ABSTRACT
BH3 mimetics like venetoclax target prosurvival Bcl-2 family proteins and are important therapeutics in the treatment of hematological malignancies. We demonstrate that endogenous Bfl-1 expression can render preclinical lymphoma tumor models insensitive to Mcl-1 and Bcl-2 inhibitors. However, suppression of Bfl-1 alone was insufficient to fully induce apoptosis in Bfl-1-expressing lymphomas, highlighting the need for targeting additional prosurvival proteins in this context. Importantly, we demonstrated that cyclin-dependent kinase 9 (CDK9) inhibitors rapidly downregulate both Bfl-1 and Mcl-1, inducing apoptosis in BH3-mimetic-resistant lymphoma cell lines in vitro and driving in vivo tumor regressions in diffuse large B-cell lymphoma patient-derived xenograft models expressing Bfl-1. These data underscore the need to clinically develop CDK9 inhibitors, like AZD4573, for the treatment of lymphomas using Bfl-1 as a selection biomarker.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Drug Resistance, Neoplasm/drug effects , Lymphoma, Large B-Cell, Diffuse/drug therapy , Macrocyclic Compounds/pharmacology , Molecular Targeted Therapy , Neoplasm Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Sulfonamides/pharmacology , Animals , Antineoplastic Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Cell Line, Tumor , Cyclin-Dependent Kinase 9/physiology , Cycloheximide/pharmacology , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Leupeptins/pharmacology , Macrocyclic Compounds/therapeutic use , Mice , Mice, Inbred NOD , Mice, SCID , Minor Histocompatibility Antigens/biosynthesis , Minor Histocompatibility Antigens/genetics , Myeloid Cell Leukemia Sequence 1 Protein/biosynthesis , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Peptide Fragments/antagonists & inhibitors , Piperazines/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Proto-Oncogene Proteins c-bcl-2/genetics , Pyridines/pharmacology , Sulfonamides/therapeutic use , Xenograft Model Antitumor AssaysABSTRACT
Spleen tyrosine kinase (SYK) is a non-receptor cytosolic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signaling, inhibition of SYK has been targeted in a variety of disease areas. Herein, we report the optimization of a series of potent and selective SYK inhibitors, focusing on improving metabolic stability, pharmacokinetics and hERG inhibition. As a result, we identified 30, which exhibited no hERG activity but unfortunately was poorly absorbed in rats and mice. We also identified a SYK chemical probe, 17, which exhibits excellent potency at SYK, and an adequate rodent PK profile to support in vivo efficacy/PD studies.
Subject(s)
Indazoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Syk Kinase/antagonists & inhibitors , Animals , Binding Sites , Caco-2 Cells , Crystallography, X-Ray , ERG1 Potassium Channel/antagonists & inhibitors , Humans , Indazoles/chemical synthesis , Indazoles/metabolism , Indazoles/pharmacokinetics , Mice , Microsomes, Liver/metabolism , Molecular Structure , Protein Binding , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacokinetics , Rats, Wistar , Structure-Activity Relationship , Syk Kinase/chemistry , Syk Kinase/metabolismABSTRACT
A key goal of cancer therapeutics is to selectively target the genetic lesions that initiate and maintain cancer cell proliferation and survival. While most cancers harbor multiple oncogenic mutations, a wealth of preclinical and clinical data supports that many cancers are sensitive to inhibition of single oncogenes, a concept referred to as 'oncogene addiction'. Herein, we describe the clinical evidence supporting oncogene addiction and discuss common mechanistic themes emerging from the response and acquired resistance to oncogene-targeted therapies. Finally, we suggest several opportunities toward exploiting oncogene addiction to achieve curative cancer therapies.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Delivery Systems/methods , Drug Resistance, Neoplasm/genetics , Neoplasms/genetics , Oncogenes/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Resistance, Neoplasm/physiology , Genes, Essential/genetics , Humans , Models, Biological , Oncogenes/geneticsABSTRACT
The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits beta-catenin-mediated transcription. XAV939 stimulates beta-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.
