Abstract 1618: Inhibition of lung tumorigenesis by a novel small molecule CA170 targeting the immune checkpoint protein VISTA

2021 ◽  
Author(s):  
Jing Pan ◽  
Yao Chen ◽  
Qi Zhang ◽  
Achia Khatun ◽  
Katie Palen ◽  
...  
Keyword(s):  
Small Molecule ◽  
Immune Checkpoint ◽  
Lung Tumorigenesis ◽  
Checkpoint Protein

207 Small molecule inhibitors of Sec61 cotranslational translocation regulate the phagocytosis checkpoint molecule CD47

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A223-A223
Author(s):  
Jennifer Whang ◽  
Andrea Fan ◽  
Christopher Kirk ◽  
Eric Lowe ◽  
Dustin McMinn ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Small Molecule ◽  
Immune Checkpoint ◽  
Small Molecule Inhibitors ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Checkpoint Proteins ◽  
Cotranslational Translocation ◽  
Macrophage Phagocytosis ◽  
Immune Checkpoint Proteins

BackgroundMany tumor cells escape immune cell clearance by overexpressing CD47, a multi-pass transmembrane protein, which binds signal regulatory protein α (SIRPα) on macrophages leading to decreased phagocytic activity. Blockade of CD47/SIRPα interactions enhances macrophage phagocytosis and is being targeted with antibody-based drugs, some of which are used in combination therapies in clinical trials. A novel method to target CD47 is through the inhibition of cotranslational translocation of transmembrane proteins. Immediately after exiting the ribosome, signal sequences that are unique to each protein are directed through the Sec61 channel into the ER for extracellular expression.1 Several Sec61-targeting compounds have been identified to suppress translocation in a signal sequence-specific manner.2 We previously described Sec61 inhibitors capable of selectively targeting immune checkpoint proteins and enhancing T cell function.3 Here, we demonstrate the blockade of CD47 expression on tumor cells and enhancement of macrophage phagocytosis with small molecule inhibitors of Sec61.MethodsSec61-dependent expression of target proteins was assayed using HEK293 cells overexpressing constructs comprised of signal sequences fused to a luciferase reporter. Stimulated PBMCs or tumor cells were incubated with Sec61 inhibitors, and surface expression of checkpoint molecules were examined by flow cytometry. Necrotic and apoptotic cells were assessed by Annexin V and 7AAD labeling. Human CD14+ monocytes were differentiated to M1- or M2-type macrophages. Jurkat or SKBR3 cells were incubated with Sec61 inhibitors, labeled with a pH sensitive dye and co-cultured with macrophages to assess phagocytosis.ResultsWe identified Sec61 inhibitors that block select immune checkpoint proteins. Compounds demonstrated either selective or multi-target profiles in transient transfection screens, which was supported by decreased protein expression on activated T cells. KZR-9275 targeted multiple checkpoint molecules, including PD-1, LAG-3 and CD73, along with a potent inhibition of the CD47 signal sequence reporter. CD47 surface expression was decreased on Jurkat and SKBR3 cells following 72 hours of compound treatment. KZR-9275 treatment of SKBR3 cells induced a minor increase in apoptotic cells, which was not detected in Jurkat cells. Increased macrophage phagocytosis, especially with M2-type macrophages, was observed when Jurkat or SKBR3 cells were pre-treated with KZR-9275.ConclusionsOur findings demonstrate that Sec61 inhibitors can block the expression of CD47, a phagocytosis checkpoint protein, on tumor cells and subsequently modulate macrophage phagocytic activity. Small molecule inhibitors of Sec61 provide an opportunity to target multiple checkpoint proteins on various cell populations. Future in vivo tumor models will assess the efficacy of Sec61 inhibitors to provide combination-like therapy.ReferencesPark E, Rapoport TA. Mechanisms of Sec61/SecY-mediated protein translocation across membranes. Annu Rev Biophys 2012; 41:1–20.Van Puyenbroeck V, Vermeire K. Inhibitors of protein translocation across membranes of the secretory pathway: novel antimicrobial and anticancer agents. Cell Mol Life Sci 2018; 75:1541–1558.Whang J, Anderl J, Fan A, Kirk C, Lowe E, McMinn D, et al. Targeting multiple immune checkpoint proteins with novel small molecule inhibitors of Sec61-dependent cotranslational translocation. 34th Annual Meeting & Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2019): part 2. J Immunother Cancer 2019; 7: 283. Abstract 815.

