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BACKGROUND: Immunotherapy based on checkpoint inhibitors is highly effective in mismatch repair deficient (MMRd) colorectal cancer (CRC). These tumors carry a high number of mutations, which are predicted to translate into a wide array of neoepitopes; however, a systematic classification of the neoantigen repertoire in MMRd CRC is lacking. Mass spectrometry peptidomics has demonstrated the existence of MHC class I associated peptides (MAPs) originating from non-coding DNA regions. Based on these premises we investigated DNA genomic regions responsible for generating MMRd-induced peptides. METHODS: We exploited mouse CRC models in which the MMR gene Mlh1 was genetically inactivated. Isogenic cell lines CT26 Mlh1+/+ and Mlh1-/- were inoculated in immunocompromised and immunocompetent mice. Whole genome and RNA sequencing data were generated from samples obtained before and after injection in murine hosts. First, peptide databases were built from transcriptomes of isogenic cell lines. We then compiled a database of peptides lost after tumor cells injection in immunocompetent mice, likely due to immune editing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matched next-generation sequencing databases were employed to identify the DNA regions from which the immune-targeted MAPs originated. Finally, we adopted in vitro T cell assays to verify whether MAP-specific T cells were part of the in vivo immune response against Mlh1-/- cells. RESULTS: Whole genome sequencing analyses revealed an unbalanced distribution of immune edited alterations across the genome in Mlh1-/- cells grown in immunocompetent mice. Specifically, untranslated (UTR) and coding regions exhibited the largest fraction of mutations leading to highly immunogenic peptides. Moreover, the integrated computational and LC-MS/MS analyses revealed that MAPs originate mainly from atypical translational events in both Mlh1+/+ and Mlh1-/- tumor cells. In addition, mutated MAPs-derived from UTRs and out-of-frame translation of coding regions-were highly enriched in Mlh1-/- cells. The MAPs trigger T-cell activation in mice primed with Mlh1-/- cells. CONCLUSIONS: Our results suggest that-in comparison to MMR proficient CRC-MMRd tumors generate a significantly higher number of non-canonical mutated peptides able to elicit T cell responses. These results reveal the importance of evaluating the diversity of neoepitope repertoire in MMRd tumors.
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Neoplasias Encefálicas , Neoplasias del Colon , Neoplasias Colorrectales , Síndromes Neoplásicos Hereditarios , Animales , Ratones , Reparación de la Incompatibilidad de ADN/genética , Cromatografía Liquida , Espectrometría de Masas en Tándem , Neoplasias Colorrectales/patología , Péptidos , Antígenos de Histocompatibilidad Clase I/genética , ADNRESUMEN
Patients affected by colorectal cancer (CRC) with DNA mismatch repair deficiency (MMRd), often respond to immune checkpoint blockade therapies, while those with mismatch repair-proficient (MMRp) tumors generally do not. Interestingly, a subset of MMRp CRCs contains variable fractions of MMRd cells, but it is unknown how their presence impacts immune surveillance. We asked whether modulation of the MMRd fraction in MMR heterogeneous tumors acts as an endogenous cancer vaccine by promoting immune surveillance. To test this hypothesis, we use isogenic MMRp (Mlh1+/+) and MMRd (Mlh1-/-) mouse CRC cells. MMRp/MMRd cells mixed at different ratios are injected in immunocompetent mice and tumor rejection is observed when at least 50% of cells are MMRd. To enrich the MMRd fraction, MMRp/MMRd tumors are treated with 6-thioguanine, which leads to tumor rejection. These results suggest that genetic and pharmacological modulation of the DNA mismatch repair machinery potentiate the immunogenicity of MMR heterogeneous tumors.
