RESUMEN
Renewable tissues exhibit heightened sensitivity to DNA damage, which is thought to result from a high level of p53. However, cell proliferation in renewable tissues requires p53 down-regulation, creating an apparent discrepancy between the p53 level and elevated sensitivity to DNA damage. Using a combination of genetic mouse models and pharmacologic inhibitors, we demonstrate that it is p53-regulated MDM2 that functions together with MDMX to regulate DNA damage sensitivity by targeting EZH2 (enhancer of zeste homolog 2) for ubiquitination/degradation. As a methyltransferase, EZH2 promotes H3K27me3, and therefore chromatin compaction, to determine sensitivity to DNA damage. We demonstrate that genetic and pharmacologic interference of the association between MDM2 and MDMX stabilizes EZH2, resulting in protection of renewable tissues from radio-/chemotherapy-induced acute injury. In cells with p53 mutation, there are diminished MDM2 levels, and thus accumulation of EZH2, underpinning the resistant phenotype. Our work uncovers an epigenetic mechanism behind tissue sensitivity to DNA damage, carrying important translation implications.
Asunto(s)
Cromatina/metabolismo , Daño del ADN , Proteína Potenciadora del Homólogo Zeste 2/genética , Epigénesis Genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas/genética , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis , Cromatina/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Ratones , Ratones Transgénicos , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , UbiquitinaciónRESUMEN
Delta-like ligand 3 (DLL3) is expressed in more than 70% of small cell lung cancers (SCLCs) and other neuroendocrine-derived tumor types. SCLC is highly aggressive and limited therapeutic options lead to poor prognosis for patients. HPN328 is a tri-specific T cell activating construct (TriTAC) consisting of three binding domains: a CD3 binder for T cell engagement, an albumin binder for half-life extension, and a DLL3 binder for tumor cell engagement. In vitro assays, rodent models and non-human primates were used to assess the activity of HPN328. HPN328 induces potent dose-dependent killing of DLL3-expressing SCLC cell lines in vitro concomitant with T cell activation and cytokine release. In an NCI-H82 xenograft model with established tumors, HPN328 treatment led to T cell recruitment and anti-tumor activity. In an immunocompetent mouse model expressing a human CD3ε epitope, mice previously treated with HPN328 withstood tumor rechallenge, demonstrating long-term anti-tumor immunity. When repeat doses were administered to cynomolgus monkeys, HPN328 was well tolerated up to 10 mg/kg. Pharmacodynamic changes, such as transient cytokine elevation, were observed, consistent with the expected mechanism of action of T cell engagers. HPN328 exhibited linear pharmacokinetic in the given dose range with a serum half-life of 78 to 187 hours, supporting weekly or less frequent administration of HPN328 in humans. Preclinical and nonclinical characterization suggests that HPN328 is a highly efficacious, safe, and novel therapeutic candidate. A phase 1/2 clinical trial is currently underway testing safety and efficacy in patients with DLL3 expressing malignancies.
