ABSTRACT
A systematic strategy to develop dual-warhead inhibitors is introduced to circumvent the limitations of conventional covalent inhibitors such as vulnerability to mutations of the corresponding nucleophilic residue. Currently, all FDA-approved covalent small molecules feature one electrophile, leaving open a facile route to acquired resistance. We conducted a systematic analysis of human proteins in the protein data bank to reveal â¼400 unique targets amendable to dual covalent inhibitors, which we term "molecular bidents". We demonstrated this strategy by targeting two kinases: MKK7 and EGFR. The designed compounds, ZNL-8162 and ZNL-0056, are ATP-competitive inhibitors that form two covalent bonds with cysteines and retain potency against single cysteine mutants. Therefore, molecular bidents represent a new pharmacological modality with the potential for improved selectivity, potency, and drug resistance profile.
ABSTRACT
Lung cancer is commonly caused by activating mutations in the epidermal growth factor receptor (EGFR). Allosteric kinase inhibitors are unaffected by common ATP-site resistance mutations and represent a promising therapeutic strategy for targeting drug-resistant EGFR variants. However, allosteric inhibitors are antagonized by kinase dimerization, and understanding this phenomenon has been limited to cellular experiments. To facilitate the study of allosteric inhibitor pharmacology, we designed and purified a constitutive EGFR kinase dimer harboring the clinically relevant L858R/T790M mutations. Kinetic characterization revealed that the EGFR kinase dimer is more active than monomeric EGFR(L858R/T790M) kinase and has the same Km,ATP Biochemical profiling of a large panel of ATP-competitive and allosteric EGFR inhibitors showed that allosteric inhibitor potency decreased by >500-fold in the kinase dimer compared with monomer, yielding IC50 values that correlate well with Ba/F3 cellular potencies. Thus, this readily purifiable constitutive asymmetric EGFR kinase dimer represents an attractive tool for biochemical evaluation of EGFR inhibitor pharmacology, in particular for allosteric inhibitors. SIGNIFICANCE STATEMENT: Drugs targeting epidermal growth factor receptor (EGFR) kinase are commonly used to treat lung cancers but are affected by receptor dimerization. Here, we describe a locked kinase dimer that can be used to study EGFR inhibitor pharmacology.
Subject(s)
ErbB Receptors , Lung Neoplasms , Humans , ErbB Receptors/metabolism , Lung Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Mutation , Adenosine Triphosphate , Drug Resistance, NeoplasmABSTRACT
Lung cancer is frequently caused by activating mutations in the epidermal growth factor receptor (EGFR). Allosteric EGFR inhibitors offer promise as the next generation of therapeutics, as they are unaffected by common ATP-site resistance mutations and synergize with the drug osimertinib. Here, we examine combinations of ATP-competitive and allosteric inhibitors to better understand the molecular basis for synergy. We identify a subset of irreversible EGFR inhibitors that display positive binding cooperativity and synergy with the allosteric inhibitor JBJ-04-125-02 in several EGFR variants. Structural analysis of these complexes reveals conformational changes occur mainly in the phosphate-binding loop (P-loop). Mutation of F723 in the P-loop reduces cooperative binding and synergy, supporting a mechanism in which F723-mediated contacts between the P-loop and the allosteric inhibitor are critical for synergy. These structural and mechanistic insights will aid in the identification and development of additional inhibitor combinations with potential clinical value.
