ABSTRACT
Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease driven by gain-of-function variants in activin receptor-like kinase 2 (ALK2), the most common variant being ALK2R206H. In FOP, ALK2 variants display increased and dysregulated signaling through the bone morphogenetic protein (BMP) pathway resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of BLU-782 (IPN60130), a small-molecule ALK2R206H inhibitor developed for the treatment of FOP. A small-molecule library was screened in a biochemical ALK2 binding assay to identify potent ALK2 binding compounds. Iterative rounds of structure-guided drug design were used to optimize compounds for ALK2R206H binding, ALK2 selectivity, and other desirable pharmacokinetic properties. BLU-782 preferentially bound to ALK2R206H with high affinity, inhibiting signaling from ALK2R206H and other rare FOP variants in cells in vitro without affecting signaling of closely related homologs ALK1, ALK3, and ALK6. In vivo efficacy of BLU-782 was demonstrated using a conditional knock-in ALK2R206H mouse model, where prophylactic oral dosing reduced edema and prevented cartilage and heterotopic ossification (HO) in both muscle and bone injury models. BLU-782 treatment preserved the normal muscle-healing response in ALK2R206H mice. Delayed dosing revealed a short 2-day window after injury when BLU-782 treatment prevented HO in ALK2R206H mice, but dosing delays of 4 days or longer abrogated HO prevention. Together, these data suggest that BLU-782 may be a candidate for prevention of HO in FOP.
Subject(s)
Disease Models, Animal , Myositis Ossificans , Ossification, Heterotopic , Animals , Myositis Ossificans/drug therapy , Myositis Ossificans/metabolism , Ossification, Heterotopic/drug therapy , Ossification, Heterotopic/metabolism , Ossification, Heterotopic/prevention & control , Mice , Humans , Activin Receptors, Type II/metabolism , Activin Receptors, Type I/metabolism , Activin Receptors, Type I/antagonists & inhibitors , Signal Transduction/drug effectsABSTRACT
Background and Aims: Fibrolamellar carcinoma (FLC) is a rare, difficult-to-treat liver cancer primarily affecting pediatric and adolescent patients, and for which precision medicine approaches have historically not been possible. The DNAJB1-PRKACA gene fusion was identified as a driver of FLC pathogenesis. We aimed to assess whether FLC tumors maintain dependency on this gene fusion and determine if PRKACA is a viable therapeutic target. Methods: FLC patient-derived xenograft (PDX) shRNA cell lines were implanted subcutaneously into female NOD-SCID mice and tumors were allowed to develop prior to randomization to doxycycline (to induce knockdown) or control groups. Tumor development was assessed every 2 days. To assess the effect of treatment with novel selective PRKACA small molecule kinase inhibitors, BLU0588 and BLU2864, FLC PDX tumor cells were implanted subcutaneously into NOD-SCID mice and tumors allowed to develop. Mice were randomized to treatment (BLU0588 and BLU2864, orally, once daily) or control groups and tumor size determined as previously. Results: Knockdown of DNAJB1-PRKACA reversed a FLC-specific gene signature and reduced PDX tumor growth in mice compared to the control group. Furthermore, FLC PDX tumor growth was significantly reduced with BLU0588 and BLU2864 treatment vs control (P = .003 and P = .0005, respectively). Conclusion: We demonstrated, using an inducible knockdown and small molecule approaches, that FLC PDX tumors were dependent upon DNAJB1-PRKACA fusion activity. In addition, this study serves as a proof-of-concept that PRKACA is a viable therapeutic target for FLC and warrants further investigation.
ABSTRACT
Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide with no clinically confirmed oncogenic driver. Although preclinical studies implicate the FGF19 receptor FGFR4 in hepatocarcinogenesis, the dependence of human cancer on FGFR4 has not been demonstrated. Fisogatinib (BLU-554) is a potent and selective inhibitor of FGFR4 and demonstrates clinical benefit and tumor regression in patients with HCC with aberrant FGF19 expression. Mutations were identified in the gatekeeper and hinge-1 residues in the kinase domain of FGFR4 upon disease progression in 2 patients treated with fisogatinib, which were confirmed to mediate resistance in vitro and in vivo. A gatekeeper-agnostic, pan-FGFR inhibitor decreased HCC xenograft growth in the presence of these mutations, demonstrating continued FGF19-FGFR4 pathway dependence. These results validate FGFR4 as an oncogenic driver and warrant further therapeutic targeting of this kinase in the clinic. SIGNIFICANCE: Our study is the first to demonstrate on-target FGFR4 kinase domain mutations as a mechanism of acquired clinical resistance to targeted therapy. This further establishes FGF19-FGFR4 pathway activation as an oncogenic driver. These findings support further investigation of fisogatinib in HCC and inform the profile of potential next-generation inhibitors.See related commentary by Subbiah and Pal, p. 1646.This article is highlighted in the In This Issue feature, p. 1631.
