ABSTRACT
XPO1 (Exportin-1) is the nuclear export protein responsible for the normal shuttling of several proteins and RNA species between the nucleocytoplasmic compartment of eukaryotic cells. XPO1 recognizes the nuclear export signal (NES) of its cargo proteins to facilitate its export. Alterations of nuclear export have been shown to play a role in oncogenesis in several types of solid tumour and haematologic cancers. Over more than a decade, there has been substantial progress in targeting nuclear export in cancer using selective XPO1 inhibitors. This has resulted in recent approval for the first-in-class drug selinexor for use in relapsed, refractory multiple myeloma and diffuse large B-cell lymphoma (DLBCL). Despite these successes, not all patients respond effectively to XPO1 inhibition and there has been lack of biomarkers for response to XPO1 inhibitors in the clinic. Using haematologic malignancy cell lines and samples from patients with myelodysplastic neoplasms treated with selinexor, we have identified XPO1, NF-κB(p65), MCL-1 and p53 protein levels as protein markers of response to XPO1 inhibitor therapy. These markers could lead to the identification of response upon XPO1 inhibition for more accurate decision-making in the personalized treatment of cancer patients undergoing treatment with selinexor.
Subject(s)
Hematologic Neoplasms , Multiple Myeloma , Humans , Karyopherins/genetics , Active Transport, Cell Nucleus , Hematologic Neoplasms/drug therapy , Hematologic Neoplasms/geneticsABSTRACT
Uveal melanoma is the most common primary intraocular malignancy in adults and has a high incidence of metastatic disease. Current treatments have shown limited clinical activity in patients with uveal melanoma with metastasis and there is an urgent need for new effective therapies. Recent findings have shown that women with uveal melanoma have better survival rates than men. The G protein-coupled estrogen receptor-1 (GPER) has distinct functions from those of the classic estrogen receptors ERα/ß and its activation by specific agonists has tumor-suppressive roles in several cancers. However, the role of GPER had not previously been investigated in uveal melanoma. We demonstrated that downregulation of GPER in uveal melanoma cells decreased expression of p53 and stimulated cell growth. In contrast, the clinical GPER agonist, LNS8801, upregulated p53 and p21, induced melanocytic differentiation markers, inhibited cell proliferation and cell migration, and induced apoptosis. Furthermore, LNS8801 treatment arrested the cells in G2-M-phase of the cell cycle with concomitant activation of mitotic markers and disruption of the mitotic spindle apparatus. LNS8801 significantly inhibited tumor growth of uveal melanoma xenografts in vivo, suggesting that GPER agonists may be a novel treatment for uveal melanoma. Significance: Current treatments against metastatic uveal melanoma have shown limited clinical activity and there is an urgent need for effective therapies. Here, we demonstrate that the GPER agonist LNS8801 induced both GPER-dependent and GPER-independent effects and elicited potent anticancer activities in vitro and in vivo. Our results complement and support the ongoing clinical trial of LNS8801 in advanced uveal melanoma.
Subject(s)
Melanoma , Tumor Suppressor Protein p53 , Male , Adult , Humans , Female , Tumor Suppressor Protein p53/pharmacology , Melanoma/drug therapy , Estrogens/metabolism , Apoptosis , Cell Line, TumorABSTRACT
Uveal melanoma (UM) is a rare subset of melanoma characterized by the presence of early initiating GNAQ/11 mutations, with downstream activation of the PKC, MAPK, and PI3Kα pathways. Activity has been observed with the PKC inhibitors sotrastaurin (AEB071) and darovasertib (IDE196) in patients with UM. Inhibition of the PI3K pathway enhances PKC inhibition in in vivo models. We therefore conducted a phase Ib study of sotrastaurin in combination with the PI3Kα inhibitor alpelisib to identify a tolerable regimen that may enhance the activity of PKC inhibition alone. Patients with metastatic uveal melanoma (n = 24) or GNAQ/11 mutant cutaneous melanoma (n = 1) were enrolled on escalating dose levels of sotrastaurin (100-400 mg BID) and alpelisib (200-350 mg QD). The primary objective was to identify the maximum tolerated dose (MTD) of these agents when administered in combination. Treatment-related adverse events (AE) occurred in 86% (any grade) and 29% (Grade 3). No Grade 4-5-related AEs occurred. Dose Level 4 (sotrastaurin 200 mg BID and alpelisib 350 mg QD) was identified as the maximum tolerated dose. Pharmacokinetic analysis demonstrated increasing concentration levels with increasing doses of sotrastaurin and alpelisib, without evidence of interaction between agents. Pharmacodynamic assessment of pMARCKS and pAKT protein expression with drug exposure suggested modest target inhibition that did not correlate with clinical response. No objective responses were observed, and median progression-free survival was 8 weeks (range, 3-51 weeks). Although a tolerable dose of sotrastaurin and alpelisib was identified with pharmacodynamic evidence of target inhibition and without evidence of a corresponding immunosuppressive effect, limited clinical activity was observed.
