ABSTRACT
Activating Janus kinase (JAK) and signal transducer and activator of transcription (STAT) mutations have been discovered in many T-cell malignancies, including anaplastic lymphoma kinase (ALK)- anaplastic large cell lymphomas (ALCLs). However, such mutations occur in a minority of patients. To investigate the clinical application of targeting JAK for ALK- ALCL, we treated ALK- cell lines of various histological origins with JAK inhibitors. Interestingly, most exogenous cytokine-independent cell lines responded to JAK inhibition regardless of JAK mutation status. JAK inhibitor sensitivity correlated with the STAT3 phosphorylation status of tumor cells. Using retroviral shRNA knockdown, we have demonstrated that these JAK inhibitor-sensitive cells are dependent on both JAK1 and STAT3 for survival. JAK1 and STAT3 gain-of-function mutations were found in some, but not all, JAK inhibitor-sensitive cells. Moreover, the mutations alone cannot explain the JAK1/STAT3 dependency, given that wild-type JAK1 or STAT3 was sufficient to promote cell survival in the cells that had either JAK1or STAT3 mutations. To investigate whether other mechanisms were involved, we knocked down upstream receptors GP130 or IL-2Rγ. Knockdown of GP130 or IL-2Rγ induced cell death in selected JAK inhibitor-sensitive cells. High expression levels of cytokines, including IL-6, were demonstrated in cell lines as well as in primary ALK- ALCL tumors. Finally, ruxolitinib, a JAK1/2 inhibitor, was effective in vivo in a xenograft ALK- ALCL model. Our data suggest that cytokine receptor signaling is required for tumor cell survival in diverse forms of ALK- ALCL, even in the presence of JAK1/STAT3 mutations. Therefore, JAK inhibitor therapy might benefit patients with ALK- ALCL who are phosphorylated STAT3.
Subject(s)
Janus Kinase 1/genetics , Lymphoma, Large-Cell, Anaplastic/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Cytokine/metabolism , STAT3 Transcription Factor/genetics , Anaplastic Lymphoma Kinase , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival , Cytokine Receptor gp130/genetics , Cytokine Receptor gp130/metabolism , Cytokines/genetics , Cytokines/metabolism , Humans , Janus Kinase 1/antagonists & inhibitors , Janus Kinase 1/metabolism , Lymphoma, Large-Cell, Anaplastic/drug therapy , Lymphoma, Large-Cell, Anaplastic/genetics , Mice , Nitriles , Phosphorylation , Piperidines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Cytokine/genetics , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Adenosine is known to be an important signaling molecule in many physiological processes and has recently been shown to be an important molecule in oncology. A fit for purpose method has been developed for the quantification of adenosine in murine tumor samples using pre-column derivatization and liquid chromatography-mass spectrometry (LC-MS/MS). To overcome adenosine quantification challenges, derivatization with dansyl chloride was employed. This derivatization technique, following protein precipitation and liquid-liquid extraction, improved the sensitivity and selectivity of adenosine in tumor samples through the reduction of endogenous interference and matrix effects. This method utilizes a mouse plasma calibration curve, qualified over a range of 0.019⯵M-37⯵M. The 15â¯min derivatization incubation time and 1â¯min chromatographic run time allow for higher throughput. The following established method overcomes challenges associated with the quantification of low molecular weight, polar, endogenous molecules, such as adenosine, using derivatization and LC-MS/MS. With the additional analysis of murine tumors, this method will contribute to the understanding of the impact adenosine plays in the tumor microenvironment and the bearing it has on targeted cancer therapies.
Subject(s)
Adenosine/blood , Brain Neoplasms/blood , Liver Neoplasms/blood , Adenosine/analogs & derivatives , Adenosine/chemistry , Animals , Brain Neoplasms/diagnosis , Calibration , Chromatography, Liquid , Liver Neoplasms/diagnosis , Mice , Tandem Mass SpectrometryABSTRACT
We have identified a class of azabenzimidazoles as potent and selective JAK1 inhibitors. Investigations into the SAR are presented along with the structural features required to achieve selectivity for JAK1 versus other JAK family members. An example from the series demonstrated highly selective inhibition of JAK1 versus JAK2 and JAK3, along with inhibition of pSTAT3 in vivo, enabling it to serve as a JAK1 selective tool compound to further probe the biology of JAK1 selective inhibitors.
