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1.
Biophys J ; 2024 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-38762754

RESUMO

Cyclin-dependent kinase 12 (CDK12) is a critical regulatory protein involved in transcription and DNA repair processes. Dysregulation of CDK12 has been implicated in various diseases, including cancer. Understanding the CDK12 interactome is pivotal for elucidating its functional roles and potential therapeutic targets. Traditional methods for interactome prediction often rely on protein structure information, limiting applicability to CDK12 characterized by partly disordered terminal C region. In this study, we present a structure-independent machine-learning model that utilizes proteins' sequence and functional data to predict the CDK12 interactome. This approach is motivated by the disordered character of the CDK12 C-terminal region mitigating a structure-driven search for binding partners. Our approach incorporates multiple data sources, including protein-protein interaction networks, functional annotations, and sequence-based features, to construct a comprehensive CDK12 interactome prediction model. The ability to predict CDK12 interactions without relying on structural information is a significant advancement, as many potential interaction partners may lack crystallographic data. In conclusion, our structure-independent machine-learning model presents a powerful tool for predicting the CDK12 interactome and holds promise in advancing our understanding of CDK12 biology, identifying potential therapeutic targets, and facilitating precision-medicine approaches for CDK12-associated diseases.

2.
PLoS Comput Biol ; 20(2): e1011873, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38335222

RESUMO

Super enhancers (SE), large genomic elements that activate transcription and drive cell identity, have been found with cancer-specific gene regulation in human cancers. Recent studies reported the importance of understanding the cooperation and function of SE internal components, i.e., the constituent enhancers (CE). However, there are no pan-cancer studies to identify cancer-specific SE signatures at the constituent level. Here, by revisiting pan-cancer SE activities with H3K27Ac ChIP-seq datasets, we report fingerprint SE signatures for 28 cancer types in the NCI-60 cell panel. We implement a mixture model to discriminate active CEs from inactive CEs by taking into consideration ChIP-seq variabilities between cancer samples and across CEs. We demonstrate that the model-based estimation of CE states provides improved functional interpretation of SE-associated regulation. We identify cancer-specific CEs by balancing their active prevalence with their capability of encoding cancer type identities. We further demonstrate that cancer-specific CEs have the strongest per-base enhancer activities in independent enhancer sequencing assays, suggesting their importance in understanding critical SE signatures. We summarize fingerprint SEs based on the cancer-specific statuses of their component CEs and build an easy-to-use R package to facilitate the query, exploration, and visualization of fingerprint SEs across cancers.


Assuntos
Neoplasias , Super Intensificadores , Humanos , Epigenômica , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Neoplasias/genética
3.
Eur J Med Chem ; 266: 116101, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38232465

RESUMO

The UNC-51-like kinase-1 (ULK1) is one of the central upstream regulators of the autophagy pathway, represents a key target for the development of molecular probes to abrogate autophagy and explore potential therapeutic avenues. Here we report the discovery, structure-activity and structure-property relationships of selective, potent, and cell-active ULK1/2 inhibitors based on a 7-azaindole scaffold. Using structure-based drug design, we have developed a series of analogs with excellent binding affinity and biochemical activity against ULK1/2 (IC50 < 25 nM). The validation of cellular target engagement for these compounds was achieved through the employment of the ULK1 NanoBRET intracellular kinase assay. Notably, we have successfully solved the crystal structure of the lead compound, MR-2088, bound to the active site of ULK1. Moreover, the combination treatment of MR-2088 with known KRAS→RAF→MEK→ERK pathway inhibitors, such as trametinib, showed promising synergistic effect in vitro using H2030 (KRASG12C) cell lines. Lastly, our findings underscore MR-2088's potential to inhibit starvation/stimuli-induced autophagic flux, coupled with its suitability for in vivo studies based on its pharmacokinetic properties.