ABSTRACT
Uveal melanoma is a rare form of melanoma with particularly poor outcomes in the metastatic setting. In contrast with cutaneous melanoma, uveal melanoma lacks BRAF mutations and demonstrates very low response rates to immune-checkpoint blockade. Our objectives were to study the transcriptomics of metastatic uveal melanoma with the intent of assessing gene pathways and potential molecular characteristics that might be nominated for further exploration as therapeutic targets. We initially analyzed transcriptional data from The Cancer Genome Atlas suggesting PI3K/mTOR and glycolysis as well as IL6 associating with poor survival. From tumor samples collected in a prospective phase II trial (A091201), we performed a transcriptional analysis of human metastatic uveal melanoma observing a novel role for epithelial-mesenchymal transition associating with survival. Specifically, we nominate and describe initial functional validation of neuropillin-1 from uveal melanoma cells as associated with poor survival and as a mediator of proliferation and migration for uveal melanoma in vitro. These results immediately nominate potential next steps in clinical research for patients with metastatic uveal melanoma.
Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Melanoma/genetics , Skin Neoplasms/genetics , Uveal Neoplasms/genetics , Humans , Melanoma/mortality , Neoplasm Metastasis , Skin Neoplasms/mortality , Survival Analysis , Transfection , Uveal Neoplasms/mortalityABSTRACT
Sarcoma is a rare cancer arising from soft tissue and bone and consists of more than 50 distinct subtypes. There is an increasing emphasis on understanding the cancer biology of individual sarcoma subtypes to inform the development of targeted and immunotherapy-based treatment approaches. While some advances have recently been made in this respect, most sarcomas are still treated with chemotherapy. The homologous recombination DNA repair pathway plays an important role in repairing highly cytotoxic double-stranded DNA breaks and restarting stalled replication forks. A subset of human cancers, notably ovarian, breast, prostate, and pancreatic cancers, harbor defects in components of the homologous recombination repair pathway, such as mutation or loss of BRCA1/2, and are sensitive to treatments which induce double stranded DNA breaks or replication fork arrest, including oral small molecule poly-ADP-ribose polymerase (PARP) inhibitors. Our understanding of DNA repair defects in sarcoma remains at an early stage. Recently, uterine leiomyosarcoma was identified as a sarcoma subtype with characteristic defects in the homologous recombination repair pathway and frequent BRCA2 loss. Preclinical data, presented here, demonstrates marked activity for the PARP inhibitor olaparib in combination with the alkylating agent temozolomide in leiomyosarcoma models. Ongoing research promises to identify other sarcomas with DNA repair defects and may offer a new opportunity for the targeted treatment of this rare, aggressive cancer.
