ABSTRACT
Ovarian cancers (OVCAs) and endometrial cancers (EMCAs) with CCNE1-amplification are often resistant to standard of care treatment and represent an unmet clinical need. Previously, synthetic-lethal screening identified loss of the CDK1 regulator, PKMYT1, as synthetically lethal with CCNE1-amplification. We hypothesized that CCNE1-amplification associated replication stress will be more effectively targeted by combining the PKMYT1 inhibitor, lunresertib (RP-6306), with the ATR inhibitor, camonsertib (RP-3500/RG6526). Low dose combination RP-6306 with RP-3500 synergistically increased cytotoxicity more in CCNE1 amplified compared to non-amplified cells. Combination treatment produced durable antitumor activity and increased survival in CCNE1 amplified patient-derived and cell line-derived xenografts. Mechanistically, low doses of RP-6306 with RP-3500 increase CDK1 activation more so than monotherapy, triggering rapid and robust induction of premature mitosis, DNA damage and apoptosis in a CCNE1-dependent manner. These findings suggest that targeting CDK1 activity by combining RP-6306 with RP-3500 is a novel therapeutic approach to treat CCNE1-amplifed OVCAs and EMCAs.
ABSTRACT
Endocrine therapies (ET) with CDK4/6 inhibition are the standard treatment for estrogen receptor-α-positive (ER+) breast cancer, however drug resistance is common. In this study, proteogenomic analyses of 22 ER+ breast cancer patient-derived xenografts (PDXs) demonstrated that PKMYT1, a WEE1 homolog, is estradiol (E2) regulated in E2-dependent PDXs and constitutively expressed when growth is E2-independent. In clinical samples, high PKMYT1 mRNA levels associated with resistance to both ET and CDK4/6 inhibition. The PKMYT1 inhibitor lunresertib (RP-6306) with gemcitabine selectively and synergistically reduced the viability of ET and palbociclib-resistant ER+ breast cancer cells without functional p53. In vitro the combination increased DNA damage and apoptosis. In palbociclib-resistant, TP53 mutant PDX organoids and xenografts, RP-6306 with low-dose gemcitabine induced greater tumor volume reduction compared to treatment with either single agent. Our study demonstrates the clinical potential of RP-6306 in combination with gemcitabine for ET and CDK4/6 inhibitor resistant TP53 mutant ER+ breast cancer.
ABSTRACT
BACKGROUND: Long-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive given the relatively intact immune system of patients with LGG and the slow tumor growth rate. However, accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) in IDH-mutated gliomas leads to suppression of inflammatory pathways in the tumor microenvironment, thereby contributing to the 'cold' tumor phenotype. Inhibiting D-2HG production presents an opportunity to generate a robust antitumor response following tumor antigen vaccination and immune checkpoint blockade. METHODS: An IDH1R132H glioma model was created in syngeneic HLA-A2/HLA-DR1-transgenic mice, allowing us to evaluate the vaccination with the human leukocyte antigens (HLA)-DR1-restricted, IDH1R132H mutation-derived neoepitope. The effects of an orally available inhibitor of mutant IDH1 and IDH2, AG-881, were evaluated as monotherapy and in combination with the IDH1R132H peptide vaccination or anti-PD-1 immune checkpoint blockade. RESULTS: The HLA-A2/HLA-DR1-syngeneic IDH1R132H cell line expressed the IDH1 mutant protein and formed D-2HG producing orthotopic gliomas in vivo. Treatment of tumor-bearing mice with AG-881 resulted in a reduction of D-2HG levels in IDH1R132H glioma cells (10 fold) and tumor-associated myeloid cells, which demonstrated high levels of intracellular D-2HG in the IDH1R132H gliomas. AG-881 monotherapy suppressed the progression of IDH1R132H gliomas in a CD4+ and CD8+ cell-dependent manner, enhanced proinflammatory IFNγ-related gene expression, and increased the number of CD4+ tumor-infiltrating T-cells. Prophylactic vaccination with the HLA-DR1-restricted IDH1R132H peptide or tumor-associated HLA-A2-restricted peptides did not enhance survival of tumor-bearing animals; however, vaccination with both HLA-A2-IDH1R132H and DR1-IDH1R132H peptides in combination with the IDH inhibitor significantly prolonged survival. Finally, tumor-bearing mice treated with both AG-881 and a PD-1 blocking antibody demonstrated improved survival when compared with either treatment alone. CONCLUSION: The development of effective IDH1R132H-targeting vaccine may be enhanced by integration with HLA class I-restricted cytotoxic T cell epitopes and AG-881. Our HLA-A2/HLA-DR1-syngeneic IDH1R132H glioma model should allow us to evaluate key translational questions related to the development of novel strategies for patients with IDH-mutant glioma.