Subject(s)
Drug Resistance, Neoplasm , Lung Neoplasms , Adenosine Triphosphate , Aniline Compounds , Drug Resistance, Neoplasm/genetics , ErbB Receptors/metabolism , Humans , Mutation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacologyABSTRACT
Epidermal growth factor receptor (EGFR) therapy using small-molecule tyrosine kinase inhibitors (TKIs) is initially efficacious in patients with EGFR-mutant lung cancer, although drug resistance eventually develops. Allosteric EGFR inhibitors, which bind to a different EGFR site than existing ATP-competitive EGFR TKIs, have been developed as a strategy to overcome therapy-resistant EGFR mutations. Here we identify and characterize JBJ-09-063, a mutant-selective allosteric EGFR inhibitor that is effective across EGFR TKI-sensitive and resistant models, including those with EGFR T790M and C797S mutations. We further uncover that EGFR homo- or heterodimerization with other ERBB family members, as well as the EGFR L747S mutation, confers resistance to JBJ-09-063, but not to ATP-competitive EGFR TKIs. Overall, our studies highlight the potential clinical utility of JBJ-09-063 as a single agent or in combination with EGFR TKIs to define more effective strategies to treat EGFR-mutant lung cancer.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Adenosine Triphosphate/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , ErbB Receptors/genetics , Humans , Lung Neoplasms/drug therapy , Mutation , Protein Kinase Inhibitors/pharmacologyABSTRACT
The C797S mutation confers resistance to covalent EGFR inhibitors used in the treatment of lung tumors with the activating L858R mutation. Isoindolinones such as JBJ-4-125-02 bind in an allosteric pocket and are active against this mutation, with high selectivity over wild-type EGFR. The most potent examples we developed from that series have a potential chemical instability risk from the combination of the amide and phenol groups. We explored a scaffold hopping approach to identify new series of allosteric EGFR inhibitors that retained good potency in the absence of the phenol group. The 5-F quinazolinone 34 demonstrated tumor regression in an H1975 efficacy model upon once daily oral dosing at 25 mg/kg.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm , ErbB Receptors/genetics , ErbB Receptors/metabolism , Humans , Lung Neoplasms/metabolism , Mutation , Phenols , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Quinazolinones/pharmacology , Quinazolinones/therapeutic useABSTRACT
Inhibitors targeting the epidermal growth factor receptor (EGFR) are an effective therapy for patients with non-small cell lung cancer harboring drug-sensitive activating mutations in the EGFR kinase domain. Drug resistance due to treatment-acquired mutations has motivated the development of successive generations of inhibitors that bind in the ATP site. The third-generation agent osimertinib is now a first-line treatment for this disease. Recently, allosteric inhibitors have been developed to overcome drug-resistant mutations that confer a resistance to osimertinib. Here, we present the structure-guided design and synthesis of a mutant-selective lead compound, which consists of a pyridinyl imidazole-fused benzylisoindolinedione scaffold that simultaneously occupies the orthosteric and allosteric sites. The compound potently inhibits enzymatic activity in L858R/T790M/C797S mutant EGFR (4.9 nM), with a significantly lower activity for wild-type EGFR (47 nM). Additionally, this compound achieves modest cetuximab-independent and mutant-selective cellular efficacies on the L858R (1.2 µM) and L858R/T790M (4.4 µM) variants.
Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Drug Design , Drug Resistance, Neoplasm/drug effects , Imidazoles/chemistry , Mutation , Protein Kinase Inhibitors/pharmacology , Acrylamides/pharmacology , Allosteric Site , Aniline Compounds/pharmacology , Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathologyABSTRACT
The clinical efficacy of epidermal growth factor receptor (EGFR)targeted therapy in EGFR-mutant nonsmall cell lung cancer is limited by the development of drug resistance. One mechanism of EGFR inhibitor resistance occurs through amplification of the human growth factor receptor (MET) proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent EGFR mutation and MET amplification are historically thought to be codependent on the activation of both oncogenes. Hence, patients whose tumors harbor both alterations are commonly treated with a combination of EGFR and MET tyrosine kinase inhibitors (TKIs). Here, we identify and characterize six patient-derived models of EGFR-mutant, MET-amplified lung cancer that have switched oncogene dependence to rely exclusively on MET activation for survival. We demonstrate in this MET-driven subset of EGFR TKI-refractory cancers that canonical EGFR downstream signaling was governed by MET, even in the presence of sustained mutant EGFR expression and activation. In these models, combined EGFR and MET inhibition did not result in greater efficacy in vitro or in vivo compared to single-agent MET inhibition. We further identified a reduced EGFR:MET mRNA expression stoichiometry as associated with MET oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitorresistant EGFR-mutant, MET-amplified patients also exhibited a reduced EGFR:MET mRNA ratio. Our findings reveal that a subset of EGFR-mutant, MET-amplified lung cancers develop dependence on MET activation alone, suggesting that such patients could be treated with a single-agent MET TKI rather than the current standard-of-care EGFR and MET inhibitor combination regimens.