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Neoplasias Encefálicas , Neoplasias Colorrectales , Animales , Ratones , Reparación de la Incompatibilidad de ADN/genética , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Inestabilidad de MicrosatélitesRESUMEN
Cyclin-dependent kinase 12 (CDK12) is a serine/threonine kinase involved in the regulation of RNA polymerase II and in the transcription of a subset of genes involved in the DNA damage response. CDK12 is one of the most mutated genes in ovarian carcinoma. These mutations result in loss-of-function and can predict the responses to PARP1/2 inhibitor and platinum. To investigate the role of CDK12 in ovarian cancer, CRISPR/Cas9 technology was used to generate a stable CDK12 knockout (KO) clone in A2780 ovarian carcinoma cells. This is the first report on a CDK12 null cell line. The clone had slower cell growth and was less clonogenic than parental cells. These data were confirmed in vivo, where CDK12 KO transplanted cells had a much longer time lag and slightly slower growth rate than CDK12-expressing cells. The slower growth was associated with a higher basal level of apoptosis, but there were no differences in the basal level of autophagy and senescence. While cell cycle distribution was similar in parental and knockout cells, there was a doubling in DNA content, with an almost double modal number of chromosomes in the CDK12 KO clone which, however did not display any increase in γH2AX, a marker of DNA damage. We found partial down-regulation of the expression of DNA repair genes at the mRNA level and, among the down-regulated genes, an enrichment in the G2/M checkpoint genes. Although the biological features of CDK12 KO cells are compatible with the function of CDK12, contrary to some reports, we could not find any difference in the sensitivity to cisplatin and olaparib between wild-type and CDK12 KO cells.
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PURPOSE: Genomic instability is a hallmark of cancer and targeting DNA damage response (DDR) is emerging as a promising therapeutic strategy in different solid tumors. The effectiveness of targeting DDR in colorectal cancer has not been extensively explored. EXPERIMENTAL DESIGN: We challenged 112 cell models recapitulating the genomic landscape of metastatic colorectal cancer with ATM, ATR, CHK1, WEE1, and DNA-PK inhibitors, in parallel with chemotherapeutic agents. We focused then on ATR inhibitors (ATRi) and, to identify putative biomarkers of response and resistance, we analyzed at multiple levels colorectal cancer models highly sensitive or resistant to these drugs. RESULTS: We found that around 30% of colorectal cancers, including those carrying KRAS and BRAF mutations and unresponsive to targeted agents, are sensitive to at least one DDR inhibitor. By investigating potential biomarkers of response to ATRi, we found that ATRi-sensitive cells displayed reduced phospho-RPA32 foci at basal level, while ATRi-resistant cells showed increased RAD51 foci formation in response to replication stress. Lack of ATM and RAD51C expression was associated with ATRi sensitivity. Analysis of mutational signatures and HRDetect score identified a subgroup of ATRi-sensitive models. Organoids derived from patients with metastatic colorectal cancer recapitulated findings obtained in cell lines. CONCLUSIONS: In conclusion, a subset of colorectal cancers refractory to current therapies could benefit from inhibitors of DDR pathways and replication stress. A composite biomarker involving phospho-RPA32 and RAD51 foci, lack of ATM and RAD51C expression, as well as analysis of mutational signatures could be used to identify colorectal cancers likely to respond to ATRi.
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Antineoplásicos , Neoplasias Colorrectales , Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Daño del ADN , Replicación del ADN , Proteína Quinasa Activada por ADN/genética , Humanos , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Mitochondria have attracted attention in cancer research as organelles associated with tumor development and response to therapy. We recently reported acquisition of resistance to cisplatin (DDP) associated with a metabolic rewiring in ovarian cancer patient-derived xenografts (PDXs) models. DDP-resistant PDXs models were obtained mimicking the clinical setting, treating mice bearing sensitive-DDP tumors with multiple cycles of DDP until the development of resistance. To further characterize the metabolic rewiring, the present study focused on tumor mitochondria. We analysed by transmission electron microscopy the mitochondria structure in two models of DDP-resistant and the corresponding DDP-sensitive PDXs and evaluated tumor mDNA content, the expression of genes and proteins involved in mitochondria functionality, and mitochondria fitness-related processes, such as autophagy. We observed a decrease in the number of mitochondria paralleled by an increased volume in DDP-resistant versus DDP-sensitive PDXs. DDP-resistant PDXs presented a higher percentage of damaged mitochondria, in particular of type 2 (concave-shape), and type 3 (cristolysis) damage. We found no difference in the mDNA content, and the expression of genes involved in mitochondrial biogenesis was similar between the sensitive and resistant PDXs. An upregulation of some genes involved in mitochondrial fitness in DDP-R versus DDP-S PDXs was observed. At protein level, no difference in the expression of proteins involved in mitochondrial function and biogenesis, and in autophagy/mitophagy was found. We here reported that the acquisition of DDP resistance is associated with morphological alterations in mitochondria, even if we couldn't find any dysregulation in the studied genes/proteins that could explain the observed differences.