RESUMEN
BACKGROUND: Prior to the introduction of tamoxifen, high dose estradiol was used to treat breast cancer patients with similar efficacy as tamoxifen, albeit with some undesirable side effects. There is renewed interest to utilize estradiol to treat endocrine resistant breast cancers, especially since findings from several preclinical models and clinical trials indicate that estradiol may be a rational second-line therapy in patients exhibiting resistance to tamoxifen and/or aromatase inhibitors. We and others reported that breast cancer patients bearing protein kinase C alpha (PKCα)- expressing tumors exhibit endocrine resistance and tumor aggressiveness. Our T47D:A18/PKCα preclinical model is tamoxifen-resistant, hormone-independent, yet is inhibited by 17ß-estradiol (E2) in vivo. We previously reported that E2-induced T47D:A18/PKCα tumor regression requires extranuclear ERα and interaction with the extracellular matrix. METHODS: T47D:A18/PKCα cells were grown in vitro using two-dimensional (2D) cell culture, three-dimensional (3D) Matrigel and in vivo by establishing xenografts in athymic mice. Immunofluoresence confocal microscopy and co-localization were applied to determine estrogen receptor alpha (ERα) subcellular localization. Co-immunoprecipitation and western blot were used to examine interaction of ERα with caveolin-1. RESULTS: We report that although T47D:A18/PKCα cells are cross-resistant to raloxifene in cell culture and in Matrigel, raloxifene induces regression of tamoxifen-resistant tumors. ERα rapidly translocates to extranuclear sites during T47D:A18/PKCα tumor regression in response to both raloxifene and E2, whereas ERα is primarily localized in the nucleus in proliferating tumors. E2 treatment induced complete tumor regression whereas cessation of raloxifene treatment resulted in tumor regrowth accompanied by re-localization of ERα to the nucleus. T47D:A18/neo tumors that do not overexpress PKCα maintain ERα in the nucleus during tamoxifen-mediated regression. An association between ERα and caveolin-1 increases in tumors regressing in response to E2. CONCLUSIONS: Extranuclear ERα plays a role in the regression of PKCα-overexpressing tamoxifen-resistant tumors. These studies underline the unique role of extranuclear ERα in E2- and raloxifene-induced tumor regression that may have implications for treatment of endocrine-resistant PKCα-expressing tumors encountered in the clinic.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos/genética , Receptor alfa de Estrógeno/metabolismo , Expresión Génica , Proteína Quinasa C-alfa/genética , Tamoxifeno/farmacología , Animales , Antineoplásicos Hormonales/administración & dosificación , Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Caveolina 1/metabolismo , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Citoplasma/metabolismo , Modelos Animales de Enfermedad , Estradiol/farmacología , Femenino , Humanos , Ratones , Unión Proteica , Transporte de Proteínas/efectos de los fármacos , Clorhidrato de Raloxifeno/administración & dosificación , Clorhidrato de Raloxifeno/farmacología , Tamoxifeno/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Mesothelin (MSLN) is a glycophosphatidylinositol-linked tumor antigen overexpressed in a variety of malignancies, including ovarian, pancreatic, lung, and triple-negative breast cancer. Early signs of clinical efficacy with MSLN-targeting agents have validated MSLN as a promising target for therapeutic intervention, but therapies with improved efficacy are still needed to address the significant unmet medical need posed by MSLN-expressing cancers. EXPERIMENTAL DESIGN: We designed HPN536, a 53-kDa, trispecific, T-cell-activating protein-based construct, which binds to MSLN-expressing tumor cells, CD3ε on T cells, and to serum albumin. Experiments were conducted to assess the potency, activity, and half-life of HPN536 in in vitro assays, rodent models, and in nonhuman primates (NHP). RESULTS: HPN536 binds to MSLN-expressing tumor cells and to CD3ε on T cells, leading to T-cell activation and potent redirected target cell lysis. A third domain of HPN536 binds to serum albumin for extension of plasma half-life. In cynomolgus monkeys, HPN536 at doses ranging from 0.1 to 10 mg/kg demonstrated MSLN-dependent pharmacologic activity, was well tolerated, and showed pharmacokinetics in support of weekly dosing in humans. CONCLUSIONS: HPN536 is potent, is well tolerated, and exhibits extended half-life in NHPs. It is currently in phase I clinical testing in patients with MSLN-expressing malignancies (NCT03872206).
Asunto(s)
Inmunoterapia/métodos , Activación de Linfocitos/inmunología , Mesotelina/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Anticuerpos de Dominio Único/farmacología , Linfocitos T/inmunología , Animales , Antígenos de Neoplasias/inmunología , Apoptosis , Proliferación Celular , Femenino , Humanos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Fragmentos de Péptidos/inmunología , Anticuerpos de Dominio Único/inmunología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cyclodextrin (CD) is a well known drug carrier and excipient for enhancing aqueous solubility. CDs themselves are anticipated to have low membrane permeability because of relatively high hydrophilicity and molecular weight. CD derivatization with 17-beta estradiol (E(2)) was explored extensively using a number of different click chemistries and the cell membrane permeability of synthetic CD-E(2) conjugate was explored by cell reporter assays and confocal fluorescence microscopy. In simile with reported dendrimer-E(2) conjugates, CD-E(2) was found to be a stable, extranuclear receptor selective estrogen that penetrated into the cytoplasm.
