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Background: The induction of reactive oxygen species (ROS) represents a viable strategy for enhancing the activity of radiotherapy. The authors hypothesized that napabucasin would increase ROS via its ability to inhibit NAD(P)H:quinone oxidoreductase 1 and potentiate the response to chemoradiotherapy in rectal cancer via distinct mechanisms. Method: Proliferation studies, colony formation assays, and ROS levels were measured in HCT116 and HT29 cell lines treated with napabucasin, chemoradiation, or their combination. DNA damage (pγH2AX), activation of STAT, and downstream angiogenesis were evaluated in both untreated and treated cell lines. Finally, the effects of napabucasin, chemoradiotherapy, and their combination were assessed in vivo with subcutaneous mouse xenograft models. Results: Napabucasin significantly potentiated the growth inhibition of chemoradiation in both cell lines. Napabucasin increased ROS generation. Inhibition of ROS by N-acetylcysteine decreased the growth inhibitory effect of napabucasin alone and in combination with chemoradiotherapy. Napabucasin significantly increased pγH2AX in comparison with chemoradiotherapy alone. Napabucasin reduced the levels of pSTAT3 and VEGF and inhibited angiogenesis through an ROS-mediated effect. Napabucasin significantly potentiated the inhibition of growth and blood vessel formation by chemoradiotherapy in mouse xenografts. Conclusion: Napabucasin is a radiosensitizer with a novel mechanism of action: increasing ROS production and inhibiting angiogenesis. Clinical trials testing the addition of napabucasin to chemoradiotherapy in rectal cancer are needed.

Background BTC is an aggressive disease exacerbated by inflammation and immune suppression. Expansion of immunosuppressive cells occurs in biliary tract cancer (BTC), yet the role of BTC-derived cytokines in this process is unclear. Methods Activated signalling pathways and cytokine production were evaluated in a panel of human BTC cell lines. Human peripheral blood mononuclear cells (PBMCs) were cultured with BTC supernatants, with and without cytokine neutralising antibodies, and analysed by flow cytometry or immunoblot. A human BTC tissue microarray (TMA, n = 69) was stained for IL-6, GM-CSF, and CD33+S100a9+ cells and correlated with clinical outcomes. Results Immunomodulatory factors (IL-6, GM-CSF, MCP-1) were present in BTC supernatants. BTC supernatants expanded CD33dimCD11b+HLA-DRlow/− myeloid-derived suppressor cells (MDSCs) from human PBMCs. Neutralisation of IL-6 and GM-CSF in BTC supernatants inhibited activation of STAT3/5, respectively, in PBMCs, with heterogeneous effects on MDSC expansion in vitro. Staining of a BTC TMA revealed a positive correlation between IL-6 and GM-CSF, with each cytokine and more CD33+S100a9+ cells. Increased CD33+S100a9+ staining positively correlated with higher tumour grade, differentiation and the presence of satellite lesions. Conclusion BTC-derived factors promote suppressive myeloid cell expansion, and higher numbers of CD33+S100a9+ cells in resectable BTC tumours correlates with more aggressive disease.

Cytokine signaling is challenging to study and therapeutically exploit as the effects of these protein are often pleiotropic. A subset of cytokines can, however, exert signal specificity via association with latency-inducing proteins which cage the cytokine until disrupted by discreet biological stimuli. Inspired by this precision, here we describe a strategy for synthetic induction of cytokine latency via modification with photo-labile polymers that mimic latency while attached, then restore protein activity in response to light, thus controlling the magnitude, duration, and location of cytokine signals. We characterize the high dynamic range of latent cytokine activity modulation and find that polymer-induced latency, alone, can prolong in vivo circulation and bias receptor subunit binding. We further show that protein de-repression can be achieved with near single-cell resolution and demonstrate the feasibility of transcutaneous photoactivation. Future extensions of this approach could enable multicolor, optical reprogramming of cytokine signaling networks and more precise immunotherapies.

Pancreatic ductal adenocarcinoma (PDAC) is exceptionally resistant to immune checkpoint inhibition (ICI). We previously reported that elevated systemic interleukin-6 (IL-6) and increased numbers of T cells positive for circulating cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) correlate with worse overall survival in patients with PDAC. We postulated that combined blockade of IL-6 and CTLA-4 would significantly enhance anti-tumor immune responses to PDAC. Dual blockade of IL-6 and CTLA-4 in immune competent mice bearing subcutaneously injected pancreatic tumors significantly inhibited tumor growth, accompanied by overwhelming T cell infiltration. Therapeutic efficacy was confirmed in an orthotopic murine model of pancreatic cancer and T cell depletion studies unveiled a unique dependence on CD4+ T cells for anti-tumor activity of dual IL-6 and CTLA-4 blockade. In vitro studies utilizing T cells from a TRP-1 transgenic mouse as an antigen-specific model system demonstrate this combination therapy elicits increased IFN-γ production by activated CD4+ T cells. Additionally, IFN-γ stimulation of pancreatic tumor cells in vitro profoundly increased tumor cell production of CXCR3 specific chemokines (CXCL10 and CXCL9). Further studies blocking CXCR3 in the presence of combined IL-6 and CTLA-4 blockade prevented orthotopic tumor regression, demonstrating a dependence on the CXCR3 axis for anti-tumor efficacy. We also found combination therapy increased intratumoral CD4+ T cells and elicited systemic changes in T-helper subsets. These data represent the first report of IL-6 and CTLA-4 blockade as a means to regress pancreatic tumors with defined operative mechanisms of efficacy. Given these results, this therapeutic combination has potential for immediate clinical translation.One Sentence SummaryBlockade of interleukin-6 in pancreatic cancer enhances CTLA-4 immune checkpoint inhibition to regress tumors in a CD4+ T cell and CXCR3-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) has dismal 5-year survival (

465 Background: Biliary tract cancers (BTCs) are a group of rare and aggressive malignancies often refractory to chemotherapeutic or targeted therapies, carrying an overall poor prognosis and high mortality. Identifying prognostic biomarkers and effective therapeutic targets for this disease therefore remains a high priority. T-cell immunoglobulin mucin domain-containing protein 3 (TIM-3), a negative regulatory immune checkpoint receptor, is known to induce immune tolerance and inhibit T-cell antitumor immunity. Its ligand, galectin-9 (Gal-9), has been shown to play a paradoxical role in tumorigenesis in a number of disease states. However, the role of Gal-9 in the setting of BTCs is not completely understood. Methods: We examined expression patterns of total cellular Gal-9 and TIM-3 in a panel of human BTC cell lines with diverse molecular profiles via immunoblot. Soluble Gal-9 (sGal-9) was measured by enzyme-linked immunoassay (ELISA). Tetrazolium-based MTT assay was utilized to evaluate cell viability following Ab-mediated Gal-9 neutralization. Pre-treatment peripheral blood was obtained from patients with metastatic BTCs enrolled in a randomized phase II clinical trial (NCI10139) and sGal-9 levels were measured. ROC curve analysis was employed to identify an optimal concentration cut-point value. Kaplan-Meier models were used to assess the association between sGal-9 levels and overall survival. Results: Human BTC cells demonstrate detectable and differential expression levels of total Gal-9 and TIM-3 proteins in vitro, with greater expression among intrahepatic cell lines (HUCCT-1, HUH28, SNU478). TIM-3 expression was not detected among extrahepatic (MzChA-1) and gallbladder cancer (WITT) cell lines. sGal-9 is secreted at variable levels between cell lines and its neutralization does not affect viability. Pre-treatment peripheral blood was obtained from 73 patients with metastatic BTCs (53% (n = 39) intrahepatic cholangiocarcinoma, 22% (n = 16) extrahepatic cholangiocarcinoma, 25% (n = 18) gallbladder cancer). There was no significant difference in sGal-9 levels between disease sites (p = 0.08). Patients were dichotomized by low and high sGal-9 levels. High baseline plasma levels of sGal-9 were associated with worse overall survival among patients with advanced BTCs (p = 0.03). Conclusions: Biliary tract cancer cells differentially express galectin-9 and its ligand, TIM-3, in vitro. Higher plasma levels of soluble galectin-9 are associated with worse overall survival, suggesting galectin-9 expression may be related to a more aggressive disease state. Additional work is needed to better inform the mechanistic role of the galectin-9/TIM-3 axis on disease progression and how best to leverage this pathway as a therapeutic target in the management of biliary tract cancers.

TPS626 Background: Interleukin-6 (IL-6) is associated with carcinogenesis, immune suppression, and poor prognosis in pancreatic adenocarcinoma (PDAC). Preclinical data demonstrated dual inhibition of IL-6 and (programmed death ligand-1) PD-L1 facilitates CD8+ T cell migration into pancreatic tumors and was effective in controlling tumor growth in syngeneic and genetically engineered PDAC mouse models. Siltuximab is a chimeric monoclonal antibody which targets the IL-6 molecule specifically and spartalizumab is a high-affinity ligand-blocking humanized IgG4 antibody against the PD-1 receptor. Based on this preclinical rationale, we developed a phase Ib/II trial to determine the recommended phase II dose (RP2D), evaluate the safety, toxicity profile, preliminary antitumor activity, and immunogenicity of the siltuximab and spartalizumab in patients with previously treated metastatic PDAC. Methods: The phase Ib trial design is standard 3+3. Primary endpoint is to determine RP2D. Siltuximab is administered intravenously (IV) in three dose levels of 6 mg/kg (DL1), 11 mg/kg (DL2), 9 mg/kg (only if 2 DLTs observed on DL2) every 3 weeks with spartalizumab at 300 mg IV every 3 weeks. Eligible patients must have stage IV PDAC who have failed at least one prior therapy age ≥18 years, ECOG PS 0-1, no prior anti PD-1 or anti-PD-L1 agent. After RP2D is established, an expansion phase will enroll 24 patients with PDAC. Pre and on-treatment biopsy will be performed in 24 patients in the expansion cohort for correlative analysis. Pre-treatment and on-treatment peripheral blood samples will be collected from all patients. In the expansion phase patients will receive initial cycle (every 3 weeks) treatment with either spartalizumab or spartalizumab plus siltuximab and then starting cycle 2 all patients receive the combination following the on-treatment research biopsy. This design will enable us to evaluate the immunological effects of spartalizumab alone versus the combination in the tumor microenvironment and peripheral blood. This study was activated in January 2020 and to date 12 patients were enrolled in dose escalation phase. The dose expansion phase has recently started accrual. Clinical trial information: NCT04191421.

