RESUMEN
Inactivation of the p53 tumor suppressor by Mdm2 is one of the most frequent events in cancer, so compounds targeting the p53-Mdm2 interaction are promising for cancer therapy. Mechanisms conferring resistance to p53-reactivating compounds are largely unknown. Here we show using CRISPR-Cas9-based target validation in lung and colorectal cancer that the activity of nutlin, which blocks the p53-binding pocket of Mdm2, strictly depends on functional p53. In contrast, sensitivity to the drug RITA, which binds the Mdm2-interacting N terminus of p53, correlates with induction of DNA damage. Cells with primary or acquired RITA resistance display cross-resistance to DNA crosslinking compounds such as cisplatin and show increased DNA cross-link repair. Inhibition of FancD2 by RNA interference or pharmacological mTOR inhibitors restores RITA sensitivity. The therapeutic response to p53-reactivating compounds is therefore limited by compound-specific resistance mechanisms that can be resolved by CRISPR-Cas9-based target validation and should be considered when allocating patients to p53-reactivating treatments.
Asunto(s)
Sistemas CRISPR-Cas , Resistencia a Antineoplásicos/efectos de los fármacos , Furanos/farmacología , Genes p53 , Terapia Molecular Dirigida/métodos , Cisplatino/farmacología , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Regulación de la Expresión Génica , Genes p53/fisiología , Células HCT116/efectos de los fármacos , Humanos , Morfolinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
TP63, a member of the p53 gene family gene, encodes the ΔNp63 protein and is one of the most frequently amplified genes in squamous cell carcinomas (SCC) of the head and neck (HNSCC) and lungs (LUSC). Using an epiallelic series of siRNAs with intrinsically different knockdown abilities, we show that the complete loss of ΔNp63 strongly impaired cell proliferation, whereas partial ΔNp63 depletion rendered cells hypersensitive to cisplatin accompanied by an accumulation of DNA damage. Expression profiling revealed wide-spread transcriptional regulation of DNA repair genes and in particular Fanconi anemia (FA) pathway components such as FANCD2 and RAD18 - known to be crucial for the repair of cisplatin-induced interstrand crosslinks. In SCC patients ΔNp63 levels significantly correlate with FANCD2 and RAD18 expression confirming ΔNp63 as a key activator of the FA pathway in vivo Mechanistically, ΔNp63 bound an upstream enhancer of FANCD2 inactive in primary keratinocytes but aberrantly activated by ΔNp63 in SCC. Consistently, depletion of FANCD2 sensitized to cisplatin similar to depletion of ΔNp63. Together, our results demonstrate that ΔNp63 directly activates the FA pathway in SCC and limits the efficacy of cisplatin treatment. Targeting ΔNp63 therefore would not only inhibit SCC proliferation but also sensitize tumors to chemotherapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/genética , Cisplatino/uso terapéutico , Reparación del ADN , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos , Elementos de Facilitación Genéticos , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Humanos , Factores de Transcripción/fisiología , Activación Transcripcional , Proteínas Supresoras de Tumor/fisiología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
p53 is known to prevent tumour formation by restricting the proliferation of damaged or oncogene-expressing cells. In contrast, how the p53 family member p73 suppresses tumour formation remains elusive. Using a step-wise transformation protocol for human cells, we show that, in premalignant stages, expression of the transactivation-competent p73 isoform TAp73 is triggered in response to pRB pathway alterations. TAp73 expression at this stage of transformation results in increased sensitivity to chemotherapeutic drugs and oxidative stress and inhibits proliferation and survival at high cell density. Importantly, TAp73 triggers a transcriptional programme to prevent anchorage-independent growth, which is considered a crucial hallmark of fully transformed cells. An essential suppressor of anchorage-independent growth is KCNK1, which is directly transactivated by TAp73 and commonly downregulated in glioma, melanoma and ovarian cancer. Oncogenic Ras switches p73 expression from TAp73 to the oncogenic deltaNp73 isoform in a phosphatidyl-inositol 3-kinase-dependent manner. Our results implicate TAp73 as a barrier to anchorage-independent growth and indicate that downregulation of TAp73 is a key transforming activity of oncogenic Ras mutants.
