CCR5 Inhibition Research Articles

Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop.

Aberrant energy metabolism is critical for cancer progression. Tumor-associated macrophages (TAMs) can stimulate tumor angiogenesis and enhance cancer metastasis; however, the metabolic interaction between cancer cells and macrophages characterized by lactate shuttles remains unclear. Here, we showed that lactate activated human macrophages to a TAM-like phenotype and stimulated the secretion of CCL5 by activation of Notch signaling in macrophages. Reciprocally, CCL5 increased cell migration, induced cancer cell EMT, and promoted aerobic glycolysis in breast cancer cells, suggesting a positive metabolic feedback loop in the co-culture system. Inhibition of CCR5, the cognate receptor of CCL5, or neutralization of CCL5, broke the metabolic loop and decreased cancer cell migration and EMT. Inhibition of aerobic glycolysis significantly reduced breast cancer cell EMT, indicated that aerobic glycolysis was necessary for the invasive phenotype of cancer cells. We further showed that TGF-β signaling regulated the expression of CCR5 in the co-culture system, and CCL5 induced glycolysis by mediation of AMPK signaling. The expression of CCL5-CCR5 axis was highly associated with macrophage infiltration, TGF-β and p-AMPK in clinical samples. CCL5-CCR5 axis promoted breast cancer metastasis in vivo. Our findings suggested a pivotal role of CCL5-CCR5 axis in the metabolic communication between cancer cells and macrophages.

CCR2 expression on circulating monocytes is associated with arterial wall inflammation assessed by 18F-FDG PET/CT in patients at risk for cardiovascular disease.

Circulating monocytes infiltrate the plaque and differentiate into macrophages, contributing to an inflammatory environment which is associated with higher risk of cardiovascular events. Although the pivotal role of circulating monocytes in plaque inflammation has been firmly established, the search continues to identify specific monocyte subsets that may be especially atherogenic. Therefore, we evaluated the relation between monocyte phenotype, particularly surface receptor expression, and arterial wall inflammation in patients at increased cardiovascular risk. We performed a multivariate linear regression analysis in 79 patients at increased cardiovascular risk who had both an 18F-fluorodeoxyglucose positron emission tomography/computed tomography to assess arterial wall inflammation and extensive monocyte characterization (using flow cytometry). We found that CCR2, a monocyte chemokine receptor essential for transmigration, significantly correlates with arterial wall inflammation. This relationship was independent of traditional cardiovascular risk factors and statin use (β = 0.429, P = 0.015). We found no relation between arterial wall inflammation and monocyte count or monocyte subsets, namely CD14+CD16-, CD14+CD16+, CD14+CD16 ++, CCR5+, CD18+, CD11b+, or CD11c+ monocytes. Monocyte CCR2 expression is associated with arterial wall inflammation in patients at increased cardiovascular risk. Our data warrant further studies to assess if inhibition of CCR2 may attenuate atherosclerotic plaque inflammation.

Differential impact of the dual CCR2/CCR5 inhibitor cenicriviroc on migration of monocyte and lymphocyte subsets in acute liver injury.

A hallmark of acute hepatic injury is the recruitment of neutrophils, monocytes and lymphocytes, including natural killer (NK) or T cells, towards areas of inflammation. The recruitment of leukocytes from their reservoirs bone marrow or spleen into the liver is directed by chemokines such as CCL2 (for monocytes) and CCL5 (for lymphocytes). We herein elucidated the impact of chemokine receptor inhibition by the dual CCR2 and CCR5 inhibitor cenicriviroc (CVC) on the composition of myeloid and lymphoid immune cell populations in acute liver injury. CVC treatment effectively inhibited the migration of bone marrow monocytes and splenic lymphocytes (NK, CD4 T-cells) towards CCL2 or CCL5 in vitro. When liver injury was induced by an intraperitoneal injection of carbon tetrachloride (CCl4) in mice, followed by repetitive oral application of CVC, flow cytometric and unbiased t-SNE analysis of intrahepatic leukocytes demonstrated that dual CCR2/CCR5 inhibition in vivo significantly decreased numbers of monocyte derived macrophages in acutely injured livers. CVC also reduced numbers of Kupffer cells (KC) or monocyte derived macrophages with a KC-like phenotype, respectively, after injury. In contrast to the inhibitory effects in vitro, CVC had no impact on the composition of hepatic lymphoid cell populations in vivo. Effective inhibition of monocyte recruitment was associated with reduced inflammatory macrophage markers and moderately ameliorated hepatic necroses at 36h after CCl4. In conclusion, dual CCR2/CCR5 inhibition primarily translates into reduced monocyte recruitment in acute liver injury in vivo, suggesting that this strategy will be effective in reducing inflammatory macrophages in conditions of liver disease.