147 Background: Over half of patients with colorectal cancer (CRC) develop liver metastases. While immunotherapy is an emerging treatment of solid tumors, its use among CRC patients is limited. Furthermore, gene expression patterns of liver-specific CRC metastases remain unclear. The purpose of this study was to identify a high-risk gene expression profile for patients with colorectal liver metastasis (CRLM) to better inform prognosis and development of novel targeted therapies. Methods: Fifty-three FFPE CRLM samples from patients who underwent complete metastatectomy from 2009-2017 were examined. Expression profiling of extracted RNA was performed using NanoString Immuno-Oncology (IO360) 750-gene panel. Statistical analyses using cutoffs of absolute log 2-fold change≥1.5 and p-value≤0.05 were performed. Patients were analyzed by extremes of outcomes: survival time in the lowest quartile compared to those still alive at last follow-up. Results: Eight differentially expressed genes were associated with poor survival. Overexpressed genes included IL6R, CXCL2, C7, MGMT, PCK2, CSF1 and LILRB4 (Table). PLA2G2A was under-expressed. Conclusions: This study demonstrates differential gene expression associated with poor survival among patients with CRLM. Specific genes of interest include IL6R, MGMT, CSF1 and LILRB4. IL6R is a known effector in tumor proliferation via IL-6 signaling from tumor-associated macrophages, myeloid-derived suppressor cells (MDSCs) and T-cells. MGMT repairs alkylating DNA damage and is implicated in carcinogenesis and response to chemotherapy. CSF1 promotes macrophage differentiation to M2 phenotype, suppressing inflammation and anti-tumor defense mechanisms. LILRB4 activation via MDSCs leads to T-cell inhibition. These overall suggest a myeloid-dominant tumor immune microenvironment and represent important potential therapeutic targets. Next steps include performing immunohistochemistry to validate findings at the protein level and investigate the tumor intrinsic role using human cell lines.[Table: see text]

This study addresses the hypothesis that IL-6/STAT3 signaling is of clinical relevance in oropharyngeal squamous cell carcinoma (OPSCC). We evaluated relationships between key components of this pathway in tumors from a unique cohort of n = 59 fully annotated, treatment-naïve patients with OPSCC. The multiplex Opal platform was utilized for immunofluorescence (IF) analysis of tissues to detect IL-6 and phosphorylated STAT3 (pSTAT3), taking into consideration its nuclear versus cytoplasmic localization. Abundant staining for both IL-6 and pSTAT3 was evident in tumor-rich regions of each specimen. IL-6 correlated with cytoplasmic pSTAT3 but not nuclear or total pSTAT3 in this cohort of OPSCC tumors, regardless of p16 status (r = 0.682, p

Background: Pancreatic ductal adenocarcinoma (PDAC) has a highly immunosuppressive tumor microenvironment (TME). The immune landscape of these tumors is further influenced by cross-talk with fibroblasts present in stroma. Importantly, genomic features also influence immune and stromal composition within PDAC and may provide opportunity for personalized immunotherapy. Given the central role of CKDN2A in PDAC (being lost in up to half of patients, and the second most commonly alteration across all metastatic disease), we hypothesize that the CDKN2A-deficient PDAC TME can be adapted for therapeutic targeting to sensitize PDAC to immunotherapy. Methods: Bioinformatic analysis of PDAC samples from the TCGA database evaluated how CDKN2A loss alters expression of T cell-inflamed gene signatures. Preclinical in vitro and in vivo studies were conducted with CDKN2A-targeted CRISPR-Cas9-edited luciferase-expressing KPC cells (KPC-luc) to generate isogenic KPC-lucΔCKDN2A cells. The BET inhibitor JQ-1 and CDK inhibitor palbociclib were used to evaluate impact on apoptosis and immune-related signaling using isogenic KPC-luc cells and patient-derived PDAC organoids. Spheroids were used to evaluate impact of CDK/BET inhibition on cross-talk with pancreatic cancer-associated fibroblast (CAF) spheroids. Finally, in vivo studies tested the ability of CDK/BET inhibition to enhance T cell migration, and delay tumor growth when combined with PD-L1 blockade. Results: CDKN2A loss was enriched in tumors lacking a T cell-inflamed gene signature. Similarly, CDKN2A loss decreased T cell migration in vitro and into the KPC-lucΔCDKN2A TME in vivo. Loss of CDKN2A rendered KPC-luc cells susceptible to direct apoptosis from dual CDK/BET inhibition. Combined CDK/BET inhibition at sublethal doses also modulated immune phenotype of tumors, inducing DNA damage and cGAS/STING to increase type I interferon and T cell migration in vitro. Additionally, treatment with BETi alone or combined with CDKi also modulated checkpoint ligands on KPC-luc cells and patient-derived organoids. In stroma, treatment of PDAC CAFs with JQ-1 polarized CAFs towards a myofibroblast CAF (myCAF) phenotype. This could also be mediated via cross-talk between tumor and CAF cells, as JQ-1-treated KPC-lucΔCDKN2A cells or supernatants cultured with CAF spheroids also induced myCAFs. This cross-talk was bidirectional, as supernatants from BETi-treated CAFs modulated expression of checkpoint ligands on KPC-luc cells. In vivo, combined BET/CDK inhibition increased T cell infiltration into PDAC tumors, and when combined with PD-L1 blockade diminished tumor growth. Conclusion: While CDKN2A loss promotes an immunosuppressive PDAC TME, it also renders tumors susceptible to BET/CDK inhibition. This approach polarizes CAF cells towards a myCAF phenotype, directing cross-talk between tumors and stroma to promote a more immunogenic phenotype. This suggests combined CDK/BET inhibition may be a personalized therapeutic approach for patients with CKDN2A-deficient PDAC, and a means to sensitize PDAC to immunotherapy. Citation Format: Brian M. Olson, Alison J. Thomas, Michael B. Ware, Gregory B. Lesinski. Combined CDK and BET inhibition reprograms the tumor and stromal compartments to enhance anti-tumor immunity in immunologically-cold CDKN2A-deficient pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-122.

Pancreatic ductal adenocarcinoma (PDAC) is unresponsive to immune checkpoint therapy largely due to a paucity of T cells within the tumor microenvironment (TME) and abundant immunosuppressive signaling pathways. In this study we show that human PDAC tumors over-express vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide that suppresses T cell effector properties and promotes the generation of regulatory T cells (Tregs). Therefore, we treated tumor-bearing mice with VIP receptor peptide antagonists and measured T cell homing, activation, and anti-tumor responses in preclinical murine models of PDAC. Pharmacological inhibition of VIP receptor (VIP-R) signaling using daily subcutaneous injections of peptide antagonists had no discernable toxicity in healthy and tumor-bearing mice. Treatment with VIP-R antagonists in combination with anti-PD-1 checkpoint blockade significantly decreased tumor burden, and improved survival in subcutaneous and orthotopic murine PDAC models. Combination therapy significantly enhanced T cell activation and proliferation and decreased frequencies of Tregs within the TME. Anti-tumor responses were T cell dependent, as the combination therapy failed to improve survival in CD4 or CD8 deficient mice using knock-out strains and antibody depletion. Furthermore, combination therapy significantly increased frequencies of tumor specific T cells (measured with a tetramer reagent) and provided protective immunity against tumor rechallenge. Combination therapy led to significant increases in the infiltration of adoptively transferred GFP+ T cells into PDAC tumors and decreased CXCR4 expression levels on T cells. Encouragingly, peptide-based VIP-R antagonists enhanced the in vitro activation of human T cells isolated from peripheral blood of PDAC patients. Human T cells cultured with VIP-R antagonists had increased proliferation, activation, and decreased proportions of T regs and exhausted T cells co-expressing PD-1, Tim-3 and Lag-3. Taken together, our findings show that VIP is a targetable mechanism of immune escape in PDAC. Inhibiting VIP receptor signaling improves T cell effector properties and synergistically improves anti-tumor responses to checkpoint inhibitors in mouse PDAC models. Additionally, as the VIP sequence is identical between human and mice, and since VIP-R antagonists have similar effects on human and murine T cells in culture, clinical translation is highly feasible. Citation Format: Sruthi Ravindranathan, Passang Tenzin, Jian Ming Li, Rohan Dhamsania, Michael Ware, Mohammad Zaidi, Shuhua Wang, Jingru Zhu, Maria Cardenas, Yuan Liu, Gaurav Joshi, Sanjeev Gumber, Brian Robinson, Anish Sen-Majumdar, Shanmuganathan Chandrakasan, Haydn Kissick, Alan Frey, Susan Thomas, Bassel El-Rayes, Gregory Lesinski, Edmund K. Waller. Inhibiting vasoactive intestinal peptide receptor signaling elicits T cell dependent anti-tumor response of pancreatic ductal adenocarcinoma to immune checkpoint therapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-072.