Assuntos
Indóis , Proteínas Proto-Oncogênicas p21(ras) , Indóis/farmacologia , Autofagia , Linhagem Celular
4.
Neuro Oncol ; 26(1): 70-84, 2024 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-37551745

RESUMO

BACKGROUND: Glioblastoma (GBM) is refractory to current treatment modalities while side effects of treatments result in neurotoxicity and cognitive impairment. Here we test the hypothesis that inhibiting CDK7 or CDK9 would effectively combat GBM with reduced neurotoxicity. METHODS: We examined the effect of a CDK7 inhibitor, THZ1, and multiple CDK9 inhibitors (SNS032, AZD4573, NVP2, and JSH150) on GBM cell lines, patient-derived temozolomide (TMZ)-resistant and responsive primary tumor cells and glioma stem cells (GSCs). Biochemical changes were assessed by western blotting, immunofluorescence, multispectral imaging, and RT-PCR. In vivo, efficacy was assessed in orthotopic and subcutaneous xenograft models. RESULTS: CDK7 and CDK9 inhibitors suppressed the viability of TMZ-responsive and resistant GBM cells and GSCs at low nanomolar concentrations, with limited cytotoxic effects in vivo. The inhibitors abrogated RNA Pol II and p70S6K phosphorylation and nascent protein synthesis. Furthermore, the self-renewal of GSCs was significantly reduced with a corresponding reduction in Sox2 and Sox9 levels. Analysis of TCGA data showed increased expression of CDK7, CDK9, SOX2, SOX9, and RPS6KB1 in GBM; supporting this, multispectral imaging of a TMA revealed increased levels of CDK9, Sox2, Sox9, phospho-S6, and phospho-p70S6K in GBM compared to normal brains. RNA-Seq results suggested that inhibitors suppressed tumor-promoting genes while inducing tumor-suppressive genes. Furthermore, the studies conducted on subcutaneous and orthotopic GBM tumor xenograft models showed that administration of CDK9 inhibitors markedly suppressed tumor growth in vivo. CONCLUSIONS: Our results suggest that CDK7 and CDK9 targeted therapies may be effective against TMZ-sensitive and resistant GBM.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioblastoma/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Linhagem Celular Tumoral , Glioma/tratamento farmacológico , Neoplasias Encefálicas/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase 9 Dependente de Ciclina/metabolismo
5.
J Biol Chem ; 300(1): 105501, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38016516

RESUMO

Inhibition of cyclin-dependent kinases (CDKs) has evolved as an emerging anticancer strategy. In addition to the cell cycle-regulating CDKs, the transcriptional kinases Cdk12 and Cdk13 have become the focus of interest as they mediate a variety of functions, including the transition from transcription initiation to elongation and termination, precursor mRNA splicing, and intronic polyadenylation. Here, we determine the crystal structure of the small molecular inhibitor SR-4835 bound to the Cdk12/cyclin K complex at 2.68 Å resolution. The compound's benzimidazole moiety is embedded in a unique hydrogen bond network mediated by the kinase hinge region with flanking hydroxy groups of the Y815 and D819 side chains. Whereas the SR-4835 head group targets the adenine-binding pocket, the kinase's glycine-rich loop is shifted down toward the activation loop. Additionally, the αC-helix adopts an inward conformation, and the phosphorylated T-loop threonine interacts with all three canonical arginines, a hallmark of CDK activation that is altered in Cdk12 and Cdk13. Dose-response inhibition measurements with recombinant CMGC kinases show that SR-4835 is highly specific for Cdk12 and Cdk13 following a 10-fold lower potency for Cdk10. Whereas other CDK-targeting compounds exhibit tighter binding affinities and higher potencies for kinase inhibition, SR-4835 can be considered a selective transcription elongation antagonist. Our results provide the basis for a rational improvement of SR-4835 toward Cdk12 inhibition and a gain in selectivity over other transcription regulating CDKs.


Assuntos
Quinases Ciclina-Dependentes , Ciclinas , Poliadenilação , Ciclinas/metabolismo , Conformação Molecular , Humanos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/química
6.
J Med Chem ; 66(11): 7162-7178, 2023 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-37204207

RESUMO

Specific inhibition of a single kinase isoform is a challenging task due to the highly conserved nature of ATP-binding sites. Casein kinase 1 (CK1) δ and ε share 97% sequence identity in their catalytic domains. From a comparison of the X-ray crystal structures of CK1δ and CK1ε, we developed a potent and highly CK1ε-isoform-selective inhibitor (SR-4133). The X-ray co-crystal structure of the CK1δ-SR-4133 complex reveals that the electrostatic surface between the naphthyl unit of SR-4133 and CK1δ is mismatched, destabilizing the interaction of SR-4133 with CK1δ. Conversely, the hydrophobic surface area resulting from the Asp-Phe-Gly motif (DFG)-out conformation of CK1ε stabilizes the binding of SR-4133 in the ATP-binding pocket of CK1ε, leading to the selective inhibition of CK1ε. The potent CK1ε-selective agents display nanomolar growth inhibition of bladder cancer cells and inhibit the phosphorylation of 4E-BP1 in T24 cells, which is a direct downstream effector of CK1ε.