750P - Phase Ib study of PF-04136309 (an oral CCR2 inhibitor) in combination with nab-paclitaxel/gemcitabine in first-line treatment of metastatic pancreatic adenocarcinoma

750P - Phase Ib study of PF-04136309 (an oral CCR2 inhibitor) in combination with nab-paclitaxel/gemcitabine in first-line treatment of metastatic pancreatic adenocarcinoma

An agent-based model of triple-negative breast cancer: the interplay between chemokine receptor CCR5 expression, cancer stem cells, and hypoxia

BackgroundTriple-negative breast cancer lacks estrogen, progesterone, and HER2 receptors and is thus not possible to treat with targeted therapies for these receptors. Therefore, a greater understanding of triple-negative breast cancer is necessary for the treatment of this cancer type. In previous work from our laboratory, we found that chemokine ligand-receptor CCL5-CCR5 axis is important for the metastasis of human triple-negative breast cancer cell MDA-MB-231 to the lymph nodes and lungs, in a mouse xenograft model. We collected relevant experimental data from our and other laboratories for numbers of cancer stem cells, numbers of CCR5+ cells, and cell migration rates for different breast cancer cell lines and different experimental conditions.ResultsUsing these experimental data we developed an in silico agent-based model of triple-negative breast cancer that considers surface receptor CCR5-high and CCR5-low cells and breast cancer stem cells, to predict the tumor growth rate and spatio-temporal distribution of cells in primary tumors. We find that high cancer stem cell percentages greatly increase tumor growth. We find that anti-stem cell treatment decreases tumor growth but may not lead to dormancy unless all stem cells get eliminated. We further find that hypoxia increases overall tumor growth and treatment with a CCR5 inhibitor maraviroc slightly decreases overall tumor growth. We also characterize 3D shapes of solid and invasive tumors using several shape metrics.ConclusionsBreast cancer stem cells and CCR5+ cells affect the overall growth and morphology of breast tumors. In silico drug treatments demonstrate limited efficacy of incomplete inhibition of cancer stem cells after which tumor growth recurs, and CCR5 inhibition causes only a slight reduction in tumor growth.

Abstract 5655: Inhibition of CCR2 potentiates checkpoint inhibitor immunotherapy in murine model of pancreatic cancer

Abstract Pancreatic cancer is an aggressive malignancy with a 5 year survival rate of less than five percent. The predominant immune cells infiltrating the tumor microenvironment are monocytes/macrophages, which are reported to support tumor growth by suppressing host immune responses to the tumor. Recruitment of monocytes to various tissues, including tumors, is dependent upon activation of the chemokine receptor CCR2 by one or more of the chemokines CCL2, CCL8 and CCL13. In preclinical and clinical studies, inhibition of CCR2 in pancreatic cancer has shown to decrease tumor progression by blocking recruitment and accumulation of monocytes/macrophages in the tumor microenvironment. Analysis of human pancreatic tumors revealed elevation of both CCL2 and CSF1, which recruit monocytes, as well as the monocyte marker CD14, in advanced pancreatic cancers. Current immunotherapy using checkpoint inhibitors are effective in some tumors, but lack efficacy in immune insensitive cancers, including pancreatic cancer. Here, we report that the inhibition of CCR2 using a small molecule antagonist potentiates anti-PD-1 immunotherapy in a syngeneic, orthotropic mouse model of pancreatic cancer. Our data reveal that blocking CCR2 decreases tumor burden by blocking monocyte infiltration and creating a microenvironment more favorable for CD8 T cells activity, and provide a mechanistic rationale for investigating the combination of a CCR2 antagonist and an immune checkpoint inhibitor in pancreatic cancer. Citation Format: Christine Janson, Heiyoun Jung, Linda Ertl, Shirley Liu, Ton Dang, Yibin Zeng, Antoni Krasinski, Jeff McMahon, Penglie Zhang, Israel Charo, Rajinder Singh, Thomas J. Schall. Inhibition of CCR2 potentiates checkpoint inhibitor immunotherapy in murine model of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5655. doi:10.1158/1538-7445.AM2017-5655

