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
Oncology drug combinations can improve therapeutic responses and increase treatment options for patients. The number of possible combinations is vast and responses can be context-specific. Systematic screens can identify clinically relevant, actionable combinations in defined patient subtypes. We present data for 109 anticancer drug combinations from AstraZeneca's oncology small molecule portfolio screened in 755 pan-cancer cell lines. Combinations were screened in a 7 × 7 concentration matrix, with more than 4 million measurements of sensitivity, producing an exceptionally data-rich resource. We implement a new approach using combination Emax (viability effect) and highest single agent (HSA) to assess combination benefit. We designed a clinical translatability workflow to identify combinations with clearly defined patient populations, rationale for tolerability based on tumor type and combination-specific "emergent" biomarkers, and exposures relevant to clinical doses. We describe three actionable combinations in defined cancer types, confirmed in vitro and in vivo, with a focus on hematologic cancers and apoptotic targets. SIGNIFICANCE: We present the largest cancer drug combination screen published to date with 7 × 7 concentration response matrices for 109 combinations in more than 750 cell lines, complemented by multi-omics predictors of response and identification of "emergent" combination biomarkers. We prioritize hits to optimize clinical translatability, and experimentally validate novel combination hypotheses. This article is featured in Selected Articles from This Issue, p. 695.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Neoplasms , Humans , Cell Line, Tumor , Neoplasms/drug therapy , Neoplasms/pathology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Drug Screening Assays, Antitumor/methods , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic useABSTRACT
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
Suppressive myeloid cells mediate resistance to immune checkpoint blockade. PI3Kγ inhibition can target suppressive macrophages, and enhance efficacy of immune checkpoint inhibitors. However, how PI3Kγ inhibitors function in different tumor microenvironments (TME) to activate specific immune cells is underexplored. The effect of the novel PI3Kγ inhibitor AZD3458 was assessed in preclinical models. AZD3458 enhanced antitumor activity of immune checkpoint inhibitors in 4T1, CT26, and MC38 syngeneic models, increasing CD8+ T-cell activation status. Immune and TME biomarker analysis of MC38 tumors revealed that AZD3458 monotherapy or combination treatment did not repolarize the phenotype of tumor-associated macrophage cells but induced gene signatures associated with LPS and type II INF activation. The activation biomarkers were present across tumor macrophages that appear phenotypically heterogenous. AZD3458 alone or in combination with PD-1-blocking antibodies promoted an increase in antigen-presenting (MHCII+) and cytotoxic (iNOS+)-activated macrophages, as well as dendritic cell activation. AZD3458 reduced IL-10 secretion and signaling in primary human macrophages and murine tumor-associated macrophages, but did not strongly regulate IL-12 as observed in other studies. Therefore, rather than polarizing tumor macrophages, PI3Kγ inhibition with AZD3458 promotes a cytotoxic switch of macrophages into antigen-presenting activated macrophages, resulting in CD8 T-cell-mediated antitumor activity with immune checkpoint inhibitors associated with tumor and peripheral immune activation.
Subject(s)
Class Ib Phosphatidylinositol 3-Kinase/metabolism , Immune Checkpoint Inhibitors/therapeutic use , Animals , Disease Models, Animal , Female , Humans , Immune Checkpoint Inhibitors/pharmacology , Macrophages/drug effects , MiceABSTRACT
Dual Bcl-2/Bcl-xL inhibitors are expected to deliver therapeutic benefit in many haematological and solid malignancies, however, their use is limited by tolerability issues. AZD4320, a potent dual Bcl-2/Bcl-xL inhibitor, has shown good efficacy however had dose limiting cardiovascular toxicity in preclinical species, coupled with challenging physicochemical properties, which prevented its clinical development. Here, we describe the design and development of AZD0466, a drug-dendrimer conjugate, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer. Mathematical modelling was employed to determine the optimal release rate of the drug from the dendrimer for maximal therapeutic index in terms of preclinical anti-tumour efficacy and cardiovascular tolerability. The optimised candidate is shown to be efficacious and better tolerated in preclinical models compared with AZD4320 alone. The AZD4320-dendrimer conjugate (AZD0466) identified, through mathematical modelling, has resulted in an improved therapeutic index and thus enabled progression of this promising dual Bcl-2/Bcl-xL inhibitor into clinical development.