BackgroundBiliary tract cancers (BTCs) are aggressive malignancies often refractory to chemotherapy and immunotherapy. There is urgent need to identify novel, data-driven therapeutic targets to change the course of disease. It is also challenging to procure substantial quantities of tissue for research from patients with advanced or metastatic disease. Thus, we posit that peripheral blood holds particular value for exploring immune landscapes in patients with advanced BTC. We hypothesized actionable immunotherapy targets can be identified from blood that are suited to improve outcomes for BTC patients.MethodsWe examined a comprehensive panel of immune biomarkers in pre-treatment blood from 79 patients with advanced BTC on a national, randomized Phase II clinical trial of atezolizumab ± cobimetinib in advanced metastatic BTC (NCI10139). A cohort of 12 healthy donors was obtained as a comparator. Blood was processed via ficoll and density gradient centrifugation. PBMCs and plasma were cryopreserved prior to analysis by bioplex assay and multiparameter flow cytometry (n=23 colors). Analysis of 45 peripheral soluble factors was performed on a Luminex 100. Flow cytometric analysis was conducted on a Cytek Aurora and analyzed using FlowJo software. Comparisons of normal versus BTC patient samples were conducted via unpaired two-tailed t tests.ResultsBioplex analysis of 45 peripheral soluble factors revealed significant elevations in BTC plasma compared to normal donors for several factors including IL-6, IL-18, eotaxin/CCL11, IP-10/CXCL10, MIP-1ß/CCL9, IFN-?, PDGF-BB, PIGF-1, and SCF (p’sAbstract 18 Figure 1Several immune biomarkers are elevated in the peripheral blood of biliary tract cancer patients compared to normal donors, including (a) percent of CD45% cells that are phenotypically defined as M2 macrophages (CD11b+CD11c+CD16+CD33+CD14+), (b) percent of CD45% cells that are BTLA+CD8+ T cells, (c) soluble MIP-1β/CCL9, and (d) soluble IL-6.ConclusionsTo our knowledge, this work represents the most comprehensive analysis of peripheral immune biomarkers in BTC patients to date. These data suggest that cytokine mediators and/or distinct immune checkpoints deserve further investigation as actionable targets in patients with BTC.Ethics ApprovalPatient samples were collected as part of a study approved by the National Cancer Institute’s (NCI) Cancer Therapy Evaluation Program (CTEP), as well as central and local institutional review boards (IRBs). All patients and healthy donors gave informed consent before participation in these studies.

BackgroundPancreatic ductal adenocarcinoma (PDAC) has yet to widely benefit from T cell-targeted immunotherapy and displays universally poor prognosis. Thus, enhancing the activity of immunotherapy is a high priority. Our laboratory recently reported that heat shock protein-90 (Hsp90) inhibition enhances the efficacy of PD-1 blockade in murine models of PDAC (Zhang Y. et al., Mol Cancer Ther, 2020). Hsp90 inhibitors can limit activation of cancer associated fibroblasts (CAF) and promote infiltration of T cells when combined with PD-1 blockade in preclinical systems.MethodsBased on these data, we are conducting a Phase Ib/II clinical trial to evaluate the combination of XL888 (Hsp90 inhibitor) and pembrolizumab in patients with metastatic pancreatic cancer. We hypothesize that this combination will be safe and elicit pronounced microenvironmental changes, leading to enhanced efficacy of checkpoint blockade in a tumor type that is otherwise refractory to this approach. During the phase II portion patients were randomized to receive a three week lead in with either pembrolizumab or pembrolizumab and XL888. Paired biopsies and blood samples were obtained at baseline and at week two on treatment and CyTOF was used to assess changes in circulating and tumor infiltrating immune populations. Further, CyTOF profiling of circulating immune cells was performed to assess impacts of XL888 on over thirty phenotypically defined immune populations (figure 1).ResultsAs of June 2021, paired liver biopsy specimens from sites of metastasis have been successfully obtained from a total of 8 patients and paired peripheral blood mononuclear cell samples have been analyzed in 24 patients. Our CyTOF analysis illustrated a surprising increase in myeloid cell populations within the tumor following treatment. Analysis of circulating immune cells illustrated a decrease in natural killer cells and Th17 populations following treatment while naïve B cells were increased. These data will be validated by immunohistochemical analysis of FFPE biopsy specimens obtained in parallel at the time of CyTOF analysis. The impact of XL888 on systemic cytokines and chemokines (n=48 total) in the peripheral blood from patients enrolled in the clinical trial is therefore being assessed as a potential mechanism to explain this observation.Abstract 403 Figure 1Clinical trial and correlative analysis schema. Patients were randomized to receive either pembrolizumab alone or in combination with the HSP90 inhibitor XL888 for a two week cycle prior to crossover to the combination arm. Plasma, peripheral blood mononuclear cells (PBMC), and biopsies were assayed to evaluate immunomodulatory effects of the therapies.ConclusionsClinical data from this trial indicates that this combination is safe in patients. As clinical data matures, changes in soluble and cellular biomarkers will be correlated with response to elucidate mechanisms of response or resistance to this combination therapy.Trial RegistrationThis clinical trial is underway and registered with the ID NCT03095781Ethics ApprovalThe study was approved by Emory University’s Ethics Board, approval IRB00087397.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Silencing of tumor suppressor and DNA repair genes through methylation plays a role in cancer development, growth and response to therapy in colorectal and pancreatic cancers. Heat shock protein 90 (HSP90) regulates transcription of DNA methyltransferase enzymes (DNMT). In addition, DNMTs are client proteins of HSP90. The aim of this study is to evaluate the effects of HSP90 inhibition on DNA methylation in colorectal and pancreatic cancer cell lines. Our data shows that inhibition of HSP90 using ganetespib resulted in downregulation of mRNA and protein expression of DNMT1, DNMT3A, and DNMT3B in HT-29 and MIA PaCa-2 cell lines. This in turn was associated with a drop in the fraction of methylated cytosine residues and re-expression of silenced genes including MLH-1, P16 and SPARC. These effects were validated in HT-29 tumors implanted subcutaneously in mice following in vivo administration of ganetespib. This work demonstrates the effectiveness of ganetespib, an HSP90 inhibitor in modulating DNA methylation through downregulation of DNMT expression.

Lipocalin-2 (LCN2) promotes malignant development in many cancer types. LCN2 is upregulated in patients with pancreatic ductal adenocarcinoma (PDAC) and in obese individuals, but whether it contributes to PDAC development is unclear. In this study, we investigated the effects of Lcn2 depletion on diet-induced obesity, inflammation, and PDAC development. Mice with acinar cell–specific expression of KrasG12D were crossed with Lcn2-depleted animals and fed isocaloric diets with varying amounts of fat content. Pancreas were collected and analyzed for inflammation, pancreatic intraepithelial neoplasia (PanIN), and PDAC. We also used a syngeneic orthotopic PDAC mouse model to study tumor growth in the presence or absence of Lcn2 expression. In addition, to understand the mechanistic role of how LCN2 could be mediating PDAC, we studied LCN2 and its specific receptor solute carrier family 22 member 17 (SLC22A17) in human pancreatic cancer stellate cells (PSC), key mediators of the PDAC stroma. Depletion of Lcn2 diminished extracellular matrix deposition, immune cell infiltration, PanIN formation, and tumor growth. Notably, it also increased survival in both obesity-driven and syngeneic orthotopic PDAC mouse models. LCN2 modulated the secretion of proinflammatory cytokines in PSC of the PDAC tumor microenvironment, whereas downregulation of LCN2-specific receptor SLC22A17 blocked these effects. Our results reveal how LCN2 acts in the tumor microenvironment links obesity, inflammation, and PDAC development. Cancer Res; 77(10); 2647–60. ©2017 AACR

Involuntary weight loss, a part of the cachexia syndrome, is a debilitating comorbidity of cancer and currently has no treatment options. Results from a recent clinical trial at our institution showed that biliary tract cancer patients treated with a MEK inhibitor exhibited poor tumor responses but surprisingly gained weight and increased their skeletal muscle mass. This implied that MEK inhibition might be anticachectic. To test this potential effect of MEK inhibition, we utilized the established Colon-26 model of cancer cachexia and the MEK1/2 inhibitor MEK162. Results showed that MEK inhibition effectively prevented muscle wasting. Importantly, MEK162 retained its ability to spare muscle loss even in mice bearing a Colon-26 clone resistant to the MEK inhibitor, demonstrating that the effects of blocking MEK are at least in part independent of the tumor. Because single-agent MEK inhibitors have been limited as a first-line targeted therapy due to compensatory activation of other oncogenic signaling pathways, we combined MEK162 with the PI3K/Akt inhibitor buparlisib. Results showed that this combinatorial treatment significantly reduced tumor growth due to a direct activity on Colon-26 tumor cells in vitro and in vivo, while also preserving skeletal muscle mass. Together, our results suggest that as a monotherapy, MEK inhibition preserves muscle mass, but when combined with a PI3K/Akt inhibitor exhibits potent antitumor activity. Thus, combinatorial therapy might serve as a new approach for the treatment of cancer cachexia. Mol Cancer Ther; 16(2); 344–56. ©2016 AACR. See related article by Kobayashi et al., p. 357

OBJECTIVE: This study exploits the intersection between molecular-targeted therapies and immune-checkpoint inhibition to define new means to treat pancreatic cancer. DESIGN: Patient-derived cell lines and xenograft models were used to define the response to CDK4/6 and MEK inhibition in the tumour compartment. Impacts relative to immunotherapy were performed using subcutaneous and orthotopic syngeneic models. Single-cell RNA sequencing and multispectral imaging were employed to delineate effects on the immunological milieu in the tumour microenvironment. RESULTS: We found that combination treatment with MEK and CDK4/6 inhibitors was effective across a broad range of PDX models in delaying tumour progression. These effects were associated with stable cell-cycle arrest, as well as the induction of multiple genes associated with interferon response and antigen presentation in an RB-dependent fashion. Using single-cell sequencing and complementary approaches, we found that the combination of CDK4/6 and MEK inhibition had a significant impact on increasing T-cell infiltration and altering myeloid populations, while potently cooperating with immune checkpoint inhibitors. CONCLUSIONS: Together, these data indicate that there are canonical and non-canonical features of CDK4/6 and MEK inhibition that impact on the tumour and immune microenvironment. This combination-targeted treatment can promote robust tumour control in combination with immune checkpoint inhibitor therapy.