Subject(s)
Repressor Proteins/metabolism , Signal Transduction/drug effects , Tankyrases/antagonists & inhibitors , Wnt Proteins/antagonists & inhibitors , Axin Protein , Cell Division/drug effects , Cell Line , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Proteomics , Repressor Proteins/chemistry , Tankyrases/metabolism , Transcription, Genetic/drug effects , Ubiquitin/metabolism , Ubiquitination , Wnt Proteins/metabolism , beta Catenin/antagonists & inhibitors , beta Catenin/metabolismABSTRACT
Antitumor immunity can be hampered by immunosuppressive mechanisms in the tumor microenvironment, including recruitment of arginase (ARG) expressing myeloid cells that deplete l-arginine essential for optimal T-cell and natural killer cell function. Hence, ARG inhibition can reverse immunosuppression enhancing antitumor immunity. We describe AZD0011, a novel peptidic boronic acid prodrug to deliver an orally available, highly potent, ARG inhibitor payload (AZD0011-PL). We demonstrate that AZD0011-PL is unable to permeate cells, suggesting that this compound will only inhibit extracellular ARG. In vivo, AZD0011 monotherapy leads to arginine increases, immune cell activation, and tumor growth inhibition in various syngeneic models. Antitumor responses increase when AZD0011 is combined with anti-PD-L1 treatment, correlating with increases in multiple tumor immune cell populations. We demonstrate a novel triple combination of AZD0011, anti-PD-L1, and anti-NKG2A, and combination benefits with type I IFN inducers, including polyI:C and radiotherapy. Our preclinical data demonstrate AZD0011's ability to reverse tumor immunosuppression and enhance immune stimulation and antitumor responses with diverse combination partners providing potential strategies to increase immuno-oncology therapies clinically.
Subject(s)
Arginase , T-Lymphocytes , Humans , Cell Line, Tumor , Immunosuppression Therapy , Immune Tolerance , Tumor MicroenvironmentABSTRACT
Herein we report the discovery of a family of novel yet simple, amino-acid derived class I HDAC inhibitors that demonstrate isoform selectivity via access to the internal acetate release channel. Isoform selectivity criteria is discussed on the basis of X-ray crystallography and molecular modeling of these novel inhibitors bound to HDAC8, potentially revealing insights into the mechanism of enzymatic function through novel structural features revealed at the atomic level.
Subject(s)
Acetic Acid/metabolism , Amino Acids/chemistry , Amino Acids/pharmacology , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Crystallography, X-Ray , Drug Design , Histone Deacetylases/chemistry , Humans , Models, Molecular , Protein Isoforms/chemistry , Protein Isoforms/metabolismABSTRACT
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
Optimization of a series of azabenzimidazoles identified from screening hit 2 and the information gained from a co-crystal structure of the azabenzimidazole-based lead 6 bound to CDK9 led to the discovery of azaindoles as highly potent and selective CDK9 inhibitors. With the goal of discovering a highly selective and potent CDK9 inhibitor administrated intravenously that would enable transient target engagement of CDK9 for the treatment of hematological malignancies, further optimization focusing on physicochemical and pharmacokinetic properties led to azaindoles 38 and 39. These compounds are highly potent and selective CDK9 inhibitors having short half-lives in rodents, suitable physical properties for intravenous administration, and the potential to achieve profound but transient inhibition of CDK9 in vivo.