Subject(s)
Imidazoles/pharmacology , Janus Kinase 1/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , STAT3 Transcription Factor/antagonists & inhibitors , Animals , Cell Cycle/drug effects , Cell Death/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Female , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , Janus Kinase 1/metabolism , Mice , Mice, Nude , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , STAT3 Transcription Factor/metabolism , Structure-Activity RelationshipABSTRACT
Proteasome inhibition with bortezomib is a validated approach to the treatment of multiple myeloma, but drug resistance often emerges and limits its utility in the retreatment setting. To begin to identify some of the mechanisms involved, we developed bortezomib-resistant myeloma cell lines that, unlike previously reported models, showed no ß5 subunit mutations. Instead, up-regulation of the insulin-like growth factor (IGF)-1 axis was identified, with increased autocrine and paracrine secretion of IGF-1, leading to increased activation of the IGF-1 receptor (IGF-1R). Exogenous IGF-1 reduced cellular sensitivity to bortezomib, whereas pharmacologic or small hairpin RNA-mediated IGF-1R suppression enhanced bortezomib sensitivity in cell lines and patient samples. In vitro studies with OSI-906, a clinically relevant dual IGF-1R and insulin receptor inhibitor, showed it acted synergistically with bortezomib, and potently resensitized bortezomib-resistant cell lines and patient samples to bortezomib. Importantly, OSI-906 in combination with bortezomib also overcame bortezomib resistance in an in vivo model of myeloma. Taken together, these data support the hypothesis that signaling through the IGF-1/IGF-1R axis contributes to acquired bortezomib resistance, and provide a rationale for combining bortezomib with IGF-1R inhibitors like OSI-906 to overcome or possibly prevent the emergence of bortezomib-refractory disease in the clinic.
Subject(s)
Antineoplastic Agents/pharmacology , Boronic Acids/pharmacology , Drug Resistance, Neoplasm , Imidazoles/pharmacology , Multiple Myeloma/drug therapy , Pyrazines/pharmacology , Receptor, IGF Type 1/antagonists & inhibitors , Animals , Antineoplastic Combined Chemotherapy Protocols , Apoptosis/drug effects , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Bortezomib , Cell Proliferation/drug effects , Drug Synergism , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Expression Profiling , Humans , Insulin-Like Growth Factor I/metabolism , Mice , Mice, SCID , Multiple Myeloma/metabolism , Multiple Myeloma/mortality , Oligonucleotide Array Sequence Analysis , Receptor, IGF Type 1/metabolism , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Background and Aims: Fibrolamellar carcinoma (FLC) is a rare, difficult-to-treat liver cancer primarily affecting pediatric and adolescent patients, and for which precision medicine approaches have historically not been possible. The DNAJB1-PRKACA gene fusion was identified as a driver of FLC pathogenesis. We aimed to assess whether FLC tumors maintain dependency on this gene fusion and determine if PRKACA is a viable therapeutic target. Methods: FLC patient-derived xenograft (PDX) shRNA cell lines were implanted subcutaneously into female NOD-SCID mice and tumors were allowed to develop prior to randomization to doxycycline (to induce knockdown) or control groups. Tumor development was assessed every 2 days. To assess the effect of treatment with novel selective PRKACA small molecule kinase inhibitors, BLU0588 and BLU2864, FLC PDX tumor cells were implanted subcutaneously into NOD-SCID mice and tumors allowed to develop. Mice were randomized to treatment (BLU0588 and BLU2864, orally, once daily) or control groups and tumor size determined as previously. Results: Knockdown of DNAJB1-PRKACA reversed a FLC-specific gene signature and reduced PDX tumor growth in mice compared to the control group. Furthermore, FLC PDX tumor growth was significantly reduced with BLU0588 and BLU2864 treatment vs control (P = .003 and P = .0005, respectively). Conclusion: We demonstrated, using an inducible knockdown and small molecule approaches, that FLC PDX tumors were dependent upon DNAJB1-PRKACA fusion activity. In addition, this study serves as a proof-of-concept that PRKACA is a viable therapeutic target for FLC and warrants further investigation.
