RESUMO
Many oncogenic insults deregulate RNA splicing, often leading to hypersensitivity of tumors to spliceosome-targeted therapies (STTs). However, the mechanisms by which STTs selectively kill cancers remain largely unknown. Herein, we discover that mis-spliced RNA itself is a molecular trigger for tumor killing through viral mimicry. In MYC-driven triple-negative breast cancer, STTs cause widespread cytoplasmic accumulation of mis-spliced mRNAs, many of which form double-stranded structures. Double-stranded RNA (dsRNA)-binding proteins recognize these endogenous dsRNAs, triggering antiviral signaling and extrinsic apoptosis. In immune-competent models of breast cancer, STTs cause tumor cell-intrinsic antiviral signaling, downstream adaptive immune signaling, and tumor cell death. Furthermore, RNA mis-splicing in human breast cancers correlates with innate and adaptive immune signatures, especially in MYC-amplified tumors that are typically immune cold. These findings indicate that dsRNA-sensing pathways respond to global aberrations of RNA splicing in cancer and provoke the hypothesis that STTs may provide unexplored strategies to activate anti-tumor immune pathways.
Assuntos
Antivirais/farmacologia , Imunidade/efeitos dos fármacos , Spliceossomos/metabolismo , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/patologia , Imunidade Adaptativa/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Feminino , Amplificação de Genes/efeitos dos fármacos , Humanos , Íntrons/genética , Camundongos , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-myc/metabolismo , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , RNA de Cadeia Dupla/metabolismo , Transdução de Sinais/efeitos dos fármacos , Spliceossomos/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of T cell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Proteínas Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Elongação da Transcrição Genética , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular , Quinase 9 Dependente de Ciclina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Feminino , Regulação Leucêmica da Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Células Jurkat , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Complexos Multiproteicos , Proteínas Nucleares/genética , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Estabilidade Proteica , Proteólise , RNA Polimerase II/metabolismo , Fatores de Tempo , Elongação da Transcrição Genética/efeitos dos fármacos , Fatores de Transcrição/genética , Transfecção , Ubiquitina-Proteína Ligases , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Small-molecule inhibitors of translation are critical tools to study the molecular mechanisms of protein synthesis. In this study, we sought to characterize how QL47, a host-targeted, small-molecule antiviral agent, inhibits steady-state viral protein expression. We demonstrate that this small molecule broadly inhibits both viral and host protein synthesis and targets a translation step specific to eukaryotic cells. We show that QL47 inhibits protein neosynthesis initiated by both canonical cap-driven and noncanonical initiation strategies, most likely by targeting an early step in translation elongation. Our findings thus establish QL47 as a new small-molecule inhibitor that can be utilized to probe the eukaryotic translation machinery and that can be further developed as a new therapeutic agent.
Assuntos
Antivirais/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Antivirais/química , Linhagem Celular , Células HEK293 , Humanos , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Proteínas Virais/metabolismo , Viroses/tratamento farmacológico , Viroses/metabolismo , Vírus/efeitos dos fármacos , Vírus/metabolismoRESUMO
Cyclin-dependent kinase 9 (CDK9), an important regulator of transcriptional elongation, is a promising target for cancer therapy, particularly for cancers driven by transcriptional dysregulation. We characterized NVP-2, a selective ATP-competitive CDK9 inhibitor, and THAL-SNS-032, a selective CDK9 degrader consisting of a CDK-binding SNS-032 ligand linked to a thalidomide derivative that binds the E3 ubiquitin ligase Cereblon (CRBN). To our surprise, THAL-SNS-032 induced rapid degradation of CDK9 without affecting the levels of other SNS-032 targets. Moreover, the transcriptional changes elicited by THAL-SNS-032 were more like those caused by NVP-2 than those induced by SNS-032. Notably, compound washout did not significantly reduce levels of THAL-SNS-032-induced apoptosis, suggesting that CDK9 degradation had prolonged cytotoxic effects compared with CDK9 inhibition. Thus, our findings suggest that thalidomide conjugation represents a promising strategy for converting multi-targeted inhibitors into selective degraders and reveal that kinase degradation can induce distinct pharmacological effects compared with inhibition.
Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/química , Peptídeo Hidrolases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Cristalografia por Raios X , Humanos , Concentração Inibidora 50 , Ligantes , Oxazóis/farmacologia , Fosforilação , Ligação Proteica , Conformação Proteica , Proteômica , Talidomida/farmacologia , Tiazóis/farmacologia , Ubiquitina-Proteína LigasesRESUMO
Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.
Assuntos
Anilidas/farmacologia , Proteína Quinase CDC2/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Cisteína/química , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Anilidas/síntese química , Anilidas/química , Proteína Quinase CDC2/química , Proteína Quinase CDC2/metabolismo , Morte Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Cisteína/metabolismo , Dano ao DNA , Humanos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirimidinas/síntese química , Pirimidinas/químicaRESUMO
More than 60% of supratentorial ependymomas harbor a ZFTA-RELA (ZRfus) gene fusion (formerly C11orf95-RELA). To study the biology of ZRfus, we developed an autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain. Integrative epigenomic and transcriptomic mapping was performed on IUE-driven ZRfus tumors by CUT&RUN, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin sequencing, and RNA sequencing and compared with human ZRfus-driven ependymoma. In addition to direct canonical NFκB pathway activation, ZRfus dictates a neoplastic transcriptional program and binds to thousands of unique sites across the genome that are enriched with PLAGL family transcription factor (TF) motifs. ZRfus activates gene expression programs through recruitment of transcriptional coactivators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZRfus target genes converge on developmental programs marked by PLAGL TF proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks. SIGNIFICANCE: Ependymomas are aggressive brain tumors. Although drivers of supratentorial ependymoma (ZFTA- and YAP1-associated gene fusions) have been discovered, their functions remain unclear. Our study investigates the biology of ZFTA-RELA-driven ependymoma, specifically mechanisms of transcriptional deregulation and direct downstream gene networks that may be leveraged for potential therapeutic testing.This article is highlighted in the In This Issue feature, p. 2113.
Assuntos
Proteínas de Ligação a DNA/genética , Ependimoma/genética , Neoplasias Supratentoriais/genética , Fator de Transcrição RelA/genética , Fatores de Transcrição/genética , Animais , Modelos Animais de Doenças , Ependimoma/patologia , Camundongos , Neoplasias Supratentoriais/patologiaRESUMO
Castration-resistant prostate cancers (CRPCs) lose sensitivity to androgen-deprivation therapies but frequently remain dependent on oncogenic transcription driven by the androgen receptor (AR) and its splice variants. To discover modulators of AR-variant activity, we used a lysate-based small-molecule microarray assay and identified KI-ARv-03 as an AR-variant complex binder that reduces AR-driven transcription and proliferation in prostate cancer cells. We deduced KI-ARv-03 to be a potent, selective inhibitor of CDK9, an important cofactor for AR, MYC, and other oncogenic transcription factors. Further optimization resulted in KB-0742, an orally bioavailable, selective CDK9 inhibitor with potent anti-tumor activity in CRPC models. In 22Rv1 cells, KB-0742 rapidly downregulates nascent transcription, preferentially depleting short half-life transcripts and AR-driven oncogenic programs. In vivo, oral administration of KB-0742 significantly reduced tumor growth in CRPC, supporting CDK9 inhibition as a promising therapeutic strategy to target AR dependence in CRPC.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Receptores Androgênicos/genética , Transcrição Gênica/efeitos dos fármacos , Antagonistas de Receptores de Andrógenos/uso terapêutico , Animais , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Neoplasias de Próstata Resistentes à Castração/genética , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Genetic depletion of cyclin-dependent kinase 12 (CDK12) or selective inhibition of an analog-sensitive CDK12 reduces DNA damage repair gene expression, but selective inhibition of endogenous CDK12 is difficult. Here, we report the development of MFH290, a novel cysteine (Cys)-directed covalent inhibitor of CDK12/13. MFH290 forms a covalent bond with Cys-1039 of CDK12, exhibits excellent kinome selectivity, inhibits the phosphorylation of serine-2 in the C-terminal domain (CTD) of RNA-polymerase II (Pol II), and reduces the expression of key DNA damage repair genes. Importantly, these effects were demonstrated to be CDK12-dependent as mutation of Cys-1039 rendered the kinase refractory to MFH290 and restored Pol II CTD phosphorylation and DNA damage repair gene expression. Consistent with its effect on DNA damage repair gene expression, MFH290 augments the antiproliferative effect of the PARP inhibitor olaparib.
Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Proteína Quinase CDC2/química , Quinases Ciclina-Dependentes/química , Humanos , Células Jurkat , Modelos Moleculares , Conformação ProteicaRESUMO
The G1/S cell cycle checkpoint is frequently dysregulated in cancer, leaving cancer cells reliant on a functional G2/M checkpoint to prevent excessive DNA damage. Wee1 regulates the G2/M checkpoint by phosphorylating CDK1 at Tyr15 to prevent mitotic entry. Previous drug development efforts targeting Wee1 resulted in the clinical-grade inhibitor, AZD1775. However, AZD1775 is burdened by dose-limiting adverse events, and has off-target PLK1 activity. In an attempt to overcome these limitations, we developed Wee1 degraders by conjugating AZD1775 to the cereblon (CRBN)-binding ligand, pomalidomide. The resulting lead compound, ZNL-02-096, degrades Wee1 while sparing PLK1, induces G2/M accumulation at 10-fold lower doses than AZD1775, and synergizes with Olaparib in ovarian cancer cells. We demonstrate that ZNL-02-096 has CRBN-dependent pharmacology that is distinct from AZD1775, which justifies further evaluation of selective Wee1 degraders.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Desenvolvimento de Medicamentos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteólise/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Talidomida/análogos & derivados , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA , Feminino , Humanos , Estrutura Molecular , Ftalazinas/química , Ftalazinas/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/química , Pirazóis/química , Pirimidinonas/química , Talidomida/química , Talidomida/farmacologiaRESUMO
Cyclin-dependent kinase 7 (CDK7) regulates both cell cycle and transcription, but its precise role remains elusive. We previously described THZ1, a CDK7 inhibitor, which dramatically inhibits superenhancer-associated gene expression. However, potent CDK12/13 off-target activity obscured CDK7s contribution to this phenotype. Here, we describe the discovery of a highly selective covalent CDK7 inhibitor. YKL-5-124 causes arrest at the G1/S transition and inhibition of E2F-driven gene expression; these effects are rescued by a CDK7 mutant unable to covalently engage YKL-5-124, demonstrating on-target specificity. Unlike THZ1, treatment with YKL-5-124 resulted in no change to RNA polymerase II C-terminal domain phosphorylation; however, inhibition could be reconstituted by combining YKL-5-124 and THZ531, a selective CDK12/13 inhibitor, revealing potential redundancies in CDK control of gene transcription. These findings highlight the importance of CDK7/12/13 polypharmacology for anti-cancer activity of THZ1 and posit that selective inhibition of CDK7 may be useful for treatment of cancers marked by E2F misregulation.
Assuntos
Ciclo Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirróis/farmacologia , Ciclo Celular/genética , Linhagem Celular , Quinases Ciclina-Dependentes/metabolismo , Humanos , Células Jurkat , Masculino , Fenótipo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazóis/química , Pirróis/química , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. The stem cell potential of human epidermal keratinocytes is retained in vitro but lost over time suggesting extrinsic and intrinsic regulation. Transcription factor-controlled regulatory circuitries govern cell identity, are sufficient to induce pluripotency and transdifferentiate cells. We investigate whether transcriptional circuitry also governs phenotypic changes within a given cell type by comparing human primary keratinocytes with intrinsically high versus low stem cell potential. Using integrated chromatin and transcriptional profiling, we implicate IRF2 as antagonistic to stemness and show that it binds and regulates active cis-regulatory elements at interferon response and antigen presentation genes. CRISPR-KD of IRF2 in keratinocytes with low stem cell potential increases self-renewal, migration and epidermis formation. These data demonstrate that transcription factor regulatory circuitries, in addition to maintaining cell identity, control plasticity within cell types and offer potential for therapeutic modulation of cell function.