Subject(s)
Cyclin-Dependent Kinase 9/antagonists & inhibitors , Drug Discovery , Indoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Cyclin-Dependent Kinase 9/metabolism , Dose-Response Relationship, Drug , Humans , Indoles/chemical synthesis , Indoles/chemistry , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
Panobinostat (LBH589) is a highly potent deacetylase inhibitor that has demonstrated clinical efficacy in patients with advanced cutaneous T-cell lymphoma (CTCL). To gain a better understanding of the compound activity in this tumor type, we investigated the cellular and molecular effects of panobinostat using both in vitro and in vivo models of CTCL. All 4 tested CTCL cell lines exhibited very high sensitivity to panobinostat-induced growth inhibition. However, only 2 of 4 lines exhibited significant response to the cytotoxic activity of panobinostat. In a CTCL xenograft mouse tumor model, panobinostat treatment resulted in complete tumor regression. The difference in cell sensitivity to panobinostat-induced death enabled us to further investigate potential mechanisms responsible for tumor sensitivity or resistance. In CTCL cell lines that were insensitive to panobinostat-induced apoptosis, constitutively activated NF-kappaB and high levels of Bcl-2 were observed. Inhibition of Bcl-2 sensitized cells to the cytotoxic activity of panobinostat. Conversely, knockdown of Bax diminished the CTCL cell sensitivity. Interestingly, panobinostat could induce cytotoxicity in vorinostat-resistant CTCL cells by downregulating phosphorylated STAT3 and STAT5 proteins. These studies suggest distinct mechanisms responsible for resistance to different deacetylase inhibitors. We show that the intrinsic apoptotic signaling plays an essential role in mediating panobinostat anticancer activity. Moreover, cancer cell sensitivity to panobinostat treatment may be further improved by combination with inhibition of anti-apoptotic factors. These data provide preclinical support that panobinostat, as a single agent or in combination with other anticancer agents, is a promising therapy for CTCL.
Subject(s)
Antineoplastic Agents/pharmacology , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/therapeutic use , Lymphoma, T-Cell, Cutaneous/drug therapy , Skin Neoplasms/drug therapy , Animals , Antineoplastic Agents/therapeutic use , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm , Female , Histone Deacetylase Inhibitors/therapeutic use , Humans , Hydroxamic Acids/pharmacology , Indoles , Mice , Mice, SCID , Panobinostat , RNA Interference , Xenograft Model Antitumor Assays , bcl-2-Associated X Protein/geneticsABSTRACT
BACKGROUND: Combinations of drug treatments based on bortezomib or lenalidomide plus steroids have resulted in very high response rates in multiple myeloma. However, most patients still relapse, indicating the need for novel combination partners to increase duration of response or to treat relapsed disease. We explored the antimyeloma activity of triple combinations of these well-established schemes with panobinostat, a novel deacetylase inhibitor with a multi-targeted profile. DESIGN AND METHODS: The activity of these combinations was explored in vitro in cell lines by using MTT and annex-in V, ex vivo by flow cytometry, and in vivo using two different murine models of human myeloma: one bearing a subcutaneous plasmacytoma and another with a disseminated myeloma. Moreover, gene expression profiling and immunohistochemical studies were performed. RESULTS: The addition of panobinostat (LBH589) to dexamethasone and either bortezomib or lenalidomide resulted in clear potentiation in multiple myeloma cell lines, freshly isolated plasma cells, and murine models of multiple myeloma. The quantification of the potency of these combinations by using the Chou-Talalay method showed synergistic combination indices for all of them. This effect derived from the deregulation of a cluster of genes that was completely different from the sum of genes affected by the single agents (895 and 1323 genes exclusively deregulated by panobinostat and dexamethasone plus bortezomib or lenalidomide, respectively). Functional experiments, such as annexin V staining, cell cycle analysis, and immunohistochemical studies also supported this potentiation. Anti-myeloma efficacy was confirmed in an extramedullary plasmacytoma model and a disseminated luciferized model, in which panobinostat also provided a marked benefit in bone disease. CONCLUSIONS: The potent activity, together with the exclusive mechanistic profile, provides the rationale for the clinical evaluation of these drug combinations in multiple myeloma.