RESUMEN
BACKGROUND: Acute myeloid leukemia (AML) is a type of leukemia in adults with a high mortality rate and poor prognosis. Although targeted therapeutics, chemotherapy, and hematopoietic stem cell transplantation can improve the prognosis, the recurrence rate is still high, with a 5-year survival rate of approximately 40%. This study aimed to develop an IgG-based asymmetric bispecific antibody that targets CLL-1 and CD3 for treating AML. METHODS: ABL602 candidates were compared in terms of binding activity, T-cell activation, and tumor-killing activities. ABL602-mediated T-cell activation and tumor-killing activities were determined by measuring the expression of activation markers, cytokines, cytolytic proteins, and the proportion of dead cells. We evaluated in vivo tumor growth inhibitory activity in two mouse models bearing subcutaneously and orthotopically engrafted human AML. Direct tumor-killing activity and T-cell activation in patient-derived AML blasts were also evaluated. RESULTS: ABL602 2+1 showed a limited CD3 binding in the absence of CLL-1, suggesting that steric hindrance on the CD3 binding arm could reduce CLL-1 expression-independent CD3 binding. Although the CD3 binding activity was attenuated compared with that of 1+1, ABL602 2+1 exhibited much stronger T-cell activation and potent tumor-killing activities in AML cell lines. ABL602 2+1 efficiently inhibited tumor progression in subcutaneously and orthotopically engrafted AML mouse models. In the orthotopic mouse model, tumor growth inhibition was observed by gross measurement of luciferase activity, as well as a reduced proportion of AML blasts in the bone marrow, as determined by flow cytometry and immunohistochemistry (IHC) staining. ABL602 2+1 efficiently activated T cells and induced the lysis of AML blasts, even at very low effector:target (E:T) ratios (eg, 1:50). Compared with the reference 1+1 antibody, ABL602 did not induce the release of cytokines including interleukin-6 and tumor necrosis factor-α in the healthy donor-derived peripheral blood mononuclear cell. CONCLUSIONS: With its potent tumor-killing activity and reduced cytokine release, ABL602 2+1 is a promising candidate for treating patients with AML and warrants further study.
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Anticuerpos Biespecíficos , Leucemia Linfocítica Crónica de Células B , Leucemia Mieloide Aguda , Ratones , Adulto , Animales , Humanos , Citocinas/metabolismo , Leucocitos Mononucleares , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéuticoRESUMEN
BAP1 is a ubiquitin C-terminal hydrolase domain-containing deubiquitinase with a wide array of biological activities. Studies in which advanced sequencing technologies were used have uncovered a link between BAP1 and human cancer. Somatic and germline mutations of the BAP1 gene have been identified in multiple human cancers, with a particularly high frequency in mesothelioma, uveal melanoma and clear cell renal cell carcinoma. BAP1 cancer syndrome highlights that all carriers of inherited BAP1-inactivating mutations develop at least one and often multiple cancers with high penetrance during their lifetime. These findings, together with substantial evidence indicating the involvement of BAP1 in many cancer-related biological activities, strongly suggest that BAP1 functions as a tumor suppressor. Nonetheless, the mechanisms that account for the tumor suppressor function of BAP1 have only begun to be elucidated. Recently, the roles of BAP1 in genome stability and apoptosis have drawn considerable attention, and they are compelling candidates for key mechanistic factors. In this review, we focus on genome stability and summarize the details of the cellular and molecular functions of BAP1 in DNA repair and replication, which are crucial for genome integrity, and discuss the implications for BAP1-associated cancer and relevant therapeutic strategies. We also highlight some unresolved issues and potential future research directions.
