ABSTRACT
Fibroblast growth factor receptor 4 (FGFR4) is thought to be a driver in several cancer types, most notably in hepatocellular carcinoma. One way to achieve high potency and isoform selectivity for FGFR4 is covalently targeting a rare cysteine (C552) in the hinge region of its kinase domain that is not present in other FGFR family members (FGFR1-3). Typically, this cysteine is addressed via classical acrylamide electrophiles. We demonstrate that noncanonical covalent "warheads" based on nucleophilic aromatic substitution (SNAr) chemistry can be employed in a rational manner to generate highly potent and (isoform-)selective FGFR4 inhibitors with a low intrinsic reactivity. Key compounds showed low to subnanomolar potency, efficient covalent inactivation kinetics, and excellent selectivity against the other FGFRs, the kinases with an equivalent cysteine, and a representative subset of the kinome. Moreover, these compounds achieved nanomolar potencies in cellular assays and demonstrated good microsomal stability, highlighting the potential of SNAr-based approaches in covalent inhibitor design.
Subject(s)
Protein Kinase Inhibitors , Receptor, Fibroblast Growth Factor, Type 4 , Receptor, Fibroblast Growth Factor, Type 4/antagonists & inhibitors , Receptor, Fibroblast Growth Factor, Type 4/metabolism , Humans , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/chemical synthesis , Structure-Activity Relationship , Microsomes, Liver/metabolismABSTRACT
Metabolic alterations occur frequently in solid tumours, but metabolic cancer therapies are limited by the complexity and plasticity of metabolic networks. We could recently show that activation of the liver X receptor alpha (LXRα) and inhibition of a Raf-1-SCD1 protein complex induces an intracellular accumulation of saturated free fatty acids leading to lethal lipotoxicity in tumour cells and allows for an efficient treatment of liver carcinomas.
Subject(s)
Liver Neoplasms/therapy , Liver X Receptors/therapeutic use , HumansABSTRACT
The success of molecular therapies targeting specific metabolic pathways in cancer is often limited by the plasticity and adaptability of metabolic networks. Here we show that pharmacologically induced lipotoxicity represents a promising therapeutic strategy for the treatment of hepatocellular carcinoma (HCC). LXRα-induced liponeogenesis and Raf-1 inhibition are synthetic lethal in HCC owing to a toxic accumulation of saturated fatty acids. Raf-1 was found to bind and activate SCD1, and conformation-changing DFG-out Raf inhibitors could disrupt this interaction, thereby blocking fatty acid desaturation and inducing lethal lipotoxicity. Studies in genetically engineered and nonalcoholic steatohepatitis-induced HCC mouse models and xenograft models of human HCC revealed that therapies comprising LXR agonists and Raf inhibitors were well tolerated and capable of overcoming therapy resistance in HCC. Conceptually, our study suggests pharmacologically induced lipotoxicity as a new mode for metabolic targeting of liver cancer.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Non-alcoholic Fatty Liver Disease , Animals , Carcinoma, Hepatocellular/drug therapy , Disease Models, Animal , Fatty Acids/metabolism , Humans , Liver Neoplasms/drug therapy , Mice , Non-alcoholic Fatty Liver Disease/metabolismABSTRACT
MYC oncoproteins are involved in the genesis and maintenance of the majority of human tumors but are considered undruggable. By using a direct in vivo shRNA screen, we show that liver cancer cells that have mutations in the gene encoding the tumor suppressor protein p53 (Trp53 in mice and TP53 in humans) and that are driven by the oncoprotein NRAS become addicted to MYC stabilization via a mechanism mediated by aurora kinase A (AURKA). This MYC stabilization enables the tumor cells to overcome a latent G2/M cell cycle arrest that is mediated by AURKA and the tumor suppressor protein p19(ARF). MYC directly binds to AURKA, and inhibition of this protein-protein interaction by conformation-changing AURKA inhibitors results in subsequent MYC degradation and cell death. These conformation-changing AURKA inhibitors, with one of them currently being tested in early clinical trials, suppressed tumor growth and prolonged survival in mice bearing Trp53-deficient, NRAS-driven MYC-expressing hepatocellular carcinomas (HCCs). TP53-mutated human HCCs revealed increased AURKA expression and a positive correlation between AURKA and MYC expression. In xenograft models, mice bearing TP53-mutated or TP53-deleted human HCCs were hypersensitive to treatment with conformation-changing AURKA inhibitors, thus suggesting a therapeutic strategy for this subgroup of human HCCs.