Subject(s)
Cancer Vaccines , Glioma , Animals , Glioma/drug therapy , Glioma/genetics , Glioma/pathology , Glutarates , HLA-A2 Antigen/genetics , HLA-DR1 Antigen/genetics , Humans , Immune Checkpoint Inhibitors , Isocitrate Dehydrogenase/genetics , Mice , Mice, Transgenic , Tumor Microenvironment , Up-Regulation , Vaccines, SubunitABSTRACT
Approximately 20% of acute myeloid leukemia (AML) patients carry mutations in IDH1 or IDH2 that result in over-production of the oncometabolite D-2-hydroxyglutarate (2-HG). Small molecule inhibitors that block 2-HG synthesis can induce complete morphological remission; however, almost all patients eventually acquire drug resistance and relapse. Using a multi-allelic mouse model of IDH1-mutant AML, we demonstrate that the clinical IDH1 inhibitor AG-120 (ivosidenib) exerts cell-type-dependent effects on leukemic cells, promoting delayed disease regression. Although single-agent AG-120 treatment does not fully eradicate the disease, it increases cycling of rare leukemia stem cells and triggers transcriptional upregulation of the pyrimidine salvage pathway. Accordingly, AG-120 sensitizes IDH1-mutant AML to azacitidine, with the combination of AG-120 and azacitidine showing vastly improved efficacy in vivo. Our data highlight the impact of non-genetic heterogeneity on treatment response and provide a mechanistic rationale for the observed combinatorial effect of AG-120 and azacitidine in patients.
Subject(s)
Isocitrate Dehydrogenase , Leukemia, Myeloid, Acute , Animals , Azacitidine/pharmacology , Enzyme Inhibitors/pharmacology , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Mice , Mutation/genetics , Stem Cells/metabolismABSTRACT
Inhibition of the S-adenosyl methionine (SAM)-producing metabolic enzyme, methionine adenosyltransferase 2A (MAT2A), has received significant interest in the field of medicinal chemistry due to its implication as a synthetic lethal target in cancers with the deletion of the methylthioadenosine phosphorylase (MTAP) gene. Here, we report the identification of novel MAT2A inhibitors with distinct in vivo properties that may enhance their utility in treating patients. Following a high-throughput screening, we successfully applied the structure-based design lessons from our first-in-class MAT2A inhibitor, AG-270, to rapidly redesign and optimize our initial hit into two new lead compounds: a brain-penetrant compound, AGI-41998, and a potent, but limited brain-penetrant compound, AGI-43192. We hope that the identification and first disclosure of brain-penetrant MAT2A inhibitors will create new opportunities to explore the potential therapeutic effects of SAM modulation in the central nervous system (CNS).
Subject(s)
Methionine Adenosyltransferase , Neoplasms , Brain/metabolism , Drug Design , Humans , Neoplasms/drug therapy , S-Adenosylmethionine/metabolismABSTRACT
The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) was recently implicated as a synthetic lethal target in cancers with deletion of the methylthioadenosine phosphorylase (MTAP) gene, which is adjacent to the CDKN2A tumor suppressor and codeleted with CDKN2A in approximately 15% of all cancers. Previous attempts to target MAT2A with small-molecule inhibitors identified cellular adaptations that blunted their efficacy. Here, we report the discovery of highly potent, selective, orally bioavailable MAT2A inhibitors that overcome these challenges. Fragment screening followed by iterative structure-guided design enabled >10â¯000-fold improvement in potency of a family of allosteric MAT2A inhibitors that are substrate noncompetitive and inhibit release of the product, S-adenosyl methionine (SAM), from the enzyme's active site. We demonstrate that potent MAT2A inhibitors substantially reduce SAM levels in cancer cells and selectively block proliferation of MTAP-null cells both in tissue culture and xenograft tumors. These data supported progressing AG-270 into current clinical studies (ClinicalTrials.gov NCT03435250).