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Neoplasias Renales , Melanoma , Mesotelioma , Humanos , Proteínas Supresoras de Tumor/genética , Mesotelioma/genética , Inestabilidad Genómica , Ubiquitina Tiolesterasa/genética , Predisposición Genética a la EnfermedadRESUMEN
BRCA1-associated protein-1 (BAP1) is a ubiquitin C-terminal hydrolase domain-containing deubiquitinase. The gene encoding BAP1 is mutated in various human cancers, including mesothelioma, uveal melanoma and renal cell carcinoma. BAP1 plays roles in many cancer-related cellular functions, including cell proliferation, cell death, and nuclear processes crucial for genome stability, such as DNA repair and replication. While these findings suggest that BAP1 functions as a tumor suppressor, recent data also suggest that BAP1 might play tumor-promoting roles in certain cancers, such as breast cancer and hematopoietic malignancies. Here, we show that BAP1 is upregulated in colon cancer cells and tissues and that BAP1 depletion reduces colon cancer cell proliferation and tumor growth. BAP1 contributes to colon cancer cell proliferation by accelerating DNA replication and suppressing replication stress and concomitant apoptosis. A recently identified BAP1 inhibitor, TG2-179-1, which seems to covalently bind to the active site of BAP1, exhibits potent cytotoxic activity against colon cancer cells, with half-maximal inhibitory concentrations of less than 10 µM, and inhibits colon tumor growth. TG2-179-1 exerts cytotoxic activity by targeting BAP1, leading to defective replication and increased apoptosis. This work therefore shows that BAP1 acts oncogenically in colon cancer and is a potential therapeutic target for this cancer. Our work also suggests that TG2-179-1 can be developed as a potential therapeutic agent for colon cancer.
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Antineoplásicos , Neoplasias del Colon , Ubiquitina Tiolesterasa , Humanos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/genéticaRESUMEN
BRCA1-associated protein-1 (BAP1) is a ubiquitin C-terminal hydrolase domain-containing deubiquitinase with tumor suppressor activity. The gene encoding BAP1 is mutated in various human cancers, with particularly high frequency in kidney and skin cancers, and BAP1 is involved in many cancer-related cellular functions, such as DNA repair and genome stability. Although BAP1 stimulates DNA double-strand break repair, whether it functions in nucleotide excision repair (NER) is unknown. Here, we show that BAP1 promotes the repair of ultraviolet (UV)-induced DNA damage via its deubiquitination activity in various cell types, including primary melanocytes. Poly(ADP-ribose) polymerase 1 (PARP1) interacts with and recruits BAP1 to damage sites, with BAP1 recruitment peaking after the DDB2 and XPC damage sensors. BAP1 recruitment also requires histone H2A monoubiquitinated at Lys119, which accumulates at damage sites. PARP1 transiently poly(ADP-ribosyl)ates (PARylates) BAP1 at multiple sites after UV damage and stimulates the deubiquitination activity of BAP1 both intrinsically and via PARylation. PARP1 also promotes BAP1 stability via crosstalk between PARylation and ubiquitination. Many PARylation sites in BAP1 are mutated in various human cancers, among which the glutamic acid (Glu) residue at position 31, with particularly frequent mutation in kidney cancer, plays a critical role in BAP1 stabilization and promotes UV-induced DNA damage repair. Glu31 also participates in reducing the viability of kidney cancer cells. This study therefore reveals that BAP1 functions in the NER pathway and that PARP1 plays a role as a novel factor that regulates BAP1 enzymatic activity, protein stability, and recruitment to damage sites. This activity of BAP1 in NER, along with its cancer cell viability-reducing activity, may account for its tumor suppressor function.