Abstract LB-194: Targeting tumor associated myeloid cells with CCR2 inhibitor PF-04136309 enhances gemcitabine/paclitaxel and doxorubicin anti-tumor activity

Abstract Purpose: MDSCs and macrophages have been reported to be increased in the tumor microenvironment by chemotherapy treatment. These cells may limit the beneficial effects of chemotherapy by limiting drug access or by suppressing the tumor targeted immune response evoked by the chemotherapy. The purpose of the current study was to investigate the ability of chemokine receptor CCR2 inhibitor, PF-04136309, to block tumor MDSC and macrophage accumulation and to investigate the combination of this treatment with standard of care chemotherapies. Experimental design: The ability of CCR2 inhibitor, PF-04136309, to reduce tumor growth in mice was evaluated using orthotopically or SC injected syngeneic colon, pancreatic and ovarian tumor cell lines. PF-04136309 was evaluated as single agent, in combination with doxorubicin, or in the combination of gemcitabine and paclitaxel. Results: In a model of ovarian carcinomatosis (ID8), where tumor progression is driven by MDSCs and macrophages, single agent PF-04136309 reduced peritoneal tumor load as well as the total volume and rate of ascites fluid production. Even delayed treatment of established tumors, at 6 weeks post tumor inoculation, was effective. Ascites volume correlated with overall tumor load in treated mice. The treatment of ovarian tumor bearing mice with PF-04136309 in combination with doxorubicin, resulted in significant reduction in tumor load and ascites formation, beyond that produced by doxorubicin alone. To evaluate the addition of PF-04136309 to the chemotherapy combination, gemcitabine/paclitaxel, we selected models of pancreatic (Pan02) and colon (MC38) carcinoma, where PF-04136309 did not show single agent efficacy. The addition of PF-04136309 resulted in significantly decreased tumor burden compared to mice treated with chemotherapy alone. To investigate the mechanism of action of PF-04136309 we selected the MC38 model. PF-04136309 treatment of MC38 tumor bearing mice resulted in a substantial reduction, as a proportion of CD45+ cells, of MDSCs (CD11b+MHCII-Ly6C+) in the circulation (80%), and of MDSCs (60%) and macrophages (CD11b+F4/80+MHCII+Ly6C/G-) (70%) in the tumor, respectively. Conclusion: PF-04136309 significantly improved the response to several chemotherapeutics, providing strong rationale for CCR2 antagonism as an addition to standard of care chemotherapies. PF-04136309 is currently being evaluated in combination with gemcitabine/nab-paclitaxel in metastatic pancreatic cancer patients. The observed inhibitory effect of PF-04136309 on ID8 ascites formation, the synergy with doxorubicin, and the known intervention of MDSC and macrophages in human ovarian peritoneal carcinomatosis, suggests potential clinical efficacy with reduction of ascites volume and synergy in combination with pegylated doxorubicin in platinum resistant ovarian cancer patients. Citation Format: Sasha Farina, Hsunhui Yang, Guang Huan Tu, Erick C. Gamelin, John C. Lin, Changyu Wang, Reid Feldman, Siler Panowski, Cecilia Oderup. Targeting tumor associated myeloid cells with CCR2 inhibitor PF-04136309 enhances gemcitabine/paclitaxel and doxorubicin anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-194. doi:10.1158/1538-7445.AM2017-LB-194

Abstract 3021: CCR5 inhibition: macrophage repolarization therapy for colorectal cancer

