Ayumi Hashimoto

Lectin-type oxidized LDL receptor-1 distinguishes population of human polymorphonuclear myeloid-derived suppressor cells in cancer patients

PMN-MDSC in cancer patients can be distinguished from neutrophils by a genomic signature and by expression of the LOX-1 receptor. Stressing myeloid-derived suppressor cells in cancer Immunotherapies for cancer have shown promising results in part because they overcome the suppressive effects of the tumor microenvironment on immune cells. Condamine et al. now report that polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) can be distinguished from neutrophils in the same cancer patient by the expression of the lipid metabolism–related molecule lectin-type oxidized LDL receptor-1 (LOX-1). LOX-1–expressing neutrophils were nearly undetectable in healthy individuals but were found prominently in tumor tissues. Moreover, exposing neutrophils from healthy individuals to endoplasmic reticulum stress resulted in up-regulation of LOX-1 and increased suppressive function. These data support the specific targeting of LOX-1–expressing PMN-MDSC for cancer immunotherapy. Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are important regulators of immune responses in cancer and have been directly implicated in the promotion of tumor progression. However, the heterogeneity of these cells and the lack of distinct markers hamper the progress in understanding the biology and clinical importance of these cells. Using partial enrichment of PMN-MDSC with gradient centrifugation, we determined that low-density PMN-MDSC and high-density neutrophils from the same cancer patients had a distinct gene profile. The most prominent changes were observed in the expression of genes associated with endoplasmic reticulum (ER) stress. Unexpectedly, low-density lipoprotein (LDL) was one of the most increased regulators, and its receptor oxidized LDL receptor 1 (OLR1) was one of the most overexpressed genes in PMN-MDSC. Lectin-type oxidized LDL receptor-1 (LOX-1) encoded by OLR1 was practically undetectable in neutrophils in peripheral blood of healthy donors, whereas 5 to 15% of total neutrophils in cancer patients and 15 to 50% of neutrophils in tumor tissues were LOX-1+. In contrast to their LOX-1− counterparts, LOX-1+ neutrophils had gene signature, potent immunosuppressive activity, up-regulation of ER stress, and other biochemical characteristics of PMN-MDSCs. Moreover, induction of ER stress in neutrophils from healthy donors up-regulated LOX-1 expression and converted these cells to suppressive PMN-MDSCs. Thus, we identified a specific marker of human PMN-MDSC associated with ER stress and lipid metabolism, which provides new insights into the biology and potential therapeutic targeting of these cells.

Cancer-Associated Fibroblasts Neutralize the Anti-tumor Effect of CSF1 Receptor Blockade by Inducing PMN-MDSC Infiltration of Tumors

Tumor-associated macrophages (TAM) contribute to all aspects of tumor progression. Use of CSF1R inhibitors to target TAM is therapeutically appealing, but has had very limited anti-tumor effects. Here, we have identified the mechanism that limited the effect of CSF1R targeted therapy. We demonstrated that carcinoma-associated fibroblasts (CAF) are major sources of chemokines that recruit granulocytes to tumors. CSF1 produced by tumor cells caused HDAC2-mediated downregulation of granulocyte-specific chemokine expression in CAF, which limited migration of these cells to tumors. Treatment with CSF1R inhibitors disrupted this crosstalk and triggered a profound increase in granulocyte recruitment to tumors. Combining CSF1R inhibitor with a CXCR2 antagonist blocked granulocyte infiltration of tumors and showed strong anti-tumor effects.

Selective Targeting of Myeloid-Derived Suppressor Cells in Cancer Patients Using DS-8273a, an Agonistic TRAIL-R2 Antibody

Purpose: Myeloid-derived suppressor cells (MDSC) are one of the major contributors to immune suppression in cancer. We recently have demonstrated in preclinical study that MDSCs are sensitive to TRAIL receptor 2 (TRAIL-R2) agonist. The goal of this study was to clinically test the hypothesis that targeting TRAIL-R2 can selectively eliminate MDSCs. Experimental Design: The TRAIL-R2 agonistic antibody (DS-8273a) has been tested in 16 patients with advanced cancers enrolled in a phase I trial. The antibody (24 mg/kg) was administered intravenously once every 3 weeks till disease progression, unacceptable toxicities, or withdrawal of consent. The safety and the presence of various populations of myeloid and lymphoid cells in peripheral blood and tumor tissues were evaluated. Results: The treatment was well tolerated with only mild to moderate adverse events attributable to the study drug. Treatment with DS-8273a resulted in reduction of the elevated numbers of MDSCs in the peripheral blood of most patients to the levels observed in healthy volunteers. However, in several patients, MDSCs rebounded back to the pretreatment level by day 42. In contrast, DS-8273a did not affect the number of neutrophils, monocytes, and other populations of myeloid and lymphoid cells. Decrease in MDSCs inversely correlated with the length of progression-free survival. In tumors, DS-8273a treatment resulted in a decrease of MDSCs in 50% of the patients who were able to provide pre- and on-treatment biopsies. Conclusions: Targeting TRAIL-R2 resulted in elimination of different populations of MDSCs without affecting mature myeloid or lymphoid cells. These data support the use of this antibody in combination immmunotherapy of cancer. Clin Cancer Res; 23(12); 2942–50. ©2016 AACR.