Subject(s)
Antineoplastic Agents , Dendrimers , Neoplasms/drug therapy , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Dendrimers/chemical synthesis , Dendrimers/chemistry , Dendrimers/pharmacokinetics , Dendrimers/therapeutic use , Dogs , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, SCID , Neoplasms/metabolism , Neoplasms/pathology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Rats , Rats, Wistar , Therapeutic Index , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , bcl-X Protein/antagonists & inhibitorsABSTRACT
The RAS-regulated RAF-MEK1/2-ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF-mutant melanoma, with limited effect of single-agent pathway inhibitors in KRAS-mutant tumors. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signaling in KRAS-mutant tumors and achieve meaningful clinical benefit. Here, we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF-mutant and KRAS-mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose- and time-dependent modulation of ERK1/2-dependent signaling biomarkers resulting in tumor regression in sensitive BRAF- and KRAS-mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor, selumetinib (AZD6244 and ARRY142886), enhances efficacy in KRAS-mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signaling pathway that is not achievable with single-agent treatment. The AZD0364 and selumetinib combination also results in significant tumor regressions in multiple KRAS-mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS-mutant tumors.
Subject(s)
Benzimidazoles/therapeutic use , Imidazoles/therapeutic use , Proto-Oncogene Proteins p21(ras)/metabolism , Pyrazines/therapeutic use , Pyrimidines/therapeutic use , Animals , Benzimidazoles/pharmacology , Disease Models, Animal , Humans , Imidazoles/pharmacology , Mice , Mice, Nude , Pyrazines/pharmacology , Pyrimidines/pharmacologyABSTRACT
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
PURPOSE: Targeting Bcl-2 family members upregulated in multiple cancers has emerged as an important area of cancer therapeutics. While venetoclax, a Bcl-2-selective inhibitor, has had success in the clinic, another family member, Bcl-xL, has also emerged as an important target and as a mechanism of resistance. Therefore, we developed a dual Bcl-2/Bcl-xL inhibitor that broadens the therapeutic activity while minimizing Bcl-xL-mediated thrombocytopenia. EXPERIMENTAL DESIGN: We used structure-based chemistry to design a small-molecule inhibitor of Bcl-2 and Bcl-xL and assessed the activity against in vitro cell lines, patient samples, and in vivo models. We applied pharmacokinetic/pharmacodynamic (PK/PD) modeling to integrate our understanding of on-target activity of the dual inhibitor in tumors and platelets across dose levels and over time. RESULTS: We discovered AZD4320, which has nanomolar affinity for Bcl-2 and Bcl-xL, and mechanistically drives cell death through the mitochondrial apoptotic pathway. AZD4320 demonstrates activity in both Bcl-2- and Bcl-xL-dependent hematologic cancer cell lines and enhanced activity in acute myeloid leukemia (AML) patient samples compared with the Bcl-2-selective agent venetoclax. A single intravenous bolus dose of AZD4320 induces tumor regression with transient thrombocytopenia, which recovers in less than a week, suggesting a clinical weekly schedule would enable targeting of Bcl-2/Bcl-xL-dependent tumors without incurring dose-limiting thrombocytopenia. AZD4320 demonstrates monotherapy activity in patient-derived AML and venetoclax-resistant xenograft models. CONCLUSIONS: AZD4320 is a potent molecule with manageable thrombocytopenia risk to explore the utility of a dual Bcl-2/Bcl-xL inhibitor across a broad range of tumor types with dysregulation of Bcl-2 prosurvival proteins.
Subject(s)
Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Hematologic Neoplasms/drug therapy , Piperidines/pharmacology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Sulfones/pharmacology , Thrombocytopenia/drug therapy , bcl-X Protein/antagonists & inhibitors , Animals , Antineoplastic Agents/therapeutic use , Apoptosis , Benzamides/therapeutic use , Cell Proliferation , Female , Hematologic Neoplasms/metabolism , Hematologic Neoplasms/pathology , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Piperidines/therapeutic use , Sulfones/therapeutic use , Thrombocytopenia/metabolism , Thrombocytopenia/pathology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
DNA-dependent protein kinase (DNA-PK) is a critical player in the DNA damage response (DDR) and instrumental in the non-homologous end-joining pathway (NHEJ) used to detect and repair DNA double-strand breaks (DSBs). We demonstrate that the potent and highly selective DNA-PK inhibitor, AZD7648, is an efficient sensitizer of radiation- and doxorubicin-induced DNA damage, with combinations in xenograft and patient-derived xenograft (PDX) models inducing sustained regressions. Using ATM-deficient cells, we demonstrate that AZD7648, in combination with the PARP inhibitor olaparib, increases genomic instability, resulting in cell growth inhibition and apoptosis. AZD7648 enhanced olaparib efficacy across a range of doses and schedules in xenograft and PDX models, enabling sustained tumour regression and providing a clear rationale for its clinical investigation. Through its differentiated mechanism of action as an NHEJ inhibitor, AZD7648 complements the current armamentarium of DDR-targeted agents and has potential in combination with these agents to achieve deeper responses to current therapies.
