RESUMO
Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/metabolismo , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , DNA Helicases/genética , Replicação do DNA/efeitos dos fármacos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/efeitos dos fármacos , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Nucleossomos/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genéticaRESUMO
Making choices about whether and when to engage cognitive effort are a common feature of everyday experience, with important consequences for academic, career, and health outcomes. Yet, despite their hypothesized importance, very little is understood about the underlying mechanisms that support this form of human cost-benefit decision-making. To investigate these mechanisms, we used the Cognitive Effort Discounting Paradigm (Cog-ED) during fMRI scanning to precisely quantify the neural encoding of varying cognitive effort demands relative to reward outcomes, within two distinct cognitive domains (working memory, speech comprehension). The findings provide strong evidence that the dorsal anterior cingulate cortex (dACC) plays a central and selective role in this decision-making process. Trial-by-trial modulations in dACC activation tracked the relative subjective value of the low-effort, low-reward option, with the strongest activity occurring when this was of greater value than the high-effort, high-reward option. In contrast, dACC activity was not modulated by decision difficulty, though such effects were found in other frontoparietal regions. Critically, dACC activity was also strongly correlated across the two decision-making task domains, and further predicted subsequent choice behavior in both. Together, the results suggest that dACC activity modulation reflects a domain-general valuation comparison mechanism, which acts to bias participants away from decisions to engage in cognitive effort, when the perceived subjective costs of such engagement outweigh the reward-related benefits. These findings complement work in other cost domains and species by pointing to a clear role of dACC in representing subjective value differences between choice options during cost-benefit decision-making.Significance Statement Despite the growing popularity of decision-making paradigms to study the neural mechanisms underlying choices to engage in cognitive effort, there is still little consensus about how such decisions are implemented in the human brain. This study used two distinct versions of a cognitive effort decision-making paradigm during fMRI scanning to identify generalized brain mechanisms engaged while participants decided whether to engage in cognitive effort. Importantly, the results provide additional evidence that the dorsal anterior cingulate cortex is a key locus of cognitive effort-based decision-making. Specifically, this brain region signals the difference in relative value across choice options, making it an attractive target for future studies aimed at investigating the altered decision-making observed in populations for which apathy is commonly observed.
RESUMO
BACKGROUND: Invasive Lobular Carcinoma (ILC) is a morphologically distinct breast cancer subtype that represents up to 15% of all breast cancers. Compared to Invasive Breast Carcinoma of No Special Type (IBC-NST), ILCs exhibit poorer long-term outcome and a unique pattern of metastasis. Despite these differences, the systematic discovery of robust prognostic biomarkers and therapeutically actionable molecular pathways in ILC remains limited. METHODS: Pathway-centric multivariable models using statistical machine learning were developed and tested in seven retrospective clinico-genomic cohorts (n = 996). Further external validation was performed using a new RNA-Seq clinical cohort of aggressive ILCs (n = 48). RESULTS AND CONCLUSIONS: mRNA dysregulation scores of 25 pathways were strongly prognostic in ILC (FDR-adjusted P < 0.05). Of these, three pathways including Cell-cell communication, Innate immune system and Smooth muscle contraction were also independent predictors of chemotherapy response. To aggregate these findings, a multivariable machine learning predictor called PSILC was developed and successfully validated for predicting overall and metastasis-free survival in ILC. Integration of PSILC with CRISPR-Cas9 screening data from breast cancer cell lines revealed 16 candidate therapeutic targets that were synthetic lethal with high-risk ILCs. This study provides interpretable prognostic and predictive biomarkers of ILC which could serve as the starting points for targeted drug discovery for this disease.
