RESUMEN
Mediator kinases (CDK8/19) are transcriptional regulators broadly implicated in cancer. Despite their central role in fine-tuning gene-expression programs, we find complete loss of CDK8/19 is tolerated in colorectal cancer (CRC) cells. Using orthogonal functional genomic and pharmacological screens, we identify BET protein inhibition as a distinct vulnerability in CDK8/19-depleted cells. Combined CDK8/19 and BET inhibition led to synergistic growth retardation in human and mouse models of CRC. Strikingly, depletion of CDK8/19 in these cells led to global repression of RNA polymerase II (Pol II) promoter occupancy and transcription. Concurrently, loss of Mediator kinase led to a profound increase in MED12 and BRD4 co-occupancy at enhancer elements and increased dependence on BET proteins for the transcriptional output of cell-essential genes. In total, this work demonstrates a synthetic lethal interaction between Mediator kinase and BET proteins and exposes a therapeutic vulnerability that can be targeted using combination therapies.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Neoplasias Colorrectales/enzimología , Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Complejo Mediador/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Sitios de Unión , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Quinasa 8 Dependiente de Ciclina/genética , Quinasas Ciclina-Dependientes/genética , Elementos de Facilitación Genéticos , Femenino , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Masculino , Complejo Mediador/antagonistas & inhibidores , Complejo Mediador/genética , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Transcripción Genética , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Colorectal cancer remains a major unmet medical need, prompting large-scale genomics efforts in the field to identify molecular drivers for which targeted therapies might be developed. We previously reported the identification of recurrent translocations in R-spondin genes present in a subset of colorectal tumours. Here we show that targeting RSPO3 in PTPRK-RSPO3-fusion-positive human tumour xenografts inhibits tumour growth and promotes differentiation. Notably, genes expressed in the stem-cell compartment of the intestine were among those most sensitive to anti-RSPO3 treatment. This observation, combined with functional assays, suggests that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumour growth and indicates that the therapeutic targeting of stem-cell properties within tumours may be a clinically relevant approach for the treatment of colorectal tumours.
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Diferenciación Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Terapia Molecular Dirigida , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Trombospondinas/metabolismo , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Anticuerpos/uso terapéutico , División Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Progresión de la Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citología , Intestinos/efectos de los fármacos , Intestinos/patología , Masculino , Ratones , Células Madre Neoplásicas/metabolismo , Células Madre/citología , Células Madre/metabolismo , Trombospondinas/antagonistas & inhibidores , Trombospondinas/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The spliceosome machinery is composed of multimeric protein complexes that generate a diverse repertoire of mRNA through coordinated splicing of heteronuclear RNAs. While somatic mutations in spliceosome components have been discovered in several cancer types, the molecular bases and consequences of spliceosome aberrations in cancer are poorly understood. Here we report for the first time that PRPF6, a member of the tri-snRNP (small ribonucleoprotein) spliceosome complex, drives cancer proliferation by preferential splicing of genes associated with growth regulation. Inhibition of PRPF6 and other tri-snRNP complex proteins, but not other snRNP spliceosome complexes, selectively abrogated growth in cancer cells with high tri-snRNP levels. High-resolution transcriptome analyses revealed that reduced PRPF6 alters the constitutive and alternative splicing of a discrete number of genes, including an oncogenic isoform of the ZAK kinase. These findings implicate an essential role for PRPF6 in cancer via splicing of distinct growth-related gene products.
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Neoplasias del Colon/genética , Neoplasias del Colon/patología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Empalme Alternativo , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Humanos , Isoformas de Proteínas , Factores de Empalme de ARN , EmpalmosomasRESUMEN
Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.