The accessibility of adoptive T-cell transfer therapies (ACT) is hindered by the cost and time required for product development. Here we describe a streamlined ACT protocol using Th17 cells expanded only 4 days ex vivo. While shortening expansion compromised cell yield, this method licensed Th17 cells to eradicate large tumors to a greater extent than cells expanded longer term. Day 4 Th17 cells engrafted, induced release of multiple cytokines including IL6, IL17, MCP-1, and GM-CSF in the tumor-bearing host, and persisted as memory cells. IL6 was a critical component for efficacy of these therapies via its promotion of long-term immunity and resistance to tumor relapse. Mechanistically, IL6 diminished engraftment of FoxP3+ donor T cells, corresponding with robust tumor infiltration by donor effector over regulatory cells for the Day 4 Th17 cell product relative to cell products expanded longer durations ex vivo. Collectively, this work describes a method to rapidly generate therapeutic T-cell products for ACT and implicates IL6 in promoting durable immunity of Th17 cells against large, established solid tumors. Significance: An abbreviated, 4-day ex vivo expansion method licenses Th17 cells to confer long-lived immunity against solid malignancies via induction of systemic IL6 in the host. See related commentary by Fiering and Ho, p. 3795

Soybeans are a rich source of isoflavones that have been linked with anti-inflammatory processes and various health benefits. However, specific mechanisms whereby soy bioactives impact immune cell subsets are unclear. Isoflavones, such as genistein and daidzein, are metabolized by microbes to bioactive metabolites as O-desmethylangolensin (O-DMA) and equol, whose presence has been linked to health benefits. We examined how soy isoflavones and metabolites impact natural killer (NK) cell signaling and function. We observe no impact of isoflavones on viability of healthy donor peripheral blood mononuclear cells (PBMCs) or NK cells, even at high (25 µM) concentrations. However, pre-treatment of PBMCs with physiologically-relevant concentrations of genistein (p = 0.0023) and equol (p = 0.006) decreases interleukin (IL)-12/IL-18-induced interferon-gamma (IFN-γ) production versus controls. Detailed cellular analyses indicate genistein and equol decrease IL-12/IL-18-induced IFN-γ production by human NK cell subsets, but do not consistently alter cytotoxicity. At the level of signal transduction, genistein decreases IL-12/IL-18-induced total phosphorylated tyrosine, and phosphorylation MAPK pathway components. Further, genistein limits IL-12/IL-18-mediated upregulation of IL-18Rα expression on NK cells (p = 0.0109). Finally, in vivo studies revealed that C57BL/6 mice fed a soy-enriched diet produce less plasma IFN-γ following administration of IL-12/IL-18 versus control-fed animals (p

4023 Background: Biliary tract cancers (BTC) are increasingly subtyped by molecular alterations, but little is known about the relationship between gain-of-function mutations and the RNA transcript expression of immune-related pathways. Methods: A sample of retrospective, clinicogenomic and transcriptomic data from de-identified records of patients with BTC in the Tempus database was selected. We then investigated the relationship between the mutational landscape and immune-related RNA signatures of different anatomic and genomic BTC subtypes. Results: The cohort included 455 samples of intrahepatic bile duct (IH) (n=267), gallbladder (GB) (n=153), and extrahepatic bile duct (EH) (n=35) cancer subtypes. Across all subtypes, we detected alterations in TP53 (43.8%), ARID1A (19.8%), KMT2C (18.2%), BAP1 (14.6%), KRAS (12.7%), TERT (12.0%), IDH1 (11.4%), KMT2D (11.0%), LRP1B (11.0%), and PBRM1 (10.7%), along with FGFR2 fusions (2.6%). Potentially actionable biomarkers ( FGFR2 and NTRK1-3 fusions, IDH1 and BRAFV600E mutations, tumor mutational burden [TMB]>10, HER2 expression, and/or microsatellite instability) were identified in 21.1% of all BTC and 28.6% of IH samples. Mutually exclusive alterations observed between subtypes were TP53 & BAP1, KRAS & BAP1, TP53 & IDH1, KRAS & IDH1, and SMAD4 & BAP1 ( P < 0.001 for all). GB was more inflamed based on RNA signatures and classical immune biomarkers, including PD-L1 and TMB. RNA signature analyses revealed a higher expression of immune-related pathways in GB than IH ( P = 0.001) with no differences in comparison with EH. PD-L1 expression and continuous TMB were elevated in GB versus the other anatomical subtypes. Significant associations were noted between particular genetic mutations and immune profiling features (table). Conclusions: BTC subtypes are diverse in DNA alterations, RNA inflammatory signatures, and immune markers. Notably, potentially actionable biomarkers were identified in a sizable portion of the cohort and varied significantly between subtypes. These results provide guidance for targeted therapy development and support the use of multimodal immune profiling for BTC. For example, GB-specific clinical trials may be considered due to the relative increase in immune-related biomarkers observed in GB and the historically limited success of BTC trials.[Table: see text]

95 Background: Preclinical data suggests metformin can improve immune exhaustion of tumor infiltrating lymphocytes and potentiate the effects of PD-1 blockade. By normalizing the hypoxic TME, metformin was shown to improve cytotoxic T cell function and efficacy of anti-PD-1 antibody in highly aggressive B16 melanoma and MC38 colon adenocarcinoma tumor models. Based on this preclinical rationale we conducted a phase II study with nivolumab and metformin combination in treatment refractory MSS metastatic colorectal cancer (mCRC). Methods: Nivolumab 480 mg IV every 4 weeks and Metformin 1000 mg po twice daily was administered in 28-day cycles following a 14-day metformin only lead-in phase.Eligible patients included stage IV metastatic treatment refractory MSS mCRC (patients must have received oxaliplatin, irinotecan, and fluoropyrimidine), age ≥18 years, ECOG PS 0-1, adequate organ function, no prior anti PD-1 agent. The primary endpoint was overall response rate (ORR). Secondary endpoints included overall survival (OS) and progression free survival (PFS). Simon’s two-stage Minimax design was employed (H0: ORR =4%; H1: ORR=15%; alpha = 0.1; power =80%). If ≥1 objective response was observed in the first evaluable 18 patients, 10 additional patients would be included in the cohort. ≥3 objective responders in 28 patients would be required to be considered positive study. Pre-treatment and on-treatment research biopsies and correlative peripheral blood specimens were collected. Results: A total of 24 patients were enrolled, 6 patients were replaced per protocol, and 18 patients had evaluable disease. Of the 18 evaluable patients 11/18 (61%) were female, median age 58 [IQR 50-67]. 2 patients had prolonged stable disease (4 and 10 cycles). No patients had objective response based on RECIST 1.1. Median OS and PFS was 5.1 months [95% CI (2-11.7)] and 2.3 months [95% CI (1.7-2.4)], respectively. Most common grade 3 and 4 toxicities were anemia (n=2) and diarrhea (n=2). Conclusions: In treatment refractory MSS mCRCnivolumab and metformin combination was well tolerated. Two patients achieved stable disease, but no objective response was seen; therefore, the study did not proceed with the second stage of enrollment. Immunologic correlative analysis of this study is ongoing. Clinical trial information: NCT03800602.

Preclinical studies have suggested that the pancreatic tumor microenvironment both inhibits and promotes tumor development and growth. Here we establish the role of stromal fibroblasts during acinar-to-ductal metaplasia (ADM), an initiating event in pancreatic cancer formation. The transcription factor V-Ets avian erythroblastosis virus E26 oncogene homolog 2 (ETS2) was elevated in smooth muscle actin–positive fibroblasts in the stroma of pancreatic ductal adenocarcinoma (PDAC) patient tissue samples relative to normal pancreatic controls. LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx-1-Cre (KPC) mice showed that ETS2 expression initially increased in fibroblasts during ADM and remained elevated through progression to PDAC. Conditional ablation of Ets- 2 in pancreatic fibroblasts in a Kras G12D -driven mouse ADM model decreased the amount of ADM events. ADMs from fibroblast Ets- 2–deleted animals had reduced epithelial cell proliferation and increased apoptosis. Surprisingly, fibroblast Ets-2 deletion significantly altered immune cell infiltration into the stroma, with an increased CD8+ T-cell population, and decreased presence of regulatory T cells (Tregs), myeloid-derived suppressor cells, and mature macrophages. The mechanism involved ETS2-dependent chemokine ligand production in fibroblasts. ETS2 directly bound to regulatory sequences for Ccl3 , Ccl4 , Cxcl4 , Cxcl5 , and Cxcl10 , a group of chemokines that act as potent mediators of immune cell recruitment. These results suggest an unappreciated role for ETS2 in fibroblasts in establishing an immune-suppressive microenvironment in response to oncogenic Kras G12D signaling during the initial stages of tumor development.

Prime-boost vaccination with recombinant (r) vaccinia(V)-CEA(6D)-TRICOM (triad of co-stimulatory molecules B7.1, ICAM-1 and LFA-3) and rFowlpox(F)-CEA(6D)-TRICOM infect antigen-presenting cells and direct expression of co-stimulatory molecules. We hypothesized that co-administration of vaccine with GM-CSF and interferon alpha (IFN-α) would have efficacy in CEA-expressing cancers. Patients with CEA-expressing cancers received the rV-CEA(6D)-TRICOM vaccine subcutaneously (s.c.) on day 1 followed by GM-CSF s.c. to the injection site on days 1-4. In Cycle 1, patients received thrice weekly s.c. injections of IFN-α-2b the week after rV-CEA(6D)-TRICOM. In Cycles 2-4, patients received thrice weekly s.c. injections of IFN-α-2b the same week that rF-CEA(6D)-TRICOM was given. The first cohort received no IFN followed by dose escalation of IFN-α in subsequent cohorts. Thirty-three patients were accrued (mean 59.8 years). Grade 3 toxicities included fatigue and hyperglycemia. Grade 4-5 adverse events (unrelated to treatment) were confusion (1), elevated aspartate transaminase (AST)/alanine transaminase (ALT) (1), and sudden death (1). No patients had a partial response, and eight patients exhibited stable disease of ≥3 months. Median progression-free survival and overall survival (OS) were 1.8 and 6.3 months, respectively. Significantly higher serum CD27 levels were observed after vaccine therapy (p = 0.006 post 1-2 cycles, p = 0.003 post 3 cycles, p = 0.03 post 4-7 cycles) and 42 % of patients assayed developed CEA-specific T cell responses. Pre-treatment levels of myeloid-derived suppressor cells correlated with overall survival (p = 0.04). Administration of IFN-α led to significantly increased OS (p = 0.02) compared to vaccine alone. While the vaccine regimen produced no clinical responses, IFN-α administration was associated with improved survival.

