggest that alterations in AMPA signaling and cytoskeletal processes occur early in AD. Most important, these deficits are prevented by Aβ immunotherapy, suggesting that existing therapies, if administered earlier, could confer functional benefits.