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Neoplasias Renales , Ubiquitina Tiolesterasa , Humanos , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Daño del ADN , Reparación del ADN , Roturas del ADN de Doble Cadena , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: Stimulation of 4-1BB with agonistic antibodies is a promising strategy for improving the therapeutic efficacy of immune checkpoint inhibitors (ICIs) or for overcoming resistance to ICIs. However, dose-dependent hepatotoxicity was observed in clinical trials with monoclonal anti-4-1BB agonistic antibodies due to the activation of 4-1BB signaling in liver resident Kupffer cells. METHODS: To avoid this on-target liver toxicity, we developed a novel bispecific antibody (4-1BB×PD-L1 bispecific antibody, termed "ABL503") uniquely designed to activate 4-1BB signaling only in the context of PD-L1, while also blocking PD-1/PD-L1 signaling. RESULTS: Functional evaluation using effector cells expressing both 4-1BB and PD-1 revealed superior biological activity of ABL503 compared with the combination of each monoclonal antibody. ABL503 also augmented T-cell activation in in vitro assays and further enhanced the anti-PD-L1-mediated reinvigoration of tumor-infiltrating CD8+ T cells from patients with cancer. Furthermore, in humanized PD-L1/4-1BB transgenic mice challenged with huPD-L1-expressing tumor cells, ABL503 induced superior anti-tumor activity and maintained an anti-tumor response against tumor rechallenge. ABL503 was well tolerated, with normal liver function in monkeys. CONCLUSION: The novel anti-4-1BB×PD-L1 bispecific antibody may exert a strong anti-tumor therapeutic efficacy with a low risk of liver toxicity through the restriction of 4-1BB stimulation in tumors.
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Anticuerpos Biespecíficos/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia/métodos , Neoplasias/tratamiento farmacológico , Animales , Anticuerpos Biespecíficos/farmacología , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Masculino , RatonesRESUMEN
The INO80 chromatin remodeling complex has roles in many essential cellular processes, including DNA replication. However, the mechanisms that regulate INO80 in these processes remain largely unknown. We previously reported that the stability of Ino80, the catalytic ATPase subunit of INO80, is regulated by the ubiquitin proteasome system and that BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase with tumor suppressor activity, stabilizes Ino80 via deubiquitination and promotes replication fork progression. However, the E3 ubiquitin ligase that targets Ino80 for proteasomal degradation was unknown. Here, we identified the C-terminus of Hsp70-interacting protein (CHIP), the E3 ubiquitin ligase that functions in cooperation with Hsp70, as an Ino80-interacting protein. CHIP polyubiquitinates Ino80 in a manner dependent on Hsp70. Contrary to our expectation that CHIP degrades Ino80, CHIP instead stabilizes Ino80 by extending its halflife. The data suggest that CHIP stabilizes Ino80 by inhibiting degradative ubiquitination. We also show that CHIP works together with BAP1 to enhance the stabilization of Ino80, leading to its chromatin binding. Interestingly, both depletion and overexpression of CHIP compromise replication fork progression with little effect on fork stalling, as similarly observed for BAP1 and Ino80, indicating that an optimal cellular level of Ino80 is important for replication fork speed but not for replication stress suppression. This work therefore idenitifes CHIP as an E3 ubiquitin ligase that stabilizes Ino80 via nondegradative ubiquitination and suggests that CHIP and BAP1 act in concert to regulate Ino80 ubiquitination to fine-tune its stability for efficient DNA replication.
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ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Sitios de Unión , Cromatina/metabolismo , Células HEK293 , Proteínas HSP70 de Choque Térmico/metabolismo , Células HT29 , Humanos , Poliubiquitina/metabolismo , Unión Proteica , Estabilidad ProteicaRESUMEN
Cancer immunotherapy with 4-1BB agonists has limited further clinical development because of dose-limiting toxicity. Here, we developed a bispecific antibody (bsAb; B7-H3×4-1BB), targeting human B7-H3 (hB7-H3) and mouse or human 4-1BB, to restrict the 4-1BB stimulation in tumors. B7-H3×m4-1BB elicited a 4-1BB-dependent antitumor response in hB7-H3-overexpressing tumor models without systemic toxicity. BsAb primarily targets CD8 T cells in the tumor and increases their proliferation and cytokine production. Among the CD8 T cell population in the tumor, 4-1BB is solely expressed on PD-1+Tim-3+ "terminally differentiated" subset, and bsAb potentiates these cells for eliminating the tumor. Furthermore, the combination of bsAb and PD-1 blockade synergistically inhibits tumor growth accompanied by further increasing terminally differentiated CD8 T cells. B7-H3×h4-1BB also shows antitumor activity in h4-1BB-expressing mice. Our data suggest that B7-H3×4-1BB is an effective and safe therapeutic agent against B7-H3-positive cancers as monotherapy and combination therapy with PD-1 blockade.