Subject(s)
ErbB Receptors , Lung Neoplasms , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , ErbB Receptors/genetics , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Mutation/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic useABSTRACT
Targeting epidermal growth factor receptor (EGFR) through an allosteric mechanism provides a potential therapeutic strategy to overcome drug-resistant EGFR mutations that emerge within the ATP binding site. Here, we develop an allosteric EGFR degrader, DDC-01-163, which can selectively inhibit the proliferation of L858R/T790M (L/T) mutant Ba/F3 cells while leaving wildtype EGFR Ba/F3 cells unaffected. DDC-01-163 is also effective against osimertinib-resistant cells with L/T/C797S and L/T/L718Q EGFR mutations. When combined with an ATP-site EGFR inhibitor, osimertinib, the anti-proliferative activity of DDC-01-163 against L858R/T790M EGFR-Ba/F3 cells is enhanced. Collectively, DDC-01-163 is a promising allosteric EGFR degrader with selective activity against various clinically relevant EGFR mutants as a single agent and when combined with an ATP-site inhibitor. Our data suggests that targeted protein degradation is a promising drug development approach for mutant EGFR.
Subject(s)
Drug Resistance, Neoplasm/drug effects , Mutation , Protein Kinase Inhibitors/pharmacology , Allosteric Regulation , Cell Line, Tumor , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , ErbB Receptors/metabolism , Humans , ProteolysisABSTRACT
Eradicating tumor dormancy that develops following epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment of EGFR-mutant non-small cell lung cancer, is an attractive therapeutic strategy but the mechanisms governing this process are poorly understood. Blockade of ERK1/2 reactivation following EGFR TKI treatment by combined EGFR/MEK inhibition uncovers cells that survive by entering a senescence-like dormant state characterized by high YAP/TEAD activity. YAP/TEAD engage the epithelial-to-mesenchymal transition transcription factor SLUG to directly repress pro-apoptotic BMF, limiting drug-induced apoptosis. Pharmacological co-inhibition of YAP and TEAD, or genetic deletion of YAP1, all deplete dormant cells by enhancing EGFR/MEK inhibition-induced apoptosis. Enhancing the initial efficacy of targeted therapies could ultimately lead to prolonged treatment responses in cancer patients.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic , Lung Neoplasms/metabolism , Transcription Factors/metabolism , Animals , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Survival , Cellular Senescence , ErbB Receptors/metabolism , Female , Gene Deletion , Humans , Lung Neoplasms/pathology , MAP Kinase Kinase 1/metabolism , Male , Mice , Mice, Knockout , Mutation , Signal Transduction , Transcription, Genetic , YAP-Signaling ProteinsABSTRACT
Allosteric kinase inhibitors represent a promising new therapeutic strategy for targeting kinases harboring oncogenic driver mutations in cancers. Here, we report the discovery, optimization, and structural characterization of allosteric mutant-selective EGFR inhibitors comprising a 5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one scaffold. Our structure-based medicinal chemistry effort yielded an inhibitor (3) of the EGFR(L858R/T790M) and EGFR(L858R/T790M/C797S) mutants with an IC50 of â¼10 nM and high selectivity, as assessed by kinome profiling. Further efforts to develop allosteric dibenzodiazepinone inhibitors may serve as the basis for new therapeutic options for targeting drug-resistant EGFR mutations.