Subject(s)
Enzyme Inhibitors/chemistry , Methionine Adenosyltransferase/antagonists & inhibitors , Purine-Nucleoside Phosphorylase/genetics , Binding Sites , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/therapeutic use , Homozygote , Humans , Methionine Adenosyltransferase/metabolism , Molecular Dynamics Simulation , Neoplasms/drug therapy , Purine-Nucleoside Phosphorylase/metabolism , S-Adenosylmethionine/metabolism , Structure-Activity RelationshipABSTRACT
The methylthioadenosine phosphorylase (MTAP) gene is located adjacent to the cyclin-dependent kinase inhibitor 2A (CDKN2A) tumor-suppressor gene and is co-deleted with CDKN2A in approximately 15% of all cancers. This co-deletion leads to aggressive tumors with poor prognosis that lack effective, molecularly targeted therapies. The metabolic enzyme methionine adenosyltransferase 2α (MAT2A) was identified as a synthetic lethal target in MTAP-deleted cancers. We report the characterization of potent MAT2A inhibitors that substantially reduce levels of S-adenosylmethionine (SAM) and demonstrate antiproliferative activity in MTAP-deleted cancer cells and tumors. Using RNA sequencing and proteomics, we demonstrate that MAT2A inhibition is mechanistically linked to reduced protein arginine methyltransferase 5 (PRMT5) activity and splicing perturbations. We further show that DNA damage and mitotic defects ensue upon MAT2A inhibition in HCT116 MTAP-/- cells, providing a rationale for combining the MAT2A clinical candidate AG-270 with antimitotic taxanes.
Subject(s)
DNA Damage/drug effects , Enzyme Inhibitors/pharmacology , Methionine Adenosyltransferase/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/genetics , Purine-Nucleoside Phosphorylase/genetics , RNA Splicing/drug effects , RNA, Messenger/genetics , Animals , Cell Line , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p16 , DNA Damage/genetics , Gene Deletion , HCT116 Cells , HEK293 Cells , Humans , Methionine Adenosyltransferase/genetics , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Neoplasms/drug therapy , Neoplasms/genetics , RNA Splicing/genetics , S-Adenosylmethionine/metabolismABSTRACT
Cell death is of broad physiological and pathological importance, making quantification of biochemical events associated with cell demise a high priority for experimental pathology. Fibrosis is a common consequence of tissue injury involving necrotic cell death. Using tissue specimens from experimental mouse models of traumatic brain injury, cardiac fibrosis, and cancer, as well as human tumor specimens assembled in tissue microarray (TMA) format, we undertook computer-assisted quantification of specific immunohistochemical and histological parameters that characterize processes associated with cell death. In this study, we demonstrated the utility of image analysis algorithms for color deconvolution, colocalization, and nuclear morphometry to characterize cell death events in tissue specimens: (a) subjected to immunostaining for detecting cleaved caspase-3, cleaved poly(ADP-ribose)-polymerase, cleaved lamin-A, phosphorylated histone H2AX, and Bcl-2; (b) analyzed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay to detect DNA fragmentation; and (c) evaluated with Masson's trichrome staining. We developed novel algorithm-based scoring methods and validated them using TMAs as a high-throughput format. The proposed computer-assisted scoring methods for digital images by brightfield microscopy permit linear quantification of immunohistochemical and histochemical stainings. Examples are provided of digital image analysis performed in automated or semiautomated fashion for successful quantification of molecular events associated with cell death in tissue sections.