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Anticuerpos Biespecíficos , Linfocitos T CD8-positivos , Linfocitos Infiltrantes de Tumor , Neoplasias , Animales , Anticuerpos Biespecíficos/farmacología , Linfocitos T CD8-positivos/inmunología , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Neoplasias/tratamiento farmacológico , Receptor de Muerte Celular Programada 1RESUMEN
Many chemotherapeutic drugs produce double-strand breaks (DSB) on cancer cell DNA, thereby inducing cell death. However, the DNA damage response (DDR) enables cancer cells to overcome DNA damage and escape cell death, often leading to therapeutic resistance and unsuccessful outcomes. It is therefore important to develop inhibitors that target DDR proteins to render cancer cells hypersensitive to DNA damage. Here, we investigated the applicability of PFI-3, a recently developed bromodomain inhibitor specifically targeting the SWI/SNF chromatin remodeler that functions to promote DSB repair, in cancer treatment. We verified that PFI-3 effectively blocks chromatin binding of its target bromodomains and dissociates the corresponding SWI/SNF proteins from chromatin. We then found that, while having little toxicity as a single agent, PFI-3 synergistically sensitizes several human cancer cell lines to DNA damage induced by chemotherapeutic drugs such as doxorubicin. This PFI-3 activity occurs only for the cancer cells that require SWI/SNF for DNA repair. Our mechanism studies show that PFI-3 exerts the DNA damage-sensitizing effect by directly blocking SWI/SNF's chromatin binding, which leads to defects in DSB repair and aberrations in damage checkpoints, eventually resulting in increase of cell death primarily via necrosis and senescence. This work therefore demonstrates the activity of PFI-3 to sensitize cancer cells to DNA damage and its mechanism of action via SWI/SNF targeting, providing an experimental rationale for developing PFI-3 as a sensitizing agent in cancer chemotherapy. IMPLICATIONS: This study, revealing the activity of PFI-3 to sensitize cancer cells to chemotherapeutic drugs, provides an experimental rationale for developing this bromodomain inhibitor as a sensitizing agent in cancer chemotherapy.
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Compuestos de Azabiciclo/antagonistas & inhibidores , Compuestos de Azabiciclo/uso terapéutico , Proteínas Cromosómicas no Histona/genética , Daño del ADN/genética , Dominios Proteicos/genética , Piridinas/antagonistas & inhibidores , Piridinas/uso terapéutico , Factores de Transcripción/genética , Compuestos de Azabiciclo/farmacología , Humanos , Piridinas/farmacologíaRESUMEN
Bromodomain (BRD), a protein module that recognizes acetylated lysine residues on histones and other proteins, has recently emerged as a promising therapeutic target for human diseases such as cancer. While most of the studies have been focused on inhibitors against BRDs of the bromo- and extra-terminal domain (BET) family proteins, non-BET family BRD inhibitors remain largely unexplored. Here, we investigated a potential anticancer activity of the recently developed non-BET family BRD inhibitor NVS-CECR2-1 that targets the cat eye syndrome chromosome region, candidate 2 (CECR2). We show that NVS-CECR2-1 inhibits chromatin binding of CECR2 BRD and displaces CECR2 from chromatin within cells. NVS-CECR2-1 exhibits cytotoxic activity against various human cancer cells, killing SW48 colon cancer cells in particular with a submicromolar half maximum inhibition value mainly by inducing apoptosis. The sensitivity of the cancer cells to NVS-CECR2-1 is reduced by CECR2 depletion, suggesting that NVS-CECR2-1 exerts its activity by targeting CECR2. Interestingly, our data show that NVS-CECR2-1 also kills cancer cells by CECR2-independent mechanism. This study reports for the first time the cancer cell cytotoxic activity for NVS-CECR2-1 and provides a possibility of this BRD inhibitor to be developed as an anticancer therapeutic agent.