Subject(s)
Antineoplastic Agents/pharmacology , DNA Repair-Deficiency Disorders/drug therapy , Recombinational DNA Repair/drug effects , Sarcoma/drug therapy , Animals , Humans , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Sarcoma/geneticsABSTRACT
The new Ru(II) chloroquine complexes [Ru(eta(6)-arene)(CQ)Cl2] (CQ = chloroquine; arene = p-cymene 1, benzene 2), [Ru(eta(6)-p-cymene)(CQ)(H2O)2][BF4]2 (3), [Ru(eta(6)-p-cymene)(CQ)(en)][PF6]2 (en = ethylenediamine) (4), and [Ru(eta(6)-p-cymene)(eta(6)-CQDP)][BF4]2 (5, CQDP = chloroquine diphosphate) have been synthesized and characterized by use of a combination of NMR and FTIR spectroscopy with DFT calculations. Each complex is formed as a single coordination isomer: In 1-4, chloroquine binds to ruthenium in the eta(1)-N mode through the quinoline nitrogen atom, whereas in 5 an unprecedented eta(6) bonding through the carbocyclic ring is observed. 1, 2, 3, and 5 are active against CQ-resistant (Dd2, K1, and W2) and CQ-sensitive (FcB1, PFB, F32, and 3D7) malaria parasites (Plasmodium falciparum); importantly, the potency of these complexes against resistant parasites is consistently higher than that of the standard drug chloroquine diphosphate. 1 and 5 also inhibit the growth of colon cancer cells, independently of the p53 status and of liposarcoma tumor cell lines with the latter showing increased sensitivity, especially to 1 (IC50 8 microM); this is significant because this type of tumor does not respond to currently employed chemotherapies.
Subject(s)
Antimalarials/chemical synthesis , Antimalarials/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Chloroquine/chemistry , Ruthenium Compounds/chemical synthesis , Ruthenium Compounds/pharmacology , Animals , Antimalarials/chemistry , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Structure , Plasmodium falciparum/drug effects , Ruthenium Compounds/chemistry , Spectrophotometry, InfraredABSTRACT
Uveal melanoma is a rare but often lethal malignancy and is the leading cause of death due to an ophthalmic condition. Uveal melanoma is often diagnosed at a late stage and has a strong propensity to hepatic metastasis. Recently, the most common driver mutations in uveal melanoma have been identified, predominantly in the G-proteins GNAQ. This pattern differs from that of cutaneous melanoma in which Braf and Nras predominate. There are no current clinically used agents that target GNAQ mutations, unlike the use of Braf inhibitors in cutaneous melanoma. We tested the novel agent Tris DBA palladium and found that it was markedly more effective against GNAQ mutant melanomas than wild type uveal melanomas. Given that ARF6 has recently been discovered as a node in GNAQ mutations, we evaluated the efficacy of Tris DBA palladium on ARF6 signaling and found that it was effective in inhibiting ARF6 activation. Finally, Tris DBA palladium was orally effective against GNAQ mutant melanoma in vivo. Tris DBA Palladium deserves further evaluation as a systemic agent for uveal melanoma.
ABSTRACT
Bromodomain and extraterminal protein inhibitors (BETi) are epigenetic therapies aimed to target dysregulated gene expression in cancer cells. Despite early successes of BETi in a range of malignancies, the development of drug resistance may limit their clinical application. Here, we evaluated the mechanisms of BETi resistance in uveal melanoma, a disease with little treatment options, using two approaches: a high-throughput combinatorial drug screen with the clinical BET inhibitor PLX51107 and RNA sequencing of BETi-resistant cells. NF-κB inhibitors synergistically sensitized uveal melanoma cells to PLX51107 treatment. Furthermore, genes involved in NF-κB signaling were upregulated in BETi-resistant cells, and the transcription factor CEBPD contributed to the mechanism of resistance. These findings suggest that inhibitors of NF-κB signaling may improve the efficacy of BET inhibition in patients with advanced uveal melanoma. SIGNIFICANCE: These findings provide evidence that inhibitors of NF-κB signaling synergize with BET inhibition in in vitro and in vivo models, suggesting a clinical utility of these targeted therapies in patients with uveal melanoma.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic/drug effects , Liver Neoplasms/drug therapy , Melanoma/drug therapy , NF-kappa B/antagonists & inhibitors , Proteins/antagonists & inhibitors , Uveal Neoplasms/drug therapy , Animals , Apoptosis , Cell Proliferation , Drug Synergism , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/secondary , Melanoma/metabolism , Melanoma/pathology , Mice , Mice, Nude , Tumor Cells, Cultured , Uveal Neoplasms/metabolism , Uveal Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