501 VISTA regulates the differentiation and suppressive function of myeloid-derived suppressor cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A536-A536
Author(s):  
Juan Dong ◽  
Cassandra Gilmore ◽  
Hieu Ta ◽  
Keman Zhang ◽  
Sarah Stone ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Signaling Pathways ◽  
Immune Checkpoint ◽  
Suppressor Cells ◽  
Myeloid Cell ◽  
Myeloid Derived Suppressor Cells ◽  
Checkpoint Protein ◽  
Suppressive Function

BackgroundV-domain immunoglobulin suppressor of T cell activation (VISTA) is a B7 family inhibitory immune checkpoint protein and is highly expressed on myeloid cells and T cells.1 VISTA acts as both an inhibitory ligand when expressed on antigen-presenting cells and a receptor when expressed on T cells. Our recent study has shown that VISTA is a myeloid cell-specific immune checkpoint and that blocking VISTA can reprogram suppressive myeloid cells and promote a T cell-stimulatory tumor microenvironment.2 In this study, we further demonstrate that VISTA blockade directly alters the differentiation and the suppressive function of myeloid-derived suppressor cells (MDSC).MethodsFlow cytometry was performed to examine VISTA expression on MDSCs in multiple murine tumor models including the B16BL6 melanoma model, MC38 colon cancer model, and the KPC pancreatic cancer models. To examine the role of VISTA in controlling the differentiation and suppressive function of MDSCs, we cultured wild type (WT) and VISTA.KO bone marrow progenitor cells with GM-CSF and IL-6 to induce BM -derived MDSCs.ResultsOur preliminary results show that VISTA is highly expressed on M-MDSCs in B16BL6, MC38 and KPC tumors. In BM-derived MDSCs, VISTA deletion significantly altered the signaling pathways and the differentiation of MDSCs. Multiple inflammatory signaling pathways were downregulated in VISTA KO MDSCs, resulting in decreased production of cytokines such as IL1 and chemokines such as CCL2/4/9, as well as significantly impaired their ability to suppress the activation of CD8+ T cells. The loss of suppressive function in VISTA KO MDSCs is correlated with significantly reduced expression of iNOS. To validate the results from BM-MDSCs, we sorted CD11b+CD11c-Ly6C+Ly6G- M-MDSCs and CD11b+CD11c-Ly6G+ G-MDSCs from B16BL6 tumor tissues and tested the ability of a VISTA-blocking mAb to reverse the suppressive effects of tumor-derived MDSCs. Our results show that blocking VISTA impaired the suppressive function of tumor-derived M-MDSC but not G-MDSCs.ConclusionsTaken together, these results demonstrate a crucial role of VISTA in regulating the differentiation and function of MDSCs, and that blocking VISTA abolishes MDSC-mediated T cell suppression, thereby boosting.Ethics ApprovalAll in vivo studies were reviewed and approved by Institutional Animal Care and Use Committee (Approval number 2019-2142).ReferencesXu W, Hire T, Malarkannan, S. et al. The structure, expression, and multifaceted role of immune-checkpoint protein VISTA as a critical regulator of anti-tumor immunity, autoimmunity, and inflammation. Cell Mol Immunol 2018;15:438–446.Xu W, Dong J, Zheng Y, et al. Immune-checkpoint protein VISTA regulates antitumor immunity by controlling myeloid cell-mediated inflammation and immunosuppression. Cancer Immunol Res 2019;7:1497–510.

scholarly journals Combined immune checkpoint protein blockade and low dose whole body irradiation as immunotherapy for myeloma

2015 ◽  
Vol 3 (1) ◽  
Author(s):  
Weiqing Jing ◽  
Jill A Gershan ◽  
James Weber ◽  
Dominique Tlomak ◽  
Laura McOlash ◽  
...  
Keyword(s):  
Immune Checkpoint ◽  
Low Dose ◽  
Whole Body ◽  
Checkpoint Protein

scholarly journals The JAK1/2 Inhibitor Ruxolitinib Downregulates the Immune Checkpoint Protein B7-H3 in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1824-1824
Author(s):  
Ning Xu ◽  
Nicole Ng ◽  
Mingjie Li ◽  
Erin Yu ◽  
Eric Sanchez ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Immune Checkpoint ◽  
Research Funding ◽  
Flow Cytometric Analysis ◽  
Cell Activity ◽  
Cytokine Signaling ◽  
Checkpoint Protein ◽  
Equity Ownership

