RESUMEN
Patient-derived xenograft (PDX) models of breast cancer are an effective discovery platform and tool for preclinical pharmacologic testing and biomarker identification. We established orthotopic PDX models of triple negative breast cancer (TNBC) from the primary breast tumors of patients prior to and following neoadjuvant chemotherapy (NACT) while they were enrolled in the ARTEMIS trial (NCT02276443). Serial biopsies were obtained from patients prior to treatment (pre-NACT), from poorly responsive disease after four cycles of Adriamycin and cyclophosphamide (AC, mid-NACT), and in cases of AC-resistance, after a 3-month course of different experimental therapies and/or additional chemotherapy (post-NACT). Our study cohort includes a total of 269 fine needle aspirates (FNAs) from 217 women, generating a total of 62 PDX models (overall success-rate = 23%). Success of PDX engraftment was generally higher from those cancers that proved to be treatment-resistant, whether poorly responsive to AC as determined by ultrasound measurements mid-NACT (p = 0.063), RCB II/III status after NACT (p = 0.046), or metastatic relapse within 2 years of surgery (p = 0.008). TNBC molecular subtype determined from gene expression microarrays of pre-NACT tumors revealed no significant association with PDX engraftment rate (p = 0.877). Finally, we developed a statistical model predictive of PDX engraftment using percent Ki67 positive cells in the patient's diagnostic biopsy, positive lymph node status at diagnosis, and low volumetric reduction of the patient's tumor following AC treatment. This novel bank of 62 PDX models of TNBC provides a valuable resource for biomarker discovery and preclinical therapeutic trials aimed at improving neoadjuvant response rates for patients with TNBC.
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The relationship between ATR/Chk1 activity and replication stress, coupled with the development of potent and tolerable inhibitors of this pathway, has led to the clinical exploration of ATR and Chk1 inhibitors (ATRi/Chk1i) as anticancer therapies for single-agent or combinatorial application. The clinical efficacy of these therapies relies on the ability to ascertain which patient populations are most likely to benefit, so there is intense interest in identifying predictive biomarkers of response. To comprehensively evaluate the components that modulate cancer cell sensitivity to replication stress induced by Chk1i, we performed a synthetic-lethal drop-out screen in a cell line derived from a patient with triple-negative breast cancer (TNBC), using a pooled barcoded shRNA library targeting ~350 genes involved in DNA replication, DNA damage repair, and cycle progression. In addition, we sought to compare the relative requirement of these genes when DNA fidelity is challenged by clinically relevant anticancer breast cancer drugs, including cisplatin and PARP1/2 inhibitors, that have different mechanisms of action. This global comparison is critical for understanding not only which agents should be used together for combinatorial therapies in breast cancer patients, but also the genetic context in which these therapies will be most effective, and when a single-agent therapy will be sufficient to provide maximum therapeutic benefit to the patient. We identified unique potentiators of response to ATRi/Chk1i and describe a new role for components of the cytosolic iron-sulfur assembly (CIA) pathway, MMS19 and CIA2B-FAM96B, in replication stress tolerance of TNBC.
RESUMEN
Checkpoint kinase inhibitors (CHKi) exhibit striking single-agent activity in certain tumors, but the mechanisms accounting for hypersensitivity are poorly understood. We screened a panel of 49 established human head and neck squamous cell carcinoma (HNSCC) cell lines and report that nearly 20% are hypersensitive to CHKi monotherapy. Hypersensitive cells underwent early S-phase arrest at drug doses sufficient to inhibit greater than 90% of CHK1 activity. Reduced rate of DNA replication fork progression and chromosomal shattering were also observed, suggesting replication stress as a root causative factor in CHKi hypersensitivity. To explore genomic underpinnings of CHKi hypersensitivity, comparative genomic analysis was performed between hypersensitive cells and cells categorized as least sensitive because they showed drug IC50 value greater than the cell panel median and lacked early S-phase arrest. Novel association between CDKN2A/p16 copy number loss, CDK2 activation, replication stress, and hypersensitivity of HNSCC cells to CHKi monotherapy was found. Restoring p16 in cell lines harboring CDKN2A/p16 genomic deletions alleviated CDK2 activation and replication stress, attenuating CHKi hypersensitivity. Taken together, our results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from CHKi therapy.Significance: These results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from therapy with CHK inhibitors. Cancer Res; 78(3); 781-97. ©2017 AACR.