Subject(s)
Carcinoma, Hepatocellular/diagnostic imaging , Drug Resistance, Neoplasm , Liver Neoplasms/diagnostic imaging , Pyrans/pharmacology , Quinazolines/pharmacology , Receptor, Fibroblast Growth Factor, Type 4/genetics , Aged, 80 and over , Animals , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Female , Fibroblast Growth Factors/metabolism , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Male , Mice , Middle Aged , Models, Molecular , Mutation , Neoplasm Transplantation , Protein Domains , Receptor, Fibroblast Growth Factor, Type 4/chemistry , Receptor, Fibroblast Growth Factor, Type 4/metabolismABSTRACT
Aberrant metabolism of cancer cells is well appreciated, but the identification of cancer subsets with specific metabolic vulnerabilities remains challenging. We conducted a chemical biology screen and identified a subset of neuroendocrine tumors displaying a striking pattern of sensitivity to inhibition of the cholesterol biosynthetic pathway enzyme squalene epoxidase (SQLE). Using a variety of orthogonal approaches, we demonstrate that sensitivity to SQLE inhibition results not from cholesterol biosynthesis pathway inhibition, but rather surprisingly from the specific and toxic accumulation of the SQLE substrate, squalene. These findings highlight SQLE as a potential therapeutic target in a subset of neuroendocrine tumors, particularly small cell lung cancers.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Delivery Systems , Drug Screening Assays, Antitumor , Squalene Monooxygenase/antagonists & inhibitors , Squalene Monooxygenase/metabolism , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cholesterol/biosynthesis , Gene Deletion , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , HumansABSTRACT
Somatic gain-of-function mutations in isocitrate dehydrogenases (IDH) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite (R)-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate-dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 mutation-positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2R140Q-mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with IDH2 mutation-positive advanced hematologic malignancies.Significance: Mutations in IDH1/2 are identified in approximately 20% of patients with AML and contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the targeted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces differentiation of malignant blasts, supporting its clinical development. Cancer Discov; 7(5); 478-93. ©2017 AACR.See related commentary by Thomas and Majeti, p. 459See related article by Shih et al., p. 494This article is highlighted in the In This Issue feature, p. 443.
Subject(s)
Aminopyridines/pharmacology , Antineoplastic Agents/pharmacology , Isocitrate Dehydrogenase/antagonists & inhibitors , Leukemia, Myeloid, Acute/genetics , Triazines/pharmacology , Animals , Cell Line, Tumor , Humans , Isocitrate Dehydrogenase/genetics , Mice , Mutation , Xenograft Model Antitumor AssaysABSTRACT
Although aberrant metabolism in tumors has been well described, the identification of cancer subsets with particular metabolic vulnerabilities has remained challenging. Here, we conducted an siRNA screen focusing on enzymes involved in the tricarboxylic acid (TCA) cycle and uncovered a striking range of cancer cell dependencies on OGDH, the E1 subunit of the alpha-ketoglutarate dehydrogenase complex. Using an integrative metabolomics approach, we identified differential aspartate utilization, via the malate-aspartate shuttle, as a predictor of whether OGDH is required for proliferation in 3D culture assays and for the growth of xenograft tumors. These findings highlight an anaplerotic role of aspartate and, more broadly, suggest that differential nutrient utilization patterns can identify subsets of cancers with distinct metabolic dependencies for potential pharmacological intervention.