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Antineoplásicos/farmacología , Indoles/farmacología , Neoplasias/tratamiento farmacológico , Piperidinas/farmacología , Proteínas/antagonistas & inhibidores , Pirimidinas/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Cromatina/efectos de los fármacos , Cromatina/metabolismo , Neoplasias del Colon/tratamiento farmacológico , Humanos , Indoles/uso terapéutico , Concentración 50 Inhibidora , Piperidinas/uso terapéutico , Pirimidinas/uso terapéutico , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismoRESUMEN
As mesothelin is overexpressed in various types of cancer, it is an attractive target for therapeutic antibodies. T-cell bispecific antibodies bind to target cells and engage T cells via binding to CD3, resulting in target cell killing by T-cell activation. However, the affinity of the CD3-binding arm may influence CD3-mediated plasma clearance or antibody trapping in T-cell-containing tissues. This may then affect the biodistribution of bispecific antibodies. In this study, we used scFab and knob-into-hole technologies to construct novel IgG-based 1 + 1 MG1122-A and 2 + 1 MG1122-B bispecific antibodies against mesothelin and CD3ε. MG1122-B was designed to be bivalent to mesothelin and monovalent to CD3ε, using a 2 + 1 head-to-tail format. Activities of the two antibodies were evaluated in mesothelin-positive tumor cells in vitro and xenograft models in vivo. Although both antibodies exhibited target cell killing efficacy and produced regression of xenograft tumors with CD8+ T-cell infiltration, the antitumor efficacy of MG1122-B was significantly higher. MG1122-B may improve tumor targeting because of its bivalency for tumor antigen. It may also reduce systemic toxicity by limiting the activation of circulating T cells. Thus, MG1122-B may be useful for treating mesothelin-positive solid tumors.
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Anticuerpos Biespecíficos , Antineoplásicos Inmunológicos , Proteínas Ligadas a GPI/inmunología , Inmunoglobulina G , Proteínas de Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Linfocitos T/inmunología , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Antineoplásicos Inmunológicos/inmunología , Antineoplásicos Inmunológicos/farmacología , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Células Jurkat , Mesotelina , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias/inmunología , Neoplasias/patología , Linfocitos T/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The recovery from replication stress by restarting stalled forks to continue DNA synthesis is crucial for maintaining genome stability and thereby preventing diseases such as cancer. We previously showed that BRCA1-associated protein 1 (BAP1), a nuclear deubiquitinase with tumor suppressor activity, promotes replication fork progression by stabilizing the INO80 chromatin remodeler via deubiquitination and recruiting it to replication forks during normal DNA synthesis. However, whether BAP1 functions in DNA replication under stress conditions is unknown. Here, we show that BAP1 depletion reduces S-phase progression and DNA synthesis after treatment with hydroxyurea (HU). BAP1-depleted cells exhibit a defect in the restart of HU-induced stalled replication forks, which is recovered by the ectopic expression of INO80. Both BAP1 and INO80 bind chromatin at replication forks upon HU treatment. BAP1 depletion abrogates the binding of INO80 to replication forks and increases the formation of RAD51 foci following HU treatment. BAP1-depleted cells show hypersensitivity to HU treatment, which is rescued by INO80 expression. These results suggest that BAP1 promotes the restart of stress-induced stalled replication forks by recruiting INO80 to the stalled forks. This function of BAP1 in replication stress recovery may contribute to its ability to suppress genome instability and cancer development.