ABSTRACT
Background: Adenosine receptor type 2 (A2AR) inhibitor, AZD4635, has been shown to reduce immunosuppressive adenosine effects within the tumor microenvironment (TME) and to enhance the efficacy of checkpoint inhibitors across various syngeneic models. This study aims at investigating anti-tumor activity of AZD4635 alone and in combination with an anti-PD-L1-specific antibody (anti-PD-L1 mAb) across various TME conditions and at identifying, via mathematical quantitative modeling, a therapeutic combination strategy to further improve treatment efficacy. Methods: The model is represented by a set of ordinary differential equations capturing: 1) antigen-dependent T cell migration into the tumor, with subsequent proliferation and differentiation into effector T cells (Teff), leading to tumor cell lysis; 2) downregulation of processes mediated by A2AR or PD-L1, as well as other immunosuppressive mechanisms; 3) A2AR and PD-L1 inhibition by, respectively, AZD4635 and anti-PD-L1 mAb. Tumor size dynamics data from CT26, MC38, and MCA205 syngeneic mice treated with vehicle, anti-PD-L1 mAb, AZD4635, or their combination were used to inform model parameters. Between-animal and between-study variabilities (BAV, BSV) in treatment efficacy were quantified using a non-linear mixed-effects methodology. Results: The model reproduced individual and cohort trends in tumor size dynamics for all considered treatment regimens and experiments. BSV and BAV were explained by variability in T cell-to-immunosuppressive cell (ISC) ratio; BSV was additionally driven by differences in intratumoral adenosine content across the syngeneic models. Model sensitivity analysis and model-based preclinical study simulations revealed therapeutic options enabling a potential increase in AZD4635-driven efficacy; e.g., adoptive cell transfer or treatments affecting adenosine-independent immunosuppressive pathways. Conclusions: The proposed integrative modeling framework quantitatively characterized the mechanistic activity of AZD4635 and its potential added efficacy in therapy combinations, across various immune conditions prevailing in the TME. Such a model may enable further investigations, via simulations, of mechanisms of tumor resistance to treatment and of AZD4635 combination optimization strategies.
Subject(s)
Adenosine A2 Receptor Antagonists/pharmacology , Antineoplastic Agents/pharmacology , Models, Biological , Receptor, Adenosine A2A/metabolism , Tumor Microenvironment/drug effects , Algorithms , Animals , Antineoplastic Agents, Immunological/pharmacology , B7-H1 Antigen/antagonists & inhibitors , Cell Line, Tumor , Disease Susceptibility , Drug Resistance, Neoplasm , Drug Therapy, Combination , Isografts , Mice , Xenograft Model Antitumor AssaysABSTRACT
The discovery and optimization of a series of pyrrolopyrimidine based protein kinase B (Pkb/Akt) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent inhibition of all three Akt isoforms and knockdown of phospho-PRAS40 levels in LNCaP cells and tumor xenografts.
Subject(s)
Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Pyrimidines/chemistry , Pyrroles/chemistry , Adaptor Proteins, Signal Transducing , Animals , Binding Sites , Cell Line , Crystallography, X-Ray , Drug Design , Drug Evaluation, Preclinical , High-Throughput Screening Assays , Humans , Mice , Phosphoproteins/deficiency , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Proto-Oncogene Proteins c-akt/metabolism , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Pyrroles/chemical synthesis , Pyrroles/pharmacology , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Herein we report the discovery and synthesis of a novel series of dihydrothieno- and dihydrofuropyrimidines (2 and 3) as potent pan Akt inhibitors. Utilizing previous SAR and analysis of the amino acid sequences in the binding site we have designed inhibitors displaying increased PKA and general kinase selectivity with improved tolerability compared to the progenitor pyrrolopyrimidine (1). A representative dihydrothieno compound (34) was advanced into a PC3-NCI prostate mouse tumor model in which it demonstrated a dose-dependent reduction in tumor growth and stasis when dosed orally daily at 200 mg/kg.
Subject(s)
Prostatic Neoplasms/drug therapy , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Pyrimidines/chemistry , Animals , Binding Sites , Disease Models, Animal , Dose-Response Relationship, Drug , Male , Mice , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Structure-Activity Relationship , Tumor Burden/drug effectsABSTRACT
Accumulation of extracellular adenosine within the microenvironment is a strategy exploited by tumors to escape detection by the immune system. Adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which promote tumor growth and limit the efficacy of immune checkpoint inhibitors. Preclinical data with A2AR inhibitors have demonstrated tumor regressions in mouse models by rescuing T cell function; however, the mechanism and role on other immune cells has not been fully elucidated. METHODS: We report here the development of a small molecule A2AR inhibitor including characterization of binding and inhibition of A2AR function with varying amounts of a stable version of adenosine. Functional activity was tested in both mouse and human T cells and dendritic cells (DCs) in in vitro assays to understand the intrinsic role on each cell type. The role of adenosine and A2AR inhibition was tested in DC differentiation assays as well as co-culture assays to access the cross-priming function of DCs. Syngeneic models were used to assess tumor growth alone and in combination with alphaprogrammed death-ligand 1 (αPD-L1). Immunophenotyping by flow cytometry was performed to examine global immune cell changes upon A2AR inhibition. RESULTS: We provide the first report of AZD4635, a novel small molecule A2AR antagonist which inhibits downstream signaling and increases T cell function as well as a novel mechanism of enhancing antigen presentation by CD103+ DCs. The role of antigen presentation by DCs, particularly CD103+ DCs, is critical to drive antitumor immunity providing rational to combine a priming agent AZD4635 with check point blockade. We find adenosine impairs the maturation and antigen presentation function of CD103+ DCs. We show in multiple syngeneic mouse tumor models that treatment of AZD4635 alone and in combination with αPD-L1 led to decreased tumor volume correlating with enhanced CD103+ function and T cell response. We extend these studies into human DCs to show that adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen-specific T cell activation. Our results support the novel role of adenosine signaling as an intrinsic negative regulator of CD103+ DCs maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. This unique mechanism of action in CD103+ DCs may contribute to clinical responses as AZD4635 is being evaluated in clinical trials with IMFINZI (durvalumab, αPD-L1) in patients with solid malignancies. CONCLUSION: We provide evidence implicating suppression of adaptive and innate immunity by adenosine as a mechanism for immune evasion by tumors. Inhibition of adenosine signaling through selective small molecule inhibition of A2AR using AZD4635 restores T cell function via an internal mechanism as well as tumor antigen cross-presentation by CD103+ DCs resulting in antitumor immunity.