// Daniel H. Ahn 1 , Hatice Gulcin Ozer 2 , Baris Hancioglu 2 , Gregory B. Lesinski 1 , Cynthia Timmers 1 and Tanios Bekaii-Saab 1 1 Department of Internal Medicine, Divison of Medical Oncology, Ohio State University Comprehensive Cancer Center, Columbus, OH, USA 2 Department of Biomedical Informatics, Ohio State University, Columbus, OH, USA Correspondence to: Tanios Bekaii-Saab, email: // Keywords : biliary cancer, whole-exome sequencing, MEK inhibition Received : August 27, 2015 Accepted : December 12, 2015 Published : December 16, 2015 Abstract We previously conducted a phase-II study with selumetinib (AZD6244), a small molecule inhibitor of MEK1/2, in advanced biliary tract cancers (BTC), where the primary endpoint was response rate. Several patients experienced objective response. These findings were confirmed with MEK162 in a similar patient population. To assess for tumor-specific genetic variants that mediate sensitivity to MEK inhibition in BTC, we performed whole-exome sequencing in patients with an objective response to selumetinib. Normal and tumor DNA from FFPE tissue from two patients who experienced an objective response underwent whole-exome sequencing. Raw sequence reads were processed with GATK workflow and tumor specific variants were identified using MuTect and VarScan2. Ensemble Variant Effect Predictor was used to determine functional consequences of these variants. Copy number changes and potential gene fusion events were also screened. Findings were compared to assess for any commonality between the two tumor samples, and whether the identified variants were intrinsic to the MAPK pathway. 1169 and 628 tumor-specific variants were identified in the two samples. Further analysis demonstrated 60 and 53 functional and novel variants, respectively. Of the identified tumor-specific variants, fusion events or copy number changes, no commonality was seen. Several variants in genes associated with ERK signaling were present in each tumor sample. Although there were no common tumor-specific variants in the two patients who exhibited an objective response to selumetinib, several genes associated with ERK signaling were identified. Confirmatory studies investigating the role of the identified genes and other potential tumor independent factors need further investigation.

Background: BTCs are aggressive cancers with a poor prognosis. In preclinical models, MEK inhibition modulates the tumor immune microenvironment and enhances responses to programmed death-ligand 1 (PD-L1) inhibition. We report a randomized, open-label, multicenter phase 2 trial of atezolizumab (anti-PD-L1) as monotherapy or in combination with cobimetinib (MEK inhibitor) in BTC (NCT03201458). Methods: Eligible patients had advanced BTC [intrahepatic cholangiocarcinoma (ICC), extrahepatic cholangiocarcinoma (ECC), and gallbladder cancer (GBC)], with 1-2 lines of prior therapy in the metastatic setting, measurable disease by RECIST v1.1, and ECOG performance status ≤1. Patients randomized to Arm A received atezolizumab 840 mg IV Q2w. Patients randomized to Arm B received oral cobimetinib 60 mg daily (21 days on/7 days off) plus atezolizumab 840 mg IV Q2w. The primary endpoint was progression free survival (PFS) using the Kaplan-Meier method and compared between groups under the assumption of Cox proportional hazards, stratified for primary tumor site. Secondary endpoints included objective response rate (ORR), safety and tolerability, and overall survival (OS). Results: 86 patients were enrolled at 23 centers in the United States; 77 patients were randomized and received at least one dose of study therapy (Arm A: n=37, ICC=21, ECC=7, GBC=11; Arm B: n=38, ICC=22, ECC=8, GBC=8). Median age was 63 (range 44-86), and 48 (62%) were female. The trial met its primary endpoint, with a median PFS of 3.65 months (cobimetinib+atezolizumab) vs 1.87 months (atezolizumab monotherapy) (p=0.027). OS data are not mature at the time of analysis. There was 1 PR (3.2%), 13 SD (41.9%), and 17 PD (54.8%) in the combination arm and 1 PR (2.9%), 10 SD (29.4%), and 23 PD (67.6%) in the atezolizumab monotherapy arm. Two patients in the combination arm remain on therapy 15+ months from enrollment. One patient in each treatment arm had known mismatch repair deficiency (MMRd), of whom 1 had PD as a best response and the other withdrew prior to response evaluation. Grade 3-4 treatment-related adverse events were similar in both arms, and there were no treatment-related deaths. 4 (10%) of patients receiving atezolizumab monotherapy and 8 (22.2%) receiving cobimetinib+atezolizumab discontinued therapy due to adverse events. Changes in tumor CD8, CD4, FoxP3, PDL1, and MHC expression from paired tumor biopsies will be presented at the conference. Conclusions: We report the first randomized trial of immunotherapy in BTC. The combination of atezolizumab plus cobimetinib met its primary endpoint and significantly prolonged PFS as compared to atezolizumab monotherapy in BTC. The combination of atezolizumab and cobimetinib had manageable toxicity and warrants further investigation in BTC. Citation Format: Mark Yarchoan, Leslie Cope, Robert A. Anders, Anne Noonan, Laura W. Goff, Lipika Goyal, Jill Lacy, Daneng Li, Anuj Patel, Aiwu R. He, Ghassan Abou-Alfa, Kristen Spencer, Edward Kim, Stephanie Xavier, Amanda Ruggieri, S. Lindsey Davis, Autumn McRee, Paul Kunk, Qingfeng Zhu, Andrea Wang-Gillam, Andrew Poklepovic, Helen Chen, Elad Sharon, Gregory B. Lesinski, Nilo Azad. A multicenter randomized phase 2 trial of atezolizumab as monotherapy or in combination with cobimetinib in biliary tract cancers (BTCs): A NCI Experimental Therapeutics Clinical Trials Network (ETCTN) study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT043.

Although many new drugs have been approved and more clinical trials are being conducted for various disease therapies in recent decades, efficient identification of reliable therapeutic targets is still a big challenge during drug development. Therefore, tremendous efforts have been put into advancing the technology to quickly determine the altered biomarkers in clinical samples. Nanostring nCounter GeoMxTM Digital Spatial Profiling (GeoMx DSP) has been used in multiple projects and exhibited its outstanding performance on collecting analyzable digital counts for up-to 96 proteins and 20,000 RNAs in a specific area on a single FFPE slide. Tissue regions of interests are identified by fluorescent staining of morphology markers, and either proteins or RNAs in the specific defined regions are collected as the digital nCounter count format. By using this state-of-art technology, spatial distributions of immune- and cancer- relevant proteins were identified in 71 tumor cell rich-, 70 immune cell rich-, and 69 stromal cell rich- area respectively in three Pancreatic cancer patient groups including surgery alone, neoadjuvant FOLFIRINOX (a combination of fluorouracil, folinic acid, irinotecan and oxaliplatin) alone or combined with stereotactic body radiation therapy (F+SBRT). Our results showed that differential regulations among immune checkpoint markers, pan-immune markers and tumor markers in these treatment groups. In tumor-rich regions, CD44 is present at higher levels in FOLFIRINOX treated patients than surgery-alone group. VISTA was significantly down-regulated in F+SBRT group. Also, several key factors from pStat3, PTEN and Bcl2 pathways are also regulated by treatment methods. In immune-rich regions, both T cells markers (CD3 and CD4) and B cell markers (CD19 and CD20) were regulated differently by treatment methods. GeoMx DSP platform provides a novel and more precise view of the spatial regulation of micro-world in the tissue, which enable us to explore more about internal regulation within sub-population of the tissue cells. GeoMxTM DSP platform is only for RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Jingjing Gong, Matthew R. Farren, Walid Shaib, Gregory Lesinski, Sarah Warren, Yan Liang. Small slide, big world: Fast detection of altered proteins and RNA levels in clinical samples using Nanostring nCounter® GeoMxTM Digital Spatial Profiler [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6499.

Pancreatic ductal adenocarcinoma (PDAC) constitutes more than 90% of all pancreatic cancers and has a 5-year survival rate of only 5-7%. A dense stroma, an immunosuppressive tumor microenvironment (TME) and scarce tumor infiltrating T cells, contribute toward the poor responsiveness of PDAC to conventional and FDA-approved checkpoint therapies. In this study, we show that vasoactive intestinal peptide (VIP) signaling is a novel immune checkpoint pathway in PDAC, which when inhibited in combination with anti-PD1 checkpoint inhibitor, significantly decreases tumor burden and enhances T cell infiltration in mouse models of PDAC. VIP is a 28-amino acid neuropeptide secreted at the nerve terminals, pancreas and the GI tract, and is commonly associated with regulating gut motility and blood pressure. Interestingly, VIP is also secreted by immune cells and is known to have immunosuppressive properties such as decreasing T cell proliferation and polarizing T cells towards Th2 phenotype. Intriguingly, according to the cancer genome atlas (TCGA), human exocrine pancreatic cancers expresses the highest level of VIP mRNA expression when compared to other solid malignancies. Similarly, we have generated preliminary data that shows enhanced VIP levels both systemically and locally in mouse models of PDAC and in human PDAC patients. Thus, taking all this into consideration, we hypothesized that enhanced VIP signaling is a potential mechanism of immune escape in PDAC and that inhibiting VIP signaling either alone or in combination with checkpoint inhibitors would significantly enhance treatment response in mouse models of PDAC. We tested the effect of inhibiting VIP signaling by using a VIP receptor antagonist (VIPR antagonist) in orthotopic mouse models of PDAC, where luciferase transfected KPC cells (mouse PDAC cell line), were implanted in the tail of the pancreas. Mice were then treated with VIPR antagonist and/or anti-PD1 seven days after tumor implantation. Our results showed that the combination treatment significantly reduced tumor burden and growth rate, as well as, enhanced the infiltration of both CD4+ and CD8+ T cells into the tumor microenvironment (as per immunohistochemistry of tumor tissues). Furthermore, analysis of tumor infiltrating T cells via Nanostring analysis showed that the combination treatment also significantly enhanced levels of IFN gamma and TNF alpha secreting T cells and costimulatory and effector molecules, including CD27 and FasL. In summary, we have identified that VIP signaling is a novel and targetable immune checkpoint pathway in PDAC, which when inhibited in combination with checkpoint inhibitors, significantly improves treatment response in mouse PDAC models. As the VIP sequence is conserved across different species including mouse and human, and since we have observed VIPR antagonists stimulate proliferation of human T cells, there is an increased potential for clinical translation of VIPR antagonists in the treatment of human PDAC. Citation Format: Sruthi Ravindranathan, Brandon Ware, Mohammad Zaidi, Jingru Zhu, Rohan K. Dhamsania, Shuhua Wang, Yiwen Li, Ishani H. Rao, Susan N. Thomas, Gregory B. Lesinski, Bassel El-Rayes, Edmund K. Waller. Turning up the heat on pancreatic ductal adenocarcinoma via inhibiting vasoactive intestinal peptide signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5571.