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ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas de Unión al ADN/metabolismo , Hidroxiurea/farmacología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fase S/efectos de los fármacos , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Supervivencia Celular/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Inestabilidad Genómica , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Unión Proteica/efectos de los fármacos , Recombinasa Rad51/metabolismo , Fase S/genética , Transfección , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genéticaRESUMEN
The INO80 chromatin-remodeling complex performs functions in many chromosomal processes that are crucial for genome stability, such as DNA replication and stalled replication fork recovery. Although these functions suggest that INO80 acts as a tumor suppressor, its specific role in tumorigenesis has remained obscure. Here, we show that a haploinsufficient mutation of Ino80, the catalytic ATPase of the INO80 complex, decreased intestinal adenomatous polyps and increased survival in an Apcmin/+ mouse model of colon cancer. Experiments using tumors obtained from Apcmin/+ mice and cells from human colon cancers showed that this Ino80 defect induced stalled replication forks, the concomitant activation of ATR-Chk1 signaling and an increase in apoptosis, suggesting that Ino80 haploinsufficiency inhibited colon cancer tumorigenesis by activating replication stress-induced ATR-Chk1 signaling to increase apoptosis. Importantly, in human colon cancer, we observed that the INO80 subunits were frequently present in high copy numbers and exhibited a high rate of amplification and increased protein expression. These results show that in contrast to our original prediction that INO80 acts as a tumor suppressor, INO80 actually functions oncogenically to promote colon tumorigenesis. INO80 therefore represents a novel therapeutic target in colon cancer. The results of this study also reinforce the emerging notion that while genomic instability can promote tumorigenesis, in certain genetic contexts, it can also act as a tumor suppressor.
RESUMEN
The EGFR-targeted monoclonal antibodies are a valid therapeutic strategy for patients with metastatic colorectal cancer (mCRC). However, only a small subset of mCRC patients has therapeutic benefits and there are high demands for EGFR therapeutics with a broader patient pool and more potent efficacy. In this study, we report GC1118 exhibiting a different character in terms of binding epitope, affinity, mode of action, and efficacy from other anti-EGFR antibodies. Structural analysis of the EGFR-GC1118 crystal complex revealed that GC1118 recognizes linear, discrete N-terminal epitopes of domain III of EGFR, critical for EGF binding but not overlapping with those of other EGFR-targeted antibodies. GC1118 exhibited superior inhibitory activity against high-affinity EGFR ligands in terms of EGFR binding, triggering EGFR signaling, and proliferation compared with cetuximab and panitumumab. EGFR signaling driven by low-affinity ligands, on the contrary, was well inhibited by all the antibodies tested. GC1118 demonstrated robust antitumor activity in tumor xenografts with elevated expression of high-affinity ligands in vivo, whereas cetuximab did not. Considering the significant role of high-affinity EGFR ligands in modulating tumor microenvironment and inducing resistance to various cancer therapeutics, our study suggests a potential therapeutic advantage of GC1118 in terms of efficacy and a range of benefited patient pool. Mol Cancer Ther; 15(2); 251-63. ©2015 AACR.
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Anticuerpos Monoclonales Humanizados/administración & dosificación , Antineoplásicos/administración & dosificación , Neoplasias Colorrectales/tratamiento farmacológico , Epítopos/metabolismo , Receptores ErbB/química , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Femenino , Humanos , Ligandos , Ratones , Modelos Moleculares , Unión Proteica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Radiotherapy treats cancer by inducing DNA double-strand breaks (DSB) in tumor cells using ionizing radiation. However, DNA repair in tumor cells often leads to radioresistance and unsuccessful outcome. Inhibition of DNA repair by targeting repair proteins can increase radiosensitivity of tumor cells. The BRG1 chromatin remodeling enzyme assists DSB repair by stimulating γ-H2AX formation and BRG1 binding to acetylated histones at DSBs via bromodomain (BRD) is critical for this activity. Here, we show that ectopic expression of BRG1-BRD inhibited γ-H2AX and DSB repair after irradiation and increased the radiosensitivity in various human cancer cells, including HT29 colon cancer. Dimerization of BRG1-BRD, increasing its chromatin binding affinity, aggravated the defects in γ-H2AX and DSB repair and further enhanced the radiosensitivity. While little affecting the upstream ATM activation, BRG1-BRD in irradiated HT29 cells inhibited the recruitment of 53BP1 to damaged chromatin, the downstream event of γ-H2AX, and compromised the G2-M checkpoint and increased apoptosis. Importantly, in a xenograft mouse model, BRG1-BRD increased the radiosensitivity of HT29 tumors, which was further enhanced by dimerization. These data suggest that BRG1-BRD radiosensitizes tumor cells by a dominant negative activity against BRG1, which disrupts γ-H2AX and its downstream 53BP1 pathways, leading to inefficient DNA repair, G2-M checkpoint defect, and increased apoptosis. This work therefore identifies BRG1-BRD as a novel tumor radiosensitizer and its action mechanism, providing the first example of chromatin remodeler as a target for improving cancer radiotherapy.