Abstract In patients with metastatic colorectal cancer (CRC), the local immune response influences the clinical course. An in-depth analysis of the invasive margin of human CRC liver metastases revealed a distinct immunological microenvironment. Within this microenvironment, two distinct subsets of myeloid cells induce an influx of T cells into the invasive margin via CXCL9/CXCL10. CCL5 is produced by these T cells and stimulates pro-tumoral effects via CCR5, creating an exploitive loop. CCR5 was found on macrophages, lymphocytes and on the vast majority of tumor cells. Inhibition of CCR5 in patient-derived functional in vitro organotypic culture models showed a promising macrophage repolarization with anti-tumoral effects. These effects are mediated by activation of an antiviral program in macrophages, leading to interferon and reactive oxygen species production and subsequent selective tumor cell death. These anti-tumoral effects were confirmed in a phase I trial with a CCR5 antagonist in 14 patients with liver metastases of advanced refractory CRC. Treatment with the oral CCR5 Inhibitor was very well tolerated and objective responses were seen, especially in combination with previously ineffective chemotherapy. Biopsies revealed mitigation of tumor-promoting inflammation within the tumor tissue, confirming the validity of the explant model and highlighting the feasbility of this approach. It furthermore shows the proof-of-concept for macrophage repolarization in cancer patients. Citation Format: Niels Halama, Inka Zoernig, Anna Berthel, Christoph Kahlert, Fee Klupp, Meggy Suarez-Carmona, Karsten Brand, Juergen Krauss, Felix Lasitschka, Alexis Ulrich, Juergen Weitz, Martin Schneider, Markus Buechler, Laurence Zitvogel, Thomas Herrmann, Axel Benner, Christina Kunz, Stephan Luecke, Christoph Springfeld, Christine S. Falk, Dirk Jaeger. CCR5 inhibition: macrophage repolarization therapy for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3021. doi:10.1158/1538-7445.AM2017-3021

CCR-2 neutralization augments murine fresh BMC activation by Staphylococcus aureus via two distinct mechanisms: at the level of ROS production and cytokine response.

CCR-2 signaling regulates recruitment of monocytes from the bone marrow into the bloodstream and then to sites of infection. We sought to determine whether CCL-2/CCR-2 signaling is involved in the killing of Staphylococcus aureus by murine bone marrow cells (BMCs). The intermittent link of reactive oxygen species (ROS)-NF-κB/p38-MAPK-mediated CCL-2 production in CCR-2 signaling prompted us to determine whether neutralization of CCR-2 augments the response of murine fresh BMCs (FBMCs) after S. aureus infection. It was observed that anti-CCR-2 Ab-treated FBMCs released fewer ROS on encountering S. aureus infection than CCR-2 non-neutralized FBMCs, also correlating with reduced killing of S. aureus in CCR-2 neutralized FBMCs. Staphylococcal catalase and SOD were also found to play a role in protecting S. aureus from the ROS-mediated killing of FBMC. S. aureus infection of CCR-2 intact FBMCs pre-treated with either NF-κB or p-38-MAPK blocker induced less CCL-2, suggesting that NF-κB or p-38-MAPK is required for CCL-2 production by FBMCs. Moreover, blocking of CCR-2 along with NF-κB or p-38-MAPK resulted in elevated CCL-2 production and reduced CCR-2 expression. Inhibition of CCR-2 impairs the response of murine BMCs to S. aureus infection by attenuation ROS production and modulating the cytokine response.

Targeting inflammatory monocytes in human metastatic colorectal cancer.

605 Background: Colorectal cancer (CRC) is the most common gastrointestinal malignancy. 60% of CRC patients are diagnosed with metastatic CRC (mCRC) and the 5-year survival is < 20%. CCR2+ inflammatory monocytes (IM) are recruited from the bone marrow to the tumor microenvironment by the CCL2/CCR2 chemokine axis. At the tumor, they become tumor associated macrophages (TAM) and play a crucial role in promoting tumor progression, metastasis, and chemoresistance. While the importance of IM has been shown in other malignancies, little is known about their role in mCRC. Methods: Flow cytometry was performed on human and murine PBMCs, adjacent normal tissue, and tumors. Qualitative RT-PCR, ELISA, and confocal microscopy were performed for CCL2 expression. T-cell suppression assays were performed using CD14+ IM isolated from patient PBMCs and liver metastasis (LM). A murine model of CRC LM was created by hemispleen injection of luciferase-labelled MC38 CRC cells. Mice were treated with a CCR2 inhibitor and/or FOLFOX. Results: Prior to liver resection, mCRC patients have a higher percentage of IM in the peripheral blood compared to healthy donors (p < 0.0001), and on multivariate analysis elevated monocytes were prognostic of poor survival. We also found higher levels of CCL2 in the serum of mCRC patients (p < 0.01). Additionally, there was increased expression of CCL2 in LM compared to uninvolved tissue (p < 0.01), with the production of CCL2 localized to mCRC cells. FACS analysis showed CCR2+ TAM were elevated in LM compared to adjacent normal liver (p < 0.05) with a paucity of effector T-cells. CD14+ TAMs isolated from mCRC inhibited T-cell proliferation, illustrating the immune suppressive phenotype of these cells. In a murine model of CRC LM, treating mice with a CCR2 inhibitor alone or in combination with FOLFOX chemotherapy resulted in decreased tumor burden. FACS analysis of treated tumors showed increased effector T-cells in mice treated with CCR2 inhibitor. Conclusions: IM are involved in the progression of mCRC. Targeting these immunosuppressive cells with a CCR2 inhibitor decreases tumor burden in a murine model. This represents a potential novel treatment for mCRC.

