Abstract Steroid-resistant asthma comprises an important source of morbidity in patient populations. TH17 cells represent a distinct population of CD4+ Th cells that mediate neutrophilic inflammation and are characterized by the production of IL-17, IL-22, and IL-6. To investigate the function of TH17 cells in the context of Ag-induced airway inflammation, we polarized naive CD4+ T cells from DO11.10 OVA-specific TCR-transgenic mice to a TH2 or TH17 phenotype by culturing in conditioned medium. In addition, we also tested the steroid responsiveness of TH2 and TH17 cells. In vitro, TH17 cytokine responses were not sensitive to dexamethasone (DEX) treatment despite immunocytochemistry confirming glucocorticoid receptor translocation to the nucleus following treatment. Transfer of TH2 cells to mice challenged with OVA protein resulted in lymphocyte and eosinophil emigration into the lung that was markedly reduced by DEX treatment, whereas TH17 transfer resulted in increased CXC chemokine secretion and neutrophil influx that was not attenuated by DEX. Transfer of TH17 or TH2 cells was sufficient to induce airway hyperresponsiveness (AHR) to methacholine. Interestingly, AHR was not attenuated by DEX in the TH17 group. These data demonstrate that polarized Ag-specific T cells result in specific lung pathologies. Both TH2 and TH17 cells are able to induce AHR, whereas TH17 cell-mediated airway inflammation and AHR are steroid resistant, indicating a potential role for TH17 cells in steroid-resistant asthma.
Abstract PIM kinases have been shown to play a key role as downstream effectors of growth factor signalling pathways including Flt3 and the Jak-STAT signalling pathways in AML, NHL and other solid tumors. AZD1208 is a novel, orally bioavailable, highly selective PIM kinase inhibitor with single nanomolar potency against all three PIM kinases and is currently undergoing Phase I testing and dose escalation studies in AML. Here we describe a multiplexed biomarker strategy measuring pBAD, p4EBP1 and p-p70S6K as downstream pharmacodynamic biomarkers for PIM kinase inhibition in clinical trials. Patient bone marrow aspirates and peripheral blood samples were collected pre- and post-AZD1208 treatment in AML patients during the Phase I dose escalation and expansions. Primary patient samples were analyzed for quantitative changes in pBAD, p4EBP1 by NanoPro and MesoScale assay platforms as well as a qualitative evaluation of p-p70S6K and other exploratory endpoints. Preclinical PK-PD modeling data with AZD1208 had suggested that greater than a 50% decrease in the levels of one of these phosphorlyated substrates would be indicative of efficacy and PIM pathway inhibition. Following a single dose of AZD1208 at 120mg, the first dose level, approximately 60-70% inhibition of pBAD, S112 was seen in the bone marrow and peripheral blasts. Taken together, the data presented here provide evidence for single agent AZD1208 activity in AML patients based on quantitative reduction in these biomarkers. Correlations of these biomarker endpoints with Phase I pharmacokinetic data underscore the therapeutic potential of Pim kinase inhibition by AZD1208 for the treatment of AML, and strongly support further investigation of this agent in other indications where PIM signaling may play a role in tumorigenesis and survival. Citation Format: Kristen A. McEachern, Yichen Cao, Rachel DuPont, Lourdes Pablo, Patricia McCoon, Jorge E. Cortes, Daniel J. DeAngelo, Mark D. Minden, Becker Hewes, Jeffrey L. Brown, Carl Barrett. AZD1208 PIM kinase inhibitor - Preliminary evidence of target pathway inhibition in Phase I clinical trials of AML. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3516. doi:10.1158/1538-7445.AM2013-3516
MET, the receptor for hepatocyte growth factor (HGF), plays an important role in signaling normal and tumor cell migration and invasion. Here, we describe a previously unrecognized mechanism that promotes MET expression in multiple tumor cell types. The levels of the Pim-1 protein kinase show a positive correlation with the levels of MET protein in human tumor cell lines and patient-derived tumor materials. Using small interfering RNA (siRNA), Pim knockout mice, small-molecule inhibitors, and overexpression of Pim-1, we confirmed this correlation and found that Pim-1 kinase activity regulates HGF-induced tumor cell migration, invasion, and cell scattering. The novel biochemical mechanism for these effects involves the ability of Pim-1 to control the translation of MET by regulating the phosphorylation of eukaryotic initiation factor 4B (eIF4B) on S406. This targeted phosphorylation is required for the binding of eIF4B to the eIF3 translation initiation complex. Importantly, Pim-1 action was validated by the evaluation of patient blood and bone marrow from a phase I clinical trial of a Pim kinase inhibitor, AZD1208. These results suggest that Pim inhibitors may have an important role in the treatment of patients where MET is driving tumor biology.