Assuntos
Caseína Quinase Idelta , Caseína Quinases/metabolismo , Isoformas de Proteínas/metabolismo , Sítios de Ligação , Trifosfato de Adenosina
7.
ACS Chem Biol ; 18(2): 251-264, 2023 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-36630201

RESUMO

Photoreactive fragment-like probes have been applied to discover target proteins that constitute novel cellular vulnerabilities and to identify viable chemical hits for drug discovery. Through forming covalent bonds, functionalized probes can achieve stronger target engagement and require less effort for on-target mechanism validation. However, the design of probe libraries, which directly affects the biological target space that is interrogated, and effective target prioritization remain critical challenges of such a chemical proteomic platform. In this study, we designed and synthesized a diverse panel of 20 fragment-based probes containing natural product-based privileged structural motifs for small-molecule lead discovery. These probes were fully functionalized with orthogonal diazirine and alkyne moieties and used for protein crosslinking in live lung cancer cells, target enrichment via "click chemistry," and subsequent target identification through label-free quantitative liquid chromatography-tandem mass spectrometry analysis. Pair-wise comparison with a blunted negative control probe and stringent prioritization via individual cross-comparisons against the entire panel identified glutathione S-transferase zeta 1 (GSTZ1) as a specific and unique target candidate. DepMap database query, RNA interference-based gene silencing, and proteome-wide tyrosine reactivity profiling suggested that GSTZ1 cooperated with different oncogenic alterations by supporting survival signaling in refractory non-small cell lung cancer cells. This finding may form the basis for developing novel GSTZ1 inhibitors to improve the therapeutic efficacy of oncogene-directed targeted drugs. In summary, we designed a novel fragment-based probe panel and developed a target prioritization scheme with improved stringency, which allows for the identification of unique target candidates, such as GSTZ1 in refractory lung cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Proteômica , Neoplasias Pulmonares/tratamento farmacológico , Proteínas , Glutationa , Glutationa Transferase/metabolismo
8.
Nucleic Acids Res ; 50(6): 3115-3127, 2022 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-35234924

RESUMO

Super enhancers (SEs) are broad enhancer domains usually containing multiple constituent enhancers that hold elevated activities in gene regulation. Disruption in one or more constituent enhancers causes aberrant SE activities that lead to gene dysregulation in diseases. To quantify SE aberrations, differential analysis is performed to compare SE activities between cell conditions. The state-of-art strategy in estimating differential SEs relies on overall activities and neglect the changes in length and structure of SEs. Here, we propose a novel computational method to identify differential SEs by weighting the combinatorial effects of constituent-enhancer activities and locations (i.e. internal dynamics). In addition to overall activity changes, our method identified four novel classes of differential SEs with distinct enhancer structural alterations. We demonstrate that these structure alterations hold distinct regulatory impact, such as regulating different number of genes and modulating gene expression with different strengths, highlighting the differentiated regulatory roles of these unexplored SE features. When compared to the existing method, our method showed improved identification of differential SEs that were linked to better discernment of cell-type-specific SE activity and functional interpretation.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Diferenciação Celular
9.
Neuro Oncol ; 24(10): 1673-1686, 2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-35213727