Subject(s)
Antigens, CD/metabolism , Antineoplastic Agents, Immunological/therapeutic use , Dendritic Cells/immunology , Integrin alpha Chains/metabolism , Neoplasms/immunology , Receptor, Adenosine A2A/metabolism , Antineoplastic Agents, Immunological/pharmacology , Cell Line, Tumor , Female , Humans , Male , Signal TransductionABSTRACT
BACKGROUND: PD1/PDL1-directed therapies have been unsuccessful for multiple myeloma (MM), an incurable cancer of plasma cells in the bone marrow (BM). Therefore, other immune checkpoints such as extracellular adenosine and its immunosuppressive receptor should be considered. CD39 and CD73 convert extracellular ATP to adenosine, which inhibits T-cell effector functions via the adenosine receptor A2A (A2AR). We set out to investigate whether blocking the adenosine pathway could be a therapy for MM. METHODS: Expression of CD39 and CD73 on BM cells from patients and T-cell proliferation were determined by flow cytometry and adenosine production by Liquid chromatograpy-mass spectrometry (HPCL/MS). ENTPD1 (CD39) mRNA expression was determined on myeloma cells from patients enrolled in the publicly available CoMMpass study. Transplantable 5T33MM myeloma cells were used to determine the effect of inhibiting CD39, CD73 and A2AR in mice in vivo. RESULTS: Elevated level of adenosine was found in BM plasma of MM patients. Myeloma cells from patients expressed CD39, and high gene expression indicated reduced survival. CD73 was found on leukocytes and stromal cells in the BM. A CD39 inhibitor, POM-1, and an anti-CD73 antibody inhibited adenosine production and reduced T-cell suppression in vitro in coculture of myeloma and stromal cells. Blocking the adenosine pathway in vivo with a combination of Sodium polyoxotungstate (POM-1), anti-CD73, and the A2AR antagonist AZD4635 activated immune cells, increased interferon gamma production, and reduced the tumor load in a murine model of MM. CONCLUSIONS: Our data suggest that the adenosine pathway can be successfully targeted in MM and blocking this pathway could be an alternative to PD1/PDL1 inhibition for MM and other hematological cancers. Inhibitors of the adenosine pathway are available. Some are in clinical trials and they could thus reach MM patients fairly rapidly.