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Animals , Boronic Acids/administration & dosage , Bortezomib , Cell Line, Tumor , Cells, Cultured , Dexamethasone/administration & dosage , Disease Models, Animal , Humans , Hydroxamic Acids/administration & dosage , Indoles , Lenalidomide , Mice , Mice, SCID , Panobinostat , Pyrazines/administration & dosage , Random Allocation , Thalidomide/administration & dosage , Thalidomide/analogs & derivatives , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: The emergence of secondary mutations is a cause of resistance to current KIT inhibitors used in the treatment of patients with gastrointestinal stromal tumors (GIST). AZD3229 is a selective inhibitor of wild-type KIT and a wide spectrum of primary and secondary mutations seen in patients with GIST. The objective of this analysis is to establish the pharmacokinetic-pharmacodynamic (PKPD) relationship of AZD3229 in a range of mouse GIST tumor models harboring primary and secondary KIT mutations, and to benchmark AZD3229 against other KIT inhibitors. EXPERIMENTAL DESIGN: A PKPD model was developed for AZD3229 linking plasma concentrations to inhibition of phosphorylated KIT using data generated from several in vivo preclinical tumor models, and in vitro data generated in a panel of Ba/F3 cell lines. RESULTS: AZD3229 drives inhibition of phosphorylated KIT in an exposure-dependent manner, and optimal efficacy is observed when >90% inhibition of KIT phosphorylation is sustained over the dosing interval. Integrating the predicted human pharmacokinetics into the mouse PKPD model predicts that an oral twice daily human dose greater than 34 mg is required to ensure adequate coverage across the mutations investigated. Benchmarking shows that compared with standard-of-care KIT inhibitors, AZD3229 has the potential to deliver the required target coverage across a wider spectrum of primary or secondary mutations. CONCLUSIONS: We demonstrate that AZD3229 warrants clinical investigation as a new treatment for patients with GIST based on its ability to inhibit both ATP-binding and A-loop mutations of KIT at clinically relevant exposures.
Subject(s)
Gastrointestinal Stromal Tumors/drug therapy , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-kit/antagonists & inhibitors , Quinazolines/pharmacology , Triazoles/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Gastrointestinal Stromal Tumors/pathology , Humans , Mice , Models, Biological , Mutation , Phosphorylation/drug effects , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-kit/genetics , Proto-Oncogene Proteins c-kit/metabolism , Quinazolines/therapeutic use , Receptor, Platelet-Derived Growth Factor alpha/genetics , Triazoles/therapeutic use , Xenograft Model Antitumor AssaysABSTRACT
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
Gastrointestinal stromal tumor (GIST) is the most common human sarcoma driven by mutations in KIT or platelet-derived growth factor α (PDGFRα). Although first-line treatment, imatinib, has revolutionized GIST treatment, drug resistance due to acquisition of secondary KIT/PDGFRα mutations develops in a majority of patients. Second- and third-line treatments, sunitinib and regorafenib, lack activity against a plethora of mutations in KIT/PDGFRα in GIST, with median time to disease progression of 4 to 6 months and inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) causing high-grade hypertension. Patients with GIST have an unmet need for a well-tolerated drug that robustly inhibits a range of KIT/PDGFRα mutations. Here, we report the discovery and pharmacological characterization of AZD3229, a potent and selective small-molecule inhibitor of KIT and PDGFRα designed to inhibit a broad range of primary and imatinib-resistant secondary mutations seen in GIST. In engineered and GIST-derived cell lines, AZD3229 is 15 to 60 times more potent than imatinib in inhibiting KIT primary mutations and has low nanomolar activity against a wide spectrum of secondary mutations. AZD3229 causes durable inhibition of KIT signaling in patient-derived xenograft (PDX) models of GIST, leading to tumor regressions at doses that showed no changes in arterial blood pressure (BP) in rat telemetry studies. AZD3229 has a superior potency and selectivity profile to standard of care (SoC) agents-imatinib, sunitinib, and regorafenib, as well as investigational agents, avapritinib (BLU-285) and ripretinib (DCC-2618). AZD3229 has the potential to be a best-in-class inhibitor for clinically relevant KIT/PDGFRα mutations in GIST.