Pancreatic ductal adenocarcinoma (PDAC) is plagued by a dismal 5-year survival rate, early onset of metastasis and limited efficacy of systemic therapies. This scenario highlights the need to fervently pursue novel therapeutic strategies to treat this disease. Recent research has uncovered complicated dynamics within the tumor microenvironment (TME) of PDAC. An abundant stroma provides a framework for interactions between cancer-associated fibroblasts, suppressive myeloid cells and regulatory lymphocytes, which together create an inhospitable environment for adaptive immune responses. This accounts for the poor infiltration and exhausted phenotypes of effector T cells within pancreatic tumors. Innovative studies in genetically engineered mouse models have established that with appropriate pharmacological modulation of suppressive elements in the TME, T cells can be prompted to regress pancreatic tumors. In light of this knowledge, innovative combinatorial strategies involving immunotherapy and targeted therapies working in concert are rapidly emerging. This review will highlight recent advances in the field related to immune suppression in PDAC, emerging preclinical data and rationale for ongoing immunotherapy clinical trials. In particular, we draw attention to foundational findings involving T-cell activity in PDAC and encourage development of novel therapeutics to improve T-cell responses in this challenging disease.

Recruitment of immunosuppressive myeloid cells into the tumor microenvironment (TME) is a critical limitation to the efficacy of immune checkpoint inhibitors (ICIs) in patients with head and neck squamous cell carcinoma (HNSCC). In preclinical models, antibody blockade of Semaphorin 4D (SEMA4D, CD100) reduced function and recruitment of immunosuppressive myeloid cells within the TME. Importantly, combinations of anti-SEMA4D with ICIs enhanced T-cell activity and tumor regression. Pepinemab, an IgG4 humanized monoclonal antibody targeting SEMA4D, is currently being evaluated in window of opportunity, integrated biomarker trials to characterize immunomodulatory effects of treatment. SEMA4D exerts multifaceted effects within the tumor microenvironment by creating a barrier at the tumor-stroma margin, restricting immune cell infiltration and promoting immunosuppressive activity of myeloid-derived cells. In preclinical in vitro and in vivo studies, blocking antibody to SEMA4D directly enhanced M1/M2 ratio and reduced both expression of chemokines that recruit MDSC and the ability of MDSC to suppress T-cell activity. SEMA4D antibody treatment simultaneously restored the ability of dendritic cells and cytotoxic T cells to infiltrate the TME and increased ratio of Teffector to Tregulatory cells. This coordinated shift from immunosuppression to tumoricidal activity complemented effects of other immunotherapies in syngeneic tumor models. At present, three biomarker-driven window of opportunity trials are recruiting patients with four resectable indications to investigate novel combinations of pepinemab with ICIs: 1) HNSCC (NCT03690986, n=36), 2) pancreatic ductal adenocarcinoma and colorectal cancer with resectable liver metastases (NCT03373188, n=32), and 3) metastatic melanoma (NCT03769155, n=36). Presurgical treatment cohorts include combinations of pepinemab with nivolumab and /or ipilimumab, single agents, or no treatment. Three to seven weeks later, surgically resected tumors are collected under the guidance of a pathologist for comparison of tumor infiltrates across treatment groups and with a predose tissue biopsy. Blood is collected for PK, PD, and additional correlative biomarker assessments. Correlative multiplex flow cytometric and immunohistochemistry panels have been established to phenotype cells in the TME and periphery; preliminary biomarker analysis will be presented. Additional objectives include evaluation of pathologic response and extension of the previously reported safety profile of pepinemab to additional ICI combination therapies. Twelve subjects, including two HNSCC patients, have been enrolled in these studies as of 01 Feb 2019. These trials will provide the first integrated clinical assessment of the use of anti-SEMA4D antibody to reprogram the TME. Citation Format: Elizabeth E. Evans, Gregory B. Lesinski, Terrence L. Fisher, Crystal Mallow, Brian Olson, Paul E. Clavijo, John E. Leonard, Desa Rae Pastore, Ernest S. Smith, Maurice Zauderer, Clint T. Allen, Michael Lowe, Ragini Kudchadkar, Christina Wu, Conor Steuer, Nabil F. Saba. Integrated biomarker trials to evaluate myeloid and lymphoid composition of HNSCC and solid tumors treated with pepinemab and combinations with checkpoint inhibitors [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A15.

10061 Background: SEMA4D has broad immunomodulatory effects in the tumor microenvironment (TME); blocking SEMA4D in combination with checkpoint inhibitors (CI) promotes immune infiltration, reduces recruitment of myeloid cells, enhances T cell activity, and promotes tumor regression. We hypothesized that adding pepinemab (VX15/2503), which targets SEMA4D, to CI would increase immunomodulatory effects and augment response in melanoma (NCT03769155). Methods: Patients with resectable stage IIIB/C/D melanoma were enrolled to control (no neoadjuvant therapy) or treatment cohorts (n = 8 in four cohorts of pepinemab plus nivolumab, ipilimumab, nivolumab/ipilimumab or alone). Here we report results from patients receiving two doses of nivolumab (360mg) and pepinemab (15mg/kg) every three weeks followed by surgery. Primary endpoint was T cell infiltration into the TME; secondary endpoints include pathologic response rates, peripheral immune profile, and safety. Results: Ten patients are reported: two were controls, eight received neoadjuvant therapy. Two patients had pathologic complete response, one had a near-complete pathologic response ( < 1% viable tumor), one had a partial response (41% viable tumor) and four had stable disease (73-90% viable tumor). All neoadjuvant patients underwent surgery without delay; one patient experienced grade 3 post-operative cellulitis. There were two treatment-related grade 3 adverse events (weakness and arthralgia). Pharmacodynamic studies confirmed saturation of PD-1 and SEMA4D in peripheral and tumor-infiltrating T cells. T/B cell (CD8+/CD20+) ratios, a surrogate for T cell infiltration, were higher in post-treatment tumors compared to pre-treatment and were higher in the tumor bed compared to normal adjacent tissue. Flow cytometric evaluation identified an increase in CD26hi CD4+ and CD8+ tumor-infiltrating effectors in treated patients compared to controls and an increase in peripheral frequencies of the PD-1-responsive effector HLA-DR+CD38+Ki67+ CD4+ and CD8+ T cells following treatment. Treatment increased infiltration of myeloid populations into the TME, increased expression of PD-L1 on TME myeloid populations, and increased expression of the SEMA4D receptor Plexin-B2 on the surface of TME CD45− and M2 macrophages and MDSC. Conclusions: Neoadjuvant nivolumab and pepinemab results in increased T cell infiltration with excellent major response rate (38%) and expected safety profile. We continue to enroll patients using other rational combinations of pepinemab and CI. Clinical trial information: NCT03769155.

TPS269 Background: Panitumumab (Pmab) is a recombinant monoclonal antibody that binds specifically to the human epidermal growth factor receptor (EGFR), and is indicated for metastatic colorectal carcinoma (mCRC). EGFR inhibition induces synthetic lethality with poly ADP ribose polymerase inhibitors (PARPi) by attenuating DNA repair pathways. This susceptibility to PARPi-induced cell death by EGFR inhibition is associated with deficient Non-homologous end joining (NHEJ), and Homologous recombination (HR) mediated DNA repair and persistence of DNA damage. Furthermore, efficacy of PARPi (such as niraparib) is highly correlated with platinum sensitivity. Cancer cell sensitivity and resistance to both PARPi and platinum have been associated with loss and restoration of HR DNA repair, indicating similar mechanisms of anticancer activity and resistance. Platinum sensitivity in CRC could therefore predict for anticancer properties of PARPi when utilized in the setting of synthetic lethality. Combining PARP and EGFR inhibition has the potential to confer synergistic benefit, while ameliorating resistance mechanism to PARPi. This study aims to evaluate the activity of the combination of niraparib and Pmab in RAS wildtype (WT) mCRC. Methods: Eligible patients for the trial include advanced, RAS WT mCRC who have been intolerant of, progressed on, or failed at least one line of systemic chemotherapy. Those currently on first line oxaliplatin-containing regimen are allowed on the trial if they have remained stable or better (PR or CR) for at least 4 months on that line of treatment, and are being considered for maintenance therapy as standard of care. Patients must also be 18 years old, ECOG PS 0-1 and measurable disease per RECIST 1.1. A safety run-in cohort of 6 eligible patients, and additional 20 patients with the same inclusion criteria will be enrolled. Pmab dose - 6 mg/kg IV on days 1 & 15 of each 28-day cycle; Niraparib - 200mg or 300mg (based on body weight and platelet count) orally continuously. Primary endpoint: clinical benefit rate (CR +PR + SD). Biomarker analysis includes skin biopsies evaluated for p-Caspace-3, PARP, p-MAPK, Ki-67, and p27. The study was activated in Sept. 2019. Clinical trial information: NCT03983993.