Assuntos
Neoplasias da Mama , Carcinoma Lobular , Humanos , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Carcinoma Lobular/tratamento farmacológico , Carcinoma Lobular/genética , Carcinoma Lobular/patologia , Carcinoma Lobular/metabolismo , Prognóstico , Estudos Retrospectivos , Biomarcadores Tumorais/genética , Aprendizado de Máquina , Pessoa de Meia-Idade , Regulação Neoplásica da Expressão Gênica , Invasividade NeoplásicaRESUMO
A combination of genetic and functional approaches has identified three independent breast cancer risk loci at 2q35. A recent fine-scale mapping analysis to refine these associations resulted in 1 (signal 1), 5 (signal 2), and 42 (signal 3) credible causal variants at these loci. We used publicly available in silico DNase I and ChIP-seq data with in vitro reporter gene and CRISPR assays to annotate signals 2 and 3. We identified putative regulatory elements that enhanced cell-type-specific transcription from the IGFBP5 promoter at both signals (30- to 40-fold increased expression by the putative regulatory element at signal 2, 2- to 3-fold by the putative regulatory element at signal 3). We further identified one of the five credible causal variants at signal 2, a 1.4 kb deletion (esv3594306), as the likely causal variant; the deletion allele of this variant was associated with an average additional increase in IGFBP5 expression of 1.3-fold (MCF-7) and 2.2-fold (T-47D). We propose a model in which the deletion allele of esv3594306 juxtaposes two transcription factor binding regions (annotated by estrogen receptor alpha ChIP-seq peaks) to generate a single extended regulatory element. This regulatory element increases cell-type-specific expression of the tumor suppressor gene IGFBP5 and, thereby, reduces risk of estrogen receptor-positive breast cancer (odds ratio = 0.77, 95% CI 0.74-0.81, p = 3.1 × 10-31).
Assuntos
Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Anotação de Sequência Molecular , Regiões Promotoras Genéticas , Neoplasias da Mama/genética , Sistemas CRISPR-Cas , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 2 , Feminino , Estudos de Associação Genética , Variação Genética , Humanos , Fatores de Risco , Deleção de SequênciaRESUMO
53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini1,2. This function of 53BP1 requires interactions with PTIP3 and RIF14-9, the latter of which recruits REV7 (also known as MAD2L2) to break sites10,11. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases12,13, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.
Assuntos
Reparo do DNA , Complexos Multiproteicos/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/genética , Feminino , Genes BRCA1 , Humanos , Switching de Imunoglobulina/genética , Camundongos , Modelos Biológicos , Complexos Multiproteicos/química , Complexos Multiproteicos/deficiência , Complexos Multiproteicos/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Telômeros/metabolismo , Proteína Supressora de Tumor p53/deficiênciaRESUMO
BACKGROUND & AIMS: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. METHODS: We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. RESULTS: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. CONCLUSIONS: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.
Assuntos
Adenocarcinoma/patologia , Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , Neoplasias Pancreáticas/patologia , Adenocarcinoma/genética , Adenocarcinoma/terapia , Biomarcadores , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Humanos , Terapia de Alvo Molecular , Organoides , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Intellectual disability (ID) is a highly heterogeneous disorder involving at least 600 genes, yet a genetic diagnosis remains elusive in â¼35%-40% of individuals with moderate to severe ID. Recent meta-analyses statistically analyzing de novo mutations in >7,000 individuals with neurodevelopmental disorders highlighted mutations in PPM1D as a possible cause of ID. PPM1D is a type 2C phosphatase that functions as a negative regulator of cellular stress-response pathways by mediating a feedback loop of p38-p53 signaling, thereby contributing to growth inhibition and suppression of stress-induced apoptosis. We identified 14 individuals with mild to severe ID and/or developmental delay and de novo truncating PPM1D mutations. Additionally, deep phenotyping revealed overlapping behavioral problems (ASD, ADHD, and anxiety disorders), hypotonia, broad-based gait, facial dysmorphisms, and periods of fever and vomiting. PPM1D is expressed during fetal brain development and in the adult brain. All mutations were located in the last or penultimate exon, suggesting escape from nonsense-mediated mRNA decay. Both PPM1D expression analysis and cDNA sequencing in EBV LCLs of individuals support the presence of a stable truncated transcript, consistent with this hypothesis. Exposure of cells derived from individuals with PPM1D truncating mutations to ionizing radiation resulted in normal p53 activation, suggesting that p53 signaling is unaffected. However, a cell-growth disadvantage was observed, suggesting a possible effect on the stress-response pathway. Thus, we show that de novo truncating PPM1D mutations in the last and penultimate exons cause syndromic ID, which provides additional insight into the role of cell-cycle checkpoint genes in neurodevelopmental disorders.