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Ensamble y Desensamble de Cromatina , ADN Helicasas/química , ADN Helicasas/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Tolerancia a Radiación , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de la radiación , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Estructura Terciaria de Proteína , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/efectos de la radiación , Radiación Ionizante , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
The INO80 chromatin-remodelling complex has been implicated in DNA replication during stress in yeast. However, its role in normal DNA replication and its underlying mechanisms remain unclear. Here, we show that INO80 binds to replication forks and promotes fork progression in human cells under unperturbed, normal conditions. We find that Ino80, which encodes the catalytic ATPase of INO80, is essential for mouse embryonic DNA replication and development. Ino80 is recruited to replication forks through interaction with ubiquitinated H2A--aided by BRCA1-associated protein-1 (BAP1), a tumour suppressor and nuclear de-ubiquitinating enzyme that also functions to stabilize Ino80. Importantly, Ino80 is downregulated in BAP1-defective cancer cells due to the lack of an Ino80 stabilization mechanism via BAP1. Our results establish a role for INO80 in normal DNA replication and uncover a mechanism by which this remodeler is targeted to replication forks, suggesting a molecular basis for the tumour-suppressing function of BAP1.
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Adenosina Trifosfatasas/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , Neoplasias/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Proteínas de Unión al ADN , Regulación hacia Abajo , Exones , Femenino , Regulación Neoplásica de la Expresión Génica , Genotipo , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Unión Proteica , ARN Interferente Pequeño/metabolismo , Transfección , Técnicas del Sistema de Dos Híbridos , Ubiquitina/químicaRESUMEN
BACKGROUND: Inhibition of the activated epidermal growth factor receptor (EGFR) with either enzymatic kinase inhibitors or anti-EGFR antibodies such as cetuximab, is an effective modality of treatment for multiple human cancers. Enzymatic EGFR inhibitors are effective for lung adenocarcinomas with somatic kinase domain EGFR mutations while, paradoxically, anti-EGFR antibodies are more effective in colon and head and neck cancers where EGFR mutations occur less frequently. In colorectal cancer, anti-EGFR antibodies are routinely used as second-line therapy of KRAS wild-type tumors. However, detailed mechanisms and genomic predictors for pharmacological response to these antibodies in colon cancer remain unclear. FINDINGS: We describe a case of colorectal adenocarcinoma, which was found to harbor a kinase domain mutation, G724S, in EGFR through whole genome sequencing. We show that G724S mutant EGFR is oncogenic and that it differs from classic lung cancer derived EGFR mutants in that it is cetuximab responsive in vitro, yet relatively insensitive to small molecule kinase inhibitors. Through biochemical and cellular pharmacologic studies, we have determined that cells harboring the colon cancer-derived G719S and G724S mutants are responsive to cetuximab therapy in vitro and found that the requirement for asymmetric dimerization of these mutant EGFR to promote cellular transformation may explain their greater inhibition by cetuximab than small-molecule kinase inhibitors. CONCLUSION: The colon-cancer derived G719S and G724S mutants are oncogenic and sensitive in vitro to cetuximab. These data suggest that patients with these mutations may benefit from the use of anti-EGFR antibodies as part of the first-line therapy.