Subject(s)
Aspartic Acid/metabolism , Ketoglutarate Dehydrogenase Complex/metabolism , Neoplasms/metabolism , Animals , Cell Line, Tumor , Cell Respiration/drug effects , Citric Acid Cycle/drug effects , Enzyme Inhibitors/pharmacology , Gene Knockdown Techniques , Humans , RNA, Small Interfering/metabolismABSTRACT
D-2-hydroxyglutaric aciduria (D2HGA) type II is a rare neurometabolic disorder caused by germline gain-of-function mutations in isocitrate dehydrogenase 2 (IDH2), resulting in accumulation of D-2-hydroxyglutarate (D2HG). Patients exhibit a wide spectrum of symptoms including cardiomyopathy, epilepsy, developmental delay and limited life span. Currently, there are no effective therapeutic interventions. We generated a D2HGA type II mouse model by introducing the Idh2R140Q mutation at the native chromosomal locus. Idh2R140Q mice displayed significantly elevated 2HG levels and recapitulated multiple defects seen in patients. AGI-026, a potent, selective inhibitor of the human IDH2R140Q-mutant enzyme, suppressed 2HG production, rescued cardiomyopathy, and provided a survival benefit in Idh2R140Q mice; treatment withdrawal resulted in deterioration of cardiac function. We observed differential expression of multiple genes and metabolites that are associated with cardiomyopathy, which were largely reversed by AGI-026. These findings demonstrate the potential therapeutic benefit of an IDH2R140Q inhibitor in patients with D2HGA type II.
Subject(s)
Brain Diseases, Metabolic, Inborn/drug therapy , Cardiomyopathies/drug therapy , Isocitrate Dehydrogenase/antagonists & inhibitors , Mutation/drug effects , Small Molecule Libraries/pharmacology , Animals , Brain Diseases, Metabolic, Inborn/genetics , Disease Models, Animal , Isocitrate Dehydrogenase/genetics , Mice , Mutation/geneticsABSTRACT
The most common congenital disorder of glycosylation (CDG), phosphomannomutase 2 (PMM2)-CDG, is caused by mutations in PMM2 that limit availability of mannose precursors required for protein N-glycosylation. The disorder has no therapy and there are no models to test new treatments. We generated compound heterozygous mice with the R137H and F115L mutations in Pmm2 that correspond to the most prevalent alleles found in patients with PMM2-CDG. Many Pmm2R137H/F115L mice died prenatally, while survivors had significantly stunted growth. These animals and cells derived from them showed protein glycosylation deficiencies similar to those found in patients with PMM2-CDG. Growth-related glycoproteins insulin-like growth factor (IGF) 1, IGF binding protein-3 and acid-labile subunit, along with antithrombin III, were all deficient in Pmm2R137H/F115L mice, but their levels in heterozygous mice were comparable to wild-type (WT) littermates. These imbalances, resulting from defective glycosylation, are likely the cause of the stunted growth seen both in our model and in PMM2-CDG patients. Both Pmm2R137H/F115L mouse and PMM2-CDG patient-derived fibroblasts displayed reductions in PMM activity, guanosine diphosphate mannose, lipid-linked oligosaccharide precursor and total cellular protein glycosylation, along with hypoglycosylation of a new endogenous biomarker, glycoprotein 130 (gp130). Over-expression of WT-PMM2 in patient-derived fibroblasts rescued all these defects, showing that restoration of mutant PMM2 activity is a viable therapeutic strategy. This functional mouse model of PMM2-CDG, in vitro assays and identification of the novel gp130 biomarker all shed light on the human disease, and moreover, provide the essential tools to test potential therapeutics for this untreatable disease.
Subject(s)
Biomarkers , Congenital Disorders of Glycosylation/genetics , Cytokine Receptor gp130/genetics , Phosphotransferases (Phosphomutases)/genetics , Animals , Congenital Disorders of Glycosylation/metabolism , Congenital Disorders of Glycosylation/pathology , Cytokine Receptor gp130/biosynthesis , Disease Models, Animal , Fibroblasts/metabolism , Gene Expression Regulation , Genotype , Glycosylation , Humans , Mannose/genetics , Mannose/metabolism , Mice , MutationABSTRACT
Homozygous deletions of p16/CDKN2A are prevalent in cancer, and these mutations commonly involve co-deletion of adjacent genes, including methylthioadenosine phosphorylase (MTAP). Here, we used shRNA screening and identified the metabolic enzyme, methionine adenosyltransferase II alpha (MAT2A), and the arginine methyltransferase, PRMT5, as vulnerable enzymes in cells with MTAP deletion. Metabolomic and biochemical studies revealed a mechanistic basis for this synthetic lethality. The MTAP substrate methylthioadenosine (MTA) accumulates upon MTAP loss. Biochemical profiling of a methyltransferase enzyme panel revealed that MTA is a potent and selective inhibitor of PRMT5. MTAP-deleted cells have reduced PRMT5 methylation activity and increased sensitivity to PRMT5 depletion. MAT2A produces the PRMT5 substrate S-adenosylmethionine (SAM), and MAT2A depletion reduces growth and PRMT5 methylation activity selectively in MTAP-deleted cells. Furthermore, this vulnerability extends to PRMT5 co-complex proteins such as RIOK1. Thus, the unique biochemical features of PRMT5 create an axis of targets vulnerable in CDKN2A/MTAP-deleted cancers.