RESUMO

BACKGROUND: Leptomeningeal disease (LMD) occurs as a late complication of several human cancers and has no rationally designed treatment options. A major barrier to developing effective therapies for LMD is the lack of cell-based or preclinical models that recapitulate human disease. Here, we describe the development of in vitro and in vivo cultures of patient-derived cerebrospinal fluid circulating tumor cells (PD-CSF-CTCs) from patients with melanoma as a preclinical model to identify exploitable vulnerabilities in melanoma LMD. METHODS: CSF-CTCs were collected from melanoma patients with melanoma-derived LMD and cultured ex vivo using human meningeal cell-conditioned media. Using immunoassays and RNA-sequencing analyses of PD-CSF-CTCs, molecular signaling pathways were examined and new therapeutic targets were tested for efficacy in PD-CSF-CTCs preclinical models. RESULTS: PD-CSF-CTCs were successfully established both in vitro and in vivo. Global RNA analyses of PD-CSF-CTCs revealed several therapeutically tractable targets. These studies complimented our prior proteomic studies highlighting IGF1 signaling as a potential target in LMD. As a proof of concept, combining treatment of ceritinib and trametinib in vitro and in vivo demonstrated synergistic antitumor activity in PD-CSF-CTCs and BRAF inhibitor-resistant melanoma cells. CONCLUSIONS: This study demonstrates that CSF-CTCs can be grown in vitro and in vivo from some melanoma patients with LMD and used as preclinical models. These models retained melanoma expression patterns and had signaling pathways that are therapeutically targetable. These novel models/reagents may be useful in developing rationally designed treatments for LMD.


Assuntos
Melanoma , Neoplasias Meníngeas , Células Neoplásicas Circulantes , Meios de Cultivo Condicionados , Humanos , Melanoma/patologia , Neoplasias Meníngeas/patologia , Proteômica , Proteínas Proto-Oncogênicas B-raf/genética , RNA
10.
PLoS One ; 17(1): e0261578, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35061724

RESUMO

We propose a model of cancer initiation and progression where tumor growth is modulated by an evolutionary coordination game. Evolutionary games of cancer are widely used to model frequency-dependent cell interactions with the most studied games being the Prisoner's Dilemma and public goods games. Coordination games, by their more obscure and less evocative nature, are left understudied, despite the fact that, as we argue, they offer great potential in understanding and treating cancer. In this paper we present the conditions under which coordination games between cancer cells evolve, we propose aspects of cancer that can be modeled as results of coordination games, and explore the ways through which coordination games of cancer can be exploited for therapy.


Assuntos
Dilema do Prisioneiro
11.
Haematologica ; 107(5): 1119-1130, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-34162179

RESUMO

Despite significant progress in the treatment of patients with diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), the prognosis of patients with relapsed disease remains poor due to the emergence of drug resistance and subsequent disease progression. Identification of novel targets and therapeutic strategies for these diseases represents an urgent need. Here, we report that both MCL and DLBCL are exquisitely sensitive to transcription-targeting drugs, in particular THZ531, a covalent inhibitor of cyclin-dependent kinase 12 (CDK12). By implementing pharmacogenomics and a cell-based drug screen, we found that THZ531 leads to inhibition of oncogenic transcriptional programs, especially the DNA damage response pathway, MYC target genes and the mTOR-4EBP1-MCL-1 axis, contributing to dramatic lymphoma suppression in vitro. We also identified de novo and established acquired THZ531-resistant lymphoma cells conferred by over-activation of the MEK-ERK and PI3K-AKT-mTOR pathways and upregulation of multidrug resistance-1 (MDR1) protein. Of note, EZH2 inhibitors reversed resistance to THZ531 by competitive inhibition of MDR1 and, in combination with THZ531, synergistically inhibited MCL and DLBCL growth in vitro. Our study indicates that CDK12 inhibitors, alone or together with EZH2 inhibitors, offer promise as novel effective approaches for difficult-to-treat DLBCL and MCL.


Assuntos
Linfoma Difuso de Grandes Células B , Linfoma de Célula do Manto , Adulto , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/uso terapêutico , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/patologia , Fosfatidilinositol 3-Quinases , Serina-Treonina Quinases TOR
12.
Cancer Immunol Res ; 10(1): 108-125, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34785506

RESUMO

The HER3/ERBB3 receptor is an oncogenic receptor tyrosine kinase that forms heterodimers with EGFR family members and is overexpressed in numerous cancers. HER3 overexpression associates with reduced survival and acquired resistance to targeted therapies, making it a potential therapeutic target in multiple cancer types. Here, we report on immunogenic, promiscuous MHC class II-binding HER3 peptides, which can generate HER3-specific CD4+ Th1 antitumor immune responses. Using an overlapping peptide screening methodology, we identified nine MHC class II-binding HER3 epitopes that elicited specific Th1 immune response in both healthy donors and breast cancer patients. Most of these peptides were not identified by current binding algorithms. Homology assessment of amino acid sequence BLAST showed >90% sequence similarity between human and murine HER3/ERBB3 peptide sequences. HER3 peptide-pulsed dendritic cell vaccination resulted in anti-HER3 CD4+ Th1 responses that prevented tumor development, significantly delayed tumor growth in prevention models, and caused regression in multiple therapeutic models of HER3-expressing murine tumors, including mammary carcinoma and melanoma. Tumors were robustly infiltrated with CD4+ T cells, suggesting their key role in tumor rejection. Our data demonstrate that class II HER3 promiscuous peptides are effective at inducing HER3-specific CD4+ Th1 responses and suggest their applicability in immunotherapies for human HER3-overexpressing tumors.