Asunto(s)
Carcinoma de Células Escamosas/patología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Inhibidores Enzimáticos/farmacología , Neoplasias de Cabeza y Cuello/patología , Fase S , Antineoplásicos/farmacología , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Proliferación Celular , Quinasa 2 Dependiente de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Replicación del ADN , Activación Enzimática , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Eliminación de Secuencia , Células Tumorales CultivadasRESUMEN
Effective targeted therapies for small-cell lung cancer (SCLC), the most aggressive form of lung cancer, remain urgently needed. Here we report evidence of preclinical efficacy evoked by targeting the overexpressed cell-cycle checkpoint kinase CHK1 in SCLC. Our studies employed RNAi-mediated attenuation or pharmacologic blockade with the novel second-generation CHK1 inhibitor prexasertib (LY2606368), currently in clinical trials. In SCLC models in vitro and in vivo, LY2606368 exhibited strong single-agent efficacy, augmented the effects of cisplatin or the PARP inhibitor olaparib, and improved the response of platinum-resistant models. Proteomic analysis identified CHK1 and MYC as top predictive biomarkers of LY2606368 sensitivity, suggesting that CHK1 inhibition may be especially effective in SCLC with MYC amplification or MYC protein overexpression. Our findings provide a preclinical proof of concept supporting the initiation of a clinical efficacy trial in patients with platinum-sensitive or platinum-resistant relapsed SCLC. Cancer Res; 77(14); 3870-84. ©2017 AACR.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Cisplatino/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Ftalazinas/farmacología , Piperazinas/farmacología , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Animales , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Cisplatino/administración & dosificación , Sinergismo Farmacológico , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Ftalazinas/administración & dosificación , Piperazinas/administración & dosificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Pirazinas/administración & dosificación , Pirazinas/farmacología , Pirazoles/administración & dosificación , Pirazoles/farmacología , Carcinoma Pulmonar de Células Pequeñas/enzimología , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/patologíaRESUMEN
Chromatin is organized in a highly ordered yet dynamic manner in the cell nucleus, but the principles governing this organization remain unclear. Similarly, it is unknown whether, and how, various proteins regulate chromatin motion and as a result influence nuclear organization. Here by studying the dynamics of different genomic regions in the nucleus of live cells, we show that the genome has highly constrained dynamics. Interestingly, depletion of lamin A strikingly alters genome dynamics, inducing a dramatic transition from slow anomalous diffusion to fast and normal diffusion. In contrast, depletion of LAP2α, a protein that interacts with lamin A and chromatin, has no such effect on genome dynamics. We speculate that chromosomal inter-chain interactions formed by lamin A throughout the nucleus contribute to chromatin dynamics, and suggest that the molecular regulation of chromatin diffusion by lamin A in the nuclear interior is critical for the maintenance of genome organization.
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Cromatina/fisiología , Lamina Tipo A/metabolismo , Interferencia de ARN/fisiología , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Humanos , Lamina Tipo A/genética , Proteínas de la Membrana/metabolismo , Ratones , Células 3T3 NIH , ARN Interferente Pequeño , TelómeroRESUMEN
Over 300 mutations in the LMNA gene, encoding A-type lamins, are associated with 15 human degenerative disorders and premature aging syndromes. Although genomic instability seems to contribute to the pathophysiology of some laminopathies, there is limited information about what mutations cause genomic instability and by which molecular mechanisms. Mouse embryonic fibroblasts depleted of A-type lamins or expressing mutants lacking exons 8-11 (Lmna(Δ8-11/Δ8-11)) exhibit alterations in telomere biology and DNA repair caused by cathepsin L-mediated degradation of 53BP1 and reduced expression of BRCA1 and RAD51. Thus, a region encompassing exons 8-11 seems essential for genome integrity. Given that deletion of lamin A exon 9 in the mouse (Lmna(Δ9/Δ9)) results in a progeria phenotype, we tested if this domain is important for genome integrity. Lmna(Δ9/Δ9) MEFs exhibit telomere shortening and heterochromatin alterations but do not activate cathepsin L-mediated degradation of 53BP1 and maintain expression of BRCA1 and RAD51. Accordingly, Lmna(Δ9/Δ9) MEFs do not present genomic instability, and expression of mutant lamin A Δexon9 in lamin-depleted cells restores DNA repair factors levels and partially rescues nuclear abnormalities. These data reveal that the domain encoded by exon 9 is important to maintain telomere homeostasis and heterochromatin structure but does not play a role in DNA repair, thus pointing to other exons in the lamin A tail as responsible for the genomic instability phenotype in Lmna(Δ8-11/Δ8-11) mice. Our study also suggests that the levels of DNA repair factors 53BP1, BRCA1 and RAD51 could potentially serve as biomarkers to identify laminopathies that present with genomic instability.