Entinostat Neutralizes Myeloid-Derived Suppressor Cells and Enhances the Antitumor Effect of PD-1 Inhibition in Murine Models of Lung and Renal Cell Carcinoma

Purpose: Recent advances in immunotherapy highlight the antitumor effects of immune checkpoint inhibition despite a relatively limited subset of patients receiving clinical benefit. The selective class I histone deacetylase inhibitor entinostat has been reported to have immunomodulatory activity including targeting of immune suppressor cells in the tumor microenvironment. Thus, we decided to assess whether entinostat could enhance anti–PD-1 treatment and investigate those alterations in the immunosuppressive tumor microenvironment that contribute to the combined antitumor activity. Experimental Design: We utilized syngeneic mouse models of lung (LLC) and renal cell (RENCA) carcinoma and assessed immune correlates, tumor growth, and survival following treatment with entinostat (5 or 10 mg/kg, p.o.) and a PD-1 inhibitor (10 and 20 mg/kg, s.c.). Results: Entinostat enhanced the antitumor effect of PD-1 inhibition in two syngeneic mouse tumor models by reducing tumor growth and increasing survival. Entinostat inhibited the immunosuppressive function of both polymorphonuclear (PMN)- and monocytic-myeloid derived suppressor cell (M-MDSC) populations. Analysis of MDSC response to entinostat revealed significantly reduced arginase-1, iNOS, and COX-2 levels, suggesting potential mechanisms for the altered function. We also observed significant alterations in cytokine/chemokine release in vivo with a shift toward a tumor-suppressive microenvironment. Conclusions: Our results demonstrate that entinostat enhances the antitumor effect of PD-1 targeting through functional inhibition of MDSCs and a transition away from an immune-suppressive tumor microenvironment. These data provide a mechanistic rationale for the clinical testing and potential markers of response of this novel combination in solid tumor patients. Clin Cancer Res; 23(17); 5187–201. ©2017 AACR.

CD38+ M-MDSC expansion characterizes a subset of advanced colorectal cancer patients

BACKGROUND Myeloid-derived suppressor cells (MDSCs) are a population of immature immune cells with several protumorigenic functions. CD38 is a transmembrane receptor-ectoenzyme expressed by MDSCs in murine models of esophageal cancer. We hypothesized that CD38 could be expressed on MDSCs in human colorectal cancer (CRC), which might allow for a new perspective on therapeutic targeting of human MDSCs with anti-CD38 monoclonal antibodies in this cancer. METHODS Blood samples were collected from 41 CRC patients and 8 healthy donors, followed by peripheral blood mononuclear cell (PBMC) separation. Polymorphonuclear (PMN-) and monocytic (M-) MDSCs and CD38 expression levels were quantified by flow cytometry. The immunosuppressive capacity of M-MDSCs from 10 CRC patients was validated in a mixed lymphocyte reaction (MLR) assay. RESULTS A significant expansion of CD38+ M-MDSCs and a trend of expansion of CD38+ PMN-MDSCs (accompanied by a trend of increased CD38 expression on both M- and PMN-MDSCs) were observed in PBMCs of CRC patients when compared with healthy donors. The CD38+ M-MDSCs from CRC patients were found to be immunosuppressive when compared with mature monocytes. CD38+ M- and PMN-MDSC frequencies were significantly higher in CRC patients who previously received treatment when compared with treatment-naive patients. CONCLUSIONS This study provides a rationale for an attempt to target M-MDSCs with an anti-CD38 monoclonal antibody in metastatic CRC patients. FUNDING NCI P01-CA14305603, the American Cancer Society, Scott and Suzi Lustgarten Family Colon Cancer Research Fund, Hansen Foundation, and Janssen Research and Development.