Sarcomas are rare cancers that make up about 1% of all cancers in adults; however, they occur more commonly among children and young adolescents. Sarcomas are genetically complex and are often difficult to treat given the lack of clinical efficacy of any of the currently available therapies. Receptor tyrosine kinases (RTK) such as c-Kit, c-Met, PDGFR, IGF-1R, as well as FGFR have all been reported to be involved in driving tumor development and progression in adult and pediatric soft-tissue sarcoma. These driver kinases often act as critical determinants of tumor cell proliferation and targeting these signal transduction pathways remains an attractive therapeutic approach. Nintedanib, a potent triple angiokinase inhibitor, targets PDGFR, VEGFR, and FGFR pathways critical for tumor angiogenesis and vasculature. In this study, we evaluated the preclinical efficacy of nintedanib in soft-tissue sarcoma cell lines. Nintedanib treatment resulted in significant antiproliferative effect in vitro in cell lines with high expression of RTK drug targets. Furthermore, treatment with nintedanib showed significant downregulation of downstream phosphorylated AKT and ERK1/2. Finally, treatment with nintedanib resulted in significant tumor growth suppression in mouse xenograft model of synovial sarcoma. Notably, both the in vitro and in vivo efficacy of nintedanib was superior to that of imatinib, another multikinase inhibitor, previously tested with minimal success in clinical trials in sarcoma. Overall, the data from this study provide a strong rationale to warrant further clinical exploration of this drug in patients with synovial sarcoma. Mol Cancer Ther; 17(11); 2329-40. ©2018 AACR.
Subject(s)
Indoles/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Sarcoma, Synovial/drug therapy , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Humans , Indoles/pharmacology , Inhibitory Concentration 50 , Mice, Nude , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/metabolism , Receptors, Fibroblast Growth Factor/metabolism , Receptors, Platelet-Derived Growth Factor/metabolism , Sarcoma, Synovial/enzymology , Sarcoma, Synovial/pathology , Signal Transduction/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Purpose: Selinexor, a small molecule that inhibits nuclear export protein XPO1, has demonstrated efficacy in solid tumors and hematologic malignancies with the evidence of clinical activity in sarcoma as a single agent. Treatment options available are very few, and hence the need to identify novel targets and strategic therapies is of utmost importance.Experimental Design: The mechanistic effects of selinexor in sarcomas as a monotherapy and in combination with proteasome inhibitor, carfilzomib, across a panel of cell lines in vitro and few in xenograft mouse models were investigated.Results: Selinexor induced IκB nuclear localization as a single agent, and the effect was enhanced by stabilization of IκB when pretreated with the proteasome inhibitor carfilzomib. This stabilization and retention of IκB in the nucleus resulted in inhibition of NFκB and transcriptional suppression of the critical antiapoptotic protein, survivin. Treatment of carfilzomib followed by selinexor caused selinexor-sensitive and selinexor-resistant cell lines to be more sensitive to selinexor as determined by an increase in apoptosis. This was successfully demonstrated in the MPNST xenograft model with enhanced tumor suppression.Conclusions: The subcellular distributions of IκB and NFκB are indicative of carcinogenesis. Inhibition of XPO1 results in intranuclear retention of IκB, which inhibits NFκB and thereby provides a novel mechanism for drug therapy in sarcoma. This effect can be further enhanced in relatively selinexor-resistant sarcoma cell lines by pretreatment with the proteasome inhibitor carfilzomib. Because of these results, a human clinical trial with selinexor in combination with a proteasome inhibitor is planned for the treatment of sarcoma. Clin Cancer Res; 23(15); 4301-11. ©2017 AACR.
Subject(s)
Hydrazines/administration & dosage , I-kappa B Proteins/genetics , Karyopherins/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Sarcoma/drug therapy , Triazoles/administration & dosage , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Survival/drug effects , Humans , Inhibitor of Apoptosis Proteins/genetics , Karyopherins/antagonists & inhibitors , Mice , NF-kappa B/genetics , Oligopeptides/administration & dosage , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Sarcoma/genetics , Sarcoma/pathology , Survivin , Transcription Factor RelA/genetics , Xenograft Model Antitumor Assays , Exportin 1 ProteinABSTRACT
Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10-18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma.