Subject(s)
Aurora Kinase A/metabolism , Carcinoma, Hepatocellular/genetics , Hepatocytes/metabolism , Liver Neoplasms, Experimental/genetics , Liver Neoplasms/genetics , Monomeric GTP-Binding Proteins/genetics , Proto-Oncogene Proteins c-myc/metabolism , Tumor Suppressor Protein p53/genetics , Animals , Aurora Kinase A/antagonists & inhibitors , Azepines/pharmacology , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/metabolism , Cell Cycle Checkpoints , Cyclin-Dependent Kinase Inhibitor p16/genetics , Gene Deletion , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/metabolism , Liver Neoplasms, Experimental/drug therapy , Liver Neoplasms, Experimental/metabolism , Mice , Mice, Knockout , Molecular Targeted Therapy , Mutation , Oncogene Protein p21(ras)/metabolism , Phenylurea Compounds/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , RNA, Small Interfering , Xenograft Model Antitumor AssaysABSTRACT
In solid tumors, resistance to therapy inevitably develops upon treatment with cytotoxic drugs or molecularly targeted therapies. Here, we describe a system that enables pooled shRNA screening directly in mouse hepatocellular carcinomas (HCC) in vivo to identify genes likely to be involved in therapy resistance. Using a focused shRNA library targeting genes located within focal genomic amplifications of human HCC, we screened for genes whose inhibition increased the therapeutic efficacy of the multikinase inhibitor sorafenib. Both shRNA-mediated and pharmacological silencing of Mapk14 (p38α) were found to sensitize mouse HCC to sorafenib therapy and prolong survival by abrogating Mapk14-dependent activation of Mek-Erk and Atf2 signaling. Elevated Mapk14-Atf2 signaling predicted poor response to sorafenib therapy in human HCC, and sorafenib resistance of p-Mapk14-expressing HCC cells could be reverted by silencing Mapk14. Our results suggest that a combination of sorafenib and Mapk14 blockade is a promising approach to overcoming therapy resistance of human HCC.
Subject(s)
Antineoplastic Agents/pharmacology , Liver Neoplasms, Experimental/drug therapy , Liver Neoplasms, Experimental/genetics , Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Mitogen-Activated Protein Kinase 14/genetics , Niacinamide/analogs & derivatives , Phenylurea Compounds/pharmacology , Activating Transcription Factor 2/metabolism , Animals , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Drug Resistance, Neoplasm/genetics , Female , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms, Experimental/metabolism , MAP Kinase Signaling System/drug effects , Male , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 14/metabolism , Niacinamide/pharmacology , Protein Kinase Inhibitors/pharmacology , RNA Interference , RNA, Small Interfering/genetics , Signal Transduction , Sorafenib , Xenograft Model Antitumor AssaysABSTRACT
The liver harbors a distinct capacity for endogenous regeneration; however, liver regeneration is often impaired in disease and therefore insufficient to compensate for the loss of hepatocytes and organ function. Here we describe a functional genetic approach for the identification of gene targets that can be exploited to increase the regenerative capacity of hepatocytes. Pools of small hairpin RNAs (shRNAs) were directly and stably delivered into mouse livers to screen for genes modulating liver regeneration. Our studies identify the dual-specific kinase MKK4 as a master regulator of liver regeneration. MKK4 silencing robustly increased the regenerative capacity of hepatocytes in mouse models of liver regeneration and acute and chronic liver failure. Mechanistically, induction of MKK7 and a JNK1-dependent activation of the AP1 transcription factor ATF2 and the Ets factor ELK1 are crucial for increased regeneration of hepatocytes with MKK4 silencing.
