Sothy Yi

Interleukin 27 limits autoimmune encephalomyelitis by suppressing the development of interleukin 17–producing T cells

Interleukin 27 (IL-27) was first characterized as a proinflammatory cytokine with T helper type 1–inducing activity. However, subsequent work has demonstrated that mice deficient in IL-27 receptor (IL-27Rα) show exacerbated inflammatory responses to a variety of challenges, suggesting that IL-27 has important immunoregulatory functions in vivo. Here we demonstrate that IL-27Rα-deficient mice were hypersusceptible to experimental autoimmune encephalomyelitis and generated more IL-17-producing T helper cells. IL-27 acted directly on effector T cells to suppress the development of IL-17-producing T helper cells mediated by IL-6 and transforming growth factor-β. This suppressive activity was dependent on the transcription factor STAT1 and was independent of interferon-γ. Finally, IL-27 suppressed IL-6-mediated T cell proliferation. These data provide a mechanistic explanation for the IL-27-mediated immune suppression noted in several in vivo models of inflammation.

Maternal embryonic leucine zipper kinase/murine protein serine-threonine kinase 38 is a promising therapeutic target for multiple cancers

To identify genes that could serve as targets for novel cancer therapeutics, we used a bioinformatic analysis of microarray data comparing gene expression between normal and tumor-derived primary human tissues. From this approach, we have found that maternal embryonic leucine zipper kinase (Melk), a member of the AMP serine/threonine kinase family, exhibits multiple features consistent with the potential utility of this gene as an anticancer target. An oligonucleotide microarray analysis of multiple human tumor samples and cell lines suggests that Melk expression is frequently elevated in cancer relative to normal tissues, a pattern confirmed by quantitative reverse transcription-PCR and Western blotting of selected primary tumor samples. In situ hybridization localized Melk expression to malignant epithelial cells in 96%, 23%, and 13% of colorectal, lung, and ovarian tissue tumor samples, respectively. Expression of this gene is also elevated in spontaneous tumors derived from the ApcMin and Apc1638N murine models of intestinal tumorigenesis. To begin addressing whether Melk is relevant for tumorigenesis, RNA interference-mediated silencing within human and murine tumor cell lines was done. We show that Melk knockdown decreases proliferation and anchorage-independent growth in vitro as well as tumor growth in a xenograft model. Together, these results suggest that Melk may provide a growth advantage for neoplastic cells and, therefore, inactivation may be therapeutically beneficial.

A novel type I cytokine receptor is expressed on monocytes, signals proliferation, and activates STAT-3 and STAT-5

Here we report the cloning of a novel type I cytokine receptor, gp130-like monocyte receptor (GLM-R), with homology to the interleukin-6 receptor signal transducing chain, gp130, and granulocyte colony-stimulating factor receptor. Human and murine GLM-R cDNAs encode open reading frames of 732 and 716 amino acids, respectively, and the corresponding genes are located in close proximity to gp130 genes on human chromosome 5 and mouse chromosome 13. GLM-R is specifically expressed on CD14-positive cells and is up-regulated more than 50-fold upon activation of those cells. To address the question of whether GLM-R is a signaling receptor, we constructed a chimeric molecule, consisting of the extracellular domain of human growth hormone (hGH) receptor, and the intracellular domain of GLM-R. When transfected into factor-dependent 32D cells, this chimeric molecule could signal for proliferation and activate signal transducer and activator of transcription (STAT)-3 and STAT-5 upon stimulation with hGH. Thus, GLM-R is a novel signaling receptor chain potentially involved in the development and function of monocytes and macrophages.