Orally administered CCR2 selective inhibitor CCX872-b clinical trial in pancreatic cancer.

276 Background: CCR2 inhibition decreases tumor-associated macrophages and Treg cells, and increases CD8+ and CD4+ T cells in pancreatic tumors. Single oral doses of 150 mg CCX872-B were well tolerated in patients with pancreatic cancer. The first stage results of the multiple dose part of this study are presented. Methods: Patients with locally advanced or metastatic pancreatic cancer, ECOG score ≤ 2 were studied. Patients received FOLFIRINOX (fluorouracil [5-FU], leucovorin, irinotecan, oxaliplatin) q2weeks for up to 24 wks plus 150 mg CCX872-B once or twice daily until disease progression or intolerance to CCX872-B. The primary efficacy endpoint is progression-free survival (PFS) at 24 wks. Tumor control rate (TCR) and the objective response rate (ORR), a secondary endpoint, are presented. Results: Fifty patients were enrolled. Baseline: Mean ± SD age: 60 ± 8.6 years, male 52%; primary tumor location: head 57%, body/tail 41%, local recurrence 2%; metastatic disease 78%, ECOG score 0: 62%, ECOG score 1: 38%. Thirty five of 50 patients completed week 12. Most common reasons for early withdrawal were subject request (5 pts) and adverse events typically associated with FOLFIRINOX (4 pts). In the pre-specified analysis population, i.e., those with ≥ 1 post baseline CT scan, tumor control at wk 12 was achieved in 32 of 41 (78%) patients, with 15 of 41 (37%) partial responders, and 17 of 41 (41%) with stable disease. The ORR was 37% (all partial responders) in the primary analysis population (30% when all enrolled patients were included). Thirteen of 21 patients with available CT scan results at week 24 were progression-free. Mean trough CCX872 plasma level was ~8 μg/mL. CCR2 was covered 88% on average by CCX872-B based on receptor occupancy assays. CCX872-B appeared to be well tolerated and the incidence of ≥ Gr 3 AEs was not increased with CCX872-B plus FOLFIRINOX vs historical data with FOLFIRINOX alone. The complete set of PFS data will be available by the end of 2016. Conclusions: CCX872-B plus FOLFIRINOX resulted in a TCR of 78% and an ORR of 30 to 37% with no safety issues ascribed to CCX872-B use. Clinical trial information: NCT02345408.

SAT-343 - The CCR2 inhibitor propagermanium attenuates diet-induced insulin resistance, adipose tissue inflammation and nonalcoholic steatohepatitis

SAT-343 - The CCR2 inhibitor propagermanium attenuates diet-induced insulin resistance, adipose tissue inflammation and nonalcoholic steatohepatitis

Abstract B037: Macrophage repolarization therapy in metastatic colorectal cancer: CCR5 inhibition

Abstract The influence of the local immune response on the clinical course of colorectal cancer (CRC) has been analyzed extensively. Analyzing the invasive margin of human CRC liver metastases, we identified a protumoral mechanism of T-cell-derived CCL5 that leads to immune cell exploitation by tumor cells. Two distinct subsets of myeloid cells produce CXCL9/CXCL10, which induce an influx of T cells into the invasive margin. CCL5 is produced by these exhausted T cells and stimulates tumor cell proliferation and invasive behavior via CCR5 on tumor cells and macrophages. CCR5 inhibition in patient-derived functional in vitro organotypic culture models induced macrophage repolarization with anti-tumoral effects. These immunomodulatory and anti-tumoral effects of CCR5 blockade then could be confirmed in a phase I trial with a CCR5 antagonist in advanced refractory CRC patients with liver metastases. Amelioration of tumor-promoting inflammation on the tumor tissue level and objective tumor responses in advanced metastatic CRC patients were observed. Citation Format: Niels Halama, Inka Zoernig, Anna Berthel, Christoph Kahlert, Fee Klupp, Meggy Suarez-Carmona, Juergen Krauss, Karsten Brand, Felix Lasitschka, Tina Lerchl, Claudia Luckner-Minden, Alexis Ulrich, Juergen Weitz, Martin Schneider, Markus W. Buechler, Laurence Zitvogel, Thomas Herrmann, Axel Benner, Christina Kunz, Stephan Luecke, Christoph Springfeld, Christine Falk, Dirk Jaeger. Macrophage repolarization therapy in metastatic colorectal cancer: CCR5 inhibition [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B037.