Asunto(s)
Transformación Celular Neoplásica , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Línea Celular , Proliferación Celular , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Humanos , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/genéticaRESUMEN
Resistance to cytarabine is a key problem in the treatment of acute myeloid leukemia (AML). To understand the molecular biology of resistance to cytarabine, a viability-based chemosensitizer screen was utilized. We screened synthetic lethal targets using 437 different small interfering RNAs (siRNAs) directed against factors involved in DNA repair mechanisms and cytarabine as the chemical compound. Three hits were identified: CUL4A, TP73, and RFC2. We show here that the ubiquitin ligase CULLIN 4A (CUL4A) and the tumor-suppressive transcription factor p73 contribute to drug resistance by modulating DNA damage response. P73 confers resistance to cytarabine therapy by transactivation of REV3L, encoding the catalytic subunit of translesion DNA polymerase ζ, and CUL4A probably by influencing proliferating cell nuclear antigen (PCNA) and the polymerase switch towards error-prone translesion DNA polymerases. Abrogation of the polymerase ζ by siRNA causes identical effects as siRNAs against CUL4A or TP73 and resensitizes cells towards cytarabine therapy in vitro. As CUL4A needs to be activated by neddylation to facilitate the degradation of several proteins including PCNA, we propose a novel explanation for the synergism between cytarabine and the neddylation inhibitor pevonedistat by inhibition of translesion synthesis. In keeping with this, in AML patients treated with cytarabine, we found high expression of CUL4A and TP73 to be associated with poor prognosis.
RESUMEN
Merkel cell carcinoma (MCC) is a rare, highly aggressive skin cancer for which immune modulation by immune checkpoint inhibitors shows remarkable response rates. However, primary or secondary resistance to immunotherapy prevents benefits in a significant proportion of patients. For MCC, one immune escape mechanism is insufficient for recognition by T cells owing to the downregulation of major histocompatibility complex I surface expression. Histone deacetylase inhibitors have been demonstrated to epigenetically reverse the low major histocompatibility complex I expression caused by the downregulation of the antigen-processing machinery. Domatinostat, an orally available small-molecule inhibitor targeting histone deacetylase class I, is currently in clinical evaluation to overcome resistance to immunotherapy. In this study, we present preclinical data on domatinostat's efficacy and mode of action in MCC. Single-cell RNA sequencing revealed a distinct gene expression signature of antigen processing and presentation, cell-cycle arrest, and execution phase of apoptosis on treatment. Accordingly, functional assays showed that domatinostat induced G2M arrest and apoptosis. In the surviving cells, antigen-processing machinery component gene transcription and translation were upregulated, consequently resulting in increased major histocompatibility complex I surface expression. Altogether, domatinostat not only exerts direct antitumoral effects but also restores HLA class I surface expression on MCC cells, therefore, restoring surviving MCC cells' susceptibility to recognition and elimination by cognate cytotoxic T cells.
Asunto(s)
Benzamidas/farmacología , Carcinoma de Células de Merkel/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Presentación de Antígeno/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/inmunología , Benzamidas/uso terapéutico , Carcinoma de Células de Merkel/genética , Carcinoma de Células de Merkel/inmunología , Carcinoma de Células de Merkel/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/inmunología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , RNA-Seq , Análisis de la Célula Individual , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Linfocitos T Citotóxicos/inmunología , Escape del Tumor/efectos de los fármacos , Escape del Tumor/genéticaRESUMEN
The p53 family is known as a family of transcription factors with functions in tumor suppression and development. Whereas the central DNA-binding domain is highly conserved among the three family members p53, p63 and p73, the C-terminal domains (CTDs) are diverse and subject to alternative splicing and post-translational modification. Here we demonstrate that the CTDs strongly influence DNA binding and transcriptional activity: while p53 and the p73 isoform p73gamma have basic CTDs and form weak sequence-specific protein-DNA complexes, the major p73 isoforms have neutral CTDs and bind DNA strongly. A basic CTD has been previously shown to enable sliding along the DNA backbone and to facilitate the search for binding sites in the complex genome. Our experiments, however, reveal that a basic CTD also reduces protein-DNA complex stability, intranuclear mobility, promoter occupancy in vivo, target gene activation and induction of cell cycle arrest or apoptosis. A basic CTD therefore provides both positive and negative regulatory functions presumably to enable rapid switching of protein activity in response to stress. The different DNA-binding characteristics of the p53 family members could therefore reflect their predominant role in the cellular stress response (p53) or developmental processes (p73).