Introduction: The JAKSTAT pathway plays a critical role in the regulation of hematopoietic pathways and immunological cytokine signaling. The JAK pathway is also involved in tumor cell proliferation and drug resistance in multiple myeloma (MM). Thus, inhibition of the JAK pathway should be a potentially effective strategy for treating MM patients. B7-H3 is an immune checkpoint protein in the B7 superfamily and has been shown overexpressed in several tumors. Immune checkpoint blockade may suppress tumor progression or enhance anti-tumor immune responses. In this study, we investigated the effects of the JAK1/2 inhibitor ruxolitinib (Rux) on B7-H3 in MM. Materials and Methods: Bone marrow mononuclear cells (BMMCs) were collected from MM patients after obtaining IRB approval. Single-cell suspensions were prepared from human MM LAGλ-1A xenografts which had been grown in severe combined immunodeficient mice. HS-5 stromal and SUP-T1 T cells were purchased from ATCC. The cells were cultured and treated with or without RUX and then subjected to qRT-PCR, flow cytometric analysis, and western blot analysis. For qRT-PCR, total RNA was extracted and applied to cDNA synthesis, followed by qPCR. Gene expression was analyzed in MM BMMCs alone or co-cultured with stromal cells or T cells with or without Rux treatment (1μM) in vitro. Results: We identified increased B7-H3 expression in MMBMMCs from patients with progressive disease (PD) patients compared to those in complete remission (CR). Rux significantly reduced B7-H3 expression in MMBMMCs in patients with PD, MM cells (U266), and BM from patients in PD when co-cultured with stromal cells (HS-5) after 48-72 hours. Rux decreased B7H3 expression in the human MM xenograft model LAGλ-1A when cultured ex vivo. In addition, Rux suppressed B7-H3 at protein levels as shown with flow cytometric analysis and western blotting, consistent with the gene expression results. Next, we tested whether B7-H3 blockade by Rux could potentially restore exhausted T cell activity against myeloma cells in MMBM. We found that Rux can increase IL-2 and CD8 gene expression in MMBM with lower plasma percentages (< 30%) but not among those with higher plasma cell percentages (>70%). Rux also elevated IL-2 and CD8 gene expression in BM when it was cocultured with T cells (SUP-T1), suggesting Rux may mediate immunological cytokine signaling. B7-H3-neutralizing antibody increased CD8 gene expression in MMBM in vitro, suggesting that one of the mechanisms through which Rux upregulates CD8 T cells in MMBM may be via downregulation of B7-H3. Conclusion: The immune checkpoint protein B7-H3 is overexpressed in MMBM in PD compared to CR patients. The JAK1/2 inhibitor Rux can decrease B7-H3 expression and increase IL-2 and CD8 expression in BM in vitro. Our results provide evidence for Rux inhibiting the immune checkpoint protein B7-H3 which may potentially restore exhausted T-cell activity in the MMBM tumoral microenvironment. Disclosures Chen: Oncotraker Inc: Equity Ownership. Berenson:Amgen: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Sanofi: Consultancy; Sanofi: Consultancy; Amag: Consultancy, Speakers Bureau; Amag: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; OncoTracker: Equity Ownership, Other: Officer; OncoTracker: Equity Ownership, Other: Officer; Bristol-Myers Squibb: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Incyte Corporation.: Consultancy, Research Funding; Incyte Corporation.: Consultancy, Research Funding; Takeda: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

scholarly journals Di-bromo-Based Small-Molecule Inhibitors of the PD-1/PD-L1 Immune Checkpoint

2020 ◽  
Vol 63 (19) ◽  
pp. 11271-11285
Author(s):  
Magdalena Konieczny ◽  
Bogdan Musielak ◽  
Justyna Kocik ◽  
Lukasz Skalniak ◽  
Dominik Sala ◽  
...  
Keyword(s):  
Small Molecule ◽  
Immune Checkpoint ◽  
Small Molecule Inhibitors

scholarly journals Preparation of Biphenyl-Conjugated Bromotyrosine for Inhibition of PD-1/PD-L1 Immune Checkpoint Interactions

2020 ◽  
Vol 21 (10) ◽  
pp. 3639 ◽  
Author(s):  
Eun-Hye Kim ◽  
Masuki Kawamoto ◽  
Roopa Dharmatti ◽  
Eiry Kobatake ◽  
Yoshihiro Ito ◽  
...  
Keyword(s):  
Small Molecule ◽  
Immune Checkpoint ◽  
Inhibitory Effect ◽  
Production Costs ◽  
Small Molecule Drugs ◽  
Programmed Cell Death 1 ◽  
Ic50 Value ◽  
In Silico Simulation ◽  
Ic50 Values ◽  
High Production

Cancer immunotherapy has been revolutionized by the development of monoclonal antibodies (mAbs) that inhibit interactions between immune checkpoint molecules, such as programmed cell-death 1 (PD-1), and its ligand PD-L1. However, mAb-based drugs have some drawbacks, including poor tumor penetration and high production costs, which could potentially be overcome by small molecule drugs. BMS-8, one of the potent small molecule drugs, induces homodimerization of PD-L1, thereby inhibiting its binding to PD-1. Our assay system revealed that BMS-8 inhibited the PD-1/PD-L1 interaction with IC50 of 7.2 μM. To improve the IC50 value, we designed and synthesized a small molecule based on the molecular structure of BMS-8 by in silico simulation. As a result, we successfully prepared a biphenyl-conjugated bromotyrosine (X) with IC50 of 1.5 μM, which was about five times improved from BMS-8. We further prepared amino acid conjugates of X (amino-X), to elucidate a correlation between the docking modes of the amino-Xs and IC50 values. The results suggested that the displacement of amino-Xs from the BMS-8 in the pocket of PD-L1 homodimer correlated with IC50 values. This observation provides us a further insight how to derivatize X for better inhibitory effect.

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