ABSTRACT
The multi-receptor tyrosine kinase inhibitor XL092 has been developed to inhibit the activity of oncogenic targets, including MET, VEGFR2, and the TAM family of kinases TYRO3, AXL and MER. Presented here is a preclinical evaluation of XL092. XL092 causes a significant decrease in tumor MET and AXL phosphorylation (P < 0.01) in murine Hs 746T xenograft models relative to vehicle, and a 96% inhibition of VEGFR2 phosphorylation in murine lungs. Dose-dependent tumor growth inhibition with XL092 was observed in various murine xenograft models, with dose-dependent tumor regression seen in the NCI-H441 model. Tumor growth inhibition was enhanced with the combination of XL092 with anti-PD-1, anti-programmed death ligand-1 (PD-L1), or anti-CTLA-4 compared with any of these agents alone in the MC38 murine syngeneic model and with anti-PD-1 in the CT26 colorectal cancer survival model. In vivo, XL092 promoted a decrease in the tumor microvasculature and significant increases of peripheral CD4+ T cells and B cells and decreases in myeloid cells versus vehicle. Significant increases in CD8+ T cells were also observed with XL092 plus anti-PD-1 or anti-PD-L1 versus vehicle. In addition, XL092 promoted M2 to M1 repolarization of macrophages in vitro and inhibited primary human macrophage efferocytosis in a dose-dependent manner. In summary, XL092 was shown to have significant antitumor and immunomodulatory activity in animal models both alone and in combination with immune checkpoint inhibitors, supporting its evaluation in clinical trials.
Subject(s)
Neoplasms , Humans , Animals , Mice , Carrier Proteins , CD8-Positive T-Lymphocytes , Receptor Protein-Tyrosine Kinases , Disease Models, Animal , Cell Line, TumorABSTRACT
Antisense oligonucleotides (ASOs) are widely accepted therapeutic agents that suppress RNA transcription. While the majority of ASOs are well tolerated in vivo, few sequences trigger inflammatory responses in absence of conventional CpG motifs. In this study, we identified non-CpG oligodeoxy-nucleotide (ODN) capable of triggering an inflammatory response resulting in B cell and macrophage activation in a MyD88- and TLR9-dependent manner. In addition, we found the receptor for advance glycation end product (RAGE) receptor to be involved in the initiation of inflammatory response to suboptimal concentrations of both CpG- and non-CpG-containing ODNs. In contrast, dosing RAGE KO mice with high doses of CpG or non-CpG ODNs lead to a stronger inflammatory response than observed in wild-type mice. Together, our data provide a previously uncharacterized in vivo mechanism contingent on ODN-administered dose, where TLR9 governs the primary response and RAGE plays a distinct and cooperative function in providing a pivotal role in balancing the immune response.
Subject(s)
Immunity, Cellular/immunology , Inflammation/immunology , Oligonucleotides, Antisense/therapeutic use , Receptor for Advanced Glycation End Products/metabolism , Toll-Like Receptor 9/metabolism , Animals , B-Lymphocytes/immunology , Cytokines/blood , Humans , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Differentiation Factor 88/metabolism , Primary Cell Culture , RNA/genetics , RNA/metabolism , Receptor for Advanced Glycation End Products/genetics , Single-Cell Analysis , Toll-Like Receptor 9/genetics , Transcription, GeneticABSTRACT
Alkyne 40, 5-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methylpent-4-yn-2-ol (EC144), is a second generation inhibitor of heat shock protein 90 (Hsp90) and is substantially more potent in vitro and in vivo than the first generation inhibitor 14 (BIIB021) that completed phase II clinical trials. Alkyne 40 is more potent than 14 in an Hsp90α binding assay (IC(50) = 1.1 vs 5.1 nM) as well as in its ability to degrade Her-2 in MCF-7 cells (EC(50) = 14 vs 38 nM). In a mouse model of gastric tumors (N87), 40 stops tumor growth at 5 mg/kg and causes partial tumor regressions at 10 mg/kg (po, qd × 5). Under the same conditions, 14 stops tumor growth only at 120 mg/kg, and does not induce partial regressions. Thus, alkyne 40 is approximately 20-fold more efficacious than 14 in mice.