Subject(s)
Cell Death , Algorithms , Animals , Apoptosis , Biomarkers/metabolism , Brain/pathology , Brain Injuries/metabolism , Brain Injuries/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , DNA Damage , Female , Fibrosis , Humans , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Mice, Nude , Myocardium/metabolism , Myocardium/pathology , Neoplasm Transplantation , Neurons/metabolism , Neurons/pathology , Staining and Labeling , Transplantation, HeterologousABSTRACT
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy for which new therapeutic approaches are required. One such potential therapeutic strategy is to target the ubiquitin-like modifier-activating enzyme 1 (UBA1), the initiating enzyme in the ubiquitylation cascade in which proteins are tagged with ubiquitin moieties to regulate their degradation or function. Here, we evaluated TAK-243, a first-in-class UBA1 inhibitor, in preclinical models of AML. In AML cell lines and primary AML samples, TAK-243 induced cell death and inhibited clonogenic growth. In contrast, normal hematopoietic progenitor cells were more resistant. TAK-243 preferentially bound to UBA1 over the related E1 enzymes UBA2, UBA3, and UBA6 in intact AML cells. Inhibition of UBA1 with TAK-243 decreased levels of ubiquitylated proteins, increased markers of proteotoxic stress and DNA damage stress. In vivo, TAK-243 reduced leukemic burden and targeted leukemic stem cells without evidence of toxicity. Finally, we selected populations of AML cells resistant to TAK-243 and identified missense mutations in the adenylation domain of UBA1. Thus, our data demonstrate that TAK-243 targets AML cells and stem cells and support a clinical trial of TAK-243 in this patient population. Moreover, we provide insight into potential mechanisms of acquired resistance to UBA1 inhibitors.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Leukemia, Myeloid, Acute/drug therapy , Nucleosides/pharmacology , Sulfonamides/pharmacology , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Xenograft Model Antitumor Assays , Animals , Cell Proliferation/drug effects , Endoplasmic Reticulum Stress/drug effects , Humans , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/pathology , Mice , Mice, SCID , Pyrazoles , Pyrimidines , Sulfides , Tumor Cells, CulturedABSTRACT
The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.
Subject(s)
Neoplasms/drug therapy , Nucleosides/pharmacology , Small Molecule Libraries/pharmacology , Sulfonamides/pharmacology , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Animals , Cell Line, Tumor , DNA Damage/drug effects , DNA Repair/drug effects , Humans , Imides/pharmacology , Mice , Neoplasms/genetics , Neoplasms/pathology , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/drug effects , Proteasome Endopeptidase Complex/genetics , Protein Binding , Pyrazoles , Pyrimidines , Sulfides , Ubiquitin/antagonists & inhibitors , Ubiquitin/chemistry , Ubiquitin/genetics , Ubiquitin-Activating Enzymes/chemistry , Ubiquitin-Activating Enzymes/genetics , Xenograft Model Antitumor AssaysABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) has been shown to induce apoptosis specifically in cancer cells while sparing normal tissues. Unfortunately not all cancer cells respond to TRAIL; therefore, TRAIL sensitizing agents are currently being explored. We have identified synthetic triterpenoids, including 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivative 1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole (CDDO-Im), which sensitize TRAIL-resistant cancer cells to TRAIL-mediated apoptosis. Here we show that TRAIL-treated T47D and MDA-MB-468 breast cancer cells fail to initiate detectable caspase-8 processing and, consequently, do not initiate TRAIL-mediated apoptosis. Concomitant treatment with CDDO or CDDO-Im reverses the TRAIL-resistant phenotype, promoting robust caspase-8 processing and induction of TRAIL-mediated apoptosis in vitro. The combination of triterpenoids and monoclonal anti-TRAIL receptor-1 (DR4) antibody also induces apoptosis of breast cancer cells in vitro. From a mechanistic standpoint, we show that CDDO and CDDO-Im down-regulate the antiapoptotic protein c-FLIP(L), and up-regulate cell surface TRAIL receptors DR4 and DR5. CDDO and CDDO-Im, when used in combination with TRAIL, have no adverse affect on cultured normal human mammary epithelial cells. Moreover, CDDO-Im and TRAIL are well tolerated in mice and the combination of CDDO-Im and TRAIL reduces tumor burden in vivo in an MDA-MB-468 tumor xenograft model. These data suggest that CDDO and CDDO-Im may be useful for selectively reversing the TRAIL-resistant phenotype in cancer but not normal cells.