Abstract A107: Inhibition of CCR2 potentiates the checkpoint inhibitor immunotherapy in pancreatic cancer

Abstract Pancreatic cancer is an aggressive malignancy with 5 year survival rate of less than 5 percent. The predominant immune cells infiltrating the tumor microenvironment are monocytes/macrophages, which are reported to support tumor growth by suppressing host immune responses to the tumor. Recruitment of monocytes to various tissues, including tumors, is dependent upon activation of the chemokine receptor CCR2 by one or more of the chemokines CCL2, CCL8 and CCL13. In preclinical and clinical studies, inhibition of CCR2 in pancreatic cancer has shown to decrease tumor progression by blocking recruitment and accumulation of monocytes/macrophages in the tumor microenvironment. Analysis of the TCGA genomic database of human pancreatic tumors revealed elevation of both CCL2 and CSF1, which recruit monocytes, as well as the monocyte marker CD14, in advanced pancreatic cancers. Current immunotherapy using checkpoint inhibitors are effective in some tumors, but lack efficacy in immune insensitive cancers, including pancreatic cancer. Here, we report that the inhibition of CCR2 using small molecule antagonist potentiates anti-PD-1/PD-L1 immunotherapy in a syngeneic, orthotopic mouse model of pancreatic cancer. The KCKO pancreatic cancer cell line, which harbors a K-ras mutation, was implanted into the tail of the pancreas, and a small molecule CCR2 antagonist was administered after a stable tumor mass had formed. Tumor weight correlated well with the per cent of circulating monocytes in the peripheral blood, and the CCR2 antagonist significantly decreased the blood monocyte count. Similar to the human tumor stroma, the KCKO mouse tumors were infiltrated with monocytes and macrophages, and CCR2 antagonist treatment decreased the infiltration of monocyte/ macrophage. PD-L1 expressions are found in the human pancreatic tumor microenvironment, but treatment of patients with anti-PD-1 has not been found to have efficacy. Similarly, anti-PD-1 treatment alone was not effective in the murine KCKO model, but combination treatment with a CCR2 antagonist resulted in significantly smaller tumors. Moreover, this effect was completely reversed by depleting CD8 T cells, suggesting that by blocking monocyte/macrophage recruitment, the CCR2 antagonist relieved suppression of the CD8 T cells. We confirmed this hypothesis by demonstrating that cells from the KCKO tumor microenvironment inhibited CD3/CD28-induced proliferation of CD8 T cells in culture, but that this inhibition was not present when the mice had received the CCR2 antagonist. Taken together, these data reveal that blocking CCR2 decreases tumor burden by blocking monocyte infiltration and creating a microenvironment more favorable for CD8 T cells activity, and provide a mechanistic rationale for investigating the combination of a CCR2 antagonist and an immune checkpoint inhibitor in pancreatic cancer. Citation Format: Heiyoun Jung, Linda Ertl, Christine Janson, Thomas Schall, Israel Charo. Inhibition of CCR2 potentiates the checkpoint inhibitor immunotherapy in pancreatic cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A107.