Abstract AZD9150 is a gen2.5 antisense oligonucleotide (ASO) targeting STAT3. Gen2.5 ASOs exhibit enhanced drug-like properties compared to previous generations of antisense therapeutics, including increased stability and resistance to nucleases, a marked decrease in proinflammatory effects, and enhanced potency. The immune suppressive effects of STAT3 signaling are well established (Kortylewski et al.; Nat. Med. 2005 and Curr. Opin. Immunol. 2008). Preclinical experiments were carried out to determine the potential for combination of a STAT3 ASO with checkpoint inhibitor therapy, and to explore the mechanism of the antitumor activity of STAT3 ASOs observed in mouse models. The antitumor activity of STAT3 ASO treatment, as a single agent and in combination with checkpoint inhibitors, was evaluated in syngeneic mouse models, including CT-26. ASO uptake and STAT3 knockdown were evaluated by immunohistochemistry (IHC), and the pharmacodynamic effects of STAT3 ASO treatment in mice were evaluated by IHC, flow cytometry and immune gene expression profiling. In contrast to many tumor cell lines, CT26 cells take up ASOs poorly, including gen2.5 ASOs, thus as expected the tumor-associated activity of STAT3 ASOs in CT26 tumors in vivo was primarily in stromal and immune compartments (including circulating PBMCs), rather than directly in tumor cells. This was observed in several murine tumor models, as well as in clinical samples. Expression analysis of CT-26 tumor lysates taken from STAT3 ASO-treated (50 mg/kg QDx5/wk) mice using the Nanostring mouse immunology panel (561 immune related genes) identified CD163 as the gene most consistently modulated after STAT3 ASO treatment, with an average knockdown of 85% relative to control treated tumors, across 3 independent experiments. This result was confirmed by immunohistochemistry, which showed a marked reduction in the number of tumor infiltrating CD163+ cells after STAT3 ASO treatment. CD163 is a marker of M2 macrophages, and the presence of M2 macrophages in a tumor can create an immune suppressive environment which has the potential to reduce the effectiveness of checkpoint inhibitor therapy. Consistent with this hypothesis, the addition of STAT3 ASO treatment enhanced the response to a checkpoint inhibitor antibody targeting PD-L1 (clone 10F.9G2). When treatment began 2 days after tumor implant, single agent STAT3 ASO and PD-L1 antibody treatments resulted in response rates (regression or no tumor growth during the course of the experiment) of 0% and 14% respectively, while the rate was 50% with the combination. The combination remained active when treatments were delayed until established tumors were present (140 mm3 average tumor volume), with single agent and combination regression rates of 0%, and 20% respectively. These results suggest the addition of AZD9150 treatment as a promising approach to enhance the response to checkpoint inhibitor therapy Citation Format: Richard D. Woessner, Patricia McCoon, Shaun Grosskurth, Paul Lyne, Kirsten Bell, Mike Collins, Nanhua Deng, Rachel DuPont, Michele Johnstone, Youngsoo Kim, Deborah Lawson, Robert MacLeod, Lourdes Pablo, Corinne Reimer, Vasu Sah, Margaret Veldman-Jones. STAT3 antisense treatment decreases M2 macrophage infiltration and enhances the activity of checkpoint inhibitors in preclinical tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A94.