Subject(s)
Anilides/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Sarcoma, Experimental/drug therapy , Sarcoma, Experimental/pathology , Signal Transduction/drug effects , Animals , Apoptosis/drug effects , Blotting, Western , Cell Proliferation/drug effects , Humans , Immunoenzyme Techniques , Mice , Mice, Inbred ICR , Mice, SCID , Phosphorylation/drug effects , Sarcoma, Experimental/enzymology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Uveal melanoma (UM) is an aggressive intraocular malignancy with limited therapeutic options. Both primary and metastatic UM are characterized by oncogenic mutations in the G-protein alpha subunit q and 11. Furthermore, nearly 40% of UM has amplification of the chromosomal arm 8q and monosomy of chromosome 3, with consequent anomalies of MYC copy number. Chromatin regulators have become attractive targets for cancer therapy. In particular, the bromodomain and extra-terminal (BET) inhibitor JQ1 has shown selective inhibition of c-Myc expression with antiproliferative activity in hematopoietic and solid tumors. Here we provide evidence that JQ1 had cytotoxic activity in UM cell lines carrying Gnaq/11 mutations, while in cells without the mutations had little effects. Using microarray analysis, we identified a large subset of genes modulated by JQ1 involved in the regulation of cell cycle, apoptosis and DNA repair. Further analysis of selected genes determined that the concomitant silencing of Bcl-xL and Rad51 represented the minimal requirement to mimic the apoptotic effects of JQ1 in the mutant cells, independently of c-Myc. In addition, administration of JQ1 to mouse xenograft models of Gnaq-mutant UM resulted in significant inhibition of tumor growth.Collectively, our results define BRD4 targeting as a novel therapeutic intervention against UM with Gnaq/Gna11 mutations.
Subject(s)
Azepines/therapeutic use , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , GTP-Binding Protein alpha Subunits/genetics , Melanoma/drug therapy , Melanoma/genetics , Nuclear Proteins/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Triazoles/therapeutic use , Uveal Neoplasms/drug therapy , Uveal Neoplasms/genetics , Animals , Cell Death/drug effects , Cell Death/genetics , Genes, myc/physiology , Humans , Melanoma/pathology , Mice , Mice, SCID , Molecular Targeted Therapy , Nuclear Proteins/genetics , Transcription Factors/genetics , Tumor Cells, Cultured , Uveal Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (sotrastaurin) and PI3K/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11-mutant cells with AEB071 versus no activity in wild-type cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of myristoylated alanine-rich C-kinase substrate, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal antiproliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11-mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ- and GNA11-mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ-mutant model. These findings suggest a new therapy treatment option for G-protein-mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy.
Subject(s)
Antineoplastic Agents/pharmacology , GTP-Binding Protein alpha Subunits/genetics , Melanoma/genetics , Mutation , Pyrroles/pharmacology , Quinazolines/pharmacology , Thiazoles/pharmacology , Uveal Neoplasms/genetics , Animals , Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Class I Phosphatidylinositol 3-Kinases , Disease Models, Animal , Dose-Response Relationship, Drug , Female , GTP-Binding Protein alpha Subunits, Gq-G11 , Gene Silencing , Humans , Melanoma/drug therapy , Melanoma/pathology , Mice , Phosphatidylinositol 3-Kinases/genetics , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase C/antagonists & inhibitors , Pyrroles/administration & dosage , Quinazolines/administration & dosage , RNA Interference , Signal Transduction/drug effects , Thiazoles/administration & dosage , Tumor Burden , Uveal Neoplasms/drug therapy , Uveal Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
Oncogenic mutations in GNAQ and GNA11 genes are found in 80% of uveal melanoma. These mutations result in the activation of the RAF/MEK signaling pathway culminating in the stimulation of ERK1/2 mitogen-activated protein kinases. In this study, using a siRNA strategy, we show that mutant GNAQ signals to both MEK and AKT, and that combined inhibition of these pathways with the MEK inhibitor selumetinib (AZD6244) and the AKT inhibitor MK2206 induced a synergistic decrease in cell viability. This effect was genotype dependent as autophagic markers like beclin1 and LC3 were induced in GNAQ-mutant cells, whereas apoptosis was the mechanism of cell death of BRAF-mutant cells, and cells without either mutation underwent cell-cycle arrest. The inhibition of MEK/ATK pathways induced activation of AMP-activated protein kinase (AMPK) in the GNAQ-mutant cells. The downregulation of AMPK by siRNA or its inhibition with compound C did not rescue the cells from autophagy, rather they died by apoptosis, defining AMPK as a key regulator of mutant GNAQ signaling and a switch between autophagy and apoptosis. Furthermore, this combination treatment was effective in inhibiting tumor growth in xenograft mouse models. These findings suggest that inhibition of MEK and AKT may represent a promising approach for targeted therapy of patients with uveal melanoma.