Subject(s)
5'-Nucleotidase/metabolism , Adenosine Triphosphate/metabolism , Adenosine/metabolism , Antigens, CD/metabolism , Apyrase/metabolism , Multiple Myeloma/pathology , Receptor, Adenosine A2A/chemistry , Animals , Female , Humans , Mice , Mice, Inbred C57BL , Multiple Myeloma/drug therapy , Multiple Myeloma/immunology , Multiple Myeloma/metabolism , Prognosis , Receptor, Adenosine A2A/metabolism , Survival RateABSTRACT
PURPOSE: Danvatirsen is a therapeutic antisense oligonucleotide (ASO) that selectively targets STAT3 and has shown clinical activity in two phase I clinical studies. We interrogated the clinical mechanism of action using danvatirsen-treated patient samples and conducted back-translational studies to further elucidate its immunomodulatory mechanism of action. EXPERIMENTAL DESIGN: Paired biopsies and blood samples from danvatirsen-treated patients were evaluated using immunohistochemistry and gene-expression analysis. To gain mechanistic insight, we used mass cytometry, flow cytometry, and immunofluorescence analysis of CT26 tumors treated with a mouse surrogate STAT3 ASO, and human immune cells were treated in vitro with danvatirsen. RESULTS: Within the tumors of treated patients, danvatirsen uptake was observed mainly in cells of the tumor microenvironment (TME). Gene expression analysis comparing baseline and on-treatment tumor samples showed increased expression of proinflammatory genes. In mouse models, STAT3 ASO demonstrated partial tumor growth inhibition and enhanced the antitumor activity when combined with anti-PD-L1. Immune profiling revealed reduced STAT3 protein in immune and stromal cells, and decreased suppressive cytokines correlating with increased proinflammatory macrophages and cytokine production. These changes led to enhanced T-cell abundance and function in combination with anti-PD-L1. CONCLUSIONS: STAT3 ASO treatment reverses a suppressive TME and promotes proinflammatory gene expression changes in patients' tumors and mouse models. Preclinical data provide evidence that ASO-mediated inhibition of STAT3 in the immune compartment is sufficient to remodel the TME and enhance the activity of checkpoint blockade without direct STAT3 inhibition in tumor cells. Collectively, these data provide a rationale for testing this combination in the clinic.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , B7-H1 Antigen/antagonists & inhibitors , Colonic Neoplasms/therapy , Neoplasms/therapy , Oligonucleotides/pharmacology , STAT3 Transcription Factor/antagonists & inhibitors , Tumor Microenvironment/immunology , Clinical Trials, Phase I as Topic , Colonic Neoplasms/immunology , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Drug Therapy, Combination , Humans , Immunomodulation , Macrophages/immunology , Neoplasms/immunology , Neoplasms/metabolism , Neoplasms/pathology , Prognosis , STAT3 Transcription Factor/genetics , T-Lymphocytes/immunology , Tumor Cells, CulturedABSTRACT
JAK1, JAK2, JAK3, and TYK2 belong to the JAK (Janus kinase) family. They play critical roles in cytokine signaling. Constitutive activation of JAK/STAT pathways is associated with a wide variety of diseases. Particularly, pSTAT3 is observed in response to the treatment with inhibitors of oncogenic signaling pathways such as EGFR, MAPK, and AKT and is associated with resistance or poorer response to agents targeting these pathways. Among the JAK family kinases, JAK1 has been shown to be the primary driver of STAT3 phosphorylation and signaling; therefore, selective JAK1 inhibition can be a viable means to overcome such treatment resistances. Herein, an account of the medicinal chemistry optimization from the promiscuous kinase screening hit 3 to the candidate drug 21 (AZD4205), a highly selective JAK1 kinase inhibitor, is reported. Compound 21 has good preclinical pharmacokinetics. Compound 21 displayed an enhanced antitumor activity in combination with an approved EGFR inhibitor, osimertinib, in a preclinical non-small-cell lung cancer (NSCLC) xenograft NCI-H1975 model.
Subject(s)
Indoles/therapeutic use , Janus Kinase 1/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , Acrylamides/pharmacology , Aniline Compounds/pharmacology , Animals , Cell Line, Tumor , Drug Design , Drug Discovery , Drug Screening Assays, Antitumor , Drug Synergism , ErbB Receptors/antagonists & inhibitors , Female , Humans , Indoles/chemical synthesis , Indoles/pharmacokinetics , Mice, Nude , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: We previously described a sub-population of epithelial ovarian cancer (EOC) cells with a functional TLR-4/MyD88/NF-kappaB pathway (Type I EOC cells), which confers the capacity to respond to Paclitaxel, a known TLR-4 ligand, by enhancing NF-kappaB activity and upregulating cytokine secretion - events that are known to promote tumor progression. It is therefore important to distinguish those patients that should not receive Paclitaxel; it is also important to identify alternative chemotherapy options that would benefit this sub-group of patients. The objective of this study is to determine if the KSP inhibitor, ARRY-520, can be a substitute for Paclitaxel in patients with Type I EOC. METHODS: EOC cells isolated from either ascites or tumor tissue were treated with increasing concentrations of ARRY-520 or Paclitaxel and cell viability determined. Activation of the apoptotic pathway was determined using Western blot analysis. Mitochondrial integrity was quantified using JC1 dye. Cytokine profiling was performed from supernatants using xMAP technology. NF-kappaB activity was measured using a Luciferase reporter system. In vivo activity was determined using a subcutaneous xenograft mouse model. RESULTS: ARRY-520 and Paclitaxel exhibited the same cytotoxic effect on Type I and II cells. The GI50 at 48 h for Type II EOC cells was 0.0015 microM and 0.2 microM for ARRY-520 and Paclitaxel, respectively. For Type I EOC cells, the GI50 at 48 h was > 3 microM and >20 microM for ARRY-520 and Paclitaxel, respectively. Decrease in the number of viable cells was accompanied by mitochondrial depolarization and caspase activation. Unlike Paclitaxel, ARRY-520 did not induce NF-kappaB activation, did not enhance cytokine secretion, nor induce ERK phosphorylation in Type I EOC cells. CONCLUSION: Administration of Paclitaxel to patients with high percentage Type I cancer cells could have detrimental effects due to Paclitaxel-induced enhancement of NF-kappaB and ERK activities, and cytokine production (e.g. IL-6), which promote chemoresistance and tumor progression. ARRY-520 has similar anti-tumor activity in EOC cells as that of Paclitaxel. However, unlike Paclitaxel, it does not induce these pro-tumor effects in Type I cells. Therefore, the KSP inhibitor ARRY-520 may represent an alternative to Paclitaxel in this subgroup of EOC patients.
Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Kinesins/antagonists & inhibitors , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Paclitaxel/therapeutic use , Apoptosis/drug effects , Ascites/pathology , Caspase 3/analysis , Caspase 3/metabolism , Caspase 7/analysis , Caspase 7/metabolism , Caspase 9/analysis , Caspase 9/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Female , Genes, Reporter , Humans , Inhibitory Concentration 50 , Luciferases/metabolism , Organ Culture Techniques , Thiadiazoles/antagonists & inhibitors , Time Factors , TransfectionABSTRACT
BACKGROUND: Radioresistance represents a major problem in the treatment of head and neck cancer (HNC) patients. To improve response, understanding tumor microenvironmental factors that contribute to radiation resistance is important. Regulatory T cells (Tregs) are enriched in numerous cancers and can dampen the response to radiation by creating an immune-inhibitory microenvironment. The purpose of this study was to investigate mechanisms of Treg modulation by radiation in HNC. METHODS: We utilized an orthotopic mouse model of HNC. Anti-CD25 was used for Treg depletion. Image-guided radiation was delivered to a dose of 10 Gy. Flow cytometry was used to analyze abundance and function of intratumoral immune cells. Enzyme-linked immunosorbent assay was performed to assess secreted factors. For immune-modulating therapies, anti-PD-L1, anti-CTLA-4, and STAT3 antisense oligonucleotide (ASO) were used. All statistical tests were two-sided. RESULTS: Treatment with anti-CD25 and radiation led to tumor eradication (57.1%, n = 4 of 7 mice), enhanced T-cell cytotoxicity compared with RT alone (CD4 effector T cells [Teff]: RT group mean = 5.37 [ 0.58] vs RT + αCD25 group mean =10.71 [0.67], P = .005; CD8 Teff: RT group mean = 9.98 [0.81] vs RT + αCD25 group mean =16.88 [2.49], P = .01) and induced tumor antigen-specific memory response (100.0%, n = 4 mice). In contrast, radiation alone or when combined with anti-CTLA4 did not lead to durable tumor control (0.0%, n = 7 mice). STAT3 inhibition in combination with radiation, but not as a single agent, improved tumor growth delay, decreased Tregs, myeloid-derived suppressor cells, and M2 macrophages and enhanced effector T cells and M1 macrophages. Experiments in nude mice inhibited the benefit of STAT3 ASO and radiation. CONCLUSION: We propose that STAT3 inhibition is a viable and potent therapeutic target against Tregs. Our data support the design of clinical trials integrating STAT3 ASO in the standard of care for cancer patients receiving radiation.
Subject(s)
Head and Neck Neoplasms/radiotherapy , Lymphocyte Depletion , Radioimmunotherapy/methods , STAT3 Transcription Factor/antagonists & inhibitors , Squamous Cell Carcinoma of Head and Neck/radiotherapy , T-Lymphocytes, Regulatory/radiation effects , Analysis of Variance , Animals , Cytotoxicity, Immunologic , Female , Forkhead Transcription Factors/genetics , Gene Expression Profiling , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/immunology , Humans , Immunologic Memory , Interleukin-2 Receptor alpha Subunit/genetics , Lymphocyte Depletion/methods , Macrophages/radiation effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , Myeloid-Derived Suppressor Cells/radiation effects , Radiation Tolerance , Radiotherapy, Image-Guided , STAT3 Transcription Factor/metabolism , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/immunology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Transforming Growth Factor beta1/genetics , Tumor MicroenvironmentABSTRACT
Activation of platelet-derived growth factor receptor alpha (PDGFRA) by genomic aberrations contributes to tumor progression in several tumor types. In this study, we characterize 16 novel PDGFRA mutations identified from different tumor types and identify three previously uncharacterized activating mutations that promote cell survival and proliferation. PDGFRA Y288C, an extracellular domain mutation, is primarily high mannose glycosylated consistent with trapping in the endoplasmic reticulum (ER). Strikingly, PDGFRA Y288C is constitutively dimerized and phosphorylated in the absence of ligand suggesting that trapping in the ER or aberrant glycosylation is sufficient for receptor activation. Importantly, PDGFRA Y288C induces constitutive phosphorylation of Akt, ERK1/2, and STAT3. PDGFRA Y288C is resistant to PDGFR inhibitors but sensitive to PI3K/mTOR and MEK inhibitors consistent with pathway activation results. Our findings further highlight the importance of characterizing functional consequences of individual mutations for precision medicine.