Subject(s)
Adenosine/analogs & derivatives , Antigens, Neoplasm/metabolism , Gene Deletion , Methionine Adenosyltransferase/metabolism , Neoplasms/enzymology , Protein Serine-Threonine Kinases/metabolism , Protein-Arginine N-Methyltransferases/metabolism , Purine-Nucleoside Phosphorylase/metabolism , Signal Transduction , Thionucleosides/metabolism , Adenosine/metabolism , Genomics , HCT116 Cells , Humans , Multiprotein Complexes/metabolism , Neoplasms/metabolism , Purine-Nucleoside Phosphorylase/deficiency , RNA, Small Interfering/metabolismABSTRACT
Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver removes toxic aldehydes including acetaldehyde, an intermediate of ethanol metabolism. Nearly 40% of East Asians inherit an inactive ALDH2*2 variant, which has a lysine-for-glutamate substitution at position 487 (E487K), and show a characteristic alcohol flush reaction after drinking and a higher risk for gastrointestinal cancers. Here we report the characterization of knockin mice in which the ALDH2(E487K) mutation is inserted into the endogenous murine Aldh2 locus. These mutants recapitulate essentially all human phenotypes including impaired clearance of acetaldehyde, increased sensitivity to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to a dominant-negative effect of the mutation. When treated with a chemical carcinogen, these mutants exhibit increased DNA damage response in hepatocytes, pronounced liver injury, and accelerated development of hepatocellular carcinoma (HCC). Importantly, ALDH2 protein levels are also significantly lower in patient HCC than in peritumor or normal liver tissues. Our results reveal that ALDH2 functions as a tumor suppressor by maintaining genomic stability in the liver, and the common human ALDH2 variant would present a significant risk factor for hepatocarcinogenesis. Our study suggests that the ALDH2*2 allele-alcohol interaction may be an even greater human public health hazard than previously appreciated.
Subject(s)
Aldehyde Dehydrogenase/genetics , Carcinoma, Hepatocellular/enzymology , Liver Neoplasms/enzymology , Liver Neoplasms/genetics , Mutation/genetics , Alcoholic Intoxication/enzymology , Alcoholic Intoxication/pathology , Aldehyde Dehydrogenase, Mitochondrial , Amino Acid Substitution , Animals , Base Sequence , Carcinogenesis/pathology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Ethanol/adverse effects , Gene Knock-In Techniques , Genotyping Techniques , Hepatocytes/enzymology , Hepatocytes/pathology , Humans , Hyperpigmentation/pathology , Immunohistochemistry , Liver/enzymology , Liver/pathology , Liver Neoplasms/pathology , Mice, Inbred C57BL , Mutant Proteins/metabolism , Polymorphism, Genetic , Proteasome Endopeptidase Complex/metabolism , Protein Stability , Skin/pathology , Survival AnalysisABSTRACT
Mutations of IDH1 and IDH2, which produce the oncometabolite 2-hydroxyglutarate (2HG), have been identified in several tumors, including acute myeloid leukemia. Recent studies have shown that expression of the IDH mutant enzymes results in high levels of 2HG and a block in cellular differentiation that can be reversed with IDH mutant-specific small-molecule inhibitors. To further understand the role of IDH mutations in cancer, we conducted mechanistic studies in the TF-1 IDH2 R140Q erythroleukemia model system and found that IDH2 mutant expression caused both histone and genomic DNA methylation changes that can be reversed when IDH2 mutant activity is inhibited. Specifically, histone hypermethylation is rapidly reversed within days, whereas reversal of DNA hypermethylation proceeds in a progressive manner over the course of weeks. We identified several gene signatures implicated in tumorigenesis of leukemia and lymphoma, indicating a selective modulation of relevant cancer genes by IDH mutations. As methylation of DNA and histones is closely linked to mRNA expression and differentiation, these results indicate that IDH2 mutant inhibition may function as a cancer therapy via histone and DNA demethylation at genes involved in differentiation and tumorigenesis.