830 Background: XL888 is a selective inhibitor of heat shock protein 90 (HSP90). It modulates several oncogenic signaling pathways, and the tumor microenvironment. In preclinical models, XL888 potentiates efficacy of PD-1 inhibition. We report the results of the dose escalation (DE) portion of a phase Ib trial of combined XL888 and pembrolizumab (P) in advanced gastrointestinal adenocarcinomas. Methods: XL888 was administered orally (PO) in three dose levels of 45 (DL1), 90 (DL2), 60 (only if DLT on DL2) mg twice weekly with P 200 mg IV on day 1, in 21-day cycles. Eligible patients included stage IV or locally advanced unresectable gastrointestinal adenocarcinomas with at least one prior therapy (patients with colorectal (CRC) adenocarcinoma must have received oxaliplatin, irinotecan, and fluoropyrimidine), age ≥18 years, ECOG PS 0-1, adequate organ function, no prior anti-PD-1 or anti-PD-L1 agent. The primary endpoint was recommended phase II dose (RP2D), while secondary endpoints included safety and tolerability. Pre-treatment and on-treatment correlative peripheral blood specimens were collected. Results: A total of 14 patients were enrolled in the DE phase. 9 male, median age 66.5. Diagnoses included CRC (6), pancreatic adenocarcinoma (5), biliary tract cancer (1), ampullary (1), and duodenal (1). Two patients were ineligible for assessing the primary endpoint (DL2) due to biliary stent obstruction and sepsis. One DLT (grade 3 autoimmune hepatitis) was observed on DL2. We enrolled three patients on DL3. Five additional patients were subsequently enrolled on DL2 with no additional DLT. Three patients (1 duodenal, 2 CRC) had prolonged stable disease (6, 9 and 15 cycles). The most common treatment-related toxicities included autoimmune hepatitis (G3; n = 1), retinopathy (G2; n = 2), nausea (G2; n = 1), constipation (G2; n = 1), and diarrhea (G2; n = 3). Conclusions: The XL888 and pembrolizumab combination had an acceptable safety profile and the RP2D of XL888 was 90 mg twice weekly combined with P 200 mg, every 3 weeks. The dose expansion portion and a robust series of immunologic correlative laboratory studies for this study is ongoing. Clinical trial information: NCT03095781.

High-quality and well-annotated biorepositories are needed to better understand the pathophysiology and biologic mechanisms of chronic pancreatitis (CP) and its consequences. We report a methodology for the development of a robust standard operating procedure (SOP) for a biorepository based on the experience of the clinical centers within the consortium to study Chronic Pancreatitis, Diabetes and Pancreas Cancer Clinical Centers (CPDPC), supported by the National Cancer Institute and the National Institute for Diabetes and Digestive and Kidney Diseases as a unique multidisciplinary model to study CP, diabetes, and pancreatic cancer in both children and adults. Standard operating procedures from the CPDPC centers were evaluated and consolidated. The literature was reviewed for standard biorepository operating procedures that facilitated downstream molecular analysis. The existing literature on biobanking practices was harmonized with the SOPs from the clinical centers to produce a biorepository for pancreatic research. This article reports the methods and basic principles behind the creation of SOPs to develop a biorepository for the CPDPC. These will serve as a guide for investigators developing biorepositories in pancreas research. Rigorous and meticulous adherence to standardized biospecimen collection will facilitate investigations to better understand the pathophysiology and biologic mechanisms of CP, diabetes, and pancreatic cancer.

Disrupting paracrine Hedgehog signaling in pancreatic cancer stroma through genetic deletion of fibroblast Smoothened leads to proteasomal degradation of fibroblast PTEN and accelerates tumor growth., The contribution of the tumor microenvironment to pancreatic ductal adenocarcinoma (PDAC) development is currently unclear. We therefore examined the consequences of disrupting paracrine Hedgehog (HH) signaling in PDAC stroma. Herein, we show that ablation of the key HH signaling gene Smoothened (Smo) in stromal fibroblasts led to increased proliferation of pancreatic tumor cells. Furthermore, Smo deletion resulted in proteasomal degradation of the tumor suppressor PTEN and activation of oncogenic protein kinase B (AKT) in fibroblasts. An unbiased proteomic screen identified RNF5 as a novel E3 ubiquitin ligase responsible for degradation of phosphatase and tensin homolog (PTEN) in Smo-null fibroblasts. Ring Finger Protein 5 (Rnf5) knockdown or pharmacological inhibition of glycogen synthase kinase 3β (GSKβ), the kinase that marks PTEN for ubiquitination, rescued PTEN levels and reversed the oncogenic phenotype, identifying a new node of PTEN regulation. In PDAC patients, low stromal PTEN correlated with reduced overall survival. Mechanistically, PTEN loss decreased hydraulic permeability of the extracellular matrix, which was reversed by hyaluronidase treatment. These results define non-cell autonomous tumor-promoting mechanisms activated by disruption of the HH/PTEN axis and identifies new targets for restoring stromal tumor-suppressive functions.

Pancreatic ductal adenocarcinoma (PDAC) is rarely detected early, instead presenting in patients as advanced metastatic disease once symptoms are evident. Given the central role of metastasis in pancreatic tumor progression and mortality, there is an urgent need for further investigation of how PDAC metastasizes and for novel treatment strategies targeting PDAC metastasis. A prominent histopathologic feature of PDAC is a desmoplastic, fibrotic stroma consisting of extracellular matrix components, immune cells, and activated fibroblasts termed “pancreatic stellate cells” (PSCs) that often compose a majority of cellular content in many PDAC tumors. Data from recent studies demonstrate multiple phenotypic PSC populations in the stroma (Ohlund et al., 2017); however, the influence of individual PSC populations on metastasis and disease progression is not fully understood. We hypothesized that PSCs promote pancreatic tumor metastasis through secretion of soluble factors and accompany these metastasizing tumor cells to distal sites using collective invasion mechanisms. Tumor spheroids were formed by centrifugation of HPAC cells (a human PDAC cell line) in low adhesion round bottom plates. Spheroids were embedded in both collagen- and Matrigel-based matrices and z-stacks were collected every 24hrs for 72hrs using a Lecia SP8 confocal microscope. Live cell imaging was performed in an incubation chamber for 72hrs using a Leica SP8 confocal microscope. Human pancreatic stellate cells were isolated from surgically resected tumor specimens at Emory University Hospital under IRB-approved protocols and grown to ~70% confluency before supernatants were collected. Utilizing innovative 3D invasion assays, we demonstrate the ability of human PSC conditioned media to initiate and promote invasion of HPAC pancreatic tumor spheroids. HPAC spheroids exposed to 10% PSC supernatants showed significantly decreased roundness in comparison to media alone. Further investigation of possible soluble factors mediating this interaction indicated a protein-based interaction, although alternative mechanisms by which metastasis may be initiated were also identified. Phenotypic analysis of invasion through live cell imaging led to observations of unique invasion mechanisms by which hyperactive cells at the leading edge seemingly guide invasion of large groups of cells. Additionally, we observed co-culture interactions between PSCs and HPAC cells. In comparison to HPAC cells alone, ratios of 10:1 PSCs to HPAC cells increased circularity (less invasive) while inverse ratios decreased circularity of spheroids (more invasive). This study supports the hypothesis that PSCs can promote collective invasion and migration of pancreatic tumor cells through both contact-dependent and -independent means. Further identification of mechanisms mediating this interaction may reveal novel therapeutic targets for the treatment of PDAC and present new strategies for targeting metastasis of this deadly disease. Citation Format: Michael B. Ware, Jerry Yue, Bassel El-Rayes, Shishir K. Maithel, Juan Sarmiento, Gregory B. Lesinski. Pancreatic stellate cells promote pancreatic cancer invasion and metastasis by secretion of soluble factors and through contact-mediated mechanisms [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A55.

Background: Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality, with dismal 5-year prognosis of 8%, in part due to poor response to available therapies. Thus, new systemic cancer control therapies are in dire need. Semaphorin 4D (SEMA4D) is a soluble and membrane bound glycoprotein that binds its cognate Plexin B1/B2 receptors, expressed on monocytic and granulocytic leukocytes. Prior work has shown that increased expression of SEMA4D and Plexin B in resected PDAC patients is associated with lymph node and distant metastasis, as well as a worse prognosis. Additionally, SEMA4D blockade has conferred improved immune checkpoint blockade response in murine models of colorectal carcinoma and head and neck squamous cell carcinoma via abrogation of myeloid-derived suppressor cell recruitment and function, as well as enhanced T-cell recruitment and activity within the tumor microenvironment. Here we study the effects of SEMA4D blockade in a murine model of PDAC. Methods: C57b/6 mice were orthotopically injected with murine PDAC line (KP2) derived from KRASG12D,TP53Flox/Wt;P48-Cre autochthonous tumors. Mice were treated with Folfirinox (5-FU, irinotecan, oxaliplatin, weekly), immune checkpoint blockade (ICB) (anti-PD1, anti-CTLA-4 mAbs bi-weekly), and anti-SEMA4D mAB (biweekly). Peripheral blood and tumor-infiltrating leukocytes from patients with PDAC undergoing pancreaticoduodenectomy were assessed for Plexin B1 via flow cytometry. Results: Human PDAC demonstrates penetration of Plexin B1 positive leukocytes, most notably tumor-associated macrophages, neutrophils, and monocytes. Mice injected with KP2 developed tumors detectable via high-frequency ultrasound and exhibited longer survival when treated with the combination of Folfirinox, ICB, and anti-SEMA4D antibody, compared to Folfirinox alone, Folfirinox plus ICB, or Folfirinox plus anti-SEMA4D antibody. Conclusions: Plexin B1+ myeloid subsets penetrate human PDAC tumors, and treatment with SEMA4D-blocking antibody improved response to ICB in combination with standard-of-care Folfirinox in preclinical murine studies. Future work will focus on understanding the immune mechanism of improved therapeutic response, as well as further characterization of the prevalence and prognostic ramifications of the SEMA4D-Plexin B axis in PDAC. Citation Format: Luis I. Ruffolo, Katherine M. Jackson, Nicholas Ullman, Alexander Chacon, Alexa Melucci, Paul Burchard, Mary Georger, Rachel Jewell, Peter Prieto, Brian Belt, Crystal Mallow, Elizabeth Evans, Terrence Fisher, Maurice Zauderer, Christina Wu, Brian Olson, Gregory B. Lesinski, David C. Linehan. Targeting the semaphorin 4D-plexin B axis to augment FOLFIRINOX in a murine model of pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B48.