Subject(s)
Antineoplastic Agents , Gastrointestinal Stromal Tumors , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Drug Resistance, Neoplasm , Gastrointestinal Stromal Tumors/drug therapy , Gastrointestinal Stromal Tumors/genetics , Humans , Mutation , Naphthyridines , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-kit/genetics , Pyrazoles , Pyrroles , Rats , Receptor, Platelet-Derived Growth Factor alpha/genetics , Triazines , Urea/analogs & derivatives , Vascular Endothelial Growth Factor AABSTRACT
PURPOSE: Cyclin-dependent kinase 9 (CDK9) is a transcriptional regulator and potential therapeutic target for many cancers. Multiple nonselective CDK9 inhibitors have progressed clinically but were limited by a narrow therapeutic window. This work describes a novel, potent, and highly selective CDK9 inhibitor, AZD4573. EXPERIMENTAL DESIGN: The antitumor activity of AZD4573 was determined across broad cancer cell line panels in vitro as well as cell line- and patient-derived xenograft models in vivo. Multiple approaches, including integrated transcriptomic and proteomic analyses, loss-of-function pathway interrogation, and pharmacologic comparisons, were employed to further understand the major mechanism driving AZD4573 activity and to establish an exposure/effect relationship. RESULTS: AZD4573 is a highly selective and potent CDK9 inhibitor. It demonstrated rapid induction of apoptosis and subsequent cell death broadly across hematologic cancer models in vitro, and MCL-1 depletion in a dose- and time-dependent manner was identified as a major mechanism through which AZD4573 induces cell death in tumor cells. This pharmacodynamic (PD) response was also observed in vivo, which led to regressions in both subcutaneous tumor xenografts and disseminated models at tolerated doses both as monotherapy or in combination with venetoclax. This understanding of the mechanism, exposure, and antitumor activity of AZD4573 facilitated development of a robust pharmacokinetic/PD/efficacy model used to inform the clinical trial design. CONCLUSIONS: Selective targeting of CDK9 enables the indirect inhibition of MCL-1, providing a therapeutic option for MCL-1-dependent diseases. Accordingly, AZD4573 is currently being evaluated in a phase I clinical trial for patients with hematologic malignancies (clinicaltrials.gov identifier: NCT03263637).See related commentary by Alcon et al., p. 761.
Subject(s)
Antineoplastic Agents , Hematologic Neoplasms , Apoptosis/drug effects , Cyclin-Dependent Kinase 9 , Humans , Myeloid Cell Leukemia Sequence 1 Protein , ProteomicsABSTRACT
A CDK9 inhibitor having short target engagement would enable a reduction of Mcl-1 activity, resulting in apoptosis in cancer cells dependent on Mcl-1 for survival. We report the optimization of a series of amidopyridines (from compound 2), focusing on properties suitable for achieving short target engagement after intravenous administration. By increasing potency and human metabolic clearance, we identified compound 24, a potent and selective CDK9 inhibitor with suitable predicted human pharmacokinetic properties to deliver transient inhibition of CDK9. Furthermore, the solubility of 24 was considered adequate to allow i.v. formulation at the anticipated effective dose. Short-term treatment with compound 24 led to a rapid dose- and time-dependent decrease of pSer2-RNAP2 and Mcl-1, resulting in cell apoptosis in multiple hematological cancer cell lines. Intermittent dosing of compound 24 demonstrated efficacy in xenograft models derived from multiple hematological tumors. Compound 24 is currently in clinical trials for the treatment of hematological malignancies.