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Plasticidad de la Célula/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Piridonas/farmacología , Tiofenos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , L-Lactato Deshidrogenasa/metabolismo , Modelos Moleculares , Estructura Molecular , Piridonas/química , Relación Estructura-Actividad , Tiofenos/químicaRESUMEN
CDK8 is a dissociable kinase module of the Mediator complex and has been shown to play an important role in transcriptional regulation in organisms as diverse as yeast and humans. Recent studies suggest that CDK8 functions as an oncoprotein in melanoma and colon cancer. Importantly, these studies were conducted using in vitro cell line models and the role of CDK8 in tumourigenesis in vivo has not been explored. We have generated a mouse with a Cdk8 conditional knockout allele and examined the consequences of Cdk8 loss on normal tissue homeostasis and tumour development in vivo. Cdk8 deletion in the young adult mouse did not induce any gross or histopathological abnormalities, implying that Cdk8 is largely dispensable for somatic cellular homeostasis. In contrast, Cdk8 deletion in the Apc(Min) intestinal tumour model shortened the animals' survival and increased tumour burden. Although Cdk8 deletion did not affect tumour initiation, intestinal tumour size and growth rate were significantly increased in Cdk8-null animals. Transcriptome analysis performed on Cdk8-null intestinal cells revealed up-regulation of genes that are governed by the Polycomb group (PcG) complex. In support of these findings, Cdk8-null intestinal cells and tumours displayed a reduction in histone H3K27 trimethylation, both globally and at the promoters of a number of PcG-regulated genes involved in oncogenic signalling. Together, our findings uncover a tumour suppressor function for CDK8 in vivo and suggest that the role of CDK8 activity in driving oncogenesis is context-specific. Sequencing data were deposited at GEO (Accession No. GSE71385).
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Carcinogénesis/genética , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Quinasa 8 Dependiente de Ciclina/genética , Regulación Neoplásica de la Expresión Génica/genética , Animales , Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Proteína Potenciadora del Homólogo Zeste 2 , Técnica del Anticuerpo Fluorescente , Genes APC , Genes Supresores de Tumor , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Noqueados , Complejo Represivo Polycomb 2/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
B7-H4 has been implicated in cancers of the female reproductive system and investigated for its possible use as a biomarker for cancer, but there are no preclinical studies to demonstrate that B7-H4 is a molecular target for therapeutic intervention of cancer. We provide evidence that the prevalence and expression levels of B7-H4 are high in different subtypes of breast cancer and that only a few normal tissues express B7-H4 on the cell membrane. These profiles of low normal expression and upregulation in cancer provide an opportunity for the use of antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, for the treatment of B7-H4 positive cancers. We have developed an ADC specific to B7-H4 that uses a linker drug consisting of a potent antimitotic, monomethyl auristatin E (MMAE), linked to engineered cysteines (THIOMAB) via a protease labile linker. We will refer to ADCs that use the THIOMAB format as TDCs to help distinguish the format from standard MC-vc-MMAE ADCs that are conjugated to the interchain disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced durable tumor regression in cell line and patient-derived xenograft models of triple-negative breast cancer. It also binds rat B7-H4 with similar affinity to human and allowed us to test for target dependent toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings similar to untargeted TDC. Our results validate B7-H4 as an ADC target for breast cancer and support the possible use of this TDC in the treatment of B7-H4(+) breast cancer.
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Antineoplásicos/uso terapéutico , Inmunoconjugados/uso terapéutico , Oligopéptidos/uso terapéutico , Animales , Antineoplásicos/química , Western Blotting , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Inmunoconjugados/química , Inmunohistoquímica , Ratones , Ratones SCID , Oligopéptidos/química , Ratas , Ratas Sprague-Dawley , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Aberrant activation of the canonical WNT/beta-catenin pathway occurs in almost all colorectal cancers and contributes to their growth, invasion and survival. Although dysregulated beta-catenin activity drives colon tumorigenesis, further genetic perturbations are required to elaborate full malignant transformation. To identify genes that both modulate beta-catenin activity and are essential for colon cancer cell proliferation, we conducted two loss-of-function screens in human colon cancer cells and compared genes identified in these screens with an analysis of copy number alterations in colon cancer specimens. One of these genes, CDK8, which encodes a member of the mediator complex, is located at 13q12.13, a region of recurrent copy number gain in a substantial fraction of colon cancers. Here we show that the suppression of CDK8 expression inhibits proliferation in colon cancer cells characterized by high levels of CDK8 and beta-catenin hyperactivity. CDK8 kinase activity was necessary for beta-catenin-driven transformation and for expression of several beta-catenin transcriptional targets. Together these observations suggest that therapeutic interventions targeting CDK8 may confer a clinical benefit in beta-catenin-driven malignancies.