The Jak/STAT pathway is activated in human pancreatic ductal adenocarcinoma (PDAC) and cooperates with mutant Kras to drive initiation and progression of PDAC in murine models. We hypothesized that the small-molecule Jak2 inhibitor (BMS-911543) would elicit anti-tumor activity against PDAC and decrease immune suppressive features of the disease. We used an aggressive genetically engineered PDAC model with mutant KrasG12D, tp53R270H, and Brca1 alleles (KPC-Brca1 mice). Mice with confirmed tumor burden were treated orally with vehicle or 30 mg/kg BMS-911543 daily for 14 days. Histologic analysis of pancreata from treated mice revealed fewer foci of adenocarcinoma and significantly decreased Ki67+ cells versus controls. In vivo administration of BMS-911543 significantly reduced pSTAT5 and FoxP3 positive cells within the pancreas, but did not alter STAT3 phosphorylation. Continuous dosing of KPC-Brca1 mice with BMS-911543 resulted in a median survival of 108 days, as compared to a median survival of 87 days in vehicle treated animals, a 23% increase (p = 0.055). In vitro experiments demonstrated that PDAC cell lines were poorly sensitive to BMS-911543, requiring high micromolar concentrations to achieve targeted inhibition of Jak/STAT signaling. Similarly, BMS-911543 had little in vitro effect on the viability of both murine and human PDAC-derived stellate cell lines. However, BMS-911543 potently inhibited phosphorylation of pSTAT3 and pSTAT5 at low micromolar doses in human PBMC and reduced in vitro differentiation of Foxp3+ T regulatory cells. These results indicate that single agent Jak2i deserves further study in preclinical models of PDAC and has distinct inhibitory effects on STAT5 mediated signaling.

Sorafenib is an oral multikinase inhibitor that was originally developed as a Raf kinase inhibitor. We hypothesized that sorafenib would also have inhibitory effects on cytokine signaling pathways in immune cells. PBMCs from normal donors were treated with varying concentrations of sorafenib and stimulated with IFN-α or IL-2. Phosphorylation of STAT1 and STAT5 was measured by flow cytometry and confirmed by immunoblot analysis. Changes in IFN-α– and IL-2–stimulated gene expression were measured by quantitative PCR, and changes in cytokine production were evaluated by ELISA. Cryopreserved PBMCs were obtained from cancer patients before and after receiving 400 mg sorafenib twice daily. Patient PBMCs were thawed, stimulated with IL-2 or IFN-α, and evaluated for phosphorylation of STAT1 and STAT5. Pretreatment of PBMCs with 10 μM sorafenib decreased STAT1 and STAT5 phosphorylation after treatment with IFN-α or IL-2. This inhibitory effect was observed in PBMCs from healthy donors over a range of concentrations of sorafenib (5–20 μM), IL-2 (2–24 nM), and IFN-α (101–106 U/ml). This effect was observed in immune cell subsets, including T cells, B cells, NK cells, regulatory T cells, and myeloid-derived suppressor cells. Pretreatment with sorafenib also inhibited PBMC expression of IFN-α– and IL-2–regulated genes and inhibited NK cell production of IFN-γ, RANTES, MIP1-α, and MIG in response to IFN-α stimulation. PBMCs from patients receiving sorafenib therapy showed decreased responsiveness to IL-2 and IFN-α treatment. Sorafenib is a Raf kinase inhibitor that could have off-target effects on cytokine-induced signal transduction in immune effector cells.

While surgical resection of pancreatic adenocarcinoma provides the only chance of cure, long-term survival remains poor. Immunotherapy may improve outcomes, especially as adjuvant to local therapies. Gene-mediated cytotoxic immunotherapy (GMCI) generates a systemic anti-tumor response through local delivery of an adenoviral vector expressing the HSV-tk gene (aglatimagene besadenovec, AdV-tk) followed by anti-herpetic prodrug. GMCI has demonstrated synergy with standard of care (SOC) in other tumor types. This is the first application in pancreatic cancer.Four dose levels (3 × 10(10) to 1 × 10(12) vector particles) were evaluated as adjuvant to surgery for resectable disease (Arm A) or to 5-FU chemoradiation for locally advanced disease (Arm B). Each patient received two cycles of AdV-tk + prodrug.Twenty-four patients completed therapy, 12 per arm, with no dose-limiting toxicities. All Arm A patients were explored, eight were resected, one was locally advanced and three had distant metastases. CD8(+) T cell infiltration increased an average of 22-fold (range sixfold to 75-fold) compared with baseline (p = 0.0021). PD-L1 expression increased in 5/7 samples analyzed. One node-positive resected patient is alive66 months without recurrence. Arm B RECIST response rate was 25 % with a median OS of 12 months and 1-year survival of 50 %. Patient-reported quality of life showed no evidence of deterioration.AdV-tk can be safely combined with pancreatic cancer SOC without added toxicity. Response and survival compare favorably to expected outcomes and immune activity increased. These results support further evaluation of GMCI with more modern chemoradiation and surgery as well as PD-1/PD-L1 inhibitors in pancreatic cancer.

Beverages and supplements prepared from mangosteen fruit are claimed to support gut health and immunity, despite the absence of supporting evidence from clinical trials. We recently reported that α-mangostin (α-MG), the most abundant xanthone in mangosteen fruit, altered the intestinal microbiome, promoted dysbiosis, and exacerbated colitis in C57BL/6J mice. The objective of this study was to determine whether induction of dysbiosis by dietary α-MG is limited to the C57BL/6J strain or represents a more generic response to chronic intake of the xanthone on the gut microbiota of mice. C3H, Balb/c, Nude FoxN1nu, and C57BL/6J mice, each demonstrating unique microbiomes, were fed standard diet or diet containing 0.1% α-MG for four weeks. Dietary α-MG significantly altered the cecal and colonic microbiota in all four strains of mice, promoting a reduction in generally assumed beneficial bacterial groups while increasing the abundance of pathogenic bacteria. Consumption of α-MG was associated with reduced abundance of Firmicutes and increased abundance of Proteobacteria. The abundance of Lachnospiraceae, Ruminococcaceae, and Lactobacillaceae was reduced in α-MG-fed mice, while that of Enterobacteriaceae and Enterococcaceae was increased. Dietary α-MG also was associated with increased proliferation of colonic epithelial cells, infiltration of immune cells, infiltration of immune cells and increased fluid content in stool. These results suggest that ingestion of pharmacologic doses of xanthones in mangosteen-containing supplements may adversely alter the gut microbiota and should be used with caution.

Inflammatory and fibrotic events that drive chronic pancreatitis (CP) are likely orchestrated via signaling of soluble cytokines and chemokines systemically and within the pancreas. However, a comprehensive summary of the expression of such factors during CP has not been reported to date. This information is important given continued interest in targeting cytokines that influence CP pathogenesis. Reported data on the expression change of soluble immunomodulatory factors in human CP patients were identified via a literature search using a single search term. Thirty-one articles meeting the pre-specified inclusion criteria were identified to generate a compiled data summary. Compiled data demonstrated up-regulation of several factors in the blood or pancreas microenvironment of CP patients. Nine factors were elevated in both compartments, including fractalkine, interferon gamma (IFN-γ), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), macrophage inhibitory cytokine 1 (MIC-1), neutrophil gelatinase-associated lipocalin (NGAL/LCN2), transforming growth factor beta (TGF-β), and tumor necrosis factor alpha (TNF-α). Most up-regulated factors could be classified into one of several functional groups, including inflammation, chemotaxis, angiogenesis, bone remodeling, extracellular matrix remodeling, and pain. Following further validation, these factors may be used as biomarkers for disease diagnosis, identification of comorbidities, or as potential therapeutic targets.

BACKGROUND Heat shock protein 90 (HSP90) and the ubiquitin-proteasome pathway play crucial roles in the homeostasis of pancreatic cancer cells. This study combined for the first time the HSP90 inhibitor ganetespib (Gan) and the proteasome inhibitor carfilzomib (Carf) to target key mechanisms of homeostasis in pancreatic cancer. It was hypothesized that Gan plus Carf would elicit potent antitumor activity by modulating complementary homeostatic processes. METHODS In vitro and in vivo effects of this combination on mechanisms of cell growth and viability were evaluated with human pancreatic cancer cell lines (MIA PaCa-2 and HPAC). RESULTS Combined treatment with Gan and Carf significantly decreased cell viability. The mechanism varied by cell line and involved G2-M cell-cycle arrest accompanied by a consistent reduction in key cell-cycle regulatory proteins and concomitant upregulation of p27. Further studies revealed increased autophagy markers, including the upregulation of autophagy related 7 and light chain 3 cleavage, and evidence of apoptosis (increased Bax expression and processing of caspase 3). Immunoblot analyses confirmed the modulation of other pathways that influence cell viability, including phosphoinositide 3-kinase/Akt and nuclear factor κB. Finally, the treatment of athymic mice bearing HPAC tumors with Gan and Carf significantly reduced tumor growth in vivo. An immunoblot analysis of freshly isolated tumors from animals at the end of the study confirmed in vivo modulation of key signaling pathways. CONCLUSIONS The results reveal Gan plus Carf to be a promising combination with synergistic antiproliferative, apoptotic, and pro-autophagy effects in preclinical studies of pancreatic cancer and will further the exploration of the utility of this treatment combination in clinical trials. Cancer 2017. © 2017 American Cancer Society.