RESUMO
Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention.
Assuntos
Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/química , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Benzamidas , Células Cultivadas , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Isoleucina/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/farmacologia , Transdução de Sinais , Domínios de Homologia de srcRESUMO
Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.
Assuntos
Histona Acetiltransferases/metabolismo , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Linhagem Celular Tumoral , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Chromosomal rearrangements involving the MDS1 and EVI1 complex locus (MECOM) on chromosome 3q26 define an aggressive subtype of acute myeloid leukemia (AML) that is associated with chemotherapy resistance and dismal prognosis. Established treatment regimens commonly fail in these patients, therefore, there is an urgent need for new therapeutic concepts that will require a better understanding of the molecular and cellular functions of the ecotropic viral integration site 1 (EVI1) oncogene. To characterize gene regulatory functions of EVI1 and associated dependencies in AML, we developed experimentally tractable human and murine disease models, investigated the transcriptional consequences of EVI1 withdrawal in vitro and in vivo, and performed the first genome-wide CRISPR screens in EVI1-dependent AML. By integrating conserved transcriptional targets with genetic dependency data, we identified and characterized the ETS transcription factor ERG as a direct transcriptional target of EVI1 that is aberrantly expressed and selectively required in both human and murine EVI1-driven AML. EVI1 controls the expression of ERG and occupies a conserved intragenic enhancer region in AML cell lines and samples from patients with primary AML. Suppression of ERG induces terminal differentiation of EVI1-driven AML cells, whereas ectopic expression of ERG abrogates their dependence on EVI1, indicating that the major oncogenic functions of EVI1 are mediated through aberrant transcriptional activation of ERG. Interfering with this regulatory axis may provide entry points for the development of rational targeted therapies.
Assuntos
Proteínas de Ligação a DNA , Leucemia Mieloide Aguda , Humanos , Animais , Camundongos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína do Locus do Complexo MDS1 e EVI1/genética , Proto-Oncogenes/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Carcinogênese/genética , Regulador Transcricional ERG/genéticaRESUMO
Gain-of-function mutations in the signal transducer and activator of transcription 3 (STAT3) gene are recurrently identified in patients with large granular lymphocytic leukemia (LGLL) and in some cases of natural killer (NK)/T-cell and adult T-cell leukemia/lymphoma. To understand the consequences and molecular mechanisms contributing to disease development and oncogenic transformation, we developed murine hematopoietic stem and progenitor cell models that express mutated STAT3Y640F. These cells show accelerated proliferation and enhanced self-renewal potential. We integrated gene expression analyses and chromatin occupancy profiling of STAT3Y640F-transformed cells with data from patients with T-LGLL. This approach uncovered a conserved set of direct transcriptional targets of STAT3Y640F. Among these, strawberry notch homolog 2 (SBNO2) represents an essential transcriptional target, which was identified by a comparative genome-wide CRISPR/Cas9-based loss-of-function screen. The STAT3-SBNO2 axis is also present in NK-cell leukemia, T-cell non-Hodgkin lymphoma, and NPM-ALK-rearranged T-cell anaplastic large cell lymphoma (T-ALCL), which are driven by STAT3-hyperactivation/mutation. In patients with NPM-ALK+ T-ALCL, high SBNO2 expression correlates with shorter relapse-free and overall survival. Our findings identify SBNO2 as a potential therapeutic intervention site for STAT3-driven hematopoietic malignancies.
Assuntos
Neoplasias Hematológicas , Fator de Transcrição STAT3 , Animais , Humanos , Camundongos , Quinase do Linfoma Anaplásico/metabolismo , Linhagem Celular Tumoral , Neoplasias Hematológicas/genética , Linfoma Anaplásico de Células Grandes/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.
Assuntos
Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-fes/química , Sequência de Aminoácidos , Cristalografia por Raios X , Ativação Enzimática , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas c-fes/metabolismoRESUMO
Fusion proteins involving Nucleoporin 98 (NUP98) are recurrently found in acute myeloid leukemia (AML) and are associated with poor prognosis. Lack of mechanistic insight into NUP98-fusion-dependent oncogenic transformation has so far precluded the development of rational targeted therapies. We reasoned that different NUP98-fusion proteins deregulate a common set of transcriptional targets that might be exploitable for therapy. To decipher transcriptional programs controlled by diverse NUP98-fusion proteins, we developed mouse models for regulatable expression of NUP98/NSD1, NUP98/JARID1A, and NUP98/DDX10. By integrating chromatin occupancy profiles of NUP98-fusion proteins with transcriptome profiling upon acute fusion protein inactivation in vivo, we defined the core set of direct transcriptional targets of NUP98-fusion proteins. Among those, CDK6 was highly expressed in murine and human AML samples. Loss of CDK6 severely attenuated NUP98-fusion-driven leukemogenesis, and NUP98-fusion AML was sensitive to pharmacologic CDK6 inhibition in vitro and in vivo. These findings identify CDK6 as a conserved, critical direct target of NUP98-fusion proteins, proposing CDK4/CDK6 inhibitors as a new rational treatment option for AML patients with NUP98-fusions.