Asunto(s)
Cromatina/genética , Exones/genética , Inestabilidad Genómica/genética , Lamina Tipo A/genética , Eliminación de Secuencia/genética , Telómero/genética , Animales , Proteína BRCA1/metabolismo , Línea Celular , Cromatina/química , Cromatina/patología , Proteínas Cromosómicas no Histona/metabolismo , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Ratones , Recombinasa Rad51/metabolismo , Telómero/patología , Acortamiento del Telómero/genética , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Loss of 53BP1 rescues BRCA1 deficiency and is associated with BRCA1-deficient and triple-negative breast cancers (TNBC) and with resistance to genotoxic drugs. The mechanisms responsible for decreased 53BP1 transcript and protein levels in tumors remain unknown. Here, we demonstrate that BRCA1 loss activates cathepsin L (CTSL)-mediated degradation of 53BP1. Activation of this pathway rescued homologous recombination repair and allowed BRCA1-deficient cells to bypass growth arrest. Importantly, depletion or inhibition of CTSL with vitamin D or specific inhibitors stabilized 53BP1 and increased genomic instability in response to radiation and poly(adenosine diphosphate-ribose) polymerase inhibitors, compromising proliferation. Analysis of human breast tumors identified nuclear CTSL as a positive biomarker for TNBC, which correlated inversely with 53BP1. Importantly, nuclear levels of CTSL, vitamin D receptor, and 53BP1 emerged as a novel triple biomarker signature for stratification of patients with BRCA1-mutated tumors and TNBC, with potential predictive value for drug response. We identify here a novel pathway with prospective relevance for diagnosis and customization of breast cancer therapy.
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Proteína BRCA1/genética , Neoplasias de la Mama/genética , Catepsina L/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Catepsina L/genética , Línea Celular Tumoral , Reparación del ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica , Mutación de Línea Germinal , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Spatial and temporal organization of the genome represents an additional step in the regulation of nuclear functions. The nuclear lamina, a polymeric meshwork formed by lamins (A/C and B type) and lamin-associated proteins, plays a key role in the maintenance of genome localization, structure and function. Specifically, mutations in the LMNA gene encoding lamins A/C or changes in its expression, either upregulation or silencing, are associated with defects in DNA replication, transcription and repair, as well as alterations in epigenetic modifications of chromatin. These data, together with the fact that defects in A-type lamins are associated with a whole variety of degenerative disorders, premature aging syndromes and cancer, support the notion that these proteins operate as caretakers of the genome. However, our understanding of their functions is limited due to the lack of well-defined mechanisms behind the genomic instability observed in lamin-related diseases. Here, we summarize our recent discovery of new pathways that are affected by the loss of A-type lamins. In particular, we found that A-type lamins control transcription and degradation of proteins with key roles in cell cycle regulation and DNA double-strand breaks (DSBs) repair by non-homologous end-joining (NHEJ) and homologous-recombination (HR). Importantly, the proteins regulated by A-type lamins--Rb family members, 53BP1, BRCA1 and RAD51--exert tumor suppressor functions, with their loss being associated with cancer susceptibility. Moreover, our studies revealed novel pathways that contribute to genomic instability and that can be activated in disease states independent of the status of A-type lamins.
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Ciclo Celular , Reparación del ADN , Laminas/fisiología , Animales , Roturas del ADN de Doble Cadena , Inestabilidad Genómica , Humanos , Laminas/genética , Laminas/metabolismo , Ratones , Modelos Genéticos , Vitamina D/metabolismo , Vitamina D/fisiologíaRESUMEN
Genomic instability due to telomere dysfunction and defective repair of DNA double-strand breaks (DSBs) is an underlying cause of ageing-related diseases. 53BP1 is a key factor in DNA DSBs repair and its deficiency is associated with genomic instability and cancer progression. Here, we uncover a novel pathway regulating the stability of 53BP1. We demonstrate an unprecedented role for the cysteine protease Cathepsin L (CTSL) in the degradation of 53BP1. Overexpression of CTSL in wild-type fibroblasts leads to decreased 53BP1 protein levels and changes in its cellular distribution, resulting in defective repair of DNA DSBs. Importantly, we show that the defects in DNA repair associated with 53BP1 deficiency upon loss of A-type lamins are due to upregulation of CTSL. Furthermore, we demonstrate that treatment with vitamin D stabilizes 53BP1 and promotes DNA DSBs repair via inhibition of CTSL, providing an as yet unsuspected link between vitamin D action and DNA repair. Given that CTSL upregulation is a hallmark of cancer and progeria, regulation of this pathway could be of great therapeutic significance for these diseases.