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Inactivation of beta-2 microglobulin (B2M) is considered a determinant of resistance to immune checkpoint inhibitors (ICPi) in melanoma and lung cancers. In contrast, B2M loss does not appear to affect response to ICPis in mismatch repair-deficient (MMRd) colorectal tumors where biallelic inactivation of B2M is frequently observed. We inactivated B2m in multiple murine MMRd cancer models. Although MMRd cells would not readily grow in immunocompetent mice, MMRd B2m null cells were tumorigenic and regressed when treated with anti-PD-1 and anti-CTLA4. The efficacy of ICPis against MMRd B2m null tumors did not require CD8+ T cells but relied on the presence of CD4+ T cells. Human tumors expressing low levels of B2M display increased intratumoral CD4+ T cells. We conclude that B2M inactivation does not blunt the efficacy of ICPi in MMRd tumors, and we identify a unique role for CD4+ T cells in tumor rejection. SIGNIFICANCE: B2M alterations, which impair antigen presentation, occur frequently in microsatellite-unstable colorectal cancers. Although in melanoma and lung cancers B2M loss is a mechanism of resistance to immune checkpoint blockade, we show that MMRd tumors respond to ICPis through CD4+ T-cell activation.This article is highlighted in the In This Issue feature, p. 1601.
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Linfocitos T CD4-Positivos/inmunología , Neoplasias Colorrectales/metabolismo , Microglobulina beta-2/metabolismo , Animales , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
Mucinous epithelial ovarian cancer (mEOC) is a rare subset of epithelial ovarian cancer. When diagnosed at a late stage, its prognosis is very poor, as it is quite chemo-resistant. To find new therapeutic options for mEOC, we performed high-throughput screening using a siRNA library directed against human protein kinases in a mEOC cell line, and polo-like kinase1 (PLK1) was identified as the kinase whose downregulation interfered with cell proliferation. Both PLK1 siRNA and two specific PLK1 inhibitors (onvansertib and volasertib) were able to inhibit cell growth, induce apoptosis and block cells in the G2/M phase of the cell cycle. We evaluated, in vitro, the combinations of PLK1 inhibitors and different chemotherapeutic drugs currently used in the treatment of mEOC, and we observed a synergistic effect of PLK1 inhibitors and antimitotic drugs. When translated into an in vivo xenograft model, the combination of onvansertib and paclitaxel resulted in stronger tumor regressions and in a longer mice survival than the single treatments. These effects were associated with a higher induction of mitotic block and induction of apoptosis, similarly to what was observed in vitro. These data suggest that the combination onvansertib/paclitaxel could represent a new active therapeutic option in mEOC.
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Vitamin C (VitC) is known to directly impair cancer cell growth in preclinical models, but there is little clinical evidence on its antitumoral efficacy. In addition, whether and how VitC modulates anticancer immune responses is mostly unknown. Here, we show that a fully competent immune system is required to maximize the antiproliferative effect of VitC in breast, colorectal, melanoma, and pancreatic murine tumors. High-dose VitC modulates infiltration of the tumor microenvironment by cells of the immune system and delays cancer growth in a T cell-dependent manner. VitC not only enhances the cytotoxic activity of adoptively transferred CD8 T cells but also cooperates with immune checkpoint therapy (ICT) in several cancer types. Combination of VitC and ICT can be curative in models of mismatch repair-deficient tumors with high mutational burden. This work provides a rationale for clinical trials combining ICT with high doses of VitC.
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Antineoplásicos , Melanoma , Animales , Antineoplásicos/farmacología , Ácido Ascórbico/farmacología , Ácido Ascórbico/uso terapéutico , Inmunoterapia , Ratones , Microambiente TumoralRESUMEN
The DNA damage response (DDR) kinases ATR, Chk1, and Wee1 play vital roles in the response to replication stress and in maintaining cancer genomic stability. Inhibitors of these kinases are currently under clinical investigation. Mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) are aggressive lymphomas whose clinical outcome is still largely unsatisfactory. These cell lymphoma subtypes are highly dependent on both Chk1 and Wee1 for survival. We investigated the activity of the ATR inhibitor AZD6738 as single agent and in combination with either Chk1 (AZD6738) or Wee1 (AZD1775) inhibitors in several preclinical models of MCL and DLBCL. This study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination, and validation experiments on in vivo models. Cellular and molecular mechanisms of the observed synergistic effect as well as pharmacodynamic analysis of in vivo samples were studied. AZD6738 exerted a strong synergistic cytotoxic effect in combination with both AZD7762 and AZD1775 in the 2 lymphoma subtypes regardless of their TP53, MYC, and ATM mutational status. These DDR inhibitor combinations, similarly to the Chk1/Wee1 inhibitor combination, caused a marked S-phase delay, with an increase in cyclin-dependent kinases (CDK) activity, increased DNA damage, and decreases in Wee1, MYC, and RRM2 protein levels. The synergistic in vitro activity translated to striking in vivo antitumor activity. DDR-DDR inhibitor combinations could potentially offer promising novel therapeutic strategies for patients with B-cell lymphoma.