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Imidazoles/pharmacology , Membrane Glycoproteins/pharmacology , Oleanolic Acid/analogs & derivatives , Oleanolic Acid/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Antibodies, Monoclonal/pharmacology , Apoptosis Regulatory Proteins , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Caspase 8 , Caspases/metabolism , Down-Regulation/drug effects , Drug Synergism , Female , Humans , Imidazoles/administration & dosage , Membrane Glycoproteins/administration & dosage , Mice , Mice, Inbred BALB C , Mice, Nude , Oleanolic Acid/administration & dosage , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/agonists , Receptors, Tumor Necrosis Factor/biosynthesis , Receptors, Tumor Necrosis Factor/immunology , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacology , TNF-Related Apoptosis-Inducing Ligand , Tumor Necrosis Factor-alpha/administration & dosage , Up-Regulation/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Previous investigations have revealed that bladder cancer cells are generally resistant to Fas-mediated apoptosis by conventional Fas agonists. However, the ability of these cell lines to undergo Fas-mediated apoptosis may have been underappreciated. As a result, we investigated the in vitro efficacy of Fas ligand gene therapy for bladder cancer. Three human bladder cancer lines (T24, J82, and 5637) were treated with the conventional Fas agonist CH-11, a monoclonal antibody to the Fas receptor. Cells were also treated with a replication-deficient adenovirus containing a modified murine Fas ligand gene fused to green fluorescent protein (GFP), AdGFPFasL. A virus containing the GFP gene alone was used to control for viral toxicity (AdGFP). Cell death was quantified using a tetrazolium-based (MTS) assay. Cells were also evaluated by Western blotting to evaluate poly (ADP-ribose) polymerase, caspase 8, and caspase 9 cleavage and by flow cytometry to determine the presence of coxsackie/adenovirus receptor (CAR). These studies confirmed bladder cancer resistance to cell death by the anti-Fas monoclonal antibody CH-11. This resistance was overcome with AdGFPFasL at a multiplicity of infection (MOI) of 1000 achieving over 80% cell death in all cell lines. Furthermore, greater than 80% cell death was evident in 5637 cells treated with low-dose AdGFPFasL (MOI=10). 5637 cells expressed significantly higher levels of surface CAR than J82 or T24 cells (P<.05). AdGFPFasL is cytotoxic to bladder cancer cells that would otherwise be considered Fas resistant, supporting its in vivo potential. Enhanced sensitivity to AdGFPFasL may be in part due to increased cell surface CAR levels.
Subject(s)
Apoptosis/genetics , Genetic Therapy , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/therapy , Adenoviridae , Antibodies/pharmacology , Antibodies, Monoclonal , Antigens, Surface , Calcium-Binding Proteins , Eye Proteins , Fas Ligand Protein , Genetic Vectors , Humans , Ligands , Lipoproteins , Membrane Glycoproteins , Recoverin , Tumor Cells, Cultured , fas ReceptorABSTRACT
Inducing Fas-mediated apoptosis in prostate cancer (PCa) is a promising new therapeutic approach with the potential to overcome delivery issues currently problematic in cancer gene therapy. We have previously demonstrated that a Fas Ligand (FasL) expressing adenovirus (AdGFPFasL(TET)) was able to induce Fas-mediated apoptosis in a panel of PCa cell lines regardless of their Fas-sensitivity as determined by the agonistic Fas antibody CH-11. We now report that AdGFPFasL(TET)-infected cells produce apoptotic bodies and cellular debris that continues to elicit FasL-mediated bystander killing in uninfected neighboring cells. Using light microscopy, we demonstrate that AdGFPFasL(TET)-infected cells release apoptotic bodies and cellular debris into the local environment and that this material will induce bystander killing in Jurkat, PPC-1, and PC-3 target cells, but not in DU145 and K-562 cells. The bystander killing mechanism is mediated through Fas/FasL interaction because it is significantly inhibited if target cells are pretreated with the pan spectrum caspase inhibitor Z-VAD-FMK or the Fas neutralizing antibody ZB-4. Coincubation of PPC-1 target cells with apoptotic bodies and cellular debris (effector material) induce nearly complete target cell killing at a ratio of 1:1 target to effector. Collectively, these data indicate that AdGFPFasL(TET)-infected PCa cells release apoptotic and cellular debris capable of inducing bystander killing in PCa and supports the development of FasL as a gene therapy agent.