Assuntos
Éxons , Deficiência Intelectual/genética , Mutação , Proteína Fosfatase 2C/genética , Adolescente , Ciclo Celular , Criança , Pré-Escolar , Humanos , Deficiência Intelectual/patologia , Adulto JovemRESUMO
Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10(-5)), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10(-4)) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10(-9)). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.
Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença/genética , Mosaicismo , Mutação , Neoplasias Ovarianas/genética , Fosfoproteínas Fosfatases/genética , Alelos , Análise por Conglomerados , Éxons , Feminino , Humanos , Isoenzimas/genética , Linfócitos/metabolismo , Proteína Fosfatase 2C , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Prostate cancer is a heterogeneous disease, but current treatments are not based on molecular stratification. We hypothesized that metastatic, castration-resistant prostate cancers with DNA-repair defects would respond to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibition with olaparib. METHODS: We conducted a phase 2 trial in which patients with metastatic, castration-resistant prostate cancer were treated with olaparib tablets at a dose of 400 mg twice a day. The primary end point was the response rate, defined either as an objective response according to Response Evaluation Criteria in Solid Tumors, version 1.1, or as a reduction of at least 50% in the prostate-specific antigen level or a confirmed reduction in the circulating tumor-cell count from 5 or more cells per 7.5 ml of blood to less than 5 cells per 7.5 ml. Targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase-chain-reaction testing were performed on samples from mandated tumor biopsies. RESULTS: Overall, 50 patients were enrolled; all had received prior treatment with docetaxel, 49 (98%) had received abiraterone or enzalutamide, and 29 (58%) had received cabazitaxel. Sixteen of 49 patients who could be evaluated had a response (33%; 95% confidence interval, 20 to 48), with 12 patients receiving the study treatment for more than 6 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2--in 16 of 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (4 with biallelic somatic loss, and 3 with germline mutations) and 4 of 5 with ATM aberrations. The specificity of the biomarker suite was 94%. Anemia (in 10 of the 50 patients [20%]) and fatigue (in 6 [12%]) were the most common grade 3 or 4 adverse events, findings that are consistent with previous studies of olaparib. CONCLUSIONS: Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. (Funded by Cancer Research UK and others; ClinicalTrials.gov number, NCT01682772; Cancer Research UK number, CRUK/11/029.).
Assuntos
Antineoplásicos/uso terapêutico , Reparo do DNA , Inibidores Enzimáticos/uso terapêutico , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases , Neoplasias da Próstata/tratamento farmacológico , Adulto , Idoso , Anemia/induzido quimicamente , Proteínas Mutadas de Ataxia Telangiectasia/genética , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/efeitos adversos , Fadiga/induzido quimicamente , Genes BRCA2 , Genes Supressores de Tumor , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Metástase Neoplásica/tratamento farmacológico , Ftalazinas/efeitos adversos , Piperazinas/efeitos adversos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologiaRESUMO
Mutations in BRCA2 confer an increased risk of cancer development, at least in part because the BRCA2 protein is required for the maintenance of genomic integrity. Here, we use proteomic profiling to identify APRIN (PDS5B), a cohesion-associated protein, as a BRCA2-associated protein. After exposure of cells to hydroxyurea or aphidicolin, APRIN and other cohesin components associate with BRCA2 in early S-phase. We demonstrate that APRIN expression is required for the normal response to DNA-damaging agents, the nuclear localisation of RAD51 and BRCA2 and efficient homologous recombination. The clinical significance of these findings is indicated by the observation that the BRCA2/APRIN interaction is compromised by BRCA2 missense variants of previously unknown significance and that APRIN expression levels are associated with histological grade in breast cancer and the outcome of breast cancer patients treated with DNA-damaging chemotherapy.