Subject(s)
Cyclin-Dependent Kinase 9/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Pyridines/chemistry , Animals , Apoptosis/drug effects , Binding Sites , Cell Line, Tumor , Cyclin-Dependent Kinase 9/metabolism , Dogs , Drug Evaluation, Preclinical , Half-Life , Hematologic Neoplasms/drug therapy , Hematologic Neoplasms/pathology , Humans , Mice , Molecular Docking Simulation , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pyridines/metabolism , Pyridines/pharmacology , Pyridines/therapeutic use , Rats , Solubility , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Direct pharmacological inhibition of RAS has remained elusive, and efforts to target CRAF have been challenging due to the complex nature of RAF signaling, downstream of activated RAS, and the poor overall kinase selectivity of putative RAF inhibitors. Herein, we describe 15 (LXH254, Aversa, R.; et al. Int. Patent WO2014151616A1, 2014), a selective B/C RAF inhibitor, which was developed by focusing on drug-like properties and selectivity. Our previous tool compound, 3 (RAF709; Nishiguchi, G. A.; et al. J. Med. Chem. 2017, 60, 4969), was potent, selective, efficacious, and well tolerated in preclinical models, but the high human intrinsic clearance precluded further development and prompted further investigation of close analogues. A structure-based approach led to a pyridine series with an alcohol side chain that could interact with the DFG loop and significantly improved cell potency. Further mitigation of human intrinsic clearance and time-dependent inhibition led to the discovery of 15. Due to its excellent properties, it was progressed through toxicology studies and is being tested in phase 1 clinical trials.
Subject(s)
Antineoplastic Agents/chemistry , Drug Discovery/methods , Mutation/genetics , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Animals , Antineoplastic Agents/pharmacology , Drug Design , Drug Discovery/trends , Humans , Molecular Docking Simulation/methods , Molecular Docking Simulation/trends , Mutation/drug effects , Protein Kinase Inhibitors/pharmacology , Xenograft Model Antitumor Assays/methodsABSTRACT
While the treatment of gastrointestinal stromal tumors (GISTs) has been revolutionized by the application of targeted tyrosine kinase inhibitors capable of inhibiting KIT-driven proliferation, diverse mutations to this kinase drive resistance to established therapies. Here we describe the identification of potent pan-KIT mutant kinase inhibitors that can be dosed without being limited by the tolerability issues seen with multitargeted agents. This effort focused on identification and optimization of an existing kinase scaffold through the use of structure-based design. Starting from a series of previously reported phenoxyquinazoline and quinoline based inhibitors of the tyrosine kinase PDGFRα, potency against a diverse panel of mutant KIT driven Ba/F3 cell lines was optimized, with a particular focus on reducing activity against a KDR driven cell model in order to limit the potential for hypertension commonly seen in second and third line GIST therapies. AZD3229 demonstrates potent single digit nM growth inhibition across a broad cell panel, with good margin to KDR-driven effects. Selectivity over KDR can be rationalized predominantly by the interaction of water molecules with the protein and ligand in the active site, and its kinome selectivity is similar to the best of the approved GIST agents. This compound demonstrates excellent cross-species pharmacokinetics, shows strong pharmacodynamic inhibition of target, and is active in several in vivo models of GIST.
Subject(s)
Drug Discovery , Gastrointestinal Stromal Tumors/drug therapy , Mutant Proteins/antagonists & inhibitors , Mutation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-kit/antagonists & inhibitors , Quinazolines/chemistry , Quinazolines/pharmacology , Triazoles/chemistry , Triazoles/pharmacology , Gastrointestinal Neoplasms/drug therapy , Gastrointestinal Neoplasms/metabolism , Gastrointestinal Neoplasms/pathology , Gastrointestinal Stromal Tumors/metabolism , Gastrointestinal Stromal Tumors/pathology , Humans , Models, Molecular , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Conformation , Protein Kinase Inhibitors/pharmacokinetics , Proto-Oncogene Proteins c-kit/genetics , Proto-Oncogene Proteins c-kit/metabolism , Quinazolines/pharmacokinetics , Tissue Distribution , Triazoles/pharmacokinetics , Tumor Cells, CulturedABSTRACT
Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAFV600 alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in in vivo tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor-derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors.Significance: In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and in vivo properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling. Cancer Res; 78(6); 1537-48. ©2018 AACR.