Subject(s)
Adenoviridae/genetics , Genetic Therapy , Membrane Glycoproteins/genetics , Prostatic Neoplasms/therapy , Apoptosis , Bystander Effect , Caspase Inhibitors , Cell Line, Tumor , Fas Ligand Protein , Genetic Vectors , Humans , Jurkat Cells , Male , Membrane Glycoproteins/metabolism , Microscopy, Fluorescence , Prostatic Neoplasms/pathologyABSTRACT
Although DU145 prostate cancer cells are resistant to exogenously applied Fas agonist CH-11 (anti-Fas monoclonal antibody), Fas-resistance can be overcome using a FasL expressing adenovirus (AdGFPFasL(TET)) [Hyer et al., Molecular Therapy, 2000; 2:348-58 (ref.12)]. The purpose of this study was to try to understand why DU145 cells are resistant to CH-11 and determine the signaling pathway utilized by AdGFPFasL(TET) to induce apoptosis in these Fas-resistant cells. Using immunoblot analysis, we show that AdGFPFasL(TET) is capable of initiating the classic Fas-mediated apoptotic pathway in DU145 cells, which includes activation of caspases-8, -3, -7, and -9, BID cleavage, cytochrome c release from mitochondria, and PARP cleavage. In contrast, CH-11 binds to Fas, but is unable to transmit the death signal beyond the plasma membrane suggesting a block at the DISC (death inducing signaling complex). The anti-apoptotic protein c-FLIP (cellular Flice-like inhibitory protein), which has been shown to inhibit Fas-mediated apoptosis at the DISC, was down-regulated following AdGFPFasL(TET) treatment prompting us to investigate its role in inhibiting CH-11-induced cell death. Using c-FLIP anti-sense oligonucleotides to down-regulate c-FLIP we sensitized DU145 cells to CH-11-induced apoptosis. These data suggest that c-FLIP may play a critical role in regulating Fas-mediated apoptosis in prostate cancer cells and that modulation of c-FLIP may enhance Fas signaling based therapies.
Subject(s)
Apoptosis , Carrier Proteins/metabolism , Down-Regulation , Intracellular Signaling Peptides and Proteins , Prostatic Neoplasms/metabolism , fas Receptor/metabolism , BH3 Interacting Domain Death Agonist Protein , Blotting, Western , CASP8 and FADD-Like Apoptosis Regulating Protein , Caspase 3 , Caspase 7 , Caspase 8 , Caspase 9 , Caspases/metabolism , Cell Death , Cell Line , Cell Separation , Cytochrome c Group/metabolism , Enzyme Activation , Fas Ligand Protein , Flow Cytometry , Humans , Immunoblotting , Jurkat Cells , Male , Membrane Glycoproteins/metabolism , Mitochondria/metabolism , Oligonucleotides, Antisense/pharmacology , Prostatic Neoplasms/pathology , Signal Transduction , Time Factors , Tumor Cells, CulturedABSTRACT
Plk1 is a checkpoint protein whose role spans all of mitosis and includes DNA repair, and is highly conserved in eukaryotes from yeast to man. Consistent with this wide array of functions for Plk1, the cellular consequences of Plk1 disruption are diverse, spanning delays in mitotic entry, mitotic spindle abnormalities, and transient mitotic arrest leading to mitotic slippage and failures in cytokinesis. In this work, we present the in vitro and in vivo consequences of Plk1 inhibition in cancer cells using potent, selective small-molecule Plk1 inhibitors and Plk1 genetic knock-down approaches. We demonstrate for the first time that cellular senescence is the predominant outcome of Plk1 inhibition in some cancer cell lines, whereas in other cancer cell lines the dominant outcome appears to be apoptosis, as has been reported in the literature. We also demonstrate strong induction of DNA double-strand breaks in all six lines examined (as assayed by γH2AX), which occurs either during mitotic arrest or mitotic-exit, and may be linked to the downstream induction of senescence. Taken together, our findings expand the view of Plk1 inhibition, demonstrating the occurrence of a non-apoptotic outcome in some settings. Our findings are also consistent with the possibility that mitotic arrest observed as a result of Plk1 inhibition is at least partially due to the presence of unrepaired double-strand breaks in mitosis. These novel findings may lead to alternative strategies for the development of novel therapeutic agents targeting Plk1, in the selection of biomarkers, patient populations, combination partners and dosing regimens.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Cellular Senescence/drug effects , Cellular Senescence/genetics , DNA Damage/drug effects , Mitosis/genetics , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Humans , Mitosis/drug effects , RNA Interference , RNA, Small Interfering/genetics , Polo-Like Kinase 1ABSTRACT