Assuntos
Antineoplásicos/administração & dosagem , Proteína BRCA2/metabolismo , Neoplasias da Mama/tratamento farmacológico , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Tratamento Farmacológico/métodos , Mapeamento de Interação de Proteínas , Fatores de Transcrição/metabolismo , Neoplasias da Mama/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Prognóstico , Proteoma/análise , Rad51 Recombinase/metabolismo , Recombinação Genética , Fatores de Transcrição/genética , Resultado do TratamentoRESUMO
Cells with loss of BRCA2 function are defective in homologous recombination (HR) and are highly sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP), which provides the basis for a new therapeutic approach. Here we show that resistance to PARP inhibition can be acquired by deletion of a mutation in BRCA2. We derived PARP-inhibitor-resistant (PIR) clones from the human CAPAN1 pancreatic cancer cell line, which carries the protein-truncating c.6174delT frameshift mutation. PIR clones could form DNA-damage-induced RAD51 nuclear foci and were able to limit genotoxin-induced genomic instability, both hallmarks of a competent HR pathway. New BRCA2 isoforms were expressed in the resistant lines as a result of intragenic deletion of the c.6174delT mutation and restoration of the open reading frame (ORF). Reconstitution of BRCA2-deficient cells with these revertant BRCA2 alleles rescued PARP inhibitor sensitivity and HR deficiency. Most of the deletions in BRCA2 were associated with small tracts of homology, and possibly arose from error-prone repair caused by BRCA2 deficiency. Similar ORF-restoring mutations were present in carboplatin-resistant ovarian tumours from c.6174delT mutation carriers. These observations have implications for understanding drug resistance in BRCA mutation carriers as well as in defining functionally important domains within BRCA2.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Genes BRCA2 , Deleção de Sequência/genética , Idoso , Alelos , Sequência de Aminoácidos , Proteína BRCA2/deficiência , Proteína BRCA2/genética , Sequência de Bases , Carboplatina/farmacologia , Linhagem Celular Tumoral , Aberrações Cromossômicas/induzido quimicamente , Feminino , Fluorbenzenos/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Mitomicina/farmacologia , Dados de Sequência Molecular , Mutação/genética , Fases de Leitura Aberta/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Ftalazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Recombinação Genética/genéticaRESUMO
The F-box and WD repeat domain containing 7 (FBXW7) tumour suppressor gene encodes a substrate-recognition subunit of Skp, cullin, F-box (SCF)-containing complexes. The tumour-suppressive role of FBXW7 is ascribed to its ability to drive ubiquitination and degradation of oncoproteins. Despite this molecular understanding, therapeutic approaches that target defective FBXW7 have not been identified. Using genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens, focussed RNA-interference screens and whole and phospho-proteome mass spectrometry profiling in multiple FBXW7 wild-type and defective isogenic cell lines, we identified a number of FBXW7 synthetic lethal targets, including proteins involved in the response to replication fork stress and proteins involved in replication origin firing, such as cell division cycle 7-related protein kinase (CDC7) and its substrate, DNA replication complex GINS protein SLD5 (GINS4). The CDC7 synthetic lethal effect was confirmed using small-molecule inhibitors. Mechanistically, FBXW7/CDC7 synthetic lethality is dependent upon the replication factor telomere-associated protein RIF1 (RIF1), with RIF1 silencing reversing the FBXW7-selective effects of CDC7 inhibition. The delineation of FBXW7 synthetic lethal effects we describe here could serve as the starting point for subsequent drug discovery and/or development in this area.