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas Nucleares/química , Proteína p53 Supresora de Tumor/química , Proteínas Supresoras de Tumor/química , Apoptosis , Sitios de Unión , Ciclo Celular , Línea Celular , Núcleo Celular/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Difusión , Humanos , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Transcripción Genética , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: The efficacy of PD-(L)1 blockade depends on the composition of the tumor immune microenvironment (TIME) and is generally higher in tumors with pre-existing cytotoxic T cells (CTL) than in those with low CTL numbers. Nonetheless, a significant proportion of patients with pre-existing immunity fail to respond, indicating a therapeutic potential for combining PD-(L)1 blockade with additional immunomodulatory agents in both CTL-high and -low immune phenotypes. Here, we evaluated domatinostat (4SC-202), a class I-selective histone deacetylase (HDAC) inhibitor, for its effect on the TIME and its antitumoral efficacy using syngeneic mouse models with CTL-high or CTL-low tumors. METHODS: Domatinostat was evaluated in PD-1 blockade-insensitive CTL-low (CT26) and CTL-high (C38) syngeneic models alone and in combination with different immune-inhibitory and -stimulatory approaches. Effects on the immunophenotype were assessed via flow cytometry and RNA-seq analyses. The changes in RNA-seq-based immune signatures determined in a murine setting were investigated in patient samples from the first-dose cohort of the SENSITIZE trial (NCT03278665) evaluating domatinostat combined with pembrolizumab in advanced-stage melanoma patients refractory/nonresponding to PD-1 blockade. RESULTS: Domatinostat increased the expression of antigen-presenting machinery (APM) genes and MHC class I and II molecules, along with CTL infiltration, in tumors of both immune phenotypes. In combination with PD-(L)1 blockade, domatinostat augmented antitumor effects substantially above the effects of single-agent therapies, displaying greater benefit in tumors with pre-existing CTLs. In this setting, the combination of domatinostat with agonistic anti-4-1BB or both PD-1 and LAG3 blockade further increased the antitumor efficacy. In CTL-low tumors, domatinostat enhanced the expression of genes known to reinforce immune responses against tumors. Specifically, domatinostat increased the expression of Ifng and genes associated with responses to pembrolizumab and nivolumab. Clinically, these findings were confirmed in patients with advanced melanoma treated with domatinostat for 14 days, who demonstrated elevated expression of APM and MHC genes, the IFNG gene, and the IFN-γ and pembrolizumab response signatures in individual tumor samples. CONCLUSION: In summary, these data suggest a promising potential of domatinostat in combination with immunotherapy to improve the outcome of refractory cancer patients.
Asunto(s)
Benzamidas/farmacología , Neoplasias/inmunología , Neoplasias/patología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Animales , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/farmacología , Biomarcadores de Tumor , Biopsia , Línea Celular Tumoral , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Inmunofenotipificación , Masculino , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
p73 is a member of the p53 family of tumor suppressors. Transactivating isoforms of p73 (TAp73) have p53-like, anti-proliferative and pro-apoptotic activities that are crucial for an efficient chemotherapy response. In line with this, genetic studies in mice have confirmed that TAp73 acts as a tumor suppressor. However, in contrast to p53, which is commonly inactivated in human cancer by point mutations, the TP73 gene is almost never mutated. Instead, the tumor suppressor activity of TAp73 is inhibited through a variety of mechanisms including epigenetic silencing and complex formation with inhibitory proteins. All these mechanisms have in common that they are in principle reversible and therefore amenable to therapeutic intervention. Here, we will review how tumor cells control the tumor suppressor activity of TAp73 and discuss possible strategies targeting p73 for reactivation.