ABSTRACT
INTRODUCTION: Osimertinib is approved for advanced EGFR-mutated NSCLC, and identification of on-target mechanisms of resistance (i.e., EGFR C797S) to this third-generation EGFR inhibitor are evolving. Whether durable control of subsequently osimertinib-resistant NSCLC with the EGFR-sensitizing mutation (SM)/C797S is possible with first-generation EGFR inhibitors (such as gefitinib or erlotinib) remains underreported, as does the resultant acquired resistance profile. METHODS: We used N-ethyl-N-nitrosourea mutagenesis to determine the profile of EGFR SM/C797S preclinical models exposed to reversible EGFR inhibitors. In addition, we retrospectively probed a case of EGFR SM lung adenocarcinoma treated with first-line osimertinib, followed by second-line erlotinib in the setting of EGFR SM/C797S. RESULTS: Use of N-ethyl-N-nitrosourea mutagenesis against the background of EGFR L858R/C797S in conjunction with administration of gefitinib revealed preferential outgrowth of cells with EGFR L858R/T790M/C797S. A patient with EGFR delE746_T751insV NSCLC was treated with osimertinib with sustained response for 10 months before acquiring EGFR C797S. The patient was subsequently treated with erlotinib, with response for a period of 4 months, but disease progression ensued. Liquid biopsy disclosed EGFR delE746_T751insV with T790M and C797S present in cis. CONCLUSION: EGFR SM NSCLC can acquire resistance to osimertinib through development of the EGFR C797S mutation. In this clinical scenario, the tumor may respond transiently to reversible first-generation EGFR inhibitors (gefitinib or erlotinib), but evolving mechanisms of on-target resistance-in clinical specimens and preclinical systems-indicate that EGFR C797S along with EGFR T790M can evolve. This report adds to the growing understanding of tumor evolution or adaptability to sequential EGFR inhibition and augments support for exploring combination therapies to delay or prevent on-target resistance.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Acrylamides/administration & dosage , Alkylating Agents/pharmacology , Aniline Compounds/administration & dosage , Animals , Carcinoma, Non-Small-Cell Lung/pathology , Cells, Cultured , Drug Resistance, Neoplasm , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , ErbB Receptors/metabolism , Erlotinib Hydrochloride/administration & dosage , Ethylnitrosourea/pharmacology , Female , Gefitinib/administration & dosage , Humans , Lung Neoplasms/pathology , Mice , Middle Aged , Mutagenesis , Mutation , Precursor Cells, B-Lymphoid/drug effects , Precursor Cells, B-Lymphoid/metabolism , Precursor Cells, B-Lymphoid/pathology , Protein Kinase Inhibitors/administration & dosage , Retrospective Studies , Treatment OutcomeABSTRACT
Allosteric kinase inhibitors offer a potentially complementary therapeutic strategy to ATP-competitive kinase inhibitors due to their distinct sites of target binding. In this study, we identify and study a mutant-selective EGFR allosteric inhibitor, JBJ-04-125-02, which as a single agent can inhibit cell proliferation and EGFRL858R/T790M/C797S signaling in vitro and in vivo. However, increased EGFR dimer formation limits treatment efficacy and leads to drug resistance. Remarkably, osimertinib, an ATP-competitive covalent EGFR inhibitor, uniquely and significantly enhances the binding of JBJ-04-125-02 for mutant EGFR. The combination of osimertinib and JBJ-04-125-02 results in an increase in apoptosis, a more effective inhibition of cellular growth, and an increased efficacy in vitro and in vivo compared with either single agent alone. Collectively, our findings suggest that the combination of a covalent mutant-selective ATP-competitive inhibitor and an allosteric EGFR inhibitor may be an effective therapeutic approach for patients with EGFR-mutant lung cancer. SIGNIFICANCE: The clinical efficacy of EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutant lung cancer is limited by acquired drug resistance, thus highlighting the need for alternative strategies to inhibit EGFR. Here, we identify a mutant EGFR allosteric inhibitor that is effective as a single agent and in combination with the EGFR TKI osimertinib.This article is highlighted in the In This Issue feature, p. 813.