Subject(s)
Autophagy/genetics , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein alpha Subunits/metabolism , Melanoma/genetics , Melanoma/metabolism , Mitogen-Activated Protein Kinases/metabolism , Signal Transduction , Uveal Neoplasms/genetics , Uveal Neoplasms/metabolism , Animals , Benzimidazoles/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , GTP-Binding Protein alpha Subunits, Gq-G11 , Gene Knockdown Techniques , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Male , Melanoma/pathology , Mice , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Tumor Burden/drug effects , Uveal Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways. MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations. In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055. While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only. In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model. We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells. For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis. Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis. These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.
Subject(s)
Melanoma/drug therapy , Melanoma/genetics , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , TOR Serine-Threonine Kinases/antagonists & inhibitors , Uveal Neoplasms/drug therapy , Uveal Neoplasms/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Apoptosis/drug effects , Benzimidazoles/pharmacology , Benzimidazoles/therapeutic use , Cell Cycle Proteins , Cell Line, Tumor , Cell Survival/drug effects , Drug Resistance, Neoplasm/drug effects , Drug Synergism , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein alpha Subunits, Gq-G11 , Genotype , Humans , Mechanistic Target of Rapamycin Complex 2 , Melanoma/enzymology , Mice , Mitogen-Activated Protein Kinase Kinases/metabolism , Models, Biological , Morpholines/pharmacology , Morpholines/therapeutic use , Multiprotein Complexes/metabolism , Mutation/genetics , Myeloid Cell Leukemia Sequence 1 Protein , Phosphoproteins/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptor, IGF Type 1/metabolism , TOR Serine-Threonine Kinases/metabolism , Uveal Neoplasms/enzymology , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Dysregulated cyclin-dependent kinases are important to the growth of some sarcomas. Flavopiridol is a pan-CDK inhibitor that has been shown to potentiate chemotherapy. As such, we explored the potentiation of doxorubicin by flavopiridol in sarcoma, in vitro and in vivo, and conducted a phase I trial of flavopiridol with doxorubicin in patients with advanced sarcomas. EXPERIMENTAL DESIGN: Sarcoma cell lines and xenografts were treated with flavopiridol alone and in combination with doxorubicin. In the phase I study, doxorubicin and flavopiridol were administered on two flavopiridol schedules; a 1-hour bolus and split dosing as a 30-minute bolus followed by a 4-hour infusion. RESULTS: Preclinically, flavopiridol potentiated doxorubicin. In vivo, doxorubicin administered 1 hour before flavopiridol was more active than doxorubicin alone. Clinically, 31 patients were enrolled on protocol and flavopiridol was escalated to target dose in two schedules (90 mg/m(2) bolus; 50 mg/m(2) bolus + 40 mg/m(2) infusion) both in combination with doxorubicin (60 mg/m(2)). Dose-limiting toxicities were neutropenia, leukopenia, and febrile neutropenia but no maximum tolerated dose was defined. Flavopiridol pharmacokinetics showed increasing C(max) with increasing dose. Response Evaluation Criteria in Solid Tumors (RECIST) responses included two partial responses, however, stable disease was seen in 16 patients. Of 12 evaluable patients with progressive well- and dedifferentiated liposarcoma, eight had stable disease greater than 12 weeks. CONCLUSIONS: The sequential combination of doxorubicin followed by flavopiridol is well tolerated on both schedules. Disease control was observed in well- and dedifferentiated liposarcoma specifically, a disease in which CDK4 is known to be amplified.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cyclin-Dependent Kinases/antagonists & inhibitors , Nerve Sheath Neoplasms/drug therapy , Sarcoma/drug therapy , Adult , Aged , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacokinetics , Dose-Response Relationship, Drug , Doxorubicin/administration & dosage , Drug Evaluation, Preclinical , Female , Flavonoids/administration & dosage , Humans , Immunoblotting , Male , Maximum Tolerated Dose , Mice , Mice, SCID , Middle Aged , Neoplasm Grading , Piperidines/administration & dosage , Tissue DistributionABSTRACT