Assuntos
Quinase 6 Dependente de Ciclina/metabolismo , Sistemas de Liberação de Medicamentos , Leucemia Mieloide Aguda/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Animais , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/genética , Perfilação da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genéticaRESUMO
Mutations in the CEBPA gene are present in 10-15% of acute myeloid leukemia (AML) patients. The most frequent type of mutations leads to the expression of an N-terminally truncated variant of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα), termed p30. While initial reports proposed that p30 represents a dominant-negative version of the wild-type C/EBPα protein, other studies show that p30 retains the capacity to actively regulate gene expression. Recent global transcriptomic and epigenomic analyses have advanced the understanding of the distinct roles of the p30 isoform in leukemogenesis. This review outlines direct and indirect effects of the C/EBPα p30 variant on oncogenic transformation of hematopoietic progenitor cells and discusses how studies of N-terminal CEBPA mutations in AML can be extrapolated to identify novel gain-of-function features in oncoproteins that arise from recurrent truncating mutations in transcription factors.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Mutação com Ganho de Função/genética , Leucemia Mieloide Aguda/genética , Animais , Regulação Neoplásica da Expressão Gênica/genética , Células-Tronco Hematopoéticas/fisiologia , HumanosRESUMO
Histone methyltransferases (HMTs) have attracted considerable attention as potential targets for pharmaceutical intervention in various malignant diseases. These enzymes are known for introducing methyl marks at specific locations of histone proteins, creating a complex system that regulates epigenetic control of gene expression and cell differentiation. Here, we describe the identification of first-generation cell-permeable non-nucleoside type inhibitors of SETD2, the only mammalian HMT that is able to tri-methylate the K36 residue of histone H3. By generating the epigenetic mark H3K36me3, SETD2 is involved in the progression of acute myeloid leukemia. We developed a structure-based virtual screening protocol that was first validated in retrospective studies. Next, prospective screening was performed on a large library of commercially available compounds. Experimental validation of 22 virtual hits led to the discovery of three compounds that showed dose-dependent inhibition of the enzymatic activity of SETD2. Compound C13 effectively blocked the proliferation of two acute myeloid leukemia (AML) cell lines with MLL rearrangements and led to decreased H3K36me3 levels, prioritizing this chemotype as a viable chemical starting point for drug discovery projects.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Descoberta de Drogas , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Algoritmos , Área Sob a Curva , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Química Farmacêutica/métodos , Bases de Dados Factuais , Progressão da Doença , Epigênese Genética , Histonas/metabolismo , Humanos , Concentração Inibidora 50 , Leucemia Mieloide Aguda/enzimologia , Ligantes , Mutação , Preparações Farmacêuticas , Reprodutibilidade dos TestesRESUMO
Recurrent gain-of-function mutations in the transcription factors STAT5A and much more in STAT5B were found in hematopoietic malignancies with the highest proportion in mature T- and natural killer-cell neoplasms (peripheral T-cell lymphoma, PTCL). No targeted therapy exists for these heterogeneous and often aggressive diseases. Given the shortage of models for PTCL, we mimicked graded STAT5A or STAT5B activity by expressing hyperactive Stat5a or STAT5B variants at low or high levels in the hematopoietic system of transgenic mice. Only mice with high activity levels developed a lethal disease resembling human PTCL. Neoplasia displayed massive expansion of CD8+ T cells and destructive organ infiltration. T cells were cytokine-hypersensitive with activated memory CD8+ T-lymphocyte characteristics. Histopathology and mRNA expression profiles revealed close correlation with distinct subtypes of PTCL. Pronounced STAT5 expression and activity in samples from patients with different subsets underline the relevance of JAK/STAT as a therapeutic target. JAK inhibitors or a selective STAT5 SH2 domain inhibitor induced cell death and ruxolitinib blocked T-cell neoplasia in vivo We conclude that enhanced STAT5A or STAT5B action both drive PTCL development, defining both STAT5 molecules as targets for therapeutic intervention.