Subject(s)
HSP90 Heat-Shock Proteins/antagonists & inhibitors , Pyrimidines/pharmacology , Pyrroles/pharmacology , Humans , X-Ray DiffractionABSTRACT
Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding and stabilizing multiple intracellular proteins that have roles in cell activation and proliferation. Many Hsp90 client proteins in tumor cells are mutated or overexpressed oncogenic proteins driving cancer cell growth, leading to the acceptance of Hsp90 as a potential therapeutic target for cancer. Because several signal transduction molecules that are dependent on Hsp90 function are also involved in activation of innate and adaptive cells of the immune system, we investigated the mechanism by which inhibiting Hsp90 leads to therapeutic efficacy in rodent models of inflammation and autoimmunity. EC144, a synthetic Hsp90 inhibitor, blocked LPS-induced TLR4 signaling in RAW 264.7 cells by inhibiting activation of ERK1/2, MEK1/2, JNK, and p38 MAPK but not NF-κB. Ex vivo LPS-stimulated CD11b(+) peritoneal exudate cells from EC144-treated mice were blocked from phosphorylating tumor progression locus 2, MEK1/2, and ERK1/2. Consequently, EC144-treated mice were resistant to LPS administration and had suppressed systemic TNF-α release. Inhibiting Hsp90 also blocked in vitro CD4(+) T cell proliferation in mouse and human MLRs. In vivo, semitherapeutic administration of EC144 blocked disease development in rat collagen-induced arthritis by suppressing the inflammatory response. In a mouse collagen-induced arthritis model, EC144 also suppressed disease development, which correlated with a suppressed Ag-specific Ab response and a block in activation of Ag-specific CD4(+) T cells. Our results describe mechanisms by which blocking Hsp90 function may be applicable to treatment of autoimmune diseases involving inflammation and activation of the adaptive immune response.
Subject(s)
Adaptive Immunity/drug effects , Autoimmune Diseases/drug therapy , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Immunity, Innate/drug effects , Immunosuppressive Agents/pharmacology , Inflammation Mediators/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Animals , Autoimmune Diseases/immunology , Autoimmune Diseases/metabolism , Cell Line , Cell Line, Transformed , Crystallography, X-Ray , Disease Models, Animal , Female , Humans , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/therapeutic use , Inflammation/drug therapy , Inflammation/immunology , Inflammation/metabolism , Inflammation Mediators/chemical synthesis , Inflammation Mediators/therapeutic use , Mice , Mice, Inbred BALB C , Mice, Inbred DBA , Pyrimidines/chemical synthesis , Pyrimidines/therapeutic use , Pyrroles/chemical synthesis , Pyrroles/therapeutic use , RatsABSTRACT
The Ets family members Spi-1 and Spi-B have been implicated in the regulation of genes important for B cell antigen receptor (BCR) signaling. Mice deficient in Spi-B exhibit reduced B cell proliferation in response to BCR cross-linking and impaired T cell-dependent immune responses. This defect is exacerbated in the presence of Spi-1 haplo-insufficiency (Spi1+/- SpiB-/-). Tyrosine phosphorylation and calcium mobilization induced by BCR engagement is diminished in Spi1+/- SpiB-/- B lymphocytes, although many key BCR signaling proteins are expressed, suggesting that Spi-1 and Spi-B regulate expression of additional, unidentified signaling molecules. We now demonstrate that expression of the adaptor protein Grap2 is impaired in Spi1+/- SpiB+/- and Spi1+/- SpiB-/- B lymphocytes. Analysis of two alternate murine Grap2 promoters revealed a functionally important Spi-1 and Spi-B DNA binding element located in the downstream promoter. Ectopic expression of Grap2 in Grap2-deficient B cells reduced the recruitment of BLNK to Igalpha and the phosphorylation of specific substrates. Regulation of BLNK recruitment was dependent upon the Grap2 proline-rich domain, while modulation of phosphorylation was dependent upon both the proline-rich and SH2 domains. These data indicate that Spi-1 and Spi-B directly regulate the expression of Grap2 and that Grap2 functions to modulate BCR signaling, but that reduced Grap2 expression is unlikely to account for the BCR signaling defects observed in Spi1+/- SpiB-/- B cells.