Safingol, the synthetic L-threo-stereoisomer of endogenous (D-erythro-) sphinganine, is an inhibitor of protein kinase C and sphingosine kinase in vitro, and in some cell types has been implicated in ceramide generation and induction of apoptosis. Utilizing electron microscopy, acridine orange staining, and immunoblot and fluorescent localization studies of the myosin light chain-associated protein (LC3), we determined that safingol induces cell death of an exclusively autophagic character and lacking any of the hallmarks of apoptosis. Safingol inhibited PKCbeta-I, PKC delta and PKC epsilon, and inhibited phosphorylation of critical components of the PI3k/Akt/mTOR pathway (Akt, p70S6k and rS6) and the MAPk pathway (ERK). Inhibition of PI3k with LY294002 or suppression of PKC delta and PKC epsilon with siRNA in HCT-116 cells induced autophagy, though not to the extent caused by safingol. Conversely, activation of PKCs with phorbol 12,13-dibutyrate (PDBu) or transient transfection of a constitutively active form of Akt each reduced safingol's autophagic induction, but not completely, indicating that Akt- and PKC-dependent pathways both contribute partially and independently to safingol-induced autophagy. Accordingly, combining siRNA depletion of PKC epsilon with LY294002 inhibition of PI3k induced autophagy to a degree comparable to safingol. Liquid chromatography, electrospray tandem mass spectrometry analysis indicated that safingol did not elevate levels of any endogenous sphingolipids previously shown to induce autophagy (ceramide, sphingosine-1-phosphate and dihydroceramide); therefore, these effects may be due to safingol per se or another metabolite. Thus, our studies establish that safingol induces autophagy through inhibition of PKCs and PI3k by safingol directly rather than via changes in endogenous sphingolipids.
Subject(s)
Autophagy/drug effects , Neoplasms/enzymology , Neoplasms/pathology , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase C/antagonists & inhibitors , Sphingosine/analogs & derivatives , Acridine Orange , Cell Count , Cell Line, Tumor , Cell Survival/drug effects , Chromatography, Liquid , Fluorescent Antibody Technique , Humans , Neoplasms/ultrastructure , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , RNA, Small Interfering/metabolism , Sphingolipids/metabolism , Sphingosine/pharmacology , TOR Serine-Threonine Kinases , Tandem Mass SpectrometryABSTRACT
Human CD39/NTPDase1 is an endothelial cell membrane-associated nucleotidase. Its large extracellular domain rapidly metabolizes nucleotides, especially ADP released from activated platelets, inhibiting further platelet activation/recruitment. Previous studies using our recombinant soluble CD39 demonstrated the importance of residues S57, D54, and D213 for enzymatic/biological activity. We now report effects of S57A, D54A, and D213A mutations on full-length (FL)CD39 function. Enzymatic activity of alanine modified FLCD39s was less than wild-type, contrasting the enhanced activity of their soluble counterparts. Furthermore, conservative substitutions D54E and D213E led to enzymes with activities greater than the alanine modified FLCD39s, but less than wild-type. Reductions in mutant activities were primarily associated with reduced catalytic rates. Differences in enzymatic activity were not attributable to gross changes in the nucleotide binding pocket or the enzyme's ability to multimerize. Thus, composition of the active site of wild-type CD39 appears optimized for ADPase function in the context of the transmembrane domains.