Subject(s)
Cell Differentiation , Hepatocytes/cytology , Hepatocytes/physiology , Liver/physiology , MAP Kinase Kinase 4/genetics , Animals , Cell Cycle , DNA Transposable Elements , Fibrosis , Gene Knockdown Techniques , Hydrolases/genetics , Hydrolases/metabolism , Liver/cytology , Liver/injuries , Liver/pathology , MAP Kinase Kinase 4/antagonists & inhibitors , MAP Kinase Kinase 4/metabolism , Mice , RNA Interference , RNA, Small Interfering/metabolismABSTRACT
Deregulated expression of the MYC oncoprotein contributes to the genesis of many human tumours, yet strategies to exploit this for a rational tumour therapy are scarce. MYC promotes cell growth and proliferation, and alters cellular metabolism to enhance the provision of precursors for phospholipids and cellular macromolecules. Here we show in human and murine cell lines that oncogenic levels of MYC establish a dependence on AMPK-related kinase 5 (ARK5; also known as NUAK1) for maintaining metabolic homeostasis and for cell survival. ARK5 is an upstream regulator of AMPK and limits protein synthesis via inhibition of the mammalian target of rapamycin 1 (mTORC1) signalling pathway. ARK5 also maintains expression of mitochondrial respiratory chain complexes and respiratory capacity, which is required for efficient glutamine metabolism. Inhibition of ARK5 leads to a collapse of cellular ATP levels in cells expressing deregulated MYC, inducing multiple pro-apoptotic responses as a secondary consequence. Depletion of ARK5 prolongs survival in MYC-driven mouse models of hepatocellular carcinoma, demonstrating that targeting cellular energy homeostasis is a valid therapeutic strategy to eliminate tumour cells that express deregulated MYC.
Subject(s)
Gene Expression Regulation, Neoplastic , Genes, myc/genetics , Protein Kinases/metabolism , Repressor Proteins/metabolism , AMP-Activated Protein Kinases/metabolism , Adenosine Triphosphate/metabolism , Animals , Apoptosis , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Respiration , Cell Survival , Cell Transformation, Neoplastic/genetics , Disease Models, Animal , Doxycycline/pharmacology , Electron Transport , Glutamine/metabolism , Homeostasis , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Mechanistic Target of Rapamycin Complex 1 , Mice , Mitochondria/metabolism , Multiprotein Complexes , Oncogene Protein p55(v-myc)/genetics , Oncogene Protein p55(v-myc)/metabolism , Protein Biosynthesis , Protein Kinases/deficiency , Protein Kinases/genetics , Proteins/antagonists & inhibitors , Proteins/metabolism , RNA Interference , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/deficiency , Repressor Proteins/genetics , Signal Transduction , TOR Serine-Threonine Kinases/metabolismABSTRACT
Upon the aberrant activation of oncogenes, normal cells can enter the cellular senescence program, a state of stable cell-cycle arrest, which represents an important barrier against tumour development in vivo. Senescent cells communicate with their environment by secreting various cytokines and growth factors, and it was reported that this 'secretory phenotype' can have pro- as well as anti-tumorigenic effects. Here we show that oncogene-induced senescence occurs in otherwise normal murine hepatocytes in vivo. Pre-malignant senescent hepatocytes secrete chemo- and cytokines and are subject to immune-mediated clearance (designated as 'senescence surveillance'), which depends on an intact CD4(+) T-cell-mediated adaptive immune response. Impaired immune surveillance of pre-malignant senescent hepatocytes results in the development of murine hepatocellular carcinomas (HCCs), thus showing that senescence surveillance is important for tumour suppression in vivo. In accordance with these observations, ras-specific Th1 lymphocytes could be detected in mice, in which oncogene-induced senescence had been triggered by hepatic expression of Nras(G12V). We also found that CD4(+) T cells require monocytes/macrophages to execute the clearance of senescent hepatocytes. Our study indicates that senescence surveillance represents an important extrinsic component of the senescence anti-tumour barrier, and illustrates how the cellular senescence program is involved in tumour immune surveillance by mounting specific immune responses against antigens expressed in pre-malignant senescent cells.