Abstract AZD9150, a gen2.5 antisense oligonucleotide (ASO) targeting human STAT3, has improved drug-like properties compared to previous generation ASO therapeutics, including increased stability and resistance to nucleases, reduced proinflammatory effects, and enhanced potency. We have previously reported that in tumors, STAT3 ASOs are taken up preferentially in stromal and immune cells of the tumor microenvironment (TME). Since AZD9150 is selective for human STAT3, we used a surrogate ASO (muSTAT3 ASO) to explore the pharmacodynamics of ASO-mediated STAT3 inhibition in syngeneic and genetically engineered mouse (GEM) tumor models, focusing on effects in the TME. In mice bearing subcutaneous CT-26 tumors, treatment with muSTAT3 ASO at 50 mg/kg, s.c., on a qdx5/wk schedule decreased STAT3 levels in immune cell subsets in the tumor and in circulating leukocytes by 40 - 60%, similar to the decrease in STAT3 achievable in circulating leukocytes in human patients after AZD9150 treatment. In a Nanostring analysis (nCounter mouse immunology panel) of CT-26 tumors from muSTAT3 ASO treated mice, CD163 (M2 immune suppressive macrophage marker) was the gene most consistently and significantly downregulated, by an average of 84% in three independent experiments, and was confirmed by immunohistochemistry (IHC). Flow cytometry analysis of myeloid subpopulations - tumor associated macrophages (F4/80+ TAMs), monocytic myeloid derived suppressor cells, and granulocytic cells - showed a decrease in TAMs averaging 69% across three independent experiments. The analysis was extended to include IHC for arginase (Arg, a marker of functional immune suppression activity). Subpopulations of cells identified included Arg+, CD163+, and Arg+CD163+. Treatment with muSTAT3 ASO decreased these populations by 79%, 88% and 97% respectively, compared to control treatment. These populations were also analyzed in two GEM tumor models - the KPC pancreatic cancer model, and a PTEN -/- prostate cancer model - which have a TME more representative of that found in tumors in the clinic. While the specific changes varied across the models, likely reflecting differences in TME makeup, a reduction in immune suppressive cell populations was present in both GEM models, including a decrease in CD163+ cells of 79% (along with modest antitumor activity) in the PTEN -/- prostate model after muSTAT3 ASO treatment. These results indicate that selective STAT3 inhibition can reduce immune suppressive cell populations in the TME, and suggest that STAT3 inhibition has the potential to enhance the antitumor activity of T-cell targeted therapies, such as those targeting the PD1-PDL1 axis. In support of this hypothesis, we observed that addition of muSTAT3 ASO to anti-PD-L1 Ab treatment significantly enhanced the antitumor activity of PD-L1 Ab treatment in two subcutaneous syngeneic tumor models, CT-26 and A20. Citation Format: Rich Woessner, Vasu Sah, Patricia McCoon, Shaun Grosskurth, Nanhua Deng, Rachel DuPont, Deborah Lawson, Lourdes Pablo, Corinne Reimer, Marco A. De Velasco, Hirotsugu Uemura, Juliana Candido, Paul Lyne. Inhibition of STAT3 by antisense oligonucleotide treatment decreases the immune suppressive tumor microenvironment in syngeneic and GEM tumor models [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 3684. doi:10.1158/1538-7445.AM2017-3684
Abstract AZD9150 is a therapeutic Generation 2.5 antisense oligonucleotide (ASO) targeting STAT3 that has completed two phase I clinical studies, in patients with HCC and DLBCL, with durable clinical responses seen in both trials. Biomarker studies using patient samples and related preclinical experiments were performed to investigate the mechanism of action of AZD9150. Patients were treated with three loading doses of AZD9150 in the first week followed by weekly dosing, at doses ranging from 1.0 to 3.0 mg/kg. In the DLBCL study, paired tumor biopsies were collected pre-treatment and on-treatment to evaluate drug uptake and target knockdown by immunohistochemistry (IHC). In the HCC study, blood samples were collected at baseline and at multiple time points on-treatment to evaluate target knockdown and gene expression changes. IHC staining of DLBCL patients’ tumor biopsies (at 2 & 3 mg/kg) demonstrated that the drug distributes to the tumor, with strongest uptake in stromal cells, including endothelium, fibroblasts, and immune cells. Pronounced decreases (absence of staining on-treatment) in STAT3 were observed in the endothelium of several samples. More limited STAT3 modulation was observed in tumor cells. Flow cytometry analysis of HCC patients’ blood samples revealed an average decrease in STAT3 protein staining of 49% across all peripheral leukocyte populations in the 1 mg/kg cohort. Clinical pharmacodynamics and mechanism of action were explored further by conducting a gene expression study with the Nanostring nCounter Human Immunology Panel v2 to evaluate STAT3 RNA knockdown and 593 additional immune genes in peripheral leukocytes collected from HCC patients. Statistically significant decreases of >30% in