Assuntos
Leucemia , Linfoma de Células T Periférico , Animais , Linfócitos T CD8-Positivos/metabolismo , Citocinas , Humanos , Linfoma de Células T Periférico/genética , Camundongos , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Proteínas Supressoras de TumorRESUMO
Ants are emerging model systems to study cellular signaling because distinct castes possess different physiologic phenotypes within the same colony. Here we studied the functionality of inotocin signaling, an insect ortholog of mammalian oxytocin (OT), which was recently discovered in ants. In Lasius ants, we determined that specialization within the colony, seasonal factors, and physiologic conditions down-regulated the expression of the OT-like signaling system. Given this natural variation, we interrogated its function using RNAi knockdowns. Next-generation RNA sequencing of OT-like precursor knock-down ants highlighted its role in the regulation of genes involved in metabolism. Knock-down ants exhibited higher walking activity and increased self-grooming in the brood chamber. We propose that OT-like signaling in ants is important for regulating metabolic processes and locomotion.-Liutkeviciute, Z., Gil-Mansilla, E., Eder, T., Casillas-Pérez, B., Di Giglio, M. G., Muratspahic, E., Grebien, F., Rattei, T., Muttenthaler, M., Cremer, S., Gruber, C. W. Oxytocin-like signaling in ants influences metabolic gene expression and locomotor activity.
RESUMO
The non-redundant histone methyltransferase SETD2 (SET domain containing 2; KMT3A) is responsible for tri-methylation of lysine 36 on histone H3 (H3K36me3). Presence of the H3K36me3 histone mark across the genome has been correlated with transcriptional activation and elongation, but also with the regulation of DNA mismatch repair, homologous recombination and alternative splicing. The role of SETD2 and the H3K36me3 histone mark in cancer is controversial. SETD2 is lost or mutated in various cancers, supporting a tumor suppressive role of the protein. Alterations in the SETD2 gene are also present in leukemia patients, where they are associated with aggressive disease and relapse. In line, heterozygous SETD2 loss caused chemotherapy resistance in leukemia cell lines and mouse models. In contrast, other studies indicate that SETD2 is critically required for the proliferation of leukemia cells. Thus, although studies of SETD2-dependent processes in cancer have contributed to a better understanding of the SETD2â»H3K36me3 axis, many open questions remain regarding its specific role in leukemia. Here, we review the current literature about critical functions of SETD2 in the context of hematopoietic malignancies.
Assuntos
Dano ao DNA , Histona-Lisina N-Metiltransferase/metabolismo , Leucemia/genética , Transcrição Gênica , Animais , Carcinogênese/genética , Carcinogênese/patologia , Humanos , Leucemia/patologia , Leucemia/terapia , Mutação/genéticaRESUMO
MOTIVATION: Measuring differential gene expression is a common task in the analysis of RNA-Seq data. To identify differentially expressed genes between two samples, it is crucial to normalize the datasets. While multiple normalization methods are available, all of them are based on certain assumptions that may or may not be suitable for the type of data they are applied on. Researchers therefore need to select an adequate normalization strategy for each RNA-Seq experiment. This selection includes exploration of different normalization methods as well as their comparison. Methods that agree with each other most likely represent realistic assumptions under the particular experimental conditions. RESULTS: We developed the NVT package, which provides a fast and simple way to analyze and evaluate multiple normalization methods via visualization and representation of correlation values, based on a user-defined set of uniformly expressed genes. AVAILABILITY AND IMPLEMENTATION: The R package is freely available under https://github.com/Edert/NVT CONTACT: thomas.rattei@univie.ac.atSupplementary information: Supplementary data are available at Bioinformatics online.
Assuntos
RNA/genética , Análise de Sequência de RNA/métodos , Software , Biologia Computacional/métodos , HumanosRESUMO
The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short (30-kDa) CCAAT-enhancer binding protein-α (C/EBPα) translational isoform, termed p30, represents the most common type of CEBPA mutation in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPα p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL (SET-domain/mixed-lineage leukemia) histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as downregulation of the latter inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a new small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30 cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML.