Secretory IgM Exacerbates Tumor Progression by Inducing Accumulations of MDSCs in Mice

In a mouse model of chronic lymphocytic leukemia, production of secretory IgM led to more MDSCs, fewer T cells, and shorter survival times for the mice. Thus, secretory IgM may aggravate the progression of this cancer. Chronic lymphocytic leukemia (CLL) cells can secrete immunoglobulin M. However, it is not clear whether secretory IgM (sIgM) plays a role in disease progression. We crossed the Eμ-TCL1 mouse model of CLL, in which the expression of human TCL1 oncogene was driven by the V(H) promoter-Ig(H)-Eμ enhancer, with MD4 mice whose B cells produced B-cell receptor (membrane-bound IgM) and sIgM with specificity for hen egg lysozyme (HEL). CLL cells that developed in these MD4/Eμ-TCL1 mice reactivated a parental Ig gene allele and secreted IgM, and did not recognize HEL. The MD4/Eμ-TCL1 mice had reduced survival, increased myeloid-derived suppressor cells (MDSC), and decreased numbers of T cells. We tested whether sIgM could contribute to the accumulation of MDSCs by crossing μS–/– mice, which could not produce sIgM, with Eμ-TCL1 mice. The μS–/–/Eμ-TCL1 mice survived longer than Eμ-TCL1 mice and developed decreased numbers of MDSCs which were less able to suppress proliferation of T cells. We targeted the synthesis of sIgM by deleting the function of XBP-1s and showed that targeting XBP-1s genetically or pharmacologically could lead to decreased sIgM, accompanied by decreased numbers and reduced functions of MDSCs in MD4/Eμ-TCL1 mice. Additionally, MDSCs from μS–/– mice grafted with Lewis lung carcinoma were inefficient suppressors of T cells, resulting in slower tumor growth. These results demonstrate that sIgM produced by B cells can upregulate the functions of MDSCs in tumor-bearing mice to aggravate cancer progression. In a mouse model of CLL, production of secretory IgM led to more MDSCs, fewer T cells, and shorter survival times for the mice. Thus, secretory IgM may aggravate the progression of this cancer. Cancer Immunol Res; 6(6); 696–710. ©2018 AACR.

Inhibition of Casein Kinase 2 Disrupts Differentiation of Myeloid Cells in Cancer and Enhances the Efficacy of Immunotherapy in Mice

The role of myeloid cells as regulators of tumor progression that significantly impact the efficacy of cancer immunotherapies makes them an attractive target for inhibition. Here we explore the effect of a novel, potent, and selective inhibitor of serine/threonine protein kinase casein kinase 2 (CK2) on modulating myeloid cells in the tumor microenvironment. Although inhibition of CK2 caused only a modest effect on dendritic cells in tumor-bearing mice, it substantially reduced the amount of polymorphonuclear myeloid-derived suppressor cells and tumor-associated macrophages. This effect was not caused by the induction of apoptosis, but rather by a block of differentiation. Our results implicated downregulation of CCAAT-enhancer binding protein-α in this effect. Although CK2 inhibition did not directly affect tumor cells, it dramatically enhanced the antitumor activity of immune checkpoint receptor blockade using anti-CTLA-4 antibody. These results suggest a potential role of CK2 inhibitors in combination therapies against cancer.Significance: These findings demonstrate the modulatory effects of casein kinase 2 inhibitors on myeloid cell differentiation in the tumor microenvironment, which subsequently synergize with the antitumor effects of checkpoint inhibitor CTLA4. Cancer Res; 78(19); 5644-55. ©2018 AACR.

Abstract CT095: The selective targeting of myeloid-derived suppressor cells in cancer patients using an agonistic TRAIL-R2 antibody