Assuntos
Neoplasias da Mama/terapia , Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Receptor ErbB-3/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Análise de Sobrevida , Células Th1/imunologia , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologia
13.
J Med Chem ; 64(16): 12109-12131, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34382808

RESUMO

Aberrant activation of Wnt/ß-catenin signaling is strongly associated with many diseases including cancer invasion and metastasis. Small-molecule targeting of the central signaling node of this pathway, ß-catenin, is a biologically rational approach to abolish hyperactivation of ß-catenin signaling but has been demonstrated to be a difficult task. Herein, we report a drug-like small molecule, ZW4864, that binds with ß-catenin and selectively disrupts the protein-protein interaction (PPI) between B-cell lymphoma 9 (BCL9) and ß-catenin while sparing the ß-catenin/E-cadherin PPI. ZW4864 dose-dependently suppresses ß-catenin signaling activation, downregulates oncogenic ß-catenin target genes, and abrogates invasiveness of ß-catenin-dependent cancer cells. More importantly, ZW4864 shows good pharmacokinetic properties and effectively suppresses ß-catenin target gene expression in the patient-derived xenograft mouse model. This study offers a selective chemical probe to explore ß-catenin-related biology and a drug-like small-molecule ß-catenin/BCL9 disruptor for future drug development.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Piperidinas/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Desenho de Fármacos , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos SCID , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/farmacocinética , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismo
14.
Cancer Immunol Res ; 9(5): 554-567, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33653716

RESUMO

Immunotherapy (IT) and targeted therapy (TT) are both effective against melanoma, but their combination is frequently toxic. Here, we investigated whether the sequence of IT (anti-PD-1)→ TT (ceritinib-trametinib or dabrafenib-trametinib) was associated with improved antitumor responses in mouse models of BRAF- and NRAS-mutant melanoma. Mice with NRAS-mutant (SW1) or BRAF-mutant (SM1) mouse melanomas were treated with either IT, TT, or the sequence of IT→TT. Tumor volumes were measured, and samples from the NRAS-mutant melanomas were collected for immune-cell analysis, single-cell RNA sequencing (scRNA-seq), and reverse phase protein analysis (RPPA). scRNA-seq demonstrated that the IT→TT sequence modulated the immune environment, leading to increased infiltration of T cells, monocytes, dendritic cells and natural killer cells, and decreased numbers of tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells. Durable responses to the IT→TT sequence were dependent on T-cell activity, with depletion of CD8+, but not CD4+, T cells abrogating the therapeutic response. An analysis of transcriptional heterogeneity in the melanoma compartment showed the sequence of IT→TT enriched for a population of melanoma cells with increased expression of MHC class I and melanoma antigens. RPPA analysis demonstrated that the sustained immune response induced by IT→TT suppressed tumor-intrinsic signaling pathways required for therapeutic escape. These studies establish that upfront IT improves the responses to TT in BRAF- and NRAS-mutant melanoma models.


Assuntos
Antineoplásicos/uso terapêutico , Melanoma/tratamento farmacológico , Proteínas Monoméricas de Ligação ao GTP/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Animais , Antineoplásicos/química , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Imidazóis/química , Imunoterapia , Melanoma/genética , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Oximas/química , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Piridonas/química , Pirimidinas/química , Pirimidinonas/química , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Sulfonas/química , Linfócitos T Reguladores/imunologia
15.
J Vis Exp ; (167)2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33586709

RESUMO

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the "Murine Ommaya." As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.