Subject(s)
DNA Methylation/genetics , Enzyme Inhibitors/pharmacology , Histones/genetics , Isocitrate Dehydrogenase/genetics , Mutation , Transcriptome/drug effects , Cell Line, Tumor , Chromatin Immunoprecipitation , Chromatography, Liquid , Histones/drug effects , Humans , Leukemia, Myeloid, Acute/genetics , Phenylurea Compounds/pharmacology , Principal Component Analysis , Reverse Transcriptase Polymerase Chain Reaction , Sulfonamides/pharmacology , Tandem Mass SpectrometryABSTRACT
Recent work has highlighted glutaminase (GLS) as a key player in cancer cell metabolism, providing glutamine-derived carbon and nitrogen to pathways that support proliferation. There is significant interest in targeting GLS for cancer therapy, although the gene is not known to be mutated or amplified in tumors. As a result, identification of tractable markers that predict GLS dependence is needed for translation of GLS inhibitors to the clinic. Herein we validate a small molecule inhibitor of GLS and show that non-small cell lung cancer cells marked by low E-cadherin and high vimentin expression, hallmarks of a mesenchymal phenotype, are particularly sensitive to inhibition of the enzyme. Furthermore, lung cancer cells induced to undergo epithelial to mesenchymal transition (EMT) acquire sensitivity to the GLS inhibitor. Metabolic studies suggest that the mesenchymal cells have a reduced capacity for oxidative phosphorylation and increased susceptibility to oxidative stress, rendering them unable to cope with the perturbations induced by GLS inhibition. These findings elucidate selective metabolic dependencies of mesenchymal lung cancer cells and suggest novel pathways as potential targets in this aggressive cancer type.
Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Glutaminase/antagonists & inhibitors , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Oxidative Stress/drug effects , Sulfides/pharmacology , Thiadiazoles/pharmacology , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Enzyme Inhibitors/pharmacology , Epithelial-Mesenchymal Transition , Genetic Association Studies , Glutaminase/metabolism , Humans , Lung Neoplasms/metabolism , Molecular Targeted TherapyABSTRACT
Postchemotherapy relapse presents a major unmet medical need in acute myeloid leukemia (AML), where treatment options are limited. CD25 is a leukemic stem cell marker and a conspicuous prognostic marker for overall/relapse-free survival in AML. Rare occurrence of genetic alterations among PIM family members imposes a substantial hurdle in formulating a compelling patient stratification strategy for the clinical development of selective PIM inhibitors in cancer. Here we show that CD25, a bona fide STAT5 regulated gene, is a mechanistically relevant predictive biomarker for sensitivity to PIM kinase inhibitors. Alone or in combination with tyrosine kinase inhibitors, PIM inhibitors can suppress STAT5 activation and significantly shorten the half-life of MYC to achieve substantial growth inhibition of high CD25-expressing AML cells. Our results highlight the importance of STAT5 and MYC in rendering cancer cells sensitive to PIM inhibitors. Because the presence of a CD25-positive subpopulation in leukemic blasts correlates with poor overall or relapse-free survival, our data suggest that a combination of PIM inhibitors with chemotherapy and tyrosine kinase inhibitors could improve long-term therapeutic outcomes in CD25-positive AML.
Subject(s)
Antineoplastic Agents/pharmacology , Blast Crisis , Gene Expression Regulation, Leukemic/drug effects , Interleukin-2 Receptor alpha Subunit/metabolism , Proteolysis/drug effects , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , STAT5 Transcription Factor/metabolism , Antineoplastic Agents/chemistry , Blast Crisis/drug therapy , Blast Crisis/genetics , Blast Crisis/metabolism , Blast Crisis/pathology , Female , HL-60 Cells , Humans , Interleukin-2 Receptor alpha Subunit/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Male , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-pim-1/genetics , Proto-Oncogene Proteins c-pim-1/metabolism , STAT5 Transcription Factor/geneticsABSTRACT
Major progress has been made in recent years in the development of Hedgehog (Hh) pathway inhibitors for the treatment of patients with cancer. Promising clinical trial results have been obtained in cancers that harbor activating mutations of the Hh pathway, such as basal cell carcinoma and medulloblastoma. However, for many cancers, in which Hh ligand overexpression is thought to drive tumor growth, results have been disappointing. Here we review the preclinical data that continue to shape our understanding of the Hh pathway in tumorigenesis and the emerging clinical experience with smoothened inhibitors.