The goal of this current study was to clinically test the hypothesis that by targeting TRAIL-R2, myeloid-derived suppressor cells (MDSCs) can be selectively eliminated. MDSC) are one of the major contributors to immune suppression in cancer. We previously demonstrated in a preclinical study that MDSCs are sensitive to a TRAIL receptor 2 (TRAIL-R2) agonist. The TRAIL-R2 agonistic antibody (DS-8273a; provided by Daiichi Sankyo, Inc.) was tested in 16 patients with advanced solid cancers or lymphomas enrolled in a phase 1 trial. The antibody (24 mg/kg) was administered IV once every 3 weeks till disease progression, unacceptable toxicities, or withdrawal of consent. The safety and the presence of various populations of myeloid and lymphoid cells in peripheral blood and tumor tissues were evaluated using flow cytometry and immunohistochemistry. Overall, the treatment was well tolerated with only mild to moderate adverse events attributable to the study drug. Furthermore, treatment with DS-8273a resulted in reduction of the elevated numbers of MDSC in the peripheral blood of most patients to the levels observed in healthy volunteers. However, in several patients, MDSC rebounded back to the pre-treatment level by day 42. In contrast, DS-8273a did not affect the number of neutrophils, monocytes, and other populations of myeloid and lymphoid cells with decreases in MDSC levels inversely correlating with the length of progression-free survival. In tumors, DS-8273a treatment resulted in a decrease of MDSC in 50% of the patients who were able to provide pre- and on-treatment biopsies. In conclusion, targeting TRAIL-R2 using DS-8273a resulted in a temporary elimination of MDSCs without affecting mature myeloid or lymphoid cells, and these data support further use of this antibody in combination with current immmunotherapies of cancer. Citation Format: George A. Dominguez, Thomas Condamine, Sridevi Mony, Ayumi Hashimoto, Fang Wang, Qin Liu, Andres Forero, Johanna C. Bendell, Robert Witt, Neil Hockstein, Prasanna Kumar, Dmitry I. Gabrilovich. The selective targeting of myeloid-derived suppressor cells in cancer patients using an agonistic TRAIL-R2 antibody [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 CT095. doi:10.1158/1538-7445.AM2017-CT095

Abstract 5595: HDAC inhibitor Entinostat disrupts function of PMN-MDSC

Myeloid-derived suppressor cells (MDSCs), consisting of subpopulations of polymorphonuclear (PMN; CD11b+Ly6G+Ly6Clo) and monocytic (M; CD11b+Ly6G-Ly6Chi) MDSCs play a major role as immune suppressors in cancer. MDSC-targeted therapy has not been developed yet. Histone deacetylases (HDAC) are critically involved in epigenetic regulation of multiple genes and may regulate MDSC number and function. The purpose of this study was to evaluate the effect of the class I HDAC inhibitor, entinostat on differentiation and function of MDSCs in mouse tumor models of lung cancer (Lewis Lung Carcinoma, LLC) and colon cancer (CT26). LLC and CT26 tumor-bearing mice were treated with entinostat (10 mg/kg, p.o. daily for two weeks). Entinostat caused significant increase in the presence of CD45+ CD11b+ myeloid cells in bone marrow, spleen and tumors. This increase was largely due to accumulation of Ly6ClowLy6G+ PMN. Unexpectedly, this was associated with a small decrease in the presence of common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) cells in bone marrow. This suggests that entinostat affects differentiation of PMN downstream of GMP. Experiments in vitro demonstrated that entinostat increased differentiation of enriched hematopoietic progenitor cells (HPC) to PMN in the presence of tumor explant supernatant (TES), which supported the in vivo result. To assess the effect of entinostat on the function of PMN-MDSCs, Ly6G+ cells were purified from spleen and tumor of LLC tumor-bearing mice treated with entinostat or vehicle alone and tested for their ability to suppress antigen-specific T cell. As expected Ly6G+ PMN-MDSC from tumor-bearing mice treated with vehicle alone had strong suppressive activity. In contrast, Ly6G+ cells from spleen and tumor of entinostat-treated LLC tumor-bearing mice show significantly reduced suppressive activity. This result was confirmed in experiments with Ly6G+ cells differentiated from HPC culture with/without entinostat treatment. To determine whether entinostat inhibition of PMN-MDSC immunosuppressive function could enhance immune checkpoint activity, entinostat combined with an anti-PD-1 antibody was evaluated using the LLC tumor-bearing mice. These mice were treated with entinostat (10 mg/kg, p.o., daily) and anti-mouse PD-1 rat antibody (10 mg/kg, i.p., twice a week). Entinostat or PD-1 antibody alone did not significantly inhibit the tumor growth. In striking contrast, the combination treatment blocked tumor progression. Our data demonstrate that entinostat increases a population of PMN-MDSCs that are no longer immunosuppressive leading to enhanced anti-tumor activity when combined with an immune checkpoint inhibitor. This novel phenomenon provides further rationale for combination therapy of entinostat with anti-PD-1 antibody or other checkpoint inhibitors in clinical settings. Citation Format: Ayumi Hashimoto, Vinit Kumar, Peter Ordentlich, Dmitry I. Gabrilovich. HDAC inhibitor Entinostat disrupts function of PMN-MDSC [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 5595. doi:10.1158/1538-7445.AM2017-5595