Assuntos
Doenças do Sistema Nervoso Central/terapia , Sistemas de Liberação de Medicamentos , Xenoenxertos/fisiologia , Modelos Biológicos , Animais , Neoplasias da Mama/patologia , Feminino , Injeções Intraventriculares , Neoplasias Meníngeas/diagnóstico , Neoplasias Meníngeas/tratamento farmacológico , Neoplasias Meníngeas/patologia , Camundongos , Metástase Neoplásica , Células Neoplásicas Circulantes/patologia , Prognóstico
16.
Future Med Chem ; 13(2): 117-141, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33295810

RESUMO

CDK12 and CDK13 are Ser/Thr protein kinases that regulate transcription and co-transcriptional processes. Genetic silencing of CDK12 is associated with genomic instability in a variety of cancers, including difficult-to-treat breast, ovarian, colorectal, brain and pancreatic cancers, and is synthetic lethal with PARP, MYC or EWS/FLI inhibition. CDK13 is amplified in hepatocellular carcinoma. Consequently, selective CDK12/13 inhibitors constitute powerful research tools as well as promising anti-cancer therapeutics, either alone or in combination therapy. Herein the authors discuss the role of CDK12 and CDK13 in normal and cancer cells, describe their utility as a biomarker and therapeutic target, review the medicinal chemistry optimization of existing CDK12/13 inhibitors and outline strategies for the rational design of CDK12/13 selective inhibitors.


Assuntos
Antineoplásicos/química , Biomarcadores Tumorais/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Animais , Antineoplásicos/farmacologia , Terapia Combinada , Ensaios de Seleção de Medicamentos Antitumorais , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Conformação Proteica , Inibidores de Proteínas Quinases/farmacologia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Relação Estrutura-Atividade
17.
Blood Adv ; 4(13): 3072-3084, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32634240

RESUMO

The in-clinic phosphatidylinositol 3-kinase (PI3K) inhibitors idelalisib (CAL-101) and duvelisib (IPI-145) have demonstrated high rates of response and progression-free survival in clinical trials of B-cell malignancies, such as chronic lymphocytic leukemia (CLL). However, a high incidence of adverse events has led to frequent discontinuations, limiting the clinical development of these inhibitors. By contrast, the dual PI3Kδ/casein kinase-1-ε (CK1ε) inhibitor umbralisib (TGR-1202) also shows high rates of response in clinical trials but has an improved safety profile with fewer severe adverse events. Toxicities typical of this class of PI3K inhibitors are largely thought to be immune mediated, but they are poorly characterized. Here, we report the effects of idelalisib, duvelisib, and umbralisib on regulatory T cells (Tregs) on normal human T cells, T cells from CLL patients, and T cells in an Eµ-TCL1 adoptive transfer mouse CLL model. Ex vivo studies revealed differential effects of these PI3K inhibitors; only umbralisib treatment sustained normal and CLL-associated FoxP3+ human Tregs. Further, although all 3 inhibitors exhibit antitumor efficacy in the Eµ-TCL1 CLL model, idelalisib- or duvelisib-treated mice displayed increased immune-mediated toxicities, impaired function, and reduced numbers of Tregs, whereas Treg number and function were preserved in umbralisib-treated CLL-bearing mice. Finally, our studies demonstrate that inhibition of CK1ε can improve CLL Treg number and function. Interestingly, CK1ε inhibition mitigated impairment of CLL Tregs by PI3K inhibitors in combination treatment. These results suggest that the improved safety profile of umbralisib is due to its role as a dual PI3Kδ/CK1ε inhibitor that preserves Treg number and function.


Assuntos
Antineoplásicos , Leucemia Linfocítica Crônica de Células B , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Camundongos , Fosfatidilinositol 3-Quinases/uso terapêutico
18.
Mol Cancer Ther ; 19(8): 1623-1635, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32430484