Subject(s)
Apoptosis/drug effects , Cell Proliferation/drug effects , DNA-Activated Protein Kinase/antagonists & inhibitors , Drug Synergism , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Pyrans/pharmacology , Radiation Tolerance/drug effects , Triazoles/pharmacology , A549 Cells , Animals , Antibiotics, Antineoplastic/pharmacology , Carcinoma, Non-Small-Cell Lung , Cell Line, Tumor , Doxorubicin/analogs & derivatives , Doxorubicin/pharmacology , Genomic Instability/drug effects , Humans , Lung Neoplasms , Mice , Phthalazines/pharmacology , Piperazines/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Polyethylene Glycols/pharmacology , Radiotherapy , Xenograft Model Antitumor AssaysABSTRACT
Previous reports have demonstrated that select cancers depend on BRD4 to regulate oncogenic gene transcriptional programs. Here we describe a novel role for BRD4 in DNA damage response (DDR). BRD4 associates with and regulates the function of pre-replication factor CDC6 and plays an indispensable part in DNA replication checkpoint signaling. Inhibition of BRD4 by JQ1 or AZD5153 resulted in a rapid, time-dependent reduction in CHK1 phosphorylation and aberrant DNA replication re-initiation. Furthermore, BRD4 inhibition sensitized cancer cells to various replication stress-inducing agents, and synergized with ATR inhibitor AZD6738 to induce cell killing across a number of cancer cell lines. The synergistic interaction between AZD5153 and AZD6738 is translatable to in vivo ovarian cell-line and patient-derived xenograft models. Taken together, our study uncovers a new biological function of BRD4 and provides mechanistic rationale for combining BET inhibitors with DDR-targeted agents for cancer therapy.
Subject(s)
DNA Damage/genetics , DNA Replication/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Animals , Cell Cycle Proteins , Cell Line, Tumor , DNA Damage/drug effects , DNA Replication/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Heterocyclic Compounds, 2-Ring/pharmacology , Humans , Indoles , Mice , Mice, SCID , Morpholines , Phosphorylation/drug effects , Phosphorylation/genetics , Piperazines/pharmacology , Pyrazoles , Pyridazines , Pyrimidines/pharmacology , Signal Transduction/drug effects , Signal Transduction/genetics , Sulfonamides , Sulfoxides/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
BRD4 is a transcriptional co-activator functioning to recruit regulatory complexes to acetylated chromatin. A subset of High-grade Serous Ovarian Cancer (HGSOC) patients are typified by focal, recurrent BRD4 gene amplifications. Despite previously described cancer dependencies, it is unclear whether BRD4 amplification events are oncogenic in HGSOC. We find that physiologically relevant levels of expression of BRD4 isoforms in non-transformed ovarian cells result in cellular transformation. Transcriptional profiling of BRD4-transformed ovarian cells, and BRD4-amplified HGSOC patient samples revealed shared expression patterns, including enriched MYC, and E2F1 gene signatures. Furthermore, we demonstrate that a novel BET inhibitor, AZD5153, is highly active in BRD4-amplified patient derived xenografts and uncover Neuregulin-1 as a novel BRD4 effector. Experiments involving Neuregulin-1 inhibition and exogenous addition, demonstrate Neuregulin-1 as necessary and sufficient for BRD4-mediated transformation. This study demonstrates the oncogenic potential of BRD4 amplification in cancer and establishes BRD4-amplified HGSOC as a potential patient population that could benefit from BET inhibitors.