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , B-Lymphocytes/metabolism , DNA-Binding Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Base Sequence , Binding Sites/genetics , Blotting, Northern , Blotting, Western , Cell Line, Tumor , DNA-Binding Proteins/genetics , Down-Regulation/genetics , Electrophoretic Mobility Shift Assay , Gene Expression/genetics , Genotype , Heterozygote , Mice , Mice, Knockout , Molecular Sequence Data , Mutation , Oligonucleotides/genetics , Oligonucleotides/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , Proto-Oncogene Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Antigen, B-Cell/metabolism , Sequence Alignment , Sequence Homology, Nucleic Acid , Signal Transduction , Spleen/cytology , Spleen/metabolism , Trans-Activators/genetics , Transcription Factors/geneticsABSTRACT
Little is known about the role of the Gads (GrpL) adaptor protein in mature T cell populations. In this study we show that the effects of Gads deficiency on murine CD4(+) and CD8(+) T cells are markedly different. Gads(-/-) CD4(+) T cells were markedly deficient in the spleen and had an activated phenotype and a rapid turnover rate. When transferred into a wild-type host, Gads(-/-) CD4(+) T cells continued to proliferate at a higher rate than wild-type CD4(+) T cells, demonstrating a defect in homeostatic proliferation. Gads(-/-) CD8(+) T cells had a memory-like phenotype, produced IFN-gamma in response to ex vivo stimulation, and underwent normal homeostatic proliferation in wild-type hosts. Gads(-/-) T cells had defective TCR-mediated calcium responses, but had normal activation of ERK. Gads(-/-) CD4(+) T cells, but not CD8(+) T cells, had a severe block of TCR-mediated proliferation and a high rate of spontaneous cell death and were highly susceptible to CD95-induced apoptosis. This suggests that the rapid turnover of Gads(-/-) CD4(+) T cells is due to a defect in cell survival. The intracellular signaling pathways that regulate homeostasis in CD4(+) and CD8(+) T cells are clearly different, and the Gads adaptor protein is critical for homeostasis of CD4(+) T cells.
Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins/physiology , T-Lymphocyte Subsets/cytology , Adoptive Transfer , Animals , Apoptosis , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/transplantation , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/metabolism , Calcium Signaling , Carrier Proteins/genetics , Cell Division , DNA, Complementary/genetics , Gene Library , Homeostasis , Lymphoid Tissue/cytology , Lymphoid Tissue/immunology , MAP Kinase Signaling System , Mice , Mice, Inbred C57BL , T-Lymphocyte Subsets/metabolism , Thymus Gland/cytology , Thymus Gland/immunology , fas Receptor/physiologyABSTRACT
We have characterized the function of Notch-regulated ankyrin-repeat protein (Nrarp) in mouse cell lines and in hematopoietic stem cells (HSCs). Nrarp overexpression is able to block Notch-induced activation of CBF-1. In AKR1010 thymoma cells, Nrarp overexpression blocks CBF-1-dependent transcriptional activation of Notch-responsive genes and inhibits phenotypic changes associated with Notch activation. Enforced expression of Nrarp in mouse HSCs results in a profound block in T lineage commitment and progression through early stages of thymocyte maturation. In contrast, Deltex-1 overexpression in HSCs can also block T lineage commitment but not progression through the early double negative stages of thymocyte maturation. The different effects of Deltex-1 and Nrarp overexpression suggest that alternate Notch signaling pathways mediate T vs B lineage commitment and thymocyte maturation.