Assuntos
Fator Regulador 2 de Interferon/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Humanos , Fator Regulador 2 de Interferon/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Ativação Transcricional/fisiologiaRESUMO
Cdk7, the CDK-activating kinase and transcription factor IIH component, is a target of inhibitors that kill cancer cells by exploiting tumor-specific transcriptional dependencies. However, whereas selective inhibition of analog-sensitive (AS) Cdk7 in colon cancer-derived cells arrests division and disrupts transcription, it does not by itself trigger apoptosis efficiently. Here, we show that p53 activation by 5-fluorouracil or nutlin-3 synergizes with a reversible Cdk7as inhibitor to induce cell death. Synthetic lethality was recapitulated with covalent inhibitors of wild-type Cdk7, THZ1, or the more selective YKL-1-116. The effects were allele specific; a CDK7as mutation conferred both sensitivity to bulky adenine analogs and resistance to covalent inhibitors. Non-transformed colon epithelial cells were resistant to these combinations, as were cancer-derived cells with p53-inactivating mutations. Apoptosis was dependent on death receptor DR5, a p53 transcriptional target whose expression was refractory to Cdk7 inhibition. Therefore, p53 activation induces transcriptional dependency to sensitize cancer cells to Cdk7 inhibition.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Fenilenodiaminas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Imidazóis/farmacologia , Piperazinas/farmacologia , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Under conditions of stress, such as limited growth factor signaling, translation is inhibited by the action of 4E-BP and PDCD4. These proteins, through inhibition of eIF4E and eIF4A, respectively, impair cap-dependent translation. Under stress conditions FOXO transcription factors activate 4E-BP expression amplifying the repression. Here we show that Drosophila FOXO binds the PDCD4 promoter and stimulates the transcription of PDCD4 in response to stress. We have shown previously that the 5' UTR of the Drosophila insulin-like receptor (dINR) supports cap-independent translation that is resistant to 4E-BP. Using hippuristanol, an eIF4A inhibitor, we find that translation of dINR UTR containing transcripts are also resistant to eIF4A inhibition. In addition, the murine insulin receptor and insulin-like growth factor receptor 5' UTRs support cap-independent translation and have a similar resistance to hippuristanol. This resistance to inhibition of eIF4E and eIF4A indicates a conserved strategy to allow translation of growth factor receptors under stress conditions. DOI:http://dx.doi.org/10.7554/eLife.00542.001.
Assuntos
Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Receptor de Insulina/fisiologia , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Animais , Sequência Conservada , Drosophila , Dados de Sequência Molecular , Capuzes de RNA , Receptor de Insulina/química , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Heterotrimeric guanine nucleotide binding proteins of the G12/13 subfamily, which includes the α-subunits Gα12 and Gα13, stimulate the monomeric G protein RhoA through interaction with a distinct subset of Rho-specific guanine nucleotide exchange factors (RhoGEFs). The structural features that mediate interaction between Gα13 and RhoGEFs have been examined in crystallographic studies of the purified complex, whereas a Gα12:RhoGEF complex has not been reported. Several signaling responses and effector interactions appear unique to Gα12 or Gα13, despite their similarity in amino acid sequence. METHODS: To comprehensively examine Gα12 for regions involved in RhoGEF interaction, we screened a panel of Gα12 cassette substitution mutants for binding to leukemia-associated RhoGEF (LARG) and for activation of serum response element mediated transcription. RESULTS: We identified several cassette substitutions that disrupt Gα12 binding to LARG and the related p115RhoGEF. These Gα12 mutants also were impaired in activating serum response element mediated signaling, a Rho-dependent response. Most of these mutants matched corresponding regions of Gα13 reported to contact p115RhoGEF, but unexpectedly, several RhoGEF-uncoupling mutations were found within the N- and C-terminal regions of Gα12. Trypsin protection assays revealed several mutants in these regions as retaining conformational activation. In addition, charge substitutions near the Gα12 N-terminus selectively disrupted binding to LARG but not p115RhoGEF. CONCLUSIONS: Several structural aspects of the Gα12:RhoGEF interface differ from the reported Gα13:RhoGEF complex, particularly determinants within the C-terminal α5 helix and structurally uncharacterized N-terminus of Gα12. Furthermore, key residues at the Gα12 N-terminus may confer selectivity for LARG as a downstream effector.