Reduction of Liver Fibrosis by CCR2 Antagonist CCX872 in Murine Models of NASH

Introduction: Liver fibrosis in non-alcoholic steatohepatitis (NASH) is caused by chronic inflammation with macrophage infiltration and cytokine production. Inflammatory macrophages express CCR2, which they use to migrate to the liver. We investigated the efficacy of a CCR2 specific inhibitor, CCX872, in two murine models of NASH. Methods: Two models of NASH were used for determining the efficacy of CCX872 in reducing fibrosis. One model used a high-fat diet (HFD) supplemented with 30% fructose in water, for 30 weeks, to induce NASH and liver fibrosis. After 22 weeks on this diet the mice were given CCX872 (30 mg/kg) once daily or vehicle, both administered subcutaneously for 8 weeks. In the second model the mice were fed a methionine-choline diet deficient (MCD) for 8 weeks to induce liver fibrosis. Mice were treated with CCX872 (30 mg/kg) once daily, or vehicle, both administered subcutaneously for 8 weeks. Liver injury was assessed by standard methods. Liver fibrosis was assessed by sirius red staining, and quantified with the computer program “Image J”. Results: In the HFD/fructose model, mice treated with CCX872 showed reduced accumulation of CD11b+ F4/80+ macrophages in the liver, as compared with vehicle treated mice (p=0.03). Mice treated with CCX872 also had improved insulin sensitivity (p < 0.001) and glucose tolerance (p < 0.001) compared with vehicle. Hepatic triglyceride accumulation was significantly lower in CCX872 treated mice (p < 0.01). Treatment with CCX872 significantly reduced both ALT and AST concentrations in HFD diet fed mice (p < 0.0001 for both ALT and AST). Moreover, 8 weeks of treatment with CCX872 significantly reduced collagen content by approximately 50% based on Sirius red staining (p=0.008), as compared with vehicle treated mice. In the MCD model, ALT was also reduced by CCX872 treatment (p=0.03). Administration of CCX872 reduced collagen content by approximately 25% (p=0.035) in MCD diet fed mice. Conclusion: We investigated the efficacy of a clinical-stage small molecule inhibitor of CCR2, CCX872 for the treatment of steatohepatitis in mice. Mice on HFD treated with CCX872 had reduced hepatic inflammation and steatosis as well as improved insulin sensitivity and glucose tolerance, compared to vehicle treated mice. Moreover, treatment with CCX872 had a marked therapeutic effect on improving liver fibrosis in both mice models of NASH. These findings suggest a potential application of CCR2 inhibitor, CCX872, for treatment of patients with NASH.

CCR5 inhibition in colorectal cancer patients

Immunotherapy in metastatic colorectal cancer is a challenge. I would like to thank Dr. Mukaida for his comprehensive commentary (1) on our work (2). A couple of points were raised that I will address in this letter. Immunotherapy with new immune checkpoint inhibitors is a different approach (3) compared to the immunotherapies based on vaccination or cytokine application. While for vaccination approaches no clear benefit was identified in colorectal cancer (4), combined chemotherapy with cytokines (GOLFIG trial) showed clinical effects, albeit at the price of autoimmune side effects (5). This situation highlights the complexities of immunotherapies quite well and shows that patients with microsatellite-instable colorectal cancer are a limited subgroup within colorectal cancer (6).

CCR5 inhibition in colorectal cancer patients

CCR5 inhibition in colorectal cancer patients

Chemokine (C-C motif) receptor 2-positive monocytes aggravate the early phase of acetaminophen-induced acute liver injury.

Infiltrating monocyte-derived macrophages aggravate APAP hepatotoxicity, and the pharmacological inhibition of either CCL2 or CCR2 might bear therapeutic potential by reducing the inflammatory reaction during the early phase of AILI. (Hepatology 2016;64:1667-1682).

Autocrine MCP-1/CCR2 signaling stimulates proliferation and migration of renal carcinoma cells.

The chemokine monocyte chemoattractant protein-1 [MCP-1; also known as chemokine (C-C motif) ligand 2] is an important mediator of monocyte recruitment during inflammatory processes. Pathologically high expression levels of MCP-1 by tumor cells have been observed in a variety of cancer types. In the majority of cases, high MCP-1 expression is associated with a poor prognosis, as infiltration of the tumor with inflammatory monocytes promotes tumor progression and metastasis. MCP-1 is also expressed in renal cell carcinoma (RCC). In the present study, the function and the regulation of MCP-1 was investigated in two RCC cell lines, CaKi-1 and 786-O. In both cell lines, expression of MCP-1 was significantly enhanced compared with non-cancerous control cells. As expected, secretion of MCP-1 into the medium facilitated the recruitment of peripheral blood monocytes via the chemokine (C-C motif) receptor type 2 (CCR2). As expression of CCR2 was also detected in 786-O and CaKi-1 cells, the effect of autocrine MCP-1/CCR2 signaling was evaluated in these cells. In proliferation assays, administration of an MCP-1 neutralizing antibody or of a CCR2 antagonist to CaKi-1 and 786-O cells significantly decreased cell growth; supplementation of the growth medium with recombinant human MCP-1 had no additional effect on proliferation. The migration ability of RCC cells was impaired by MCP-1 neutralization or pharmacological CCR2 inhibition, while it was stimulated by the addition of recombinant human MCP-1, compared with untreated control cells. Finally, substantial differences in the regulation of MCP-1 expression were observed between RCC cell lines. In CaKi-1 cells, expression of MCP-1 appears to be largely mediated by the transcription factor nuclear factor of activated T cells 5, while in 786-O cells, deletion of the tumor suppressor gene Von-Hippel-Lindau appeared to be responsible for MCP-1 upregulation, as suggested by previous studies. Taken together, the results of the current study indicate that expression of MCP-1 in RCC cells promotes tumor progression and metastasis not only by paracrine, but also by autocrine, MCP-1/CCR2 signaling events, enhancing cell proliferation and migration ability. Therefore, the present findings suggest the MCP-1/CCR2 axis is a potential target for future therapeutic strategies in the treatment of metastatic RCC.