Assuntos
Proteínas de Ciclo Celular , Neoplasias , Humanos , Proteína 7 com Repetições F-Box-WD/genética , Linhagem Celular Tumoral , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ubiquitinação , Interferência de RNA , Domínios Proteicos , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Cromossômicas não Histona/genéticaRESUMO
Biallelic loss of cyclin-dependent kinase 12 (CDK12) defines a unique molecular subtype of metastatic castration-resistant prostate cancer (mCRPC). It remains unclear, however, whether CDK12 loss per se is sufficient to drive prostate cancer development-either alone, or in the context of other genetic alterations-and whether CDK12-mutant tumors exhibit sensitivity to specific pharmacotherapies. Here, we demonstrate that tissue-specific Cdk12 ablation is sufficient to induce preneoplastic lesions and robust T cell infiltration in the mouse prostate. Allograft-based CRISPR screening demonstrated that Cdk12 loss is positively associated with Trp53 inactivation but negatively associated with Pten inactivation-akin to what is observed in human mCRPC. Consistent with this, ablation of Cdk12 in prostate organoids with concurrent Trp53 loss promotes their proliferation and ability to form tumors in mice, while Cdk12 knockout in the Pten-null prostate cancer mouse model abrogates tumor growth. Bigenic Cdk12 and Trp53 loss allografts represent a new syngeneic model for the study of androgen receptor (AR)-positive, luminal prostate cancer. Notably, Cdk12/Trp53 loss prostate tumors are sensitive to immune checkpoint blockade. Cdk12-null organoids (either with or without Trp53 co-ablation) and patient-derived xenografts from tumors with CDK12 inactivation are highly sensitive to inhibition or degradation of its paralog kinase, CDK13. Together, these data identify CDK12 as a bona fide tumor suppressor gene with impact on tumor progression and lends support to paralog-based synthetic lethality as a promising strategy for treating CDK12-mutant mCRPC.
RESUMO
Y-box binding protein-1 (YB-1) is the first reported oncogenic transcription factor to induce the tumor-initiating cell (TIC) surface marker CD44 in triple-negative breast cancer (TNBC) cells. In order for CD44 to be induced, YB-1 must be phosphorylated at S102 by p90 ribosomal S6 kinase (RSK). We therefore questioned whether RSK might be a tractable molecular target to eliminate TICs. In support of this idea, injection of MDA-MB-231 cells expressing Flag-YB-1 into mice increased tumor growth as well as enhanced CD44 expression. Despite enrichment for TICs, these cells were sensitive to RSK inhibition when treated ex vivo with BI-D1870. Targeting RSK2 with small interfering RNA (siRNA) or small molecule RSK kinase inhibitors (SL0101 and BI-D1870) blocked TNBC monolayer cell growth by â¼100%. In a diverse panel of breast tumor cell line models RSK2 siRNA predominantly targeted models of TNBC. RSK2 inhibition decreased CD44 promoter activity, CD44 mRNA, protein expression, and mammosphere formation. CD44(+) cells had higher P-RSK(S221/227) , P-YB-1(S102) , and mitotic activity relative to CD44(-) cells. Importantly, RSK2 inhibition specifically suppressed the growth of TICs and triggered cell death. Moreover, silencing RSK2 delayed tumor initiation in mice. In patients, RSK2 mRNA was associated with poor disease-free survival in a cohort of 244 women with breast cancer that had not received adjuvant treatment, and its expression was highest in the basal-like breast cancer subtype. Taking this further, we report that P-RSK(S221/227) is present in primary TNBCs and correlates with P-YB-1(S102) as well as CD44. In conclusion, RSK2 inhibition provides a novel therapeutic avenue for TNBC and holds the promise of eliminating TICs.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzopiranos/farmacologia , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Monossacarídeos/farmacologia , Regiões Promotoras Genéticas/genética , Pteridinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteína 1 de Ligação a Y-Box/genéticaRESUMO
Although PARP inhibitors (PARPi) now form part of the standard-of-care for the treatment of homologous recombination defective cancers, de novo and acquired resistance limits their overall effectiveness. Previously, overexpression of the BRCA1-∆11q splice variant has been shown to cause PARPi resistance. How cancer cells achieve increased BRCA1-∆11q expression has remained unclear. Using isogenic cells with different BRCA1 mutations, we show that reduction in HUWE1 leads to increased levels of BRCA1-∆11q and PARPi resistance. This effect is specific to cells able to express BRCA1-∆11q (e.g. BRCA1 exon 11 mutant cells) and is not seen in BRCA1 mutants that cannot express BRCA1-∆11q, nor in BRCA2 mutant cells. As well as increasing levels of BRCA1-∆11q protein in exon 11 mutant cells, HUWE1 silencing also restores RAD51 nuclear foci and platinum salt resistance. HUWE1 catalytic domain mutations were also seen in a case of PARPi resistant, BRCA1 exon 11 mutant, high grade serous ovarian cancer. These results suggest how elevated levels of BRCA1-∆11q and PARPi resistance can be achieved, identify HUWE1 as a candidate biomarker of PARPi resistance for assessment in future clinical trials and illustrate how some PARPi resistance mechanisms may only operate in patients with particular BRCA1 mutations.