Subject(s)
Antineoplastic Agents/pharmacology , Gene Expression Regulation, Neoplastic , Heterocyclic Compounds, 2-Ring/pharmacology , Nuclear Proteins/genetics , Ovarian Neoplasms/genetics , Piperazines/pharmacology , Transcription Factors/genetics , Algorithms , Animals , Carcinogenesis/genetics , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation , Cystadenocarcinoma, Serous/genetics , Female , Gene Expression , Gene Expression Profiling , Humans , Mice , Neoplasm Transplantation , Neuregulin-1/metabolism , Nuclear Proteins/metabolism , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Proteins/genetics , Proteins/metabolism , Pyrazoles , Pyridazines , Signal Transduction , Transcription Factors/metabolism , Xenograft Model Antitumor AssaysABSTRACT
: PARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p-S345 Chk1 accompanied by defects in mitotic signaling. Together, these results establish proof-of-concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP-ribosylation of Chk1 and modulation of its activity. SIGNIFICANCE: These findings describe a new inhibitor of PARP6 and identify a novel function of PARP6 in regulating activation of Chk1 in breast cancer cells.
Subject(s)
ADP Ribose Transferases/antagonists & inhibitors , Breast Neoplasms/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Spindle Apparatus/drug effects , Spindle Apparatus/metabolism , Animals , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Checkpoint Kinase 1/metabolism , Disease Models, Animal , Female , Humans , Mice , Poly(ADP-ribose) Polymerase Inhibitors/chemistry , Signal Transduction/drug effects , Substrate Specificity , Xenograft Model Antitumor AssaysABSTRACT
Mcl-1 is a member of the Bcl-2 family of proteins that promotes cell survival by preventing induction of apoptosis in many cancers. High expression of Mcl-1 causes tumorigenesis and resistance to anticancer therapies highlighting the potential of Mcl-1 inhibitors as anticancer drugs. Here, we describe AZD5991, a rationally designed macrocyclic molecule with high selectivity and affinity for Mcl-1 currently in clinical development. Our studies demonstrate that AZD5991 binds directly to Mcl-1 and induces rapid apoptosis in cancer cells, most notably myeloma and acute myeloid leukemia, by activating the Bak-dependent mitochondrial apoptotic pathway. AZD5991 shows potent antitumor activity in vivo with complete tumor regression in several models of multiple myeloma and acute myeloid leukemia after a single tolerated dose as monotherapy or in combination with bortezomib or venetoclax. Based on these promising data, a Phase I clinical trial has been launched for evaluation of AZD5991 in patients with hematological malignancies (NCT03218683).
Subject(s)
Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Leukemia, Myeloid, Acute/drug therapy , Multiple Myeloma/drug therapy , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Animals , Bortezomib/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Crystallography, X-Ray , Humans , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred C57BL , Mice, SCID , Multiple Myeloma/pathology , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Rats , Rats, Nude , Sulfonamides/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Genetic changes in epithelial cells initiate the development of prostatic adenocarcinomas. As nascent tumors grow and undergo progression, epithelial tumor cells are intimately associated with stromal cells. Stromal cells within the tumor microenvironment acquire new properties, including the capacity to promote phenotypic and genetic progression in adjacent epithelial cells. Affymetrix microarrays were used to identify 119 genes differentially expressed between normal-derived and carcinoma-derived prostatic stromal cells. These included 31 genes encoding extracellular proteins that may act as stromal-to-epithelial paracrine signals. Further investigation of one of these genes, secreted frizzled related protein 1 (SFRP1), revealed that its expression parallels prostatic growth with high expression during prostatic development, low expression in the adult prostate, and elevated expression in prostatic tumor stroma. In addition, as prostatic epithelial cells progressed to a tumorigenic state under the influence of tumor stroma, SFRP1 became overexpressed in the progressed epithelial cells. To further understand the roles of SFRP1 in the prostate, we tested the affects of increased SFRP1 levels on prostatic tissues and cells. Treatment of developing prostates with SFRP1 in culture led to increased organ growth. Treatment of a human prostatic epithelial cell line with SFRP1 led to increased proliferation, decreased apoptosis, and decreased signaling through the Wnt/beta-catenin pathway in vitro and increased proliferation in vivo. These data suggest that overexpression of SFRP1 by prostatic tumor stroma may account for the previously reported capacity of prostatic tumor stroma to provide a pro-proliferative paracrine signal to adjacent epithelial cells.