Abstract 4150: Blockade of CXCR2 mediated granulocytic MDSC recruitment synergizes with CCR2 inhibition of inflammatory monocytes and restores anti-tumor immunity in pancreatic adenocarcinoma

Abstract Introduction: Pancreatic cancer (PC) is characterized by a dense tumor stroma with a heavy leukocytic infiltrate, comprised predominately of immunosuppressive bone marrow (BM) derived cells. We have previously demonstrated in a phase Ib clinical trial that CCR2 inhibition (CCR2i) prevents inflammatory monocyte (IM) recruitment from the BM and results in a significant reduction of tumor associated macrophages (TAM) and an increase in treatment efficacy. However, granulocytic myeloid derived suppressor cells (G-MDSC) remain in the tumor microenvironment (TME) following CCR2i. Herein, we explored the impact of targeting G-MDSC recruitment to PC tumors both alone and in combination with CCR2i. Methods: Human BM, blood, and tumor was collected under an IRB approved protocol. A tissue microarray (TMA) from resected PC patients was analyzed for immune infiltrate. Mice were injected orthotopically with 2.5×106 syngeneic PC cells. CXCR2 and CCR2 inhibitors (Tocris) were given twice daily. Tumor growth was assessed and specimens obtained for analysis by flow cytometry, RNAseq, and IHC. Results: Human PC overexpresses CXCL5 and CXCL8, corresponding with an abundance of tumor infiltrating CXCR2+ G-MDSC. Furthermore, the ratio of CD8 to G-MDSC correlates with survival in human PC patients. In an orthotopic murine model that recapitulates human disease, ΣCXCL ligands were also increased. Either Ly6G depletion or targeted blockade with a CXCR2 inhibitor decreased G-MDSC and reduced tumor burden. Intriguingly, blockade of IM from the BM did not reduce G-MDSC and paradoxically resulted in a modest increase in this population within the tumors from human patients following CCR2i. Thus, we explored the combination of CCR2/CXCR2 blockade both with and without FOLFIRINOX chemotherapy. This resulted in a synergistic impact when both BM derived populations were targeted and dual therapy was further enhanced by FOLFIRINOX. RNAseq analysis of tumors following monotherapy or dual inhibition revealed alterations in the TME favoring an anti-tumor immune response. To test the hypothesis that this effect was mediated by restoration of anti-tumor immunity we analyzed the tumor infiltrating lymphocyte (TIL) populations and found a significant increase in the relative and absolute numbers of CD8+ and C4+ TIL. Analysis of the activation status of these cells demonstrated an increase in effector CD8+ T-cell phenotype (IFNγ+, CD69+, CD44+). Using Nur77GFP T-cell receptor reporter mice, we showed an increase in GFP expressing CD8+ TIL following dual blockade. CD8 depletion resulted in a loss of therapeutic efficacy of myeloid blockade, further confirming our hypothesis. Conclusion: These findings suggest that combinatorial blockade strategies preventing tumor infiltration by myeloid cells may restore anti-tumor immunity in PC. Citation Format: Timothy M. Nywening, Brian A. Belt, Roheena Z. Panni, Darren Cullinan, Dominic E. Sanford, Ryan C. Fields, William G. Hawkins, David G. DeNardo, William E. Gillanders, Peter Goedegebuure, David C. Linehan. Blockade of CXCR2 mediated granulocytic MDSC recruitment synergizes with CCR2 inhibition of inflammatory monocytes and restores anti-tumor immunity in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4150.