Subject(s)
Acrylamides/pharmacology , Aniline Compounds/pharmacology , Benzeneacetamides/pharmacology , Lung Neoplasms/drug therapy , Mutation , Protein Kinase Inhibitors/pharmacology , Thiazoles/pharmacology , Allosteric Regulation , Animals , Antineoplastic Combined Chemotherapy Protocols , Cell Line, Tumor , Cell Survival/drug effects , Drug Resistance, Neoplasm , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Humans , Lung Neoplasms/enzymology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice , Mice, Transgenic , NIH 3T3 Cells , Xenograft Model Antitumor AssaysABSTRACT
The mutational activations of anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR) are validated oncogenic events and the targets of approved drugs to treat non-small cell lung cancer (NSCLC). Here we report highly potent dual small molecule inhibitors of both ALK and EGFR, particularly the T790M mutant which confers resistance to first generation EGFR inhibitors. Dual ALK/EGFR inhibitors may provide an efficient approach to prevent resistance that arises as a consequence of clinically reported reciprocal activation mechanisms. Our lead compound 7c displayed remarkable inhibitory activities against both ALK and EGFR in enzymatic and cellular assays. We demonstrate that 7c is capable of recapitulating the signaling effects and antiproliferative activity of combined treatment with the approved ALK inhibitor ceritinib and T790M EGFR inhibitor osimertinib against patient-derived non-small cell lung cancer cell line, DFCI032 which harbors both EML4-ALK and activated EGFR.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery , ErbB Receptors/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Anaplastic Lymphoma Kinase , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , ErbB Receptors/metabolism , Humans , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Receptor Protein-Tyrosine Kinases/metabolism , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity RelationshipABSTRACT
Synthetic triterpenoids are a class of anti-cancer compounds that target many cellular functions, including apoptosis and cell growth in both cell culture and animal models. We have shown that triterpenoids inhibit cell migration in part by interfering with Arp2/3-dependent branched actin polymerization in lamellipodia (To et al., 2010). Our current studies reveal that Glycogen Synthase Kinase 3 beta (GSK3ß), a kinase that regulates many cellular processes, including cell adhesion dynamics, is a triterpenoid-binding protein. Furthermore, triterpenoids were observed to inhibit GSK3ß activity and increase cellular focal adhesion size. To further examine whether these effects on focal adhesions in triterpenoid-treated cells were GSK3ß-dependent, GSK3ß inhibitors (lithium chloride and SB216763) were used to examine cell adhesion and morphology as well as cell migration. Our results showed that GSK3ß inhibitors also altered cell adhesion sizes. Moreover, these inhibitors blocked cell migration and displaced proteins at the leading edge of migrating cells, consistent with what was observed in triterpenoid-treated cells. Therefore, the triterpenoids may affect cell migration via a mechanism that targets and alters the activity and localization of GSK3ß.