Abstract B060: Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK

Introduction: In normal tissue homeostasis, interaction of phosphatidylserine externalized on apoptotic cells (ACs) with the TAM (Tyro3, Axl MerTK) family RTK MerTK, via its ligands Gas6 and Protein S, leads to AC phagocytosis (efferocytosis). The resulting clearance of AC antigens, immunosuppressive M2 macrophage polarization, and suppression of pro-inflammatory cytokine production promotes tolerance to AC-derived self-antigens. This homeostatic response is coopted in tumors, which are abundant in both ACs and TAM ligands, leading to a blunted anti-tumor immune response. Syngeneic tumors implanted in MerTK -/- mice exhibit poor growth and metastasis, correlating with enhanced production of pro-inflammatory cytokines, splenocyte proliferation, and decreased IL-10 compared with those implanted in WT mice. Moreover, MerTK aberrantly expressed on hematological and epithelial malignancies promotes survival and chemoresistance. Thus, pharmacological inhibition of MerTK may have clinical benefit by increasing availability of dead tumor cell antigens and promoting an anti-tumor immune response, or by directly blocking tumor cell survival. We have therefore developed small molecule inhibitors of MerTK. Methods: MerTK kinase activity was assayed using ADP-Glo. Cellular MerTK activity was stimulated in HUVEC or H1299 cells using anti-MerTK crosslinking and measured by immunoprecipitation followed by anti-phospho-MerTK blot, or by downstream phospho-Akt Ser 473 using HTRF. A high content assay was used to measure cell number, apoptosis and proliferation. Effects on immune function were tested in human primary dendritic cells (LPS-induced IL-23 production) and human primary T cells (anti-CD3/CD28-induced IL-2 production or IL-2 induced phospho-STAT5). Efferocytosis of apoptotic Jurkat cells by human primary macrophages was detected by flow cytometry. For the PD assay, MerTK expressing tumors were grown in nude mice. 30-60 minutes post-compound dosing, MerTK was stimulated in vivo for 1hr. Tumors were snap frozen and tumor lysates were blotted with anti-phospho-MerTK antibodies. Anti-tumor efficacy was studied in syngeneic models. Results: Here we describe novel MerTK-selective and Mer-Axl small molecule inhibitors that potently block MerTK in biochemical assays. These compounds exhibit selectivity for TAM family members in an in vitro kinase panel. In cells, antibody-induced MerTK phosphorylation as well as downstream phosphorylation of Akt was inhibited by both classes of compounds with EC50 in vivo . Anti-tumor activity of these novel MerTK inhibitors is under investigation in syngeneic mouse models both as single agents and in combinations. Conclusions: We have discovered potent and novel small molecule inhibitors of MerTK that may have clinical benefit by both direct anti-tumor effects and by enhancing the anti-tumor immune response. Note: This abstract was not presented at the conference. Citation Format: Sacha J. Holland, Alexander M. Owyang, Sylvia Braselmann, Chrystelle Lamagna, Sothy Yi, Chi Young, Roy Frances, Arthur Bagos, Meagan Chan, Ernest Tai, Stacey Siu, Gary Park, David Lau, Matt Duan, Rao Kolluri, Jiaxin Yu, Ihab Darwish, Somasekhar Bhamidipati, Donald G. Payan, Esteban Masuda. Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK [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 B060.

Abstract 346: Potential role for R191, potent and selective IRAK4 kinase inhibitor, in treatment of hematologic malignancies