Subject(s)
Hedgehog Proteins/antagonists & inhibitors , Hedgehog Proteins/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Animals , Antineoplastic Combined Chemotherapy Protocols , Drug Discovery , Drug Resistance, Neoplasm/genetics , Hedgehog Proteins/genetics , Humans , Ligands , Models, Biological , Molecular Targeted Therapy , Mutation , Neoplasms/genetics , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Smoothened Receptor , Transcription Factors/metabolism , Translational Research, Biomedical , Zinc Finger Protein GLI1ABSTRACT
In glioblastoma, phosphatidylinositol 3-kinase (PI3K) signaling is frequently activated by loss of the tumor suppressor phosphatase and tensin homolog (PTEN). However, it is not known whether inhibiting PI3K represents a selective and effective approach for treatment. We interrogated large databases and found that sonic hedgehog (SHH) signaling is activated in PTEN-deficient glioblastoma. We demonstrate that the SHH and PI3K pathways synergize to promote tumor growth and viability in human PTEN-deficient glioblastomas. A combination of PI3K and SHH signaling inhibitors not only suppressed the activation of both pathways but also abrogated S6 kinase (S6K) signaling. Accordingly, targeting both pathways simultaneously resulted in mitotic catastrophe and tumor apoptosis and markedly reduced the growth of PTEN-deficient glioblastomas in vitro and in vivo. The drugs tested here appear to be safe in humans; therefore, this combination may provide a new targeted treatment for glioblastoma.
Subject(s)
Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Glioblastoma/drug therapy , Glioblastoma/metabolism , Hedgehog Proteins/metabolism , PTEN Phosphohydrolase/deficiency , Phosphatidylinositol 3-Kinases/metabolism , Aminopyridines/administration & dosage , Animals , Biphenyl Compounds/administration & dosage , Brain Neoplasms/genetics , Cell Line, Tumor , Enzyme Inhibitors/administration & dosage , Glioblastoma/genetics , Hedgehog Proteins/antagonists & inhibitors , Humans , Mice , Mice, Nude , Morpholines/administration & dosage , PTEN Phosphohydrolase/genetics , Phosphoinositide-3 Kinase Inhibitors , Pyridines/administration & dosage , Ribosomal Protein S6 Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Xenograft Model Antitumor AssaysABSTRACT
PRP4 kinase is known for its roles in regulating pre-mRNA splicing and beyond. Therefore, a wider spectrum of PRP4 kinase substrates could be expected. The role of PRP4 kinase in cancer is also yet to be fully elucidated. Attaining specific and potent PRP4 inhibitors would greatly facilitate the study of PRP4 biological function and its validation as a credible cancer target. In this report, we verified the requirement of enzymatic activity of PRP4 in regulating cancer cell growth and identified an array of potential novel substrates through orthogonal proteomics approaches. The ensuing effort in structural biology unveiled for the first time unique features of PRP4 kinase domain and its potential mode of interaction with a low molecular weight inhibitor. These results provide new and important information for further exploration of PRP4 kinase function in cancer.
Subject(s)
Neoplasm Proteins , Neoplasms , Protein Kinase Inhibitors , Ribonucleoprotein, U4-U6 Small Nuclear , Cell Line, Tumor , Humans , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplasms/drug therapy , Neoplasms/enzymology , Neoplasms/genetics , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Proteomics/methods , Ribonucleoprotein, U4-U6 Small Nuclear/antagonists & inhibitors , Ribonucleoprotein, U4-U6 Small Nuclear/chemistry , Ribonucleoprotein, U4-U6 Small Nuclear/genetics , Ribonucleoprotein, U4-U6 Small Nuclear/metabolismSubject(s)
Pyrazines/chemistry , Pyridazines/chemistry , Receptors, G-Protein-Coupled/antagonists & inhibitors , Animals , Cell Line , Disease Models, Animal , Dogs , Drug Evaluation, Preclinical , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/metabolism , Half-Life , Humans , Mice , Pyrazines/chemical synthesis , Pyrazines/pharmacokinetics , Pyridazines/chemical synthesis , Pyridazines/pharmacokinetics , Rats , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Smoothened Receptor , Solubility , Structure-Activity RelationshipABSTRACT
A number of human cancers harbor somatic point mutations in the genes encoding isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). These mutations alter residues in the enzyme active sites and confer a gain-of-function in cancer cells, resulting in the accumulation and secretion of the oncometabolite (R)-2-hydroxyglutarate (2HG). We developed a small molecule, AGI-6780, that potently and selectively inhibits the tumor-associated mutant IDH2/R140Q. A crystal structure of AGI-6780 complexed with IDH2/R140Q revealed that the inhibitor binds in an allosteric manner at the dimer interface. The results of steady-state enzymology analysis were consistent with allostery and slow-tight binding by AGI-6780. Treatment with AGI-6780 induced differentiation of TF-1 erythroleukemia and primary human acute myelogenous leukemia cells in vitro. These data provide proof-of-concept that inhibitors targeting mutant IDH2/R140Q could have potential applications as a differentiation therapy for cancer.