Assuntos
Antineoplásicos , Neoplasias , Neoplasias Ovarianas , Humanos , Feminino , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Antineoplásicos/farmacologia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Mutação , Neoplasias/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The PSMC3IP-MND1 heterodimer promotes meiotic D loop formation before DNA strand exchange. In genome-scale CRISPR-Cas9 mutagenesis and interference screens in mitotic cells, depletion of PSMC3IP or MND1 causes sensitivity to poly (ADP-Ribose) polymerase inhibitors (PARPi) used in cancer treatment. PSMC3IP or MND1 depletion also causes ionizing radiation sensitivity. These effects are independent of PSMC3IP/MND1's role in mitotic alternative lengthening of telomeres. PSMC3IP- or MND1-depleted cells accumulate toxic RAD51 foci in response to DNA damage, show impaired homology-directed DNA repair, and become PARPi sensitive, even in cells lacking both BRCA1 and TP53BP1. Epistasis between PSMC3IP-MND1 and BRCA1/BRCA2 defects suggest that abrogated D loop formation is the cause of PARPi sensitivity. Wild-type PSMC3IP reverses PARPi sensitivity, whereas a PSMC3IP p.Glu201del mutant associated with D loop defects and ovarian dysgenesis does not. These observations suggest that meiotic proteins such as MND1 and PSMC3IP have a greater role in mitotic DNA repair.
Assuntos
Antineoplásicos , Inibidores de Poli(ADP-Ribose) Polimerases , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Reparo do DNA , Dano ao DNA , Proteína BRCA1/genética , Reparo de DNA por Recombinação , Linhagem Celular TumoralRESUMO
The multidrug resistance-associated protein 2 (MRP2/ABCC2) is involved in the efflux of endogenous and xenobiotic substrates, including several anticancer and antiviral drugs. The functional consequences of ABCC2 protein variants remain inconsistent, which may be due to shortcomings of the in vitro assays used. To study systematically the functional consequences of nonsynonymous ABCC2 variants, we used a novel "Screen and Insert" (ScIn) technology to achieve stable and highly reproducible expression of 13 ABCC2 variants in HT1080 cells. Western blotting revealed lower (30-65%) ABCC2 expression for D333G, R1174H, and R1181L as compared with wild type (WT; 100%), whereas the linked variant V1188E/C1515Y resulted in higher expression (150%). R1174H caused mislocalization of ABCC2 to the cytoplasm with an endoplasmic reticulum-like distribution. Variants N1244K and R1174H decreased transport of glutathione-methylfluorescein (GS-MF) and glutathione-monochlorobimane (GS-MCB) by 80% and 50%, respectively, whereas R1181L and P1291L reduced only GS-MCB transport by 50% as compared with WT. Contrary to protein data, the double variant V1188E/C1515Y decreased specific transport activity for GS-MF and GS-MCB by 40%. The ScIn approach is a feasible and reliable method to functionally characterize systematically ABCC2 variants. D333G, R1174H, R1181L, N1244K, P1291L, and double variant V1188E/C1515Y have been identified as most promising for further clinical evaluation.
Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Negro ou Afro-Americano/genética , Asiático/genética , Linhagem Celular Tumoral , Cloraminas/metabolismo , Fibrossarcoma/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Variação Genética , Células HEK293 , Haplótipos , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Mutação de Sentido Incorreto , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tetraciclina/farmacologiaRESUMO
Inhibitors of poly (ADP-ribose)-polymerase-1 (PARP) are highly lethal to cells with deficiencies in BRCA1, BRCA2 or other components of the homologous recombination pathway. This has led to PARP inhibitors entering clinical trials as a potential therapy for cancer in carriers of BRCA1 and BRCA2 mutations. To discover new determinants of sensitivity to these drugs, we performed a PARP-inhibitor synthetic lethal short interfering RNA (siRNA) screen. We identified a number of kinases whose silencing strongly sensitised to PARP inhibitor, including cyclin-dependent kinase 5 (CDK5), MAPK12, PLK3, PNKP, STK22c and STK36. How CDK5 silencing mediates sensitivity was investigated. Previously, CDK5 has been suggested to be active only in a neuronal context, but here we show that CDK5 is required in non-neuronal cells for the DNA-damage response and, in particular, intra-S and G(2)/M cell-cycle checkpoints. These results highlight the potential of synthetic lethal siRNA screens with chemical inhibitors to define new determinants of sensitivity and potential therapeutic targets.