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Daño del ADN/efectos de los fármacos , Linfoma de Células B/tratamiento farmacológico , Animales , Línea Celular Tumoral , Femenino , Humanos , Linfoma de Células B/patología , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Epithelial ovarian cancer is the most lethal gynecological cancer and the high mortality is due to the frequent presentation at advanced stage, and to primary or acquired resistance to platinum-based therapy. METHODS: We developed three new models of ovarian cancer patient-derived xenografts (ovarian PDXs) resistant to cisplatin (cDDP) after multiple in vivo drug treatments. By different and complementary approaches based on integrated metabolomics (both targeted and untargeted mass spectrometry-based techniques), gene expression, and functional assays (Seahorse technology) we analyzed and compared the tumor metabolic profile in each sensitive and their corresponding cDDP-resistant PDXs. RESULTS: We found that cDDP-sensitive and -resistant PDXs have a different metabolic asset. In particular, we found, through metabolomic and gene expression approaches, that glycolysis, tricarboxylic acid cycle and urea cycle pathways were deregulated in resistant versus sensitive PDXs. In addition, we observed that oxygen consumption rate and mitochondrial respiration were higher in resistant PDXs than in sensitive PDXs under acute stress conditions. An increased oxidative phosphorylation in cDDP-resistant sublines led us to hypothesize that its interference could be of therapeutic value. Indeed, in vivo treatment of metformin and cDDP was able to partially reverse platinum resistance. CONCLUSIONS: Our data strongly reinforce the idea that the development of acquired cDDP resistance in ovarian cancer can bring about a rewiring of tumor metabolism, and that this might be exploited therapeutically.
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BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma with an unfavorable clinical course. Besides deregulation of the cell cycle, B cell receptor (BCR) signaling, essential for MCL proliferation and survival, is also often deregulated due to constitutive activation of Bruton's tyrosine kinase (BTK). The BTK inhibitor ibrutinib has been approved as a therapy for refractory MCL, and while it shows some clinical activity, patients frequently develop primary or secondary ibrutinib resistance and have very poor outcomes after relapsing following ibrutinib treatment. OBJECTIVE: To overcome ibrutinib resistance, new therapeutic approaches are needed. As checkpoint kinase 1 (Chk1) inhibitors have recently been shown to be effective as single agents in MCL, we assessed the combination of ibrutinib with Chk1 inhibitors. METHODS: We examined the activity of ibrutinib combined with the Chk1 inhibitor PF-00477736 in eight MCL cell lines and analyzed underlying cellular and molecular effects. RESULTS: The combination was synergistic in all tested cell lines through different mechanisms. The treatment induced apoptosis in ibrutinib-sensitive cell lines, while in ibrutinib-resistant cells the effect was mainly cytostatic and occurred at micromolar concentrations of ibrutinib. CONCLUSIONS: The pharmacological approach of simultaneously targeting cell cycle checkpoints (by Chk1 inhibitors) and pro-survival pathways (by ibrutinib) might offer a promising new therapeutic strategy for MCL patients.
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Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Linfoma de Células del Manto/tratamiento farmacológico , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Adenina/análogos & derivados , Línea Celular Tumoral , Humanos , Linfoma de Células del Manto/patología , Piperidinas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazoles/farmacología , Pirimidinas/farmacologíaRESUMEN
A xenobank of patient-derived (PDX) ovarian tumor samples has been established consisting of tumors with different sensitivity to cisplatin (DDP), from very responsive to resistant. As the DNA repair pathway is an important driver in tumor response to DDP, we analyzed the mRNA expression of 20 genes involved in the nucleotide excision repair, fanconi anemia, homologous recombination, base excision repair, mismatch repair and translesion repair pathways and the methylation patterns of some of these genes. We also investigated the correlation with the response to platinum-based therapy. The mRNA levels of the selected genes were evaluated by Real Time-PCR (RT-PCR) with ad hoc validated primers and gene promoter methylation by pyrosequencing. All the DNA repair genes were variably expressed in all 42 PDX samples analyzed, with no particular histotype-specific pattern of expression. In high-grade serous/endometrioid PDXs, the CDK12 mRNA expression levels positively correlated with the expression of TP53BP1, PALB2, XPF and POLB. High-grade serous/endometrioid PDXs with TP53 mutations had significantly higher levels of POLQ, FANCD2, RAD51 and POLB than high-grade TP53 wild type PDXs. The mRNA levels of CDK12, PALB2 and XPF inversely associated with the in vivo DDP antitumor activity; higher CDK12 mRNA levels were associated with a higher recurrence rate in ovarian patients with low residual tumor. These data support the important role of CDK12 in the response to a platinum based therapy in ovarian patients.