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Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Regulación Neoplásica de la Expresión Génica , Oncogenes , beta Catenina/metabolismo , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Neoplasias Colorrectales/patología , Quinasa 8 Dependiente de Ciclina , Quinasas Ciclina-Dependientes/deficiencia , Dosificación de Gen , Humanos , Proteínas Oncogénicas/deficiencia , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Interferencia de ARN , Transcripción GenéticaRESUMEN
Pediatric high-grade gliomas (pHGG) are malignant and usually fatal central nervous system (CNS) WHO Grade 4 tumors. The majority of pHGG consist of diffuse midline gliomas (DMG), H3.3 or H3.1 K27 altered, or diffuse hemispheric gliomas (DHG) (H3.3 G34-mutant). Due to diffuse tumor infiltration of eloquent brain areas, especially for DMG, surgery has often been limited and chemotherapy has not been effective, leaving fractionated radiation to the involved field as the current standard of care. pHGG has only been classified as molecularly distinct from adult HGG since 2012 through Next-Generation sequencing approaches, which have shown pHGG to be epigenetically regulated and specific tumor sub-types to be representative of dysregulated differentiating cells. To translate discovery research into novel therapies, improved pre-clinical models that more adequately represent the tumor biology of pHGG are required. This review will summarize the molecular characteristics of different pHGG sub-types, with a specific focus on histone K27M mutations and the dysregulated gene expression profiles arising from these mutations. Current and emerging pre-clinical models for pHGG will be discussed, including commonly used patient-derived cell lines and in vivo modeling techniques, encompassing patient-derived xenograft murine models and genetically engineered mouse models (GEMMs). Lastly, emerging techniques to model CNS tumors within a human brain environment using brain organoids through co-culture will be explored. As models that more reliably represent pHGG continue to be developed, targetable biological and genetic vulnerabilities in the disease will be more rapidly identified, leading to better treatments and improved clinical outcomes.
RESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0002020.].
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Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma; DIPG), are uniformly fatal brain tumors that lack effective treatment. Analysis of CRISPR/Cas9 loss-of-function gene deletion screens identified PIK3CA and MTOR as targetable molecular dependencies across patient derived models of DIPG, highlighting the therapeutic potential of the blood-brain barrier-penetrant PI3K/Akt/mTOR inhibitor, paxalisib. At the human-equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic glucose feedback and increased insulin levels commensurate with patients using PI3K inhibitors. To exploit genetic dependence and overcome resistance while maintaining compliance and therapeutic benefit, we combined paxalisib with the antihyperglycemic drug metformin. Metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending survival of orthotopic models. DIPG models treated with paxalisib increased calcium-activated PKC signaling. The brain penetrant PKC inhibitor enzastaurin, in combination with paxalisib, synergistically extended the survival of multiple orthotopic patient-derived and immunocompetent syngeneic allograft models; benefits potentiated in combination with metformin and standard-of-care radiotherapy. Therapeutic adaptation was assessed using spatial transcriptomics and ATAC-Seq, identifying changes in myelination and tumor immune microenvironment crosstalk. Collectively, this study has identified what we believe to be a clinically relevant DIPG therapeutic combinational strategy.