Aurora A kinase orchestrates multiple key activities, allowing cells to transit successfully into and through mitosis. MLN8237 (alisertib) is a selective Aurora A inhibitor that is being evaluated as an anticancer agent in multiple solid tumors and heme-lymphatic malignancies. The antitumor activity of MLN8237 when combined with docetaxel or paclitaxel was evaluated in in vivo models of triple-negative breast cancer grown in immunocompromised mice. Additive and synergistic antitumor activity occurred at multiple doses of MLN8237 and taxanes. Moreover, significant tumor growth delay relative to the single agents was achieved after discontinuing treatment; notably, durable complete responses were observed in some mice. The tumor growth inhibition data generated with multiple dose levels of MLN8237 and paclitaxel were used to generate an exposure-efficacy model. Exposures of MLN8237 and paclitaxel achieved in patients were mapped onto the model after correcting for mouse-to-human variation in plasma protein binding and maximum tolerated exposures. This allowed rank ordering of various combination doses of MLN8237 and paclitaxel to predict which pair would lead to the greatest antitumor activity in clinical studies. The model predicted that 60 and 80 mg/m(2) of paclitaxel (every week) in patients lead to similar levels of efficacy, consistent with clinical observations in some cancer indications. The model also supported using the highest dose of MLN8237 that can be achieved, regardless of whether it is combined with 60 or 80 mg/m(2) of paciltaxel. The modeling approaches applied in these studies can be used to guide dose-schedule optimization for combination therapies using other therapeutic agents.
Subject(s)
Aurora Kinase A/antagonists & inhibitors , Azepines/administration & dosage , Neoplasms, Experimental/drug therapy , Pyrimidines/administration & dosage , Taxoids/administration & dosage , Animals , Antineoplastic Agents/administration & dosage , Area Under Curve , Cell Line, Tumor , Docetaxel , Drug Administration Schedule , Female , Humans , Mice , Mice, Inbred BALB C , Mice, SCID , Neoplasm Transplantation , Paclitaxel/administration & dosage , Translational Research, BiomedicalABSTRACT
Diffuse large B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas, accounting for up to 30% of all newly diagnosed lymphoma cases. Current treatment options for this disease are effective, but not always curative; therefore, experimental therapies continue to be investigated. We have discovered an experimental, potent, and selective small-molecule inhibitor of PLK1, MLN0905, which inhibits cell proliferation in a broad range of human tumor cells including DLBCL cell lines. In our report, we explored the pharmacokinetic, pharmacodynamic, and antitumor properties of MLN0905 in DLBCL xenograft models grown in mice. These studies indicate that MLN0905 modulates the pharmacodynamic biomarker phosphorylated histone H3 (pHisH3) in tumor tissue. The antitumor activity of MLN0905 was evaluated in three human subcutaneous DLBCL xenograft models, OCI LY-10, OCI LY-19, and PHTX-22L (primary lymphoma). In each model, MLN0905 yielded significant antitumor activity on both a continuous (daily) and intermittent dosing schedule, underscoring dosing flexibility. The antitumor activity of MLN0905 was also evaluated in a disseminated xenograft (OCI LY-19) model to better mimic human DLBCL disease. In the disseminated model, MLN0905 induced a highly significant survival advantage. Finally, MLN0905 was combined with a standard-of-care agent, rituximab, in the disseminated OCI LY-19 xenograft model. Combining rituximab and MLN0905 provided both a synergistic antitumor effect and a synergistic survival advantage. Our findings indicate that PLK1 inhibition leads to pharmacodynamic pHisH3 modulation and significant antitumor activity in multiple DLBCL models. These data strongly suggest evaluating PLK1 inhibitors as DLBCL anticancer agents in the clinic.