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ARQ 087 is a multi-tyrosine kinase inhibitor with potent activity against the FGFR receptor family, currently in Phase I clinical studies for the treatment of advanced solid tumors. The compound has a very safe profile and induces tumor regressions in FGFR-driven models. The feasibility of combining ARQ 087 with chemotherapy was investigated in FGFR deregulated human xenografts. Nude mice were transplanted subcutaneously with H1581, and when tumor masses reached 150 mg, were randomized to receive vehicle, ARQ 087, paclitaxel, carboplatin as single agents or in combination. Similar experimental conditions were applied in nude mice bearing SNU16 and MFE296 xenografts, with the inclusion of capecitabine in the former xenograft model. In the different xenograft models, the drugs given as single agents ranged from very active to partially active. The double combinations were more active than the single ones, but the triple combinations were the most active. In particular, the combination of ARQ 087 + paclitaxel + carboplatin in H1581 bearing mice was able to induce tumor regression in all the mice, with 6/8 mice tumor free at day 140 after tumor transplant. Of note, no toxic deaths nor premature stopping or delaying of drug administration were observed. The data herein reported demonstrated the feasibility of using xenografts models for poli-chemotherapeutic trials mimicking the best standard of care in treatment of specific tumor type and that ARQ 087, a new pan-FGFR inhibitor, can be safely combined with standard cytotoxic chemotherapeutic drugs with apparently no sign of cumulative toxicity and an associated increased antitumor effect.
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The fibroblast growth factor receptor (FGFR) pathway has been implicated both as an escape mechanism from anti-angiogenic therapy and as a driver oncogene in different tumor types. Lucitanib is a small molecule inhibitor of vascular endothelial growth factor (VEGF) receptors 1 to 3 (VEGFR1 to 3), platelet derived growth factor α/ß (PDGFRα/ß) and FGFR1-3 tyrosine kinases and has demonstrated activity in a phase I/II clinical study, with objective RECIST responses in breast cancer patients with FGFR1 or FGF3/4/19 gene amplification, as well as in patients anticipated to benefit from anti-angiogenic agents. We report here the in vitro and in vivo antitumor activity of lucitanib in experimental models with or without FGFR1/2 amplification or mutations. In cell assays, lucitanib potently inhibited the growth of tumor cell lines with amplified FGFR1 or mutated/amplified FGFR2. In all xenograft models studied, lucitanib demonstrated marked tumor growth inhibition due to potent inhibition of angiogenesis. Notably, in two lung cancer models with FGFR1 amplification, the antitumor efficacy was higher, suggesting that the simultaneous inhibition of VEGF and FGF receptors in FGFR1 dependent tumors can be therapeutically advantageous. Similar antitumor activity was observed in FGFR2 wild-type and amplified or mutated xenograft models. Pharmacokinetic studies showed lucitanib plasma concentrations in the micro/sub-micromolar range demonstrated drug accumulation following repeated lucitanib administration.
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Amplificación de Genes , Mutación , Naftalenos/farmacología , Quinolinas/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Dosificación de Gen , Humanos , Concentración 50 Inhibidora , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Phosphorylation of the RNA polymerase II C-terminal domain by cyclin-dependent kinases (CDKs) is important for productive transcription. Deregulated transcription-CDKs have been reported in different human cancers. Until recently CDK9 was the only transcription-CDK with a causative role in cancer, but evidence is cumulating of the importance of CDK12. This review summarizes the role of CDK12 in transcription and RNA processing, in maintaining genomic stability/integrity and in tumorigenesis. CDK12 mutations have been reported in many cancers and have been suggested as a cause of defective DNA repair in ovarian carcinoma. CDK12 may have a role as a new therapeutic target in oncology.