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Catepsina L/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Lamina Tipo A/fisiología , Vitamina D/fisiología , Animales , Calcitriol/farmacología , Catepsina L/antagonistas & inhibidores , Catepsina L/biosíntesis , Catepsina L/genética , Línea Celular , Precursores Enzimáticos/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Lamina Tipo A/deficiencia , Lamina Tipo A/genética , Leupeptinas/farmacología , Ratones , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteínas Recombinantes de Fusión/fisiología , Especificidad de la Especie , Transfección , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
A-type lamins are emerging as regulators of nuclear organization and function. Changes in their expression are associated with cancer and mutations are linked to degenerative diseases -laminopathies-. Although a correlation exists between alterations in lamins and genomic instability, the molecular mechanisms remain largely unknown. We previously found that loss of A-type lamins leads to degradation of 53BP1 protein and defective long-range non-homologous end-joining (NHEJ) of dysfunctional telomeres. Here, we determined how loss of A-type lamins affects the repair of short-range DNA double-strand breaks (DSBs) induced by ionizing radiation (IR). We find that lamins deficiency allows activation of the DNA damage response, but compromises the accumulation of 53BP1 at IR-induced foci (IRIF), hindering the fast phase of repair corresponding to classical-NHEJ. Importantly, reconstitution of 53BP1 is sufficient to rescue long-range and short-range NHEJ. Moreover, we demonstrate an unprecedented role for A-type lamins in the maintenance of homologous recombination (HR). Depletion of lamins compromises HR by a mechanism involving transcriptional downregulation of BRCA1 and RAD51 by the repressor complex formed by the Rb family member p130 and E2F4. In line with the DNA repair defects, lamins-deficient cells exhibit increased radiosensitivity. This study demonstrates that A-type lamins promote genomic stability by maintaining the levels of proteins with key roles in DNA DSBs repair by NHEJ and HR. Our results suggest that silencing of A-type lamins by DNA methylation in some cancers could contribute to the genomic instability that drives malignancy. In addition, lamins-deficient tumor cells could represent a good target for radiation therapy.
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Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Lamina Tipo A/metabolismo , Animales , Proteína BRCA1/metabolismo , Línea Celular , Inestabilidad Cromosómica , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción E2F4/metabolismo , Recombinación Homóloga , Humanos , Lamina Tipo A/antagonistas & inhibidores , Ratones , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Recombinasa Rad51/metabolismo , Radiación Ionizante , Proteína p130 Similar a la del Retinoblastoma/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Research performed in the last few years has revealed important roles for the spatial and temporal organization of the genome on genome function and integrity. A challenge in the field is to determine the molecular mechanisms involved in the organization of genome function. A-type lamins, key structural components of the nucleus, have been implicated in the maintenance of nuclear architecture and chromatin structure. Interestingly, alterations of A-type lamins lead to defects in DNA replication and repair as well as gene transcription and silencing. Elucidating the functions of these proteins is a topical subject since alterations of A-type lamins are associated with a variety of human diseases, ranging from muscular dystrophies and premature aging syndromes to cancer. Here, we discuss novels roles for A-type lamins in the maintenance of telomere structure, length and function as well as in the stabilization of a key DNA damage response factor. These studies support the notion that increased genomic instability due to defects in telomere biology and DNA repair contribute to the pathogenesis of lamin-related diseases.
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Inestabilidad Genómica , Lamina Tipo A/fisiología , Daño del ADN , Reparación del ADN , Marcación de Gen/métodos , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Telómero/metabolismo , Telómero/fisiologíaRESUMEN
A-type lamins are intermediate filament proteins that provide a scaffold for protein complexes regulating nuclear structure and function. Mutations in the LMNA gene are linked to a variety of degenerative disorders termed laminopathies, whereas changes in the expression of lamins are associated with tumourigenesis. The molecular pathways affected by alterations of A-type lamins and how they contribute to disease are poorly understood. Here, we show that A-type lamins have a key role in the maintenance of telomere structure, length and function, and in the stabilization of 53BP1, a component of the DNA damage response (DDR) pathway. Loss of A-type lamins alters the nuclear distribution of telomeres and results in telomere shortening, defects in telomeric heterochromatin, and increased genomic instability. In addition, A-type lamins are necessary for the processing of dysfunctional telomeres by non-homologous end joining, putatively through stabilization of 53BP1. This study shows new functions for A-type lamins in the maintenance of genomic integrity, and suggests that alterations of telomere biology and defects in DDR contribute to the pathogenesis of lamin-related diseases.