Assuntos
Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Di-Hidropiridinas/farmacologia , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Sequência de Aminoácidos , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Terapia de Alvo Molecular , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Células Tumorais CultivadasRESUMO
The treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) induces granulocytic differentiation. This process renders APL cells resistant to cytotoxic chemotherapies. Epigenetic regulators of the histone deacetylases (HDACs) family, which comprise four classes (I-IV), critically control the development and progression of APL. We set out to clarify the parameters that determine the interaction between ATRA and histone deacetylase inhibitors (HDACi). Our assays included drugs against class I HDACs (MS-275, VPA, and FK228), pan-HDACi (LBH589, SAHA), and the novel HDAC6-selective compound Marbostat-100. We demonstrate that ATRA protects APL cells from cytotoxic effects of SAHA, MS-275, and Marbostat-100. However, LBH589 and FK228, which have a superior substrate-inhibitor dissociation constant (Ki) for the class I deacetylases HDAC1, 2, 3, are resistant against ATRA-dependent cytoprotective effects. We further show that HDACi evoke DNA damage, measured as induction of phosphorylated histone H2AX and by the comet assay. The ability of ATRA to protect APL cells from the induction of p-H2AX by HDACi is a readout for the cytoprotective effects of ATRA. Moreover, ATRA increases the fraction of cells in the G1 phase, together with an accumulation of the cyclin-dependent kinase inhibitor p21 and a reduced expression of thymidylate synthase (TdS). In contrast, the ATRA-dependent activation of the transcription factors STAT1, NF-κB, and C/EBP hardly influences the responses of APL cells to HDACi. We conclude that the affinity of HDACi for class I HDACs determines whether such drugs can kill naïve and maturated APL cells.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Leucemia/tratamento farmacológico , Leucemia/patologia , Tretinoína/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Leucemia/metabolismo , NF-kappa B/metabolismo , Piridinas/farmacologia , Fator de Transcrição STAT1/metabolismo , Tretinoína/administração & dosagemRESUMO
In the past decades, studies of the Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) signaling have uncovered highly conserved programs linking cytokine signaling to the regulation of essential cellular mechanisms such as proliferation, invasion, survival, inflammation and immunity. Inhibitors of the JAK/STAT pathway are used for treatment of autoimmune diseases, such as rheumatoid arthritis or psoriasis. Aberrant JAK/STAT signaling has been identified to contribute to cancer progression and metastatic development. Targeting of JAK/STAT pathway is currently one of the most promising therapeutic strategies in prostate cancer (PCa), hematopoietic malignancies and sarcomas. Notably, newly identified regulators of JAK/STAT signaling, the non-coding RNAs transcripts and their role as important targets and potential clinical biomarkers are highlighted in this review. In addition to the established role of the JAK/STAT signaling pathway in traditional cytokine signaling the non-coding RNAs add yet another layer of hidden regulation and function. Understanding the crosstalk of non-coding RNA with JAK/STAT signaling in cancer is of critical importance and may result in better patient stratification not only in terms of prognosis but also in the context of therapy.
Assuntos
Citocinas/metabolismo , Janus Quinases/metabolismo , Neoplasias/metabolismo , RNA não Traduzido/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Animais , Genoma , Humanos , Masculino , Camundongos , Neoplasias/terapia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/terapia , Sarcoma/metabolismo , Sarcoma/terapiaRESUMO
We hypothesized that elucidating the interactome of epidermal growth factor receptor (EGFR) forms that are mutated in lung cancer, via global analysis of protein-protein interactions, phosphorylation, and systematically perturbing the ensuing network nodes, should offer a new, more systems-level perspective of the molecular etiology. Here, we describe an EGFR interactome of 263 proteins and offer a 14-protein core network critical to the viability of multiple EGFR-mutated lung cancer cells. Cells with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) had differential dependence of the core network proteins based on the underlying molecular mechanisms of resistance. Of the 14 proteins, 9 are shown to be specifically associated with survival of EGFR-mutated lung cancer cell lines. This included EGFR, GRB2, MK12, SHC1, ARAF, CD11B, ARHG5, GLU2B, and CD11A. With the use of a drug network associated with the core network proteins, we identified two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib. Our results, enabled by interactome mapping, suggest new targets and combination therapies that could circumvent EGFR TKI resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Mutação , Proteínas de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Carbazóis/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Cloridrato de Erlotinib , Furanos , Humanos , Proteínas de Neoplasias/genética , Fosforilação , Mapas de Interação de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Estaurosporina/análogos & derivados , Estaurosporina/farmacologiaRESUMO
Constitutive activation of STAT5 is critical for the maintenance of chronic myeloid leukemia (CML) characterized by the BCR-ABL oncoprotein. Tyrosine kinase inhibitors (TKIs) for the STAT5-activating kinase JAK2 have been discussed as a treatment option for CML patients. Using murine leukemia models combined with inducible ablation of JAK2, we show JAK2 dependence for initial lymphoid transformation, which is lost once leukemia is established. In contrast, initial myeloid transformation and leukemia maintenance were independent of JAK2. Nevertheless, several JAK2 TKIs induced apoptosis in BCR-ABL(+) cells irrespective of the presence of JAK2. This is caused by the previously unknown direct 'off-target' inhibition of BCR-ABL. Cellular and enzymatic analyses suggest that BCR-ABL phosphorylates STAT5 directly. Our findings suggest uncoupling of the canonical JAK2-STAT5 module upon BCR-ABL expression, thereby making JAK2 targeting dispensable. Thus, attempts to pharmacologically target STAT5 in BCR-ABL(+) diseases need to focus on STAT5 itself.
Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Janus Quinase 2/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Animais , Antineoplásicos/farmacologia , Benzamidas , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Células HEK293 , Humanos , Mesilato de Imatinib , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/deficiência , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Piperazinas/farmacologia , Pirimidinas/farmacologia , Fator de Transcrição STAT5/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Células U937RESUMO
Occurrence of the BCR-ABL(T315I) gatekeeper mutation is among the most pressing challenges in the therapy of chronic myeloid leukemia (CML). Several BCR-ABL inhibitors have multiple targets and pleiotropic effects that could be exploited for their synergistic potential. Testing combinations of such kinase inhibitors identified a strong synergy between danusertib and bosutinib that exclusively affected CML cells harboring BCR-ABL(T315I). To elucidate the underlying mechanisms, we applied a systems-level approach comprising phosphoproteomics, transcriptomics and chemical proteomics. Data integration revealed that both compounds targeted Mapk pathways downstream of BCR-ABL, resulting in impaired activity of c-Myc. Using pharmacological validation, we assessed that the relative contributions of danusertib and bosutinib could be mimicked individually by Mapk inhibitors and collectively by downregulation of c-Myc through Brd4 inhibition. Thus, integration of genome- and proteome-wide technologies enabled the elucidation of the mechanism by which a new drug synergy targets the dependency of BCR-ABL(T315I) CML cells on c-Myc through nonobvious off targets.
Assuntos
Compostos de Anilina/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzamidas/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Nitrilas/farmacologia , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Quinolinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Proteômica , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Relação Estrutura-Atividade , Biologia de SistemasRESUMO
The persistent growth of cancer cells is underscored by complex metabolic reprogramming, with mitochondria playing a key role in the transition to aerobic glycolysis and representing new therapeutic targets. Mitochondrial uncoupling protein 2 (UCP2) has attracted interest because of its abundance in rapidly proliferating cells, including cancer cells, and its involvement in cellular metabolism. However, the specific contributions of UCP2 to cancer biology remain poorly defined. Our investigation of UCP2 expression in various human and mouse cancer cell lines aimed to elucidate its links to metabolic states, proliferation, and adaptation to environmental stresses such as hypoxia and nutrient deprivation. We observed significant variability in UCP2 expression across cancer types, with no direct correlation to their metabolic activity or proliferation rates. UCP2 abundance was also differentially affected by nutrient availability in different cancer cells, but UCP2 was generally downregulated under hypoxia. These findings challenge the notion that UCP2 is a marker of malignant potential and suggest its more complex involvement in the metabolic landscape of cancer.
RESUMO
Signal transducer and activator of transcription 3 (STAT3) is frequently overexpressed in patients with acute myeloid leukemia (AML). STAT3 exists in two distinct alternatively spliced isoforms, the full-length isoform STAT3α and the C-terminally truncated isoform STAT3ß. While STAT3α is predominantly described as an oncogenic driver, STAT3ß has been suggested to act as a tumor suppressor. To elucidate the role of STAT3ß in AML, we established a mouse model of STAT3ß-deficient, MLL-AF9-driven AML. STAT3ß deficiency significantly shortened survival of leukemic mice confirming its role as a tumor suppressor. Furthermore, RNA sequencing revealed enhanced STAT1 expression and interferon (IFN) signaling upon loss of STAT3ß. Accordingly, STAT3ß-deficient leukemia cells displayed enhanced sensitivity to blockade of IFN signaling through both an IFNAR1 blocking antibody and the JAK1/2 inhibitor Ruxolitinib. Analysis of human AML patient samples confirmed that elevated expression of IFN-inducible genes correlated with poor overall survival and low STAT3ß expression. Together, our data corroborate the tumor suppressive role of STAT3ß in a mouse model in vivo. Moreover, they provide evidence that its tumor suppressive function is linked to repression of the STAT1-mediated IFN response. These findings suggest that the STAT3ß/α mRNA ratio is a significant prognostic marker in AML and holds crucial information for targeted treatment approaches. Patients displaying a low STAT3ß/α mRNA ratio and unfavorable prognosis could benefit from therapeutic interventions directed at STAT1/IFN signaling.