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Adenocarcinoma/genética , Anticuerpos Monoclonales Humanizados/uso terapéutico , Neoplasias Colorrectales/genética , Receptores ErbB/genética , Mutación , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Antineoplásicos/uso terapéutico , Cetuximab , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/química , Receptores ErbB/metabolismo , Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inhibidores de Proteínas Quinasas/uso terapéutico , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
The human INO80 chromatin remodeling complex, comprising the Ino80 ATPase (hIno80) and the associated proteins such as Tip49a, has been implicated in a variety of nuclear processes other than transcription. We previously have found that hIno80 interacts with tubulin and co-localizes with the mitotic spindle and is required for spindle formation. To better understand the role of hIno80 in spindle formation, we further investigated the interaction between hIno80 and microtubule. Here, we show that the N-terminal domain, dispensable for the nucleosome remodeling activity, is important for hIno80 to interact with tubulin and co-localize with the spindle. The hIno80 N-terminal domain binds to monomeric tubulin and polymerized microtubule in vitro, and the E-hook of tubulin, involved in the polymerization of microtubule, is critical for this binding. Tip49a, which has been reported to associate with the spindle, does not bind to microtubule in vitro and dispensable for spindle formation in vivo. These results suggest that hIno80 can play a direct role in the spindle assembly independent of its chromatin remodeling activity.
Asunto(s)
ADN Helicasas/metabolismo , Microtúbulos/metabolismo , Huso Acromático/metabolismo , Tubulina (Proteína)/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Ensamble y Desensamble de Cromatina , ADN Helicasas/genética , Proteínas de Unión al ADN , Células HEK293 , Humanos , Conformación Proteica , Eliminación de Secuencia , Tubulina (Proteína)/químicaRESUMEN
BACKGROUND: Vascular endothelial growth factor A (VEGFA), a critical mediator of tumor angiogenesis, is a well-characterized target of hypoxia-inducible factor 1 (HIF-1). Murine arrest-defective protein 1A (mARD1A(225)) acetylates HIF-1alpha, triggering its degradation, and thus may play a role in decreased expression of VEGFA. METHODS: We generated Apc(Min/+)/mARD1A(225) transgenic mice and quantified growth of intestinal polyps. Human gastric MKN74 and murine melanoma B16F10 cells overexpressing mARD1A(225) were injected into mice, and tumor growth and metastasis were measured. VEGFA expression and microvessel density in tumors were assessed using immunohistochemistry. To evaluate the role of mARD1A(225) acetylation of Lys532 in HIF-1alpha, we injected B16F10-mARD1A(225) cell lines stably expressing mutant HIF-1alpha/K532R into mice and measured metastasis. All statistical tests were two-sided, and P values less than .05 were considered statistically significant. RESULTS: Apc(Min/+)/mARD1A(225) transgenic mice (n = 25) had statistically significantly fewer intestinal polyps than Apc(Min/+) mice (n = 21) (number of intestinal polyps per mouse: Apc(Min/+) mice vs Apc(Min/+)/mARD1A(225) transgenic mice, mean = 83.4 vs 38.0 polyps, difference = 45.4 polyps, 95% confidence interval [CI] = 41.8 to 48.6; P < .001). The growth and metastases of transplanted tumors were also statistically significantly reduced in mice injected with mARD1A(225)-overexpressing cells than in mice injected with control cells (P < .01). Moreover, overexpression of mARD1A(225) decreased VEGFA expression and microvessel density in tumor xenografts (P < .04) and Apc(Min/+) intestinal polyps (P = .001). Mutation of lysine 532 of HIF-1alpha in B16F10-mARD1A(225) cells prevented HIF-1alpha degradation and inhibited the antimetastatic effect of mARD1A(225) (P < .001). CONCLUSION: mARD1A(225) may be a novel upstream target that blocks VEGFA expression and tumor-related angiogenesis.