Subject(s)
2,2'-Dipyridyl/analogs & derivatives , Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Proto-Oncogene Proteins B-raf/genetics , raf Kinases/antagonists & inhibitors , ras Proteins/genetics , 2,2'-Dipyridyl/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/drug effects , Female , Humans , MAP Kinase Kinase Kinases/antagonists & inhibitors , Mice, Nude , Mutation , Protein Kinase Inhibitors/pharmacology , Protein Multimerization , Xenograft Model Antitumor Assays , raf Kinases/metabolismABSTRACT
BACKGROUND: The NCOA7 gene product is an estrogen receptor associated protein that is highly similar to the human OXR1 gene product, which functions in oxidation resistance. OXR genes are conserved among all sequenced eukaryotes from yeast to humans. In this study we examine if NCOA7 has an oxidation resistance function similar to that demonstrated for OXR1. We also examine NCOA7 expression in response to oxidative stress and its subcellular localization in human cells, comparing these properties with those of OXR1. RESULTS: We find that NCOA7, like OXR1 can suppress the oxidative mutator phenotype when expressed in an E. coli strain that exhibits an oxidation specific mutator phenotype. Moreover, NCOA7's oxidation resistance function requires expression of only its carboxyl-terminal domain and is similar in this regard to OXR1. We find that, in human cells, NCOA7 is constitutively expressed and is not induced by oxidative stress and appears to localize to the nucleus following estradiol stimulation. These properties of NCOA7 are in striking contrast to those of OXR1, which is induced by oxidative stress, localizes to mitochondria, and appears to be excluded, or largely absent from nuclei. CONCLUSION: NCOA7 most likely arose from duplication. Like its homologue, OXR1, it is capable of reducing the DNA damaging effects of reactive oxygen species when expressed in bacteria, indicating the protein has an activity that can contribute to oxidation resistance. Unlike OXR1, it appears to localize to nuclei and interacts with the estrogen receptor. This raises the possibility that NCOA7 encodes the nuclear counterpart of the mitochondrial OXR1 protein and in mammalian cells it may reduce the oxidative by-products of estrogen metabolite-mediated DNA damage.
Subject(s)
Intracellular Signaling Peptides and Proteins/physiology , Oxidative Stress , Proteins/chemistry , Cell Nucleus/metabolism , Conserved Sequence , DNA Damage , Escherichia coli , Eukaryotic Cells , Gene Expression Regulation/drug effects , Humans , Hydrogen Peroxide/pharmacology , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mitochondrial Proteins , Multigene Family , Nuclear Receptor Coactivators , Protein Structure, Tertiary/physiology , Proteins/genetics , Proteins/physiology , Tumor Cells, CulturedABSTRACT
We report here the identification and characterization of a novel nuclear receptor coactivator, ERAP140. ERAP140 was isolated in a screen for ER alpha-interacting proteins using the ER alpha ligand binding domain as a probe. The ERAP140 protein shares no sequence and has little structural homology with other nuclear receptor cofactors. However, homologues of ERAP140 have been identified in mouse, Drosophila, and Caenorhabditis elegans. The expression of ERAP140 is cell and tissue type specific and is most abundant in the brain, where its expression is restricted to neurons. In addition to interacting with ER alpha, ERAP140 also binds ER beta, TR beta, PPAR gamma, and RAR alpha. ERAP140 interacts with ER alpha via a noncanonical interaction motif. The ER alpha-ERAP140 association can be competed by coactivator NR boxes, indicating ERAP140 binds ER alpha on a surface similar to that of other coactivators. ERAP140 can enhance the transcriptional activities of nuclear receptors with which it interacts. In vivo, ERAP140 is recruited by estrogen-bound ER alpha to the promoter region of endogenous ER alpha target genes. Furthermore, the E(2)-induced recruitment of ERAP140 to the promoter follows a cyclic pattern similar to that of other coactivators. Our results suggest that ERAP140 represents a distinct class of nuclear receptor coactivators that mediates receptor signaling in specific target tissues.