Subject(s)
Drug Resistance, Neoplasm/genetics , Extracellular Space/chemistry , Molecular Targeted Therapy , Mutation/genetics , Receptor, Platelet-Derived Growth Factor alpha/chemistry , Receptor, Platelet-Derived Growth Factor alpha/genetics , Animals , Cell Line , Cell Proliferation , Endoplasmic Reticulum/metabolism , Glycosylation , Golgi Apparatus/metabolism , Humans , Mice , Phenotype , Protein Domains , Protein Kinase Inhibitors/pharmacology , Signal TransductionABSTRACT
Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Transcriptomic analysis of stem and progenitor populations in MDS and AML demonstrated overexpression of STAT3 that was validated in an independent cohort. STAT3 overexpression was predictive of a shorter survival and worse clinical features in a large MDS cohort. High STAT3 expression signature in MDS CD34+ cells was similar to known preleukemic gene signatures. Functionally, STAT3 inhibition by a clinical, antisense oligonucleotide, AZD9150, led to reduced viability and increased apoptosis in leukemic cell lines. AZD9150 was rapidly incorporated by primary MDS/AML stem and progenitor cells and led to increased hematopoietic differentiation. STAT3 knockdown also impaired leukemic growth in vivo and led to decreased expression of MCL1 and other oncogenic genes in malignant cells. These studies demonstrate that STAT3 is an adverse prognostic factor in MDS/AML and provide a preclinical rationale for studies using AZD9150 in these diseases.
Subject(s)
Leukemia, Myeloid, Acute , Myelodysplastic Syndromes , Neoplasm Proteins , Neoplastic Stem Cells , Oligonucleotides/pharmacology , STAT3 Transcription Factor , Animals , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Female , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Male , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Myelodysplastic Syndromes/drug therapy , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/metabolism , Myelodysplastic Syndromes/pathology , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Janus kinases (JAKs) have been demonstrated to be critical in cytokine signaling and have thus been implicated in both cancer and inflammatory diseases. The JAK family consists of four highly homologous members: JAK1-3 and TYK2. The development of small-molecule inhibitors that are selective for a specific family member would represent highly desirable tools for deconvoluting the intricacies of JAK family biology. Herein, we report the discovery of a potent JAK1 inhibitor, 24, which displays â¼1000-fold selectivity over the other highly homologous JAK family members (determined by biochemical assays), while also possessing good selectivity over other kinases (determined by panel screening). Moreover, this compound was demonstrated to be orally bioavailable and possesses acceptable pharmacokinetic parameters. In an in vivo study, the compound was observed to dose dependently modulate the phosphorylation of STAT3 (a downstream marker of JAK1 inhibition).
Subject(s)
Janus Kinase 1/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Administration, Oral , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Biological Availability , Cell Line , Crystallography, X-Ray , Humans , Janus Kinase 1/chemistry , Janus Kinase 1/metabolism , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/metabolism , Janus Kinase 3/metabolism , Mice , Phosphorylation/drug effects , STAT3 Transcription Factor/metabolism , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: The Janus kinase (JAK) and signal transduction and activation of transcription (STAT) signaling pathway is an attractive target in multiple cancers. Activation of the JAK-STAT pathway is important in both tumorigenesis and activation of immune responses. In diffuse large B-cell lymphoma (DLBCL), the transcription factor STAT3 has been associated with aggressive disease phenotype and worse overall survival. While multiple therapies inhibit upstream signaling, there has been limited success in selectively targeting STAT3 in patients. Antisense oligonucleotides (ASOs) represent a compelling therapeutic approach to target difficult to drug proteins such as STAT3 through of mRNA targeting. We report the evaluation of a next generation STAT3 ASO (AZD9150) in a non-Hodgkin's lymphoma population, primarily consisting of patients with DLBCL. METHODS: Patients with relapsed or treatment refractory lymphoma were enrolled in this expansion cohort. AZD9150 was administered at 2 mg/kg and the 3 mg/kg (MTD determined by escalation cohort) dose levels with initial loading doses in the first week on days 1, 3, and 5 followed by weekly dosing. Patients were eligible to remain on therapy until unacceptable toxicity or progression. Blood was collected pre- and post-treatment for analysis of peripheral immune cells. RESULTS: Thirty patients were enrolled, 10 at 2 mg/kg and 20 at 3 mg/kg dose levels. Twenty-seven patients had DLBCL. AZD9150 was safe and well tolerated at both doses. Common drug-related adverse events included transaminitis, fatigue, and thrombocytopenia. The 3 mg/kg dose level is the recommended phase 2 dose. All responses were seen among DLBCL patients, including 2 complete responses with median duration of response 10.7 months and 2 partial responses. Peripheral blood cell analysis of three patients without a clinical response to therapy revealed a relative increase in proportion of macrophages, CD4+, and CD8+ T cells; this trend did not reach statistical significance. CONCLUSIONS: AZD9150 was well tolerated and demonstrated efficacy in a subset of heavily pretreated patients with DLBCL. Studies in combination with checkpoint immunotherapies are ongoing. TRIAL REGISTRATION: Registered at ClinicalTrials.gov: NCT01563302 . First submitted 2/13/2012.