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Carcinogénesis/genética , Quinasas Ciclina-Dependientes/genética , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica/genética , Neoplasias/genética , ARN Polimerasa II/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Variaciones en el Número de Copia de ADN , Reparación del ADN/genética , Femenino , Recombinación Homóloga/genética , Humanos , Terapia Molecular Dirigida , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , FosforilaciónRESUMEN
MTAP is a ubiquitously expressed gene important for adenine and methionine salvage. The gene is located at 9p21, a chromosome region often deleted in breast carcinomas, similar to CDKN2A, a recognized tumor suppressor gene. Several research groups have shown that MTAP acts as a tumor suppressor, and some therapeutic approaches were proposed based on a tumors´ MTAP status. We analyzed MTAP and CDKN2A gene (RT-qPCR) and protein (western-blotting) expression in seven breast cancer cell lines and evaluated their promoter methylation patterns to better characterize the contribution of these genes to breast cancer. Cytotoxicity assays with inhibitors of de novo adenine synthesis (5-FU, AZA and MTX) after MTAP gene knockdown showed an increased sensitivity, mainly to 5-FU. MTAP expression was also evaluated in two groups of samples from breast cancer patients, fresh tumors and paired normal breast tissue, and from formalin-fixed paraffin embedded (FFPE) core breast cancer samples diagnosed as Luminal-A tumors and triple negative breast tumors (TNBC). The difference of MTAP expression between fresh tumors and normal tissues was not statistically significant. However, MTAP expression was significantly higher in Luminal-A breast tumors than in TNBC, suggesting the lack of expression in more aggressive breast tumors and the possibility of using the new approaches based on MTAP status in TNBC.
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Adenocarcinoma Mucinoso/genética , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Regulación Neoplásica de la Expresión Génica , Purina-Nucleósido Fosforilasa/genética , Neoplasias de la Mama Triple Negativas/genética , Adenocarcinoma Mucinoso/tratamiento farmacológico , Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patología , Antineoplásicos/farmacología , Azacitidina/farmacología , Carcinoma Ductal de Mama/tratamiento farmacológico , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Carcinoma Lobular/tratamiento farmacológico , Carcinoma Lobular/metabolismo , Carcinoma Lobular/patología , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Metilación de ADN , Receptor alfa de Estrógeno/deficiencia , Receptor alfa de Estrógeno/genética , Femenino , Fluorouracilo/farmacología , Humanos , Metástasis Linfática , Metotrexato/farmacología , Especificidad de Órganos , Regiones Promotoras Genéticas , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor ErbB-2/deficiencia , Receptor ErbB-2/genética , Receptores de Progesterona/deficiencia , Receptores de Progesterona/genética , Transducción de Señal , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the chromosomal translocation t(11;14) that leads to constitutive expression of cyclin D1, a master regulator of the G1-S phase. Chk1 inhibitors have been recently shown to be strongly effective as single agents in MCL. To investigate molecular mechanisms at the basis of Chk1 inhibitor activity, a MCL cell line resistant to the Chk1 inhibitor PF-00477736 (JEKO-1 R) was obtained and characterized. The JEKO-1 R cell line was cross resistant to another Chk1 inhibitor (AZD-7762) and to the Wee1 inhibitor MK-1775. It displayed a shorter doubling time than parental cell line, likely due to a faster S phase. Cyclin D1 expression levels were decreased in resistant cell line and its re-overexpression partially re-established PF-00477736 sensitivity. Gene expression profiling showed an enrichment in gene sets involved in pro-survival pathways in JEKO-1 R. Dasatinib treatment partly restored PF-00477736 sensitivity in resistant cells suggesting that the pharmacological interference of pro-survival pathways can overcome the resistance to Chk1 inhibitors. These data further corroborate the involvement of the t(11;14) in cellular sensitivity to Chk1 inhibitors, fostering the clinical testing of Chk1 inhibitors as single agents in MCL.