Subject(s)
Intercellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Animals , Apoptosis/genetics , Cell Growth Processes/genetics , Disease Progression , Epithelial Cells/metabolism , Epithelial Cells/pathology , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Intercellular Signaling Peptides and Proteins/biosynthesis , Male , Membrane Proteins/biosynthesis , Mice , Prostatic Neoplasms/metabolism , Signal Transduction/genetics , Stromal Cells/metabolism , Stromal Cells/pathology , Transfection , Tumor Cells, Cultured , Wnt Proteins/biosynthesis , Wnt Proteins/genetics , beta Catenin/biosynthesis , beta Catenin/geneticsABSTRACT
The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563-74. ©2016 AACR.
Subject(s)
Antineoplastic Agents/pharmacology , Hematologic Neoplasms/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Animals , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Biomarkers , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , E2F Transcription Factors/genetics , E2F Transcription Factors/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Hematologic Neoplasms/drug therapy , Hematologic Neoplasms/genetics , Hematologic Neoplasms/pathology , Humans , Mice , Molecular Targeted Therapy , Nuclear Proteins/chemistry , Protein Binding , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Transcription Factors/chemistry , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Papillary renal cell carcinoma (PRCC) is the second most common cancer of the kidney and carries a poor prognosis for patients with nonlocalized disease. The HGF receptor MET plays a central role in PRCC and aberrations, either through mutation, copy number gain, or trisomy of chromosome 7 occurring in the majority of cases. The development of effective therapies in PRCC has been hampered in part by a lack of available preclinical models. We determined the pharmacodynamic and antitumor response of the selective MET inhibitor AZD6094 in two PRCC patient-derived xenograft (PDX) models. EXPERIMENTAL DESIGN: Two PRCC PDX models were identified and MET mutation status and copy number determined. Pharmacodynamic and antitumor activity of AZD6094 was tested using a dose response up to 25 mg/kg daily, representing clinically achievable exposures, and compared with the activity of the RCC standard-of-care sunitinib (in RCC43b) or the multikinase inhibitor crizotinib (in RCC47). RESULTS: AZD6094 treatment resulted in tumor regressions, whereas sunitinib or crizotinib resulted in unsustained growth inhibition. Pharmacodynamic analysis of tumors revealed that AZD6094 could robustly suppress pMET and the duration of target inhibition was dose related. AZD6094 inhibited multiple signaling nodes, including MAPK, PI3K, and EGFR. Finally, at doses that induced tumor regression, AZD6094 resulted in a dose- and time-dependent induction of cleaved PARP, a marker of cell death. CONCLUSIONS: Data presented provide the first report testing therapeutics in preclinical in vivo models of PRCC and support the clinical development of AZD6094 in this indication.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Papillary/metabolism , Carcinoma, Papillary/pathology , Carcinoma, Renal Cell/metabolism , Carcinoma, Renal Cell/pathology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Pyrazines/pharmacology , Triazines/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Carcinoma, Papillary/drug therapy , Carcinoma, Papillary/genetics , Carcinoma, Renal Cell/drug therapy , Carcinoma, Renal Cell/genetics , Cell Line, Tumor , Crizotinib , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Humans , Indoles/pharmacology , Protein Kinase Inhibitors/administration & dosage , Proto-Oncogene Proteins c-met/genetics , Pyrazines/administration & dosage , Pyrazoles/pharmacology , Pyridines/pharmacology , Pyrroles/pharmacology , Sunitinib , Triazines/administration & dosage , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Dinaciclib is a potent CDK1, 2, 5 and 9 inhibitor being developed for the treatment of cancer. Additional understanding of antitumor mechanisms and identification of predictive biomarkers are important for its clinical development. Here we demonstrate that while dinaciclib can effectively block cell cycle progression, in vitro and in vivo studies, coupled with mouse and human pharmacokinetics, support a model whereby induction of apoptosis is a main mechanism of dinaciclib's antitumor effect and relevant to the clinical duration of exposure. This was further underscored by kinetics of dinaciclib-induced downregulation of the antiapoptotic BCL2 family member MCL1 and correlation of sensitivity with the MCL1-to-BCL-xL mRNA ratio or MCL1 amplification in solid tumor models in vitro and in vivo. This MCL1-dependent apoptotic mechanism was additionally supported by synergy with the BCL2, BCL-xL and BCL-w inhibitor navitoclax (ABT-263). These results provide the rationale for investigating MCL1 and BCL-xL as predictive biomarkers for dinaciclib antitumor response and testing combinations with BCL2 family member inhibitors.