Asunto(s)
Ciclodextrinas/química , Estradiol/química , Estrógenos/química , Permeabilidad de la Membrana Celular , Células Cultivadas , Cristalografía por Rayos X , Ciclodextrinas/síntesis química , Ciclodextrinas/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Estradiol/metabolismo , Estrógenos/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , EstereoisomerismoRESUMEN
ZBTB7A, a member of the POZ/BTB and Krüppel (POK) family of transcription factors, has been shown to have a context-dependent role in cancer development and progression. The role of ZBTB7A in estrogen receptor alpha (ERα)-positive breast cancer is largely unknown. Approximately 70% of breast cancers are classified as ERα-positive. ERα carries out the biological effects of estrogen and its expression level dictates response to endocrine therapies and prognosis for breast cancer patients. In this study, we find that ZBTB7A transcriptionally regulates ERα expression in ERα-positive breast cancer cell lines by binding to the ESR1 promoter leading to increased transcription of ERα. Inhibition of ZBTB7A in ERα-positive cells results in decreased estrogen responsiveness as demonstrated by diminished estrogen-response element-driven luciferase reporter activity, induction of estrogen target genes, and estrogen-stimulated growth. We also report that ERα potentiates ZBTB7A expression via a post-translational mechanism, suggesting the presence of a positive feedback loop between ZBTB7A and ERα, conferring sensitivity to estrogen in breast cancer. Clinically, we find that ZBTB7A and ERα are often co-expressed in breast cancers and that high ZBTB7A expression correlates with improved overall and relapse-free survival for breast cancer patients. Importantly, high ZBTB7A expression predicts a more favorable outcome for patients treated with endocrine therapies. Together, these findings demonstrate that ZBTB7A contributes to the transcriptional program maintaining ERα expression and potentially an endocrine therapy-responsive phenotype in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Unión al ADN/genética , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción/genética , Animales , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Ratones , Regiones Promotoras Genéticas , Activación TranscripcionalRESUMEN
Endocrine-resistant breast cancer is a major clinical obstacle. The use of 17ß-estradiol (E2) has reemerged as a potential treatment option following exhaustive use of tamoxifen or aromatase inhibitors, although side effects have hindered its clinical usage. Protein kinase C alpha (PKCα) expression was shown to be a predictor of disease outcome for patients receiving endocrine therapy and may predict a positive response to an estrogenic treatment. Here, we have investigated the use of novel benzothiophene selective estrogen mimics (SEM) as an alternative to E2 for the treatment of tamoxifen-resistant breast cancer. Following in vitro characterization of SEMs, a panel of clinically relevant PKCα-expressing, tamoxifen-resistant models were used to investigate the antitumor effects of these compounds. SEM treatment resulted in growth inhibition and apoptosis of tamoxifen-resistant cell lines in vitro. In vivo SEM treatment induced tumor regression of tamoxifen-resistant T47D:A18/PKCα and T47D:A18-TAM1 tumor models. T47D:A18/PKCα tumor regression was accompanied by translocation of estrogen receptor (ER) α to extranuclear sites, possibly defining a mechanism through which these SEMs initiate tumor regression. SEM treatment did not stimulate growth of E2-dependent T47D:A18/neo tumors. In addition, unlike E2 or tamoxifen, treatment with SEMs did not stimulate uterine weight gain. These findings suggest the further development of SEMs as a feasible therapeutic strategy for the treatment of endocrine-resistant breast cancer without the side effects associated with E2.