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Antineoplásicos/farmacología , Benzodiazepinonas/farmacología , Ciclo Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Linfoma de Células del Manto/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Pirazoles/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Ciclina D1/genética , Ciclina D1/metabolismo , Dasatinib/farmacología , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Linfoma de Células del Manto/enzimología , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/patología , Terapia Molecular Dirigida , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Pirimidinas/farmacología , Pirimidinonas , Transducción de Señal/efectos de los fármacos , Tiofenos/farmacología , Factores de Tiempo , Urea/análogos & derivados , Urea/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismoRESUMEN
Mantle cell lymphoma (MCL) is an aggressive, incurable disease, characterized by a deregulated cell cycle. Chk1 and Wee1 are main regulators of cell cycle progression and recent data on solid tumors suggest that simultaneous inhibition of these proteins has a strong synergistic cytotoxic effect. The effects of a Chk1 inhibitor (PF-00477736) and a Wee1 inhibitor (MK-1775) have been herein investigated in a large panel of mature B-cell lymphoma cell lines. We found that MCL cells were the most sensitive to the Chk1 inhibitor PF-00477736 and Wee1 inhibitor MK-1775 as single agents. Possible involvement of the translocation t(11;14) in Chk1 inhibitor sensitivity was hypothesized. The combined inhibition of Chk1 and Wee1 was strongly synergistic in MCL cells, leading to deregulation of the cell cycle, with increased activity of CDK2 and CDK1, and activation of apoptosis. In vivo treatment with the drug combination of mice bearing JeKo-1 xenografts (MCL) had a marked antitumor effect with tumor regressions observed at non-toxic doses (best T/C%=0.54%). Gene expression profiling suggested effect on genes involved in apoptosis. The strong synergism observed by combining Chk1 and Wee1 inhibitors in preclinical models of MCL provides the rationale for testing this combination in the clinical setting.
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Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Benzodiazepinonas/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Linfoma de Células del Manto/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína Quinasa CDC2 , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Perfilación de la Expresión Génica , Humanos , Concentración 50 Inhibidora , Linfoma de Células del Manto/enzimología , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/patología , Ratones Desnudos , Terapia Molecular Dirigida , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Quinasas/genética , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Pirimidinonas , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chk1 is implicated in several checkpoints of the cell cycle acting as a key player in the signal transduction pathway activated in response to DNA damage and crucial for the maintenance of genomic stability. Chk1 also plays a role in the mitotic spindle checkpoint, which ensures the fidelity of mitotic segregation during mitosis, preventing chromosomal instability and aneuploidy. Mad2 is one of the main mitotic checkpoint components and also exerts a role in the cellular response to DNA damage. To investigate a possible crosslink existing between Chk1 and Mad2, we studied Mad2 protein levels after Chk1 inhibition either by specific siRNAs or by a specific and selective Chk1 inhibitor (PF-00477736), and we found that after Chk1 inhibition, Mad2 protein levels decrease only in tumor cells sensitive to Chk1 depletion. We then mapped six Chk1's phosphorylatable sites on Mad2 protein, and found that Chk1 is able to phosphorylate Mad2 in vitro on more than one site, while it is incapable of phoshorylating the Mad2 form mutated on all six phosphorylatable sites. Moreover our studies demonstrate that Chk1 co-localizes and physically associates with Mad2 in cells both under unstressed conditions and after DNA damage, thus providing new and interesting evidence on Chk1 and Mad2 crosstalk in the DNA damage checkpoint and in the mitotic spindle checkpoint.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Proteínas Quinasas/metabolismo , Proteínas Represoras/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Benzodiazepinonas/farmacología , Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Humanos , Puntos de Control de la Fase M del Ciclo Celular , Proteínas Mad2 , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/química , Proteínas Quinasas/genética , Pirazoles/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/genéticaRESUMEN
Bone is the most common metastatic site for breast cancer. There is a significant need to understand the molecular mechanisms controlling the engraftment and growth of tumor cells in bone and to discover novel effective therapeutic strategies. The aim of this study was to assess the effects of tivantinib and Zoledronic Acid (ZA) in combination in a breast xenograft model of bone metastases. Cancer cells were intracardially implanted into immunodeficient mice and the effects of drugs alone or in combination on bone metastasis were evaluated by in vivo non-invasive optical and micro-CT imaging technologies. Drugs were administered either before (preventive regimen) or after (therapeutic regimen) bone metastases were detectable. In the preventive regimen, the combination of tivantinib plus ZA was much more effective than single agents in delaying bone metastatic tumor growth. When administered in the therapeutic schedule, the combination delayed metastatic progression and was effective in improving survival. These effects were not ascribed to a direct cytotoxic effect of the combined therapy on breast cancer cells in vitro. The results of this study provide the rationale for the design of new combinatorial strategies with tivantinib and ZA for the treatment of breast cancer bone metastases.