STAT3 expression were observed in 14/32 patients by the fourth week of treatment. These STAT3 changes are accompanied by +/- 40% changes in expression by additional genes associated with decreased myeloid trafficking and function, increased antigen presentation, and increased CD8 effector cell function. These data provide evidence that AZD9150 treatment may remove or reprogram immunosuppressive elements employed by tumors, leading to therapeutic benefit. Preclinical studies were carried out to investigate immune cell changes within tumors and the benefit of combining STAT3 ASO with PDL1 blockade. Monotherapy STAT3 ASO treatment resulted in CT26 tumor growth inhibition (80%) when tested in immune competent Balb/c but not immune-deficient NSG mice, and was associated with two-fold increases in CD45+ and CD8+ cell infiltrate into tumors. Mice treated with STAT3 ASO and anti-PD-L1 blocking antibody resulted in a 50% response rate for the combination treatment, vs. only 14% with anti-PD-L1 Ab alone. These data suggest that the effects of STAT3 ASO are mechanistically complementary to immune checkpoint inhibitors and that the combination with AZD9150 could broaden clinical responses to these important therapies. This hypothesis will be tested in upcoming clinical trials with AZD9150 and MEDI4736. Citation Format: Patricia E. Mccoon, Rich Woessner, Shaun Grosskurth, Chris Womack, Mason Yamashita, Gene Hung, Robert MacLeod, Kirsten Bell, Mike Collins, Rachel DuPont, Vivian Jacobs, Michele Johnstone, Margaret Veldman-Jones, Paul Lyne. Clinical and preclinical evidence of an immune modulating role for the STAT3-targeting ASO AZD9150 and potential to enhance clinical responses to anti-PDL1 therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT239. doi:10.1158/1538-7445.AM2015-CT239
Abstract The multifaceted role of STAT3 in enhancing tumor growth by tumor cell intrinsic as well as extrinsic (stromal and immune modulatory) mechanisms is well documented. Elevated STAT3 in tumor cells can lead to increased expression of cytokines, such as IL-6, which can create an immune suppressive environment in the tumor stroma. In addition, STAT3 signaling in cells of the immune system has the potential to contribute to an immune suppressive environment by several mechanisms, including suppression of dendritic cell (DC) maturation and function, enhanced myeloid derived suppressor cell (MDSC) activity, and suppression of T-cell mediated antitumor activity. An immune suppressive environment can reduce the antitumor activity of checkpoint inhibitors such as anti-PD-L1 antibodies by suppressing the ability of T-cell mediated immunity to respond to checkpoint reversal. Therefore, we hypothesized that inhibition of STAT3 has the potential to increase the anti-tumor effects of immune checkpoint inhibitors. To investigate this hypothesis, we explored the ability of a mouse STAT3 targeted antisense oligonucleotide (ASO) to enhance the antitumor activity of an anti-PD-L1 mAb in a syngeneic murine tumor model. In mice bearing subcutaneous CT-26 tumors, the combination of the STAT3 ASO plus an anti-PD-L1 mAb provided greater antitumor activity than either agent alone. Increased activity was observed when treatment was initiated soon (2 days) after tumor implantation, as well as when treatment was initiated after tumors were established (~150 mm3). When treatment was initiated soon after tumor implantation, single agents as well as the combination were initially effective, but the combination led to long term suppression of tumor growth in a greater percentage of mice (80% for the combination, vs. 20% for single agents). When treatment was initiated after tumors were established, STAT3 ASO or anti-PD-L1 antibody as single agents had significant initial antitumor activity (55% after 14 days of treatment). However, the single agent activity was transient, with the tumor growth rate returning to that of vehicle control (5 day doubling time) after two weeks of treatment. In contrast, the combination treatment led to a sustained reduction in mean tumor growth rate (15 days doubling time), and regression in 20 - 30% of tumors after four weeks of treatment. Treatment with the STAT3 ASO was associated with tumor infiltrating leukocyte changes consistent with enhanced antitumor immunity, including an increase in CD8+ effector T-cells. Combination benefit for STAT3 ASO plus anti-PD-L1 antibody was also observed in other immunocompetent murine tumor models. The data indicate that, in these models, inhibition of STAT3 has immunomodulatory activity, and can enhance the activity of immune checkpoint inhibitors, such as those targeting PD-L1. Citation Format: Rich Woessner, Patricia McCoon, Kirsten Bell, Rachel DuPont, Mike Collins, Deborah Lawson, Prasad Nadella, Lourdes Pablo, Corinne Reimer, Vasu Sah, Paul Lyne. STAT3 inhibition enhances the activity of immune checkpoint inhibitors in murine syngeneic tumor models by creating a more immunogenic tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A93.
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