Subject(s)
Cell Movement/drug effects , Focal Adhesions/drug effects , Glycogen Synthase Kinase 3 beta/metabolism , Imidazoles/pharmacology , Oleanolic Acid/analogs & derivatives , Animals , Cell Culture Techniques , Fibroblasts/metabolism , Focal Adhesions/metabolism , Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Indoles/pharmacology , Lithium Chloride/pharmacology , Maleimides/pharmacology , Oleanolic Acid/pharmacology , Protein Transport , RatsABSTRACT
Lung cancer accounts for the highest number of cancer-related deaths in the USA, highlighting the need for better prevention and therapy. Activation of the Nrf2 pathway detoxifies harmful insults and reduces oxidative stress, thus preventing carcinogenesis in various preclinical models. However, constitutive activation of the Nrf2 pathway has been detected in numerous cancers, which confers a survival advantage to tumor cells and a poor prognosis. In our study, we compared the effects of two clinically relevant classes of Nrf2 activators, dimethyl fumarate (DMF) and the synthetic oleanane triterpenoids, CDDO-imidazolide (CDDO-Im) and CDDO-methyl ester (CDDO-Me) in RAW 264.7 mouse macrophage-like cells, in VC1 lung cancer cells and in the A/J model of lung cancer. Although the triterpenoids and DMF both activated the Nrf2 pathway, CDDO-Im and CDDO-Me were markedly more potent than DMF. All of these drugs reduced the production of reactive oxygen species and inhibited nitric oxide production in RAW264.7 cells, but the triterpenoids were 100 times more potent than DMF in these assays. Microarray analysis revealed that only 52 of 99 Nrf2-target genes were induced by all three compounds, and each drug regulated a unique subset of Nrf2 genes. These drugs also altered the expression of other genes important in lung cancer independent of Nrf2. Although all three compounds enhanced the phosphorylation of CREB, only DMF increased the phosphorylation of Akt. CDDO-Me, at either 12.5 or 50mg/kg of diet, was the most effective drug in our lung cancer mouse model. Specifically, CDDO-Me significantly reduced the average tumor number, size and burden compared with the control group (P < 0.05). Additionally, 52% of the tumors in the control group were high-grade tumors compared with only 14% in the CDDO-Me group. Though less potent, CDDO-Im had similar activity as CDDO-Me. In contrast, 61-63% of the tumors in the DMF groups (400-1200mg/kg diet) were high-grade tumors compared with 52% for the controls (P < 0.05). Additionally, DMF significantly increased the average number of tumors compared with the controls (P < 0.05). Thus, in contrast to the triterpenoids, which effectively reduced pathogenesis in A/J mice, DMF enhanced the severity of lung carcinogenesis in these mice. Collectively, these results suggest that although CDDO-Im, CDDO-Me and DMF all activate the Nrf2 pathway, they target distinct genes and signaling pathways, resulting in opposite effects for the prevention of experimental lung cancer.
Subject(s)
Fumarates/pharmacology , Imidazoles/pharmacology , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Oleanolic Acid/analogs & derivatives , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Dimethyl Fumarate , Female , Gene Expression Regulation, Neoplastic/drug effects , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice, Inbred Strains , Mice, Knockout , Molecular Targeted Therapy , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Neoplasms, Experimental , Oleanolic Acid/pharmacology , Oligonucleotide Array Sequence Analysis , Oxidative Stress/drug effectsABSTRACT
Poly-ADP ribose polymerase (PARP) inhibitors are effective for the treatment of BRCA-deficient tumors. Women with these mutations have an increased risk of developing breast cancer and would benefit from effective chemoprevention. This study examines whether the PARP inhibitors, veliparib and olaparib, delay mammary gland tumor development in a BRCA1-deficient (BRCA1(Co/Co);MMTV-Cre;p53(+/-)) mouse model. In dose de-escalation studies, mice were fed with control, veliparib (100 mg/kg diet), or olaparib (200, 100, 50, or 25 mg/kg diet) continuously for up to 43 weeks. For intermittent dosing studies, mice cycled through olaparib (200 mg/kg diet) for 2 weeks followed by a 4-week rest period on control diet. To examine biomarkers, mice were fed with olaparib using the intermittent dosing regimen and mammary glands were evaluated by immunohistochemistry. In mice treated with veliparib or olaparib (200 mg/kg diet), the average age of the first detectable tumor was delayed by 2.4 and 6.5 weeks, respectively, compared with controls. Olaparib also increased the average lifespan of mice by 7 weeks. In dose de-escalation studies, lower concentrations of olaparib delayed tumor development but were less effective than the highest dose. When fed intermittently, olaparib delayed the onset of the first palpable tumor by 5.7 weeks and significantly reduced proliferation and induced apoptosis in hyperplastic mammary glands. In summary, veliparib and olaparib are effective for delaying tumor development and extending the lifespan of BRCA1-deficient mice, and intermittent dosing with olaparib was as effective as continuous dosing. These results suggest that the use of PARP inhibitors is a promising chemopreventive option.