Subject(s)
Lymphoma/drug therapy , Oligonucleotides, Antisense/therapeutic use , Oligonucleotides/therapeutic use , Adult , Aged , Aged, 80 and over , Female , Humans , Lymphoma/pathology , Male , Middle Aged , Oligonucleotides/pharmacology , Oligonucleotides, Antisense/pharmacology , STAT3 Transcription Factor , Young AdultABSTRACT
Purpose: Neuroblastoma is a pediatric tumor of peripheral sympathoadrenal neuroblasts. The long-term event-free survival of children with high-risk neuroblastoma is still poor despite the improvements with current multimodality treatment protocols. Activated JAK/STAT3 pathway plays an important role in many human cancers, suggesting that targeting STAT3 is a promising strategy for treating high-risk neuroblastoma.Experimental Design: To evaluate the biologic consequences of specific targeting of STAT3 in neuroblastoma, we assessed the effect of tetracycline (Tet)-inducible STAT3 shRNA and the generation 2.5 antisense oligonucleotide AZD9150 which targets STAT3 in three representative neuroblastoma cell line models (AS, NGP, and IMR32).Results: Our data indicated that Tet-inducible STAT3 shRNA and AZD9150 inhibited endogenous STAT3 and STAT3 target genes. Tet-inducible STAT3 shRNA and AZD9150 decreased cell growth and tumorigenicity. In vivo, STAT3 inhibition by Tet-inducible STAT3 shRNA or AZD9150 alone had little effect on growth of established tumors. However, when treated xenograft tumor cells were reimplanted into mice, there was a significant decrease in secondary tumors in the mice receiving AZD9150-treated tumor cells compared with the mice receiving ntASO-treated tumor cells. This indicates that inhibition of STAT3 decreases the tumor-initiating potential of neuroblastoma cells. Furthermore, inhibition of STAT3 significantly increased neuroblastoma cell sensitivity to cisplatin and decreased tumor growth and increased the survival of tumor-bearing mice in vivoConclusions: Our study supports the development of strategies targeting STAT3 inhibition in combination with conventional chemotherapy for patients with high-risk neuroblastoma. Clin Cancer Res; 23(7); 1771-84. ©2016 AACR.
Subject(s)
Neuroblastoma/drug therapy , Oligonucleotides, Antisense/administration & dosage , Oligonucleotides/administration & dosage , STAT3 Transcription Factor/genetics , Animals , Antineoplastic Combined Chemotherapy Protocols , Apoptosis/drug effects , Carcinogenesis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cisplatin/administration & dosage , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Neuroblastoma/genetics , Neuroblastoma/pathology , Xenograft Model Antitumor AssaysABSTRACT
INTRODUCTION: Janus kinases (JAKs) are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling and are strongly linked to both cancer and inflammatory diseases. There are currently two launched JAK inhibitors for the treatment of human conditions: tofacitinib for Rheumatoid arthritis (RA) and ruxolitinib for myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera. Areas covered: This review covers patents claiming activity against one or more JAK family members in the period 2013-2015 inclusive, and covers 95 patents from 42 applicants, split over two parts. The authors have ordered recent patents according to the primary applicant's name, with part 2 covering J through Z. Expert opinion: Inhibition of JAK-family kinases is an area of growing interest, catalysed by the maturity of data on marketed inhibitors ruxolitinib and tofacitinib in late stage clinical trials. Many applicants are pursuing traditional fast-follower strategies around these inhibitors, with a range of chemical strategies adopted. The challenge will be to show sufficient differentiation to the originator compounds, since dose limiting toxicities with such agents appear to be on target and mechanism-related and also considering that such agents may be available as generic compounds by the time follower agents reach market.