Assuntos
Inibidores de Poli(ADP-Ribose) Polimerases , RNA Interferente Pequeno/genética , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Dano ao DNA , Reparo do DNA , Citometria de Fluxo , Imunofluorescência , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Proteína Quinase 12 Ativada por Mitógeno/genética , Proteína Quinase 12 Ativada por Mitógeno/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transfecção , Proteínas Supressoras de TumorRESUMO
Poly(ADP-ribose) polymerase (PARP) is an enzyme involved in DNA repair. PARP inhibitors can act as chemosensitizers, or operate on the principle of synthetic lethality when used as single agent. Clinical trials have shown drugs in this class to be promising for BRCA mutation carriers. We postulated that inability to demonstrate response in non-BRCA carriers in which BRCA is inactivated by other mechanisms or with deficiency in homologous recombination for DNA repair is due to lack of molecular markers that define a responding subpopulation. We identified candidate markers for this purpose for olaparib (AstraZeneca) by measuring inhibitory effects of nine concentrations of olaparib in 22 breast cancer cell lines and identifying features in transcriptional and genome copy number profiles that were significantly correlated with response. We emphasized in this discovery process genes involved in DNA repair. We found that the cell lines that were sensitive to olaparib had a significant lower copy number of BRCA1 compared to the resistant cell lines (p value 0.012). In addition, we discovered seven genes from DNA repair pathways whose transcriptional levels were associated with response. These included five genes (BRCA1, MRE11A, NBS1, TDG, and XPA) whose transcript levels were associated with resistance and two genes (CHEK2 and MK2) whose transcript levels were associated with sensitivity. We developed an algorithm to predict response using the seven-gene transcription levels and applied it to 1,846 invasive breast cancer samples from 8 U133A/plus 2 (Affymetrix) data sets and found that 8-21 % of patients would be predicted to be responsive to olaparib. A similar response frequency was predicted in 536 samples analyzed on an Agilent platform. Importantly, tumors predicted to respond were enriched in basal subtype tumors. Our studies support clinical evaluation of the utility of our seven-gene signature as a predictor of response to olaparib.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasia de Células Basais/tratamento farmacológico , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA/genética , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Feminino , Expressão Gênica , Humanos , Concentração Inibidora 50 , Poli(ADP-Ribose) Polimerase-1 , Estatísticas não Paramétricas , TranscriptomaRESUMO
Previous research has linked working memory capacity (WMC) with enhanced proactive control. However, it remains unclear the extent to which this relationship reflects the influence of WMC on the tendency to engage proactive control, or rather, the ability to implement it. The current study sought to clarify this ambiguity by leveraging the Dual Mechanisms of Cognitive Control (DMCC) version of the AX-CPT task, in which the mode of cognitive control is experimentally manipulated across distinct testing sessions. To adjudicate between competing hypotheses, Bayesian mixed modeling was used to conduct sequential analyses involving two separate data sets. Posterior parameter estimates obtained from the initial analysis were entered as informed priors during the replication analysis to evaluate the influence of new data on previous estimates. Results yielded strong evidence demonstrating that the influence of WMC on proactive control is most robust under experimentally controlled conditions, during which use of proactive control is standardized across participants via explicit training and instruction. Critically, the observed pattern of findings suggests that the relationship between WMC and proactive control may be better characterized as individual differences in the ability to implement proactive control, rather than a more generalized tendency to engage it. (PsycInfo Database Record (c) 2022 APA, all rights reserved).