Subject(s)
Apoptosis/drug effects , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Neoplasms/metabolism , Pyridinium Compounds/pharmacology , bcl-X Protein/metabolism , Aniline Compounds/pharmacology , Animals , Antineoplastic Agents/pharmacology , Apoptosis/genetics , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Cyclic N-Oxides , Disease Models, Animal , Diterpenes/pharmacology , Drug Resistance, Neoplasm/genetics , Drug Synergism , Epoxy Compounds/pharmacology , Female , Gene Dosage , Humans , Indolizines , Male , Mice , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Neoplasms/genetics , Phenanthrenes/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sulfonamides/pharmacology , Xenograft Model Antitumor Assays , bcl-X Protein/geneticsABSTRACT
Inhibition of the DNA damage checkpoint kinase WEE1 potentiates genotoxic chemotherapies by abrogating cell-cycle arrest and proper DNA repair. However, WEE1 is also essential for unperturbed cell division in the absence of extrinsic insult. Here, we investigate the anticancer potential of a WEE1 inhibitor, independent of chemotherapy, and explore a possible cellular context underlying sensitivity to WEE1 inhibition. We show that MK-1775, a potent and selective ATP-competitive inhibitor of WEE1, is cytotoxic across a broad panel of tumor cell lines and induces DNA double-strand breaks. MK-1775-induced DNA damage occurs without added chemotherapy or radiation in S-phase cells and relies on active DNA replication. At tolerated doses, MK-1775 treatment leads to xenograft tumor growth inhibition or regression. To begin addressing potential response markers for MK-1775 monotherapy, we focused on PKMYT1, a kinase functionally related to WEE1. Knockdown of PKMYT1 lowers the EC(50) of MK-1775 by five-fold but has no effect on the cell-based response to other cytotoxic drugs. In addition, knockdown of PKMYT1 increases markers of DNA damage, γH2AX and pCHK1(S345), induced by MK-1775. In a post hoc analysis of 305 cell lines treated with MK-1775, we found that expression of PKMYT1 was below average in 73% of the 33 most sensitive cell lines. Our findings provide rationale for WEE1 inhibition as a potent anticancer therapy independent of a genotoxic partner and suggest that low PKMYT1 expression could serve as an enrichment biomarker for MK-1775 sensitivity.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Damage/drug effects , Disease Models, Animal , Drug Evaluation, Preclinical , Drug Resistance, Neoplasm/genetics , Female , Gene Knockdown Techniques , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Pyrazoles/administration & dosage , Pyrimidines/administration & dosage , Pyrimidinones , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
The alpha(v)beta(6) integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of alpha(v)beta(6) expression in 10 human tumor types showed increased expression relative to normal tissues. Squamous carcinomas of the cervix, skin, esophagus, and head and neck exhibited the highest frequency of expression, with positive immunostaining in 92% (n = 46), 84% (n = 49), 68% (n = 56), and 64% (n = 100) of cases, respectively. We studied the role of alpha(v)beta(6) in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent alpha(v)beta(6) expression was detected on tumor xenografts at the tumor-stroma interface resembling the expression on human head and neck carcinomas. Nonetheless, coculturing cells in vitro with matrix proteins did not up-regulate alpha(v)beta(6) expression. Detroit 562 cells showed alpha(v)beta(6)-dependent adhesion and activation of transforming growth factor-beta (TGF-beta) that was inhibited >90% with an alpha(v)beta(6) blocking antibody, 6.3G9. Although both recombinant soluble TGF-beta receptor type-II (rsTGF-beta RII-Fc) and 6.3G9 inhibited TGF-beta-mediated Smad2/3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-beta RII-Fc inhibited xenograft tumor growth by 50% (n = 10, P < 0.05) and >90% (n = 10, P < 0.001), respectively, suggesting a role for the microenvironment in this response. However, stromal collagen and smooth muscle actin content in xenograft sections were unchanged with treatments. Although further studies are required to consolidate in vitro and in vivo results and define the mechanisms of tumor inhibition by alpha(v)beta(6) antibodies, our findings support a role for alpha(v)beta(6) in human cancer and underscore the therapeutic potential of function blocking alpha(v)beta(6) antibodies.