Subject(s)
Antibodies, Monoclonal, Murine-Derived/administration & dosage , Antineoplastic Agents/administration & dosage , Benzazepines/administration & dosage , Cell Cycle Proteins/antagonists & inhibitors , Lymphoma, Large B-Cell, Diffuse/drug therapy , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Thiones/administration & dosage , Administration, Oral , Animals , Antibodies, Monoclonal, Murine-Derived/pharmacology , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Benzazepines/pharmacokinetics , Benzazepines/pharmacology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Synergism , Female , Gene Knockdown Techniques , Histones/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA Interference , Rituximab , Thiones/pharmacokinetics , Thiones/pharmacology , Tumor Burden/drug effects , Xenograft Model Antitumor Assays , Polo-Like Kinase 1ABSTRACT
This article describes the discovery of a series of potent inhibitors of Polo-like kinase 1 (PLK1). Optimization of this benzolactam-derived chemical series produced an orally bioavailable inhibitor of PLK1 (12c, MLN0905). In vivo pharmacokinetic-pharmacodynamic experiments demonstrated prolonged mitotic arrest after oral administration of 12c to tumor bearing nude mice. A subsequent efficacy study in nude mice achieved tumor growth inhibition or regression in a human colon tumor (HT29) xenograft model.
Subject(s)
Antineoplastic Agents/chemical synthesis , Benzazepines/chemical synthesis , Cell Cycle Proteins/antagonists & inhibitors , Lactams/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Thiones/chemical synthesis , Administration, Oral , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Benzazepines/pharmacokinetics , Benzazepines/pharmacology , Biological Availability , Cell Line, Tumor , Cell Survival/drug effects , Crystallography, X-Ray , Drug Screening Assays, Antitumor , Humans , Lactams/pharmacokinetics , Lactams/pharmacology , Mice , Mice, Nude , Mitosis , Models, Molecular , Neoplasm Transplantation , Protein Conformation , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Thiones/pharmacokinetics , Thiones/pharmacology , Transplantation, Heterologous , Polo-Like Kinase 1ABSTRACT
PURPOSE: Small-molecule inhibitors of Aurora A (AAK) and B (ABK) kinases, which play important roles in mitosis, are currently being pursued in oncology clinical trials. We developed three novel assays to quantitatively measure biomarkers of AAK inhibition in vivo. Here, we describe preclinical characterization of alisertib (MLN8237), a selective AAK inhibitor, incorporating these novel pharmacodynamic assays. EXPERIMENTAL DESIGN: We investigated the selectivity of alisertib for AAK and ABK and studied the antitumor and antiproliferative activity of alisertib in vitro and in vivo. Novel assays were used to assess chromosome alignment and mitotic spindle bipolarity in human tumor xenografts using immunofluorescent detection of DNA and alpha-tubulin, respectively. In addition, 18F-3'-fluoro-3'-deoxy-l-thymidine positron emission tomography (FLT-PET) was used to noninvasively measure effects of alisertib on in vivo tumor cell proliferation. RESULTS: Alisertib inhibited AAK over ABK with a selectivity of more than 200-fold in cells and produced a dose-dependent decrease in bipolar and aligned chromosomes in the HCT-116 xenograft model, a phenotype consistent with AAK inhibition. Alisertib inhibited proliferation of human tumor cell lines in vitro and produced tumor growth inhibition in solid tumor xenograft models and regressions in in vivo lymphoma models. In addition, a dose of alisertib that caused tumor stasis, as measured by volume, resulted in a decrease in FLT uptake, suggesting that noninvasive imaging could provide value over traditional measurements of response. CONCLUSIONS: Alisertib is a selective and potent inhibitor of AAK. The novel methods of measuring Aurora A pathway inhibition and application of tumor imaging described here may be valuable for clinical evaluation of small-molecule inhibitors.