Subject(s)
Antineoplastic Agents/therapeutic use , BRCA1 Protein/physiology , Benzimidazoles/therapeutic use , Mammary Neoplasms, Animal/prevention & control , Phthalazines/therapeutic use , Piperazines/therapeutic use , Poly(ADP-ribose) Polymerase Inhibitors , Animals , Apoptosis/drug effects , Biomarkers/analysis , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Female , Humans , Mammary Neoplasms, Animal/genetics , Mammary Neoplasms, Animal/pathology , Mice , Mice, Knockout , Tumor Suppressor Protein p53/physiologyABSTRACT
Synthetic triterpenoids are anti-tumor agents that affect numerous cellular functions including apoptosis and growth inhibition. Here, we used mass spectrometric and protein array approaches and uncovered that triterpenoids associate with proteins of the actin cytoskeleton, including actin-related protein 3 (Arp3). Arp3, a subunit of the Arp2/3 complex, is involved in branched actin polymerization and the formation of lamellipodia. 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)-Im and CDDO-Me were observed to 1) inhibit the localization of Arp3 and actin at the leading edge of cells, 2) abrogate cell polarity, and 3) inhibit Arp2/3-dependent branched actin polymerization. We confirmed our drug effects with siRNA targeting of Arp3 and observed a decrease in Rat2 cell migration. Taken together, our data suggest that synthetic triterpenoids target Arp3 and branched actin polymerization to inhibit cell migration.
Subject(s)
Actin-Related Protein 2-3 Complex/metabolism , Actins/chemistry , Actins/metabolism , Protein Multimerization/drug effects , Triterpenes/pharmacology , Actin-Related Protein 2-3 Complex/antagonists & inhibitors , Animals , Biological Transport , Cell Movement/drug effects , Cell Polarity/drug effects , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , Imidazoles/metabolism , Imidazoles/pharmacology , Models, Molecular , Oleanolic Acid/analogs & derivatives , Oleanolic Acid/chemical synthesis , Oleanolic Acid/chemistry , Oleanolic Acid/metabolism , Oleanolic Acid/pharmacology , Protein Structure, Quaternary , Rats , Triterpenes/chemical synthesis , Triterpenes/chemistry , Triterpenes/metabolismABSTRACT
The anti-tumor synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)-imidazolide (CDDO-Im) ectopically activates the transforming growth factor beta (TGFbeta)-Smad pathway and extends the duration of signaling by an undefined mechanism. Here we show that CDDO-Imdependent persistence of Smad2 phosphorylation is independent of Smad2 phosphatase activity and correlates with delayed TGFbeta receptor degradation and trafficking. Altered TGFbeta trafficking parallels the dispersal of EEA1-positive endosomes from the perinuclear region of CDDO-Im-treated cells. The effect of CDDO-Im on the EEA1 compartment led to an analysis of the cytoskeleton, and we observed that CDDO-Im alters microtubule dynamics by disrupting the microtubule-capping protein, Clip-170. Interestingly, biotinylated triterpenoid was found to localize to the polarity complex at the leading edge of migrating cells. Furthermore, CDDO-Im disrupted the localization of IQGAP1, PKCzeta, Par6, and TGFbeta receptors from the leading edge of migrating cells and inhibited TGFbeta-dependent cell migration. Thus, the synthetic triterpenoid CDDO-Im interferes with TGFbeta receptor trafficking and turnover and disrupts cell migration by severing the link between members of the polarity complex and the microtubule network.