RESUMO

Although gemcitabine is the cornerstone of care for pancreatic ductal adenocarcinoma (PDA), patients lack durable responses and relapse is inevitable. While the underlying mechanisms leading to gemcitabine resistance are likely to be multifactorial, there is a strong association between activating gemcitabine metabolism pathways and clinical outcome. This study evaluated casein kinase 1 delta (CK1δ) as a potential therapeutic target for PDA and bladder cancer, in which CK1δ is frequently overexpressed. We assessed the antitumor effects of genetically silencing or pharmacologically inhibiting CK1δ using our in-house CK1δ small-molecule inhibitor SR-3029, either alone or in combination with gemcitabine, on the proliferation and survival of pancreatic and bladder cancer cell lines and orthotopic mouse models. Genetic studies confirmed that silencing CK1δ or treatment with SR-3029 induced a significant upregulation of deoxycytidine kinase (dCK), a rate-limiting enzyme in gemcitabine metabolite activation. The combination of SR-3029 with gemcitabine induced synergistic antiproliferative activity and enhanced apoptosis in both pancreatic and bladder cancer cells. Furthermore, in an orthotopic pancreatic tumor model, we observed improved efficacy with combination treatment concomitant with increased dCK expression. This study demonstrates that CK1δ plays a role in gemcitabine metabolism, and that the combination of CK1δ inhibition with gemcitabine holds promise as a future therapeutic option for metastatic PDA as well as other cancers with upregulated CK1δ expression.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Caseína Quinase Idelta/antagonistas & inibidores , Desoxicitidina Quinase/metabolismo , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Desoxicitidina/farmacologia , Desoxicitidina Quinase/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Gencitabina , Neoplasias Pancreáticas
19.
Cell Death Differ ; 27(4): 1200-1213, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31506606

RESUMO

Cellular DNA is constantly under threat from internal and external insults, consequently multiple pathways have evolved to maintain chromosomal fidelity. Our previous studies revealed that chronic stress, mediated by continuous stimulation of the ß2-adrenergic-ßarrestin-1 signaling axis suppresses activity of the tumor suppressor p53 and impairs genomic integrity. In this pathway, ßarrestin-1 (ßarr1) acts as a molecular scaffold to promote the binding and degradation of p53 by the E3-ubiquitin ligase, MDM2. We sought to determine whether ßarr1 plays additional roles in the repair of DNA damage. Here we demonstrate that in mice ßarr1 interacts with p53-binding protein 1 (53BP1) with major consequences for the repair of DNA double-strand breaks. 53BP1 is a principle component of the DNA damage response, and when recruited to the site of double-strand breaks in DNA, 53BP1 plays an important role coordinating repair of these toxic lesions. Here, we report that ßarr1 directs 53BP1 degradation by acting as a scaffold for the E3-ubiquitin ligase Rad18. Consequently, knockdown of ßarr1 stabilizes 53BP1 augmenting the number of 53BP1 DNA damage repair foci following exposure to ionizing radiation. Accordingly, ßarr1 loss leads to a marked increase in irradiation resistance both in cells and in vivo. Thus, ßarr1 is an important regulator of double strand break repair, and disruption of the ßarr1/53BP1 interaction offers an attractive strategy to protect cells against high levels of exposure to ionizing radiation.


Assuntos
Reparo do DNA , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , beta-Arrestina 1/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Dano ao DNA , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Intestinos/patologia , Camundongos Endogâmicos C57BL , Ligação Proteica/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Tolerância a Radiação/efeitos da radiação , Radiação Ionizante
20.
Nat Commun ; 10(1): 5017, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676756

RESUMO

Autophagy is an intracellular catabolic process prominent in starvation, aging and disease. Neuronal autophagy is particularly important, as it affects the development and function of the nervous system, and is heavily implicated in neurodegenerative disease. Nonetheless, how autophagy is regulated in neurons remains poorly understood. Using an unbiased proteomics approach, we demonstrate that the primary initiator of autophagy, the UNC-51/ULK kinase, is negatively regulated by the ubiquitin ligase RPM-1. RPM-1 ubiquitin ligase activity restricts UNC-51 and autophagosome formation within specific axonal compartments, and exerts effects broadly across the nervous system. By restraining UNC-51 activity, RPM-1 inhibits autophagosome formation to affect axon termination, synapse maintenance and behavioral habituation. These results demonstrate how UNC-51 and autophagy are regulated subcellularly in axons, and unveils a mechanism for restricting initiation of autophagy across the nervous system. Our findings have important implications beyond nervous system development, given growing links between altered autophagy regulation and neurodegenerative diseases.


Assuntos
Autofagia/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Animais Geneticamente Modificados , Autofagossomos/metabolismo , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Axônios/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular Tumoral , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Doenças Neurodegenerativas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Sinapses/genética , Sinapses/metabolismo , Ubiquitina-Proteína Ligases/genética
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