Asunto(s)
Neoplasias del Tronco Encefálico , Glioma Pontino Intrínseco Difuso , Glioma , Metformina , Humanos , Ratones , Animales , Glioma Pontino Intrínseco Difuso/tratamiento farmacológico , Glioma Pontino Intrínseco Difuso/genética , Fosfatidilinositol 3-Quinasas/genética , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Neoplasias del Tronco Encefálico/genética , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/patología , Serina-Treonina Quinasas TOR/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , Glucosa , Metformina/farmacología , Microambiente TumoralRESUMEN
Melanocytes uniquely express specialized genes required for pigment formation, some of which are maintained following their transformation to melanoma. Here we exploit this property to selectively target melanoma with an antibody drug conjugate (ADC) specific to PMEL17, the product of the SILV pigment-forming gene. We describe new PMEL17 antibodies that detect the endogenous protein. These antibodies help define the secretory fate of PMEL17 and demonstrate its utility as an ADC target. Although newly synthesized PMEL17 is ultimately routed to the melanosome, we find substantial amounts accessible to our antibodies at the cell surface that undergo internalization and routing to a LAMP1-enriched, lysosome-related organelle. Accordingly, an ADC reactive with PMEL17 exhibits target-dependent tumor cell killing in vitro and in vivo.
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Anticuerpos/uso terapéutico , Melanocitos/metabolismo , Melanoma/tratamiento farmacológico , Melanosomas/metabolismo , Antígeno gp100 del Melanoma/metabolismo , Animales , Anticuerpos/química , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citometría de Flujo , Humanos , Inmunohistoquímica , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Microscopía Fluorescente , Oligopéptidos/química , Oligopéptidos/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Antígeno gp100 del Melanoma/genéticaRESUMEN
Pediatric cell line models are important for basic and translational research. However, their establishment has been hampered by low success rates and the lack of a unified approach. Here, we present a protocol to establish pediatric cancer cell lines from rare childhood tumors. We describe the requirements for successful establishment, including an optimized dissociation technique, and the appropriate media conditions necessary for several types of rare but lethal forms of childhood cancers. For complete details on the use and execution of this protocol, please refer to Sun et al.1.
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Neoplasias , Humanos , Niño , Línea CelularRESUMEN
Colorectal cancer (CRC) is one of the most common forms of cancer worldwide and treatment options for advanced CRC, which has a low 5-year survival rate, remain limited. Integrin-linked kinase (ILK), a multifunctional, scaffolding, pseudo-kinase regulating many integrin-mediated cellular processes, is highly expressed in many cancers. However, the role of ILK in cancer progression is yet to be fully understood. We have previously uncovered a pro-inflammatory role for myeloid-specific ILK in dextran sodium sulfate (DSS)-induced colitis. To establish a correlation between chronic intestinal inflammation and colorectal cancer (CRC), we investigated the role of myeloid-ILK in mouse models of CRC. When myeloid-ILK deficient mice along with the WT control mice were subjected to colitis-associated and APCmin/+-driven CRC, tumour burden was reduced by myeloid-ILK deficiency in both models. The tumour-promoting phenotype of macrophages, M2 polarization, in vitro was impaired by the ILK deficiency and the number of M2-specific marker CD206-expressing tumour-associated macrophages (TAMs) in vivo were significantly diminished in myeloid-ILK deficient mice. Myeloid-ILK deficient mice showed enhanced tumour infiltration of CD8+ T cells and reduced tumour infiltration of FOXP3+ T cells in colitis-associated and APCmin/+-driven CRC, respectively, with an overall elevated CD8+/FOXP3+ ratio suggesting an anti-tumour immune phenotypes. In patient CRC tissue microarrays we observed elevated ILK+ myeloid (ILK+ CD11b+) cells in tumour sections compared to adjacent normal tissues, suggesting a conserved role for myeloid-ILK in CRC development in both human and animal models. This study identifies myeloid-specific ILK expression as novel driver of CRC, which could be targeted as a potential therapeutic option for advanced disease.
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Colitis , Neoplasias Colorrectales , Humanos , Animales , Ratones , Carcinogénesis , Transformación Celular Neoplásica , Neoplasias Colorrectales/patología , Células Mieloides/patología , Factores de Transcripción ForkheadRESUMEN
BACKGROUND: Point mutations in histone variant H3.3 (H3.3K27M, H3.3G34R) and the H3.3-specific ATRX/DAXX chaperone complex are frequent events in pediatric gliomas. These H3.3 point mutations affect many chromatin modifications but the exact oncogenic mechanisms are currently unclear. Histone H3.3 is known to localize to nuclear compartments known as promyelocytic leukemia (PML) nuclear bodies, which are frequently mutated and confirmed as oncogenic drivers in acute promyelocytic leukemia. RESULTS: We find that the pediatric glioma-associated H3.3 point mutations disrupt the formation of PML nuclear bodies and this prevents differentiation down glial lineages. Similar to leukemias driven by PML mutations, H3.3-mutated glioma cells are sensitive to drugs that target PML bodies. We also find that point mutations in IDH1/2-which are common events in adult gliomas and myeloid leukemias-also disrupt the formation of PML bodies. CONCLUSIONS: We identify PML as a contributor to oncogenesis in a subset of gliomas and show that targeting PML bodies is effective in treating these H3.3-mutated pediatric gliomas.
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Neoplasias Encefálicas , Glioma , Histonas , Adulto , Niño , Humanos , Neoplasias Encefálicas/genética , Glioma/genética , Histonas/genética , Mutación , Cuerpos Nucleares de la Leucemia Promielocítica/genética , Cuerpos Nucleares de la Leucemia Promielocítica/patologíaRESUMEN
Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9-11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA-mutations showed increased sensitivity to ONC201, while those harboring TP53-mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992.
RESUMEN
Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA mutations showed increased sensitivity to ONC201, whereas those harboring TP53 mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992. SIGNIFICANCE: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib.
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T cell-engaging bispecific antibodies (TCEs) are clinically effective treatments for hematological cancers. While the utility of TCEs in solid malignancies is being explored, toxicities arising from antigen expression on normal tissues have slowed or halted several clinical trials. Here, we describe the development of TCEs that preferentially drive T cell-mediated death against target cells co-expressing two tumor-associated antigens. We show that Ly6E and B7-H4 are simultaneously expressed on approximately 50% of breast cancers, whereas normal tissue expression is limited and mostly orthogonal. Traditional bispecific TCEs targeting a singular antigen, either Ly6E or B7-H4, are active when paired with high-affinity CD3-engagers, but normal tissue expression presents a toxicity risk. Treatment with a murine cross-reactive B7-H4-TCE results in rapid and severe weight loss in mice along with damage to B7-H4-expressing tissues. To overcome on-target toxicity, we designed trispecific antibodies co-targeting Ly6E, B7-H4, and CD3 and characterized the impact of dual-antigen binding and the relative placement of each binding domain on tumor killing in vitro and in vivo. In vitro killing of tumor cells co-expressing both antigens correlates to the placement of the higher affinity B7-H4 binding domain, with only modest enhancements seen upon addition of Ly6E binding. In xenograft models, avid binding of appropriately designed trispecific TCEs enables tumor growth inhibition while evading the poor tolerability seen with active bispecific TCEs. Collectively these data highlight the potential for dual-antigen targeting to improve safety and efficacy, and expand the scope of tumors that may effectively be treated by TCEs.Abbreviations: Chimeric antigen receptor T cells (CAR-Ts), dual-antigen targeted T cell engagers (DAT-TCE), Fragment antigen-binding (Fab), Hematoxylin and eosin (H&E), Institutional Animal Care and Use Committee (IACUC), Immunoglobulin G (IgG), immunohistochemistry (IHC), NOD SCID gamma (NSG), peripheral blood mononuclear cells (PBMCs), surface plasmon resonance (SPR), T cell-engagers (TCEs).
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Anticuerpos Biespecíficos , Receptores Quiméricos de Antígenos , Animales , Anticuerpos Biespecíficos/farmacología , Línea Celular Tumoral , Eosina Amarillenta-(YS) , Hematoxilina , Humanos , Inmunoglobulina G , Leucocitos Mononucleares , Ratones , Ratones SCID , Linfocitos T , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Initiation and maintenance of transcriptional states are critical for controlling normal tissue homeostasis and differentiation. The cyclin dependent kinases CDK8 and CDK19 (Mediator kinases) are regulatory components of Mediator, a highly conserved complex that orchestrates enhancer-mediated transcriptional output. While Mediator kinases have been implicated in the transcription of genes necessary for development and growth, its function in mammals has not been well defined. Using genetically defined models and pharmacological inhibitors, we showed that CDK8 and CDK19 function in a redundant manner to regulate intestinal lineage specification in humans and mice. The Mediator kinase module bound and phosphorylated key components of the chromatin remodeling complex switch/sucrose non-fermentable (SWI/SNF) in intestinal epithelial cells. Concomitantly, SWI/SNF and MED12-Mediator colocalized at distinct lineage-specifying enhancers in a CDK8/19-dependent manner. Thus, these studies reveal a transcriptional mechanism of intestinal cell specification, coordinated by the interaction between the chromatin remodeling complex SWI/SNF and Mediator kinase.
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Ensamble y Desensamble de Cromatina , Sacarosa , Animales , Cromatina/genética , Quinasa 8 Dependiente de Ciclina/genética , Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Homeostasis , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Ratones , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Aberrant activation of Wnt/ß-catenin pathway is a key driver of colorectal cancer (CRC) growth and of great therapeutic importance. In this study, we performed comprehensive CRISPR screens to interrogate the regulatory network of Wnt/ß-catenin signaling in CRC cells. We found marked discrepancies between the artificial TOP reporter activity and ß-catenin-mediated endogenous transcription and redundant roles of T cell factor/lymphoid enhancer factor transcription factors in transducing ß-catenin signaling. Compiled functional genomic screens and network analysis revealed unique epigenetic regulators of ß-catenin transcriptional output, including the histone lysine methyltransferase 2A oncoprotein (KMT2A/Mll1). Using an integrative epigenomic and transcriptional profiling approach, we show that KMT2A loss diminishes the binding of ß-catenin to consensus DNA motifs and the transcription of ß-catenin targets in CRC. These results suggest that KMT2A may be a promising target for CRCs and highlight the broader potential for exploiting epigenetic modulation as a therapeutic strategy for ß-catenin-driven malignancies.
Asunto(s)
Neoplasias Colorrectales , beta Catenina , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Factores de Transcripción TCF/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
BACKGROUND: We recently reported on preferential deposition of bare fluorescent diamond particles FDP-NV-700/800nm (FDP-NV) in the liver following intravenous administration to rats. The pharmacokinetics of FDP-NV in that species indicated short residency in the circulation by rapid clearance by the liver. Retention of FDP-NV in the liver was not associated with any pathology. These observations suggested that cancer therapeutics, such as doxorubicin, linked to FDP-NV, could potentially serve for anti-cancer treatment while sparing toxicities of peripheral organs. PURPOSE: To generate proof-of-concept (POC) and detail mechanisms of action of doxorubicin-coated FDP-NV-700/800nm (FDP-DOX) as a prospective chemotherapeutic for metastatic liver cancer. METHODS: FDP-DOX was generated by adsorption chemistry. Experimental design included concentration and time-dependent efficacy studies as compared with naïve (baren) FDP-NV in in vitro liver cancer cells models. Uptake of FDP-NV and FDP-DOX by HepG-2, Hep-3B and hCRC organoids were demonstrated by flow-cytometry and fluorescent microscopy. FDP-DOX pharmacodynamic effects included metabolic as well as cell death biomarkers Annexin V, TUNEL and LDH leakage. DOX desorpted from FDP-DOX was assessed by confocal microscopy and chemical assay of cells fractions. RESULTS: FDP-DOX efficacy was dose- and time-dependent and manifested in both liver cancer cell lines and human CRC organoids. FDP-DOX was rapidly internalized into cancer cells/organoids leading to cancer growth inhibition and apoptosis. FDP-DOX disrupted cell membrane integrity as evident by LDH release and suppressing mitochondrial metabolic pathways (AlamarBlue assay). Access of free DOX to the nuclei was confirmed by direct UV-Visible fluorescent assay and confocal microscopy of DOX fluorescence. CONCLUSION: The rapid uptake and profound cancer inhibition observed using FDP-DOX in clinically relevant cancer models, highlight FDP-DOX promise for cancer chemotherapeutics. We also conclude that the in vitro data justify further investment in in vivo POC studies.