Recent advances in genome sequencing and tumor proteome and transcriptome analysis uncovered a key role for MyD88-dependent Toll-Like Receptor (TLR) and Interleukin-1 Receptor (IL-1R)-mediated signaling pathways in multiple hematologic malignancies. A third of activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL) and nearly 100% of Waldenstrom macroglobulinemia (WD) patients carry an activating Myd88 L265P mutation. Overexpression of multiple TLR pathway components, associated with deregulation of innate immune system and subsequent induction of proinflammatory bone marrow environment, are prominent features of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). IRAK4 kinase is a crucial enzyme in all MyD88-dependent signaling pathways, potentially making it an ideal target for disease modification with small molecule inhibitors. Through cell-based screening, we identified a potent small molecule IRAK1/4 kinase inhibitor, R191. R191 blocks TLR- and IL-1R-induced cytokine production in primary cells with potencies below 50nM while sparing unrelated pathways with at least 20-fold window. R191 is extremely potent in vitro against IRAK4 kinase (3 nM), yet exhibits good selectivity against a broad panel of kinases. In vivo, it decreases serum IL-6 in an acute mouse model of IL-1β-induced cytokine release and blocks joint inflammation in the collagen-induce arthritis model. R191 exhibited strong synergy with Bcl2 and BCR pathway inhibitors against a number of DLBCL lines in vitro. In multiple AML lines, R191 potently inhibited expression of PD-L1, potentially promoting recognition of AML blasts by the immune system. Based on the potential critical role of IRAK kinases in MDS, AML and Myd88 L265P lymphomas, R191 could provide a novel therapeutic modality in the management of multiple inflammation-driven hematologic malignancies. Citation Format: Vadim V. Markovtsov, Chrystelle Lamagna, Meagan Chan, Sothy Yi, Chi Young, Roy Frances, Stacey Siu, Sylvia Braselmann, Hui Li, Rajinder Singh, Gary Park, Esteban Masuda, Vanessa Taylor, Donald G. Payan. Potential role for R191, potent and selective IRAK4 kinase inhibitor, in treatment of hematologic malignancies. [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 346.

Abstract 4869: Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK

Introduction: MerTK, a TAM (Tyro3, Axl, MerTK) family RTK, is expressed on phagocytic myeloid and epithelial cells. Its normal function is to dampen innate immune responses to self-antigens. MerTK is an indirect phosphatidylserine (PtdSer) receptor: PtdSer-binding TAM ligands, Gas6 or Protein S, bridge interactions between MerTK and PtdSer externalized on apoptotic cells (ACs), resulting in AC internalization (efferocytosis). Ensuing MerTK signaling leads to anti-inflammatory M2 macrophage polarization, suppression of pro-inflammatory cytokine production, and a tolerogenic outcome. Tumors are rich in ACs and TAM ligands. Syngeneic tumors implanted in MerTK -/- mice exhibit impaired growth and metastasis compared with those implanted in WT mice, correlating with enhanced production of pro-inflammatory cytokines, splenocyte proliferation, and decreased IL-10. Moreover, MerTK aberrantly expressed on hematological and epithelial malignancies promotes survival and chemoresistance. Thus, pharmacological inhibition of MerTK may have clinical benefit by increasing availability of dead tumor cell antigens, blocking tumor induced immunosuppression, or directly promoting tumor cell survival. We have therefore developed small molecule inhibitors of MerTK. Methods: MerTK kinase activity was assayed using ADP-Glo. Cellular MerTK activity was stimulated in HUVEC or H1299 cells using anti-MerTK crosslinking and measured by immunoprecipitation followed by anti-phospho-MerTK blot, or by downstream phospho-Akt Ser 473 using HTRF. A high content assay was used to measure cell proliferation, DNA content and apoptosis. Immune effector assays were LPS-induced IL-23 production in human primary dendritic cells and anti-CD3/CD28-induced IL-2 production or IL-2 induced phospho-STAT5 in human primary T cells. Efferocytosis of CFSE-labeled apoptotic Jurkat cells by anti-CD14-labeled human primary macrophages was detected by flow cytometry. Results: Here we describe novel MerTK-selective and Mer-Axl small molecule inhibitors that potently block MerTK in biochemical assays. These compounds exhibit selectivity for TAM family members in an in vitro kinase panel. In cells, antibody-induced MerTK phosphorylation as well as downstream phosphorylation of Akt was inhibited by both compounds with EC50 Conclusions: We have discovered potent and novel small molecule inhibitors of MerTK that may have clinical benefit by both direct anti-tumor effects and by enhancing the anti-tumor immune response. Citation Format: Sacha J. Holland, Alex M. Owyang, Sothy Yi, Chi Young, Sylvia Braselmann, Roy Frances, Arthur Bagos, Ernest Tai, Stacey Siu, Gary Park, David Lau, Matt Duan, Rao Kolluri, Somasekhar Bhamidipati, Ihab Darwish, Matthew Duncton, Rajinder Singh, Esteban Masuda, Donald G. Payan. Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK. [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 4869.