Subject(s)
Enzyme Inhibitors/pharmacology , Hematopoiesis/drug effects , Isocitrate Dehydrogenase/antagonists & inhibitors , Isocitrate Dehydrogenase/genetics , Leukemia, Myeloid, Acute/enzymology , Phenylurea Compounds/pharmacology , Sulfonamides/pharmacology , Allosteric Site , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Catalytic Domain , Cell Line, Tumor , Cell Proliferation , Cells, Cultured , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Erythropoiesis/drug effects , Gene Expression Regulation, Leukemic , Glutarates/metabolism , Humans , Isocitrate Dehydrogenase/chemistry , Isocitrate Dehydrogenase/metabolism , Leukemia, Erythroblastic, Acute , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Molecular Targeted Therapy , Mutant Proteins/antagonists & inhibitors , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Phenylurea Compounds/chemistry , Phenylurea Compounds/metabolism , Point Mutation , Protein Multimerization , Protein Structure, Secondary , Small Molecule Libraries , Sulfonamides/chemistry , Sulfonamides/metabolismABSTRACT
Following the discovery of NVP-BEZ235, our first dual pan-PI3K/mTOR clinical compound, we sought to identify additional phosphoinositide 3-kinase (PI3K) inhibitors from different chemical classes with a different selectivity profile. The key to achieve these objectives was to couple a structure-based design approach with intensive pharmacologic evaluation of selected compounds during the medicinal chemistry optimization process. Here, we report on the biologic characterization of the 2-morpholino pyrimidine derivative pan-PI3K inhibitor NVP-BKM120. This compound inhibits all four class I PI3K isoforms in biochemical assays with at least 50-fold selectivity against other protein kinases. The compound is also active against the most common somatic PI3Kα mutations but does not significantly inhibit the related class III (Vps34) and class IV (mTOR, DNA-PK) PI3K kinases. Consistent with its mechanism of action, NVP-BKM120 decreases the cellular levels of p-Akt in mechanistic models and relevant tumor cell lines, as well as downstream effectors in a concentration-dependent and pathway-specific manner. Tested in a panel of 353 cell lines, NVP-BKM120 exhibited preferential inhibition of tumor cells bearing PIK3CA mutations, in contrast to either KRAS or PTEN mutant models. NVP-BKM120 shows dose-dependent in vivo pharmacodynamic activity as measured by significant inhibition of p-Akt and tumor growth inhibition in mechanistic xenograft models. NVP-BKM120 behaves synergistically when combined with either targeted agents such as MEK or HER2 inhibitors or with cytotoxic agents such as docetaxel or temozolomide. The pharmacological, biologic, and preclinical safety profile of NVP-BKM120 supports its clinical development and the compound is undergoing phase II clinical trials in patients with cancer.
Subject(s)
Aminopyridines/pharmacology , Morpholines/pharmacology , Neoplasms/drug therapy , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Administration, Oral , Aminopyridines/chemistry , Aminopyridines/pharmacokinetics , Animals , Biological Availability , Blotting, Western , Cell Line, Tumor , Dose-Response Relationship, Drug , HCT116 Cells , HT29 Cells , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/metabolism , Mice , Mice, Nude , Models, Molecular , Molecular Structure , Morpholines/chemistry , Morpholines/pharmacokinetics , Mutation , Neoplasms/metabolism , Neoplasms/pathology , Phosphatidylinositol 3-Kinase/chemistry , Phosphatidylinositol 3-Kinase/metabolism , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Protein Structure, Tertiary , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Rats , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment. Using a mouse model in which deletion of Rb1 and Trp53 in the lung epithelium of adult mice induces SCLC, we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rb1 and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC.