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1.
bioRxiv ; 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38826447

RESUMO

Ovulation is a spatiotemporally coordinated process that involves several tightly controlled events, including oocyte meiotic maturation, cumulus expansion, follicle wall rupture and repair, and ovarian stroma remodeling. To date, no studies have detailed the precise window of ovulation at single-cell resolution. Here, we performed parallel single-cell RNA-seq and spatial transcriptomics on paired mouse ovaries across an ovulation time course to map the spatiotemporal profile of ovarian cell types. We show that major ovarian cell types exhibit time-dependent transcriptional states enriched for distinct functions and have specific localization profiles within the ovary. We also identified gene markers for ovulation-dependent cell states and validated these using orthogonal methods. Finally, we performed cell-cell interaction analyses to identify ligand-receptor pairs that may drive ovulation, revealing previously unappreciated interactions. Taken together, our data provides a rich and comprehensive resource of murine ovulation that can be mined for discovery by the scientific community.

2.
Cell Death Differ ; 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38802657

RESUMO

Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is one of the deadliest forms of cancer with limited therapy options. Overexpression of the heat shock protein 70 (HSP70) is a hallmark of cancer that is strongly associated with aggressive disease and worse clinical outcomes. However, the underlying mechanisms by which HSP70 allows tumor cells to thrive under conditions of continuous stress have not been fully described. Here, we report that PDAC has the highest expression of HSP70 relative to normal tissue across all cancers analyzed. Furthermore, HSP70 expression is associated with tumor grade and is further enhanced in metastatic PDAC. We show that genetic or therapeutic ablation of HSP70 alters mitochondrial subcellular localization, impairs mitochondrial dynamics, and promotes mitochondrial swelling to induce apoptosis. Mechanistically, we find that targeting HSP70 suppresses the PTEN-induced kinase 1 (PINK1) mediated phosphorylation of dynamin-related protein 1 (DRP1). Treatment with the HSP70 inhibitor AP-4-139B was efficacious as a single agent in primary and metastatic mouse models of PDAC. In addition, we demonstrate that HSP70 inhibition promotes the AMP-activated protein kinase (AMPK) mediated phosphorylation of Beclin-1, a key regulator of autophagic flux. Accordingly, we find that the autophagy inhibitor hydroxychloroquine (HCQ) enhances the ability of AP-4-139B to mediate anti-tumor activity in vivo. Collectively, our results suggest that HSP70 is a multi-functional driver of tumorigenesis that orchestrates mitochondrial dynamics and autophagy. Moreover, these findings support the rationale for concurrent inhibition of HSP70 and autophagy as a novel therapeutic approach for HSP70-driven PDAC.

3.
Methods Mol Biol ; 2797: 211-225, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38570462

RESUMO

Missense mutations in the RAS family of oncogenes (HRAS, KRAS, and NRAS) are present in approximately 20% of human cancers, making RAS a valuable therapeutic target (Prior et al., Cancer Res 80:2969-2974, 2020). Although decades of research efforts to develop therapeutic inhibitors of RAS were unsuccessful, there has been success in recent years with the entrance of FDA-approved KRASG12C-specific inhibitors to the clinic (Skoulidis et al., N Engl J Med 384:2371-2381, 2021; Jänne et al., N Engl J Med 387:120-131, 2022). Additionally, KRASG12D-specific inhibitors are presently undergoing clinical trials (Wang et al., J Med Chem 65:3123-3133, 2022). The advent of these allele specific inhibitors has disproved the previous notion that RAS is undruggable. Despite these advancements in RAS-targeted therapeutics, several RAS mutants that frequently arise in cancers remain without tractable drugs. Thus, it is critical to further understand the function and biology of RAS in cells and to develop tools to identify novel therapeutic vulnerabilities for development of anti-RAS therapeutics. To do this, we have exploited the use of monobody (Mb) technology to develop specific protein-based inhibitors of selected RAS isoforms and mutants (Spencer-Smith et al., Nat Chem Biol 13:62-68, 2017; Khan et al., Cell Rep 38:110322, 2022; Wallon et al., Proc Natl Acad Sci USA 119:e2204481119, 2022; Khan et al., Small GTPases 13:114-127, 2021; Khan et al., Oncogene 38:2984-2993, 2019). Herein, we describe our combined use of Mbs and NanoLuc Binary Technology (NanoBiT) to analyze RAS protein-protein interactions and to screen for RAS-binding small molecules in live-cell, high-throughput assays.


Assuntos
Luciferases , Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Oncogenes , Comunicação Celular , Mutação
4.
Biochem Soc Trans ; 52(1): 1-13, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38174740

RESUMO

Intersectin (ITSN) is a multi-domain scaffold protein with a diverse array of functions including regulation of endocytosis, vesicle transport, and activation of various signal transduction pathways. There are two ITSN genes located on chromosomes 21 and 2 encoding for proteins ITSN1 and ITSN2, respectively. Each ITSN gene encodes two major isoforms, ITSN-Long (ITSN-L) and ITSN-Short (ITSN-S), due to alternative splicing. ITSN1 and 2, collectively referred to as ITSN, are implicated in many physiological and pathological processes, such as neuronal maintenance, actin cytoskeletal rearrangement, and tumor progression. ITSN is mis-regulated in many tumors, such as breast, lung, neuroblastomas, and gliomas. Altered expression of ITSN is also found in several neurodegenerative diseases, such as Down Syndrome and Alzheimer's disease. This review summarizes recent studies on ITSN and provides an overview of the function of this important family of scaffold proteins in various biological processes.


Assuntos
Síndrome de Down , Transdução de Sinais , Humanos , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Síndrome de Down/genética , Síndrome de Down/metabolismo , Síndrome de Down/patologia , Endocitose/fisiologia
5.
Cancer Biomark ; 38(3): 287-300, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37955079

RESUMO

Pancreatic adenocarcinoma (PDAC) is one of the deadliest cancers, with five-year survival rates of 9%. We hypothesized that secreted frizzled-related protein 2 (SFRP2) may influence stromal growth in pancreatic cancer, since it increases fibrosis and collagen production in non-neoplastic pathologies. We assessed SFRP2 value as a biomarker and assessed its function in PDAC. SFRP2 gene expression in patients with PDAC was analyzed using TCGA data. Disease free survival (DFS) was analyzed using Kaplan Meier test. The effect of KRAS inhibition on SFRP2 expression in PDAC cells was assessed. The associations of stromal content with SFPR2 mRNA and protein with fibrosis were analyzed. The role of SFRP2 in mesenchymal transformation was assessed by western blot in fibroblasts. Of all cancers in TCGA, SFRP2 levels were highest in PDAC, and higher in PDAC than normal tissues (n= 234, p= 0.0003). High SFRP2 levels correlated with decreased DFS (p= 0.0097). KRAS inhibition reduced SFRP2 levels. Spearman correlation was 0.81 between stromal RNA and SFRP2 in human PDAC, and 0.75 between fibrosis and SFRP2 levels in PDAC tumors. SFRP2-treated fibroblasts displayed mesenchymal characteristics. SFRP2 is prognostic for PDAC survival, regulated by KRAS, and associated with PDAC fibrosis.


Assuntos
Adenocarcinoma , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/genética , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética
6.
Proc Natl Acad Sci U S A ; 120(28): e2302485120, 2023 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-37399416

RESUMO

The G12D mutation is among the most common KRAS mutations associated with cancer, in particular, pancreatic cancer. Here, we have developed monobodies, small synthetic binding proteins, that are selective to KRAS(G12D) over KRAS(wild type) and other oncogenic KRAS mutations, as well as over the G12D mutation in HRAS and NRAS. Crystallographic studies revealed that, similar to other KRAS mutant-selective inhibitors, the initial monobody bound to the S-II pocket, the groove between switch II and α3 helix, and captured this pocket in the most widely open form reported to date. Unlike other G12D-selective polypeptides reported to date, the monobody used its backbone NH group to directly recognize the side chain of KRAS Asp12, a feature that closely resembles that of a small-molecule inhibitor, MTRX1133. The monobody also directly interacted with H95, a residue not conserved in RAS isoforms. These features rationalize the high selectivity toward the G12D mutant and the KRAS isoform. Structure-guided affinity maturation resulted in monobodies with low nM KD values. Deep mutational scanning of a monobody generated hundreds of functional and nonfunctional single-point mutants, which identified crucial residues for binding and those that contributed to the selectivity toward the GTP- and GDP-bound states. When expressed in cells as genetically encoded reagents, these monobodies engaged selectively with KRAS(G12D) and inhibited KRAS(G12D)-mediated signaling and tumorigenesis. These results further illustrate the plasticity of the S-II pocket, which may be exploited for the design of next-generation KRAS(G12D)-selective inhibitors.


Assuntos
Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Mutação , Transformação Celular Neoplásica/genética , Carcinogênese , Neoplasias Pancreáticas/genética
7.
Bioinformatics ; 39(6)2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37267161

RESUMO

MOTIVATION: Imaging Spatial Transcriptomics techniques characterize gene expression in cells in their native context by imaging barcoded probes for mRNA with single molecule resolution. However, the need to acquire many rounds of high-magnification imaging data limits the throughput and impact of existing methods. RESULTS: We describe the Joint Sparse method for Imaging Transcriptomics, an algorithm for decoding lower magnification Imaging Spatial Transcriptomics data than that used in standard experimental workflows. Joint Sparse method for Imaging Transcriptomics incorporates codebook knowledge and sparsity assumptions into an optimization problem, which is less reliant on well separated optical signals than current pipelines. Using experimental data obtained by performing Multiplexed Error-Robust Fluorescence in situ Hybridization on tissue from mouse brain, we demonstrate that Joint Sparse method for Imaging Transcriptomics enables improved throughput and recovery performance over standard decoding methods. AVAILABILITY AND IMPLEMENTATION: Software implementation of JSIT, together with example files, is available at https://github.com/jpbryan13/JSIT.


Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Animais , Camundongos , Hibridização in Situ Fluorescente/métodos , Perfilação da Expressão Gênica/métodos , Software , Algoritmos
8.
J Biol Chem ; 298(12): 102661, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36334633

RESUMO

Mutations in one of the three RAS genes (HRAS, KRAS, and NRAS) are present in nearly 20% of all human cancers. These mutations shift RAS to the GTP-loaded active state due to impairment in the intrinsic GTPase activity and disruption of GAP-mediated GTP hydrolysis, resulting in constitutive activation of effectors such as RAF. Because activation of RAF involves dimerization, RAS dimerization has been proposed as an important step in RAS-mediated activation of effectors. The α4-α5 allosteric lobe of RAS has been proposed as a RAS dimerization interface. Indeed, the NS1 monobody, which binds the α4-α5 region within the RAS G domain, inhibits RAS-dependent signaling and transformation as well as RAS nanoclustering at the plasma membrane. Although these results are consistent with a model in which the G domain dimerizes through the α4-α5 region, the isolated G domain of RAS lacks intrinsic dimerization capacity. Furthermore, prior studies analyzing α4-α5 point mutations have reported mixed effects on RAS function. Here, we evaluated the activity of a panel of single amino acid substitutions in the α4-α5 region implicated in RAS dimerization. We found that these proposed "dimerization-disrupting" mutations do not significantly impair self-association, signaling, or transformation of oncogenic RAS. These results are consistent with a model in which activated RAS protomers cluster in close proximity to promote the dimerization of their associated effector proteins (e.g., RAF) without physically associating into dimers mediated by specific molecular interactions. Our findings suggest the need for a nonconventional approach to developing therapeutics targeting the α4-α5 region.


Assuntos
Genes ras , Transdução de Sinais , Humanos , Ligação Proteica , Transdução de Sinais/genética , Mutação , Guanosina Trifosfato/genética
9.
Proc Natl Acad Sci U S A ; 119(43): e2204481119, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36252024

RESUMO

RAS mutants are major therapeutic targets in oncology with few efficacious direct inhibitors available. The identification of a shallow pocket near the Switch II region on RAS has led to the development of small-molecule drugs that target this site and inhibit KRAS(G12C) and KRAS(G12D). To discover other regions on RAS that may be targeted for inhibition, we have employed small synthetic binding proteins termed monobodies that have a strong propensity to bind to functional sites on a target protein. Here, we report a pan-RAS monobody, termed JAM20, that bound to all RAS isoforms with nanomolar affinity and demonstrated limited nucleotide-state specificity. Upon intracellular expression, JAM20 potently inhibited signaling mediated by all RAS isoforms and reduced oncogenic RAS-mediated tumorigenesis in vivo. NMR and mutation analysis determined that JAM20 bound to a pocket between Switch I and II, which is similarly targeted by low-affinity, small-molecule inhibitors, such as BI-2852, whose in vivo efficacy has not been demonstrated. Furthermore, JAM20 directly competed with both the RAF(RBD) and BI-2852. These results provide direct validation of targeting the Switch I/II pocket for inhibiting RAS-driven tumorigenesis. More generally, these results demonstrate the utility of tool biologics as probes for discovering and validating druggable sites on challenging targets.


Assuntos
Produtos Biológicos , Proteínas Proto-Oncogênicas p21(ras) , Carcinogênese/genética , Genes ras , Humanos , Mutação , Nucleotídeos , Proteínas Proto-Oncogênicas p21(ras)/genética
10.
Leukemia ; 36(7): 1907-1915, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35513703

RESUMO

Allogeneic hematopoietic cell transplantation (allo-HCT) is an effective immunotherapy for various hematologic malignancies, predominantly through potent graft-versus-leukemia (GVL) effect. However, the mortality after allo-HCT is because of relapse of primary malignancy and followed by graft-vs-host-disease (GVHD) as a major cause of transplant-related mortality. Hence, strategies to limit GVHD while preserving the GVL effect are highly desirable. Ceramide, which serves a central role in sphingolipid metabolism, is generated by ceramide synthases (CerS1-6). In this study, we found that genetic or pharmacologic targeting of CerS6 prevented and reversed chronic GVHD (cGVHD). Furthermore, specific inhibition of CerS6 with ST1072 significantly ameliorated acute GVHD (aGVHD) while preserving the GVL effect, which differed from FTY720 that attenuated aGVHD but impaired GVL activity. At the cellular level, blockade of CerS6 restrained donor T cells from migrating into GVHD target organs and preferentially reduced activation of donor CD4 T cells. At the molecular level, CerS6 was required for optimal TCR signaling, CD3/PKCθ co-localization, and subsequent N-RAS activation and ERK signaling, especially on CD4+ T cells. The current study provides rationale and means for targeting CerS6 to control GVHD and leukemia relapse, which would enhance the efficacy of allo-HCT as an immunotherapy for hematologic malignancies in the clinic.


Assuntos
Doença Enxerto-Hospedeiro , Neoplasias Hematológicas , Transplante de Células-Tronco Hematopoéticas , Leucemia , Ceramidas/farmacologia , GTP Fosfo-Hidrolases/metabolismo , Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Leucemia , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Oxirredutases , Recidiva , Linfócitos T , Transplante Homólogo
11.
Cell Rep ; 38(6): 110322, 2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35139380

RESUMO

RAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells. R15 inhibits the signaling and transforming activity of a subset of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., fast exchange mutants). Intracellular expression of R15 reduces the tumor-forming capacity of cancer cell lines driven by select RAS mutants and KRAS(G12D)-mutant patient-derived xenografts (PDXs). Thus, our approach establishes an opportunity to selectively inhibit a subset of RAS mutants by targeting the apo state with drug-like molecules.


Assuntos
Genes ras/genética , Mutação/genética , Nucleotídeos/metabolismo , Neoplasias Pancreáticas/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Camundongos , Neoplasias Pancreáticas/patologia , Transdução de Sinais/genética
12.
Adv Cancer Res ; 153: 237-266, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35101232

RESUMO

RAS proteins represent critical drivers of tumor development and thus are the focus of intense efforts to pharmacologically inhibit these proteins in human cancer. Although recent success has been attained in developing clinically efficacious inhibitors to KRASG12C, there remains a critical need for developing approaches to inhibit additional mutant RAS proteins. A number of anti-RAS biologics have been developed which reveal novel and potentially therapeutically targetable vulnerabilities in oncogenic RAS. This review will discuss the growing field of anti-RAS biologics and potential development of these reagents into new anti-RAS therapies.


Assuntos
Produtos Biológicos , Neoplasias , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Humanos , Mutação , Neoplasias/patologia , Proteínas ras/metabolismo
13.
Adv Cancer Res ; 153: xiii-xiv, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35101237
14.
Small GTPases ; 13(1): 114-127, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-33949915

RESUMO

RAS is the most frequently mutated oncogene in human cancer with nearly ~20% of cancer patients possessing mutations in one of three RAS genes (K, N or HRAS). However, KRAS is mutated in nearly 90% of pancreatic ductal carcinomas (PDAC). Although pharmacological inhibition of RAS has been challenging, KRAS(G12C)-specific inhibitors have recently entered the clinic. While KRAS(G12C) is frequently expressed in lung cancers, it is rare in PDAC. Thus, more broadly efficacious RAS inhibitors are needed for treating KRAS mutant-driven cancers such as PDAC. A RAS-specific tool biologic, NS1 Monobody, inhibits HRAS- and KRAS-mediated signalling and oncogenic transformation both in vitro and in vivo by targeting the α4-α5 allosteric site of RAS and blocking RAS self-association. Here, we evaluated the efficacy of targeting the α4-α5 interface of KRAS as an approach to inhibit PDAC development using an immunocompetent orthotopic mouse model. Chemically regulated NS1 expression inhibited ERK and AKT activation in KRAS(G12D) mutant KPC PDAC cells and reduced the formation and progression of pancreatic tumours. NS1-expressing tumours were characterized by increased infiltration of CD4 + T helper cells. These results suggest that targeting the #x3B1;4-#x3B1;5 allosteric site of KRAS may represent a viable therapeutic approach for inhibiting KRAS-mutant pancreatic tumours.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Camundongos , Animais , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinogênese/patologia , Neoplasias Pancreáticas
15.
Dev Cell ; 56(19): 2752-2764.e6, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34610330

RESUMO

Neuroblastoma (NB), the most common cancer in the first year of life, presents almost exclusively in the trunk. To understand why an early-onset cancer would have such a specific localization, we xenotransplanted human NB cells into discrete neural crest (NC) streams in zebrafish embryos. Here, we demonstrate that human NB cells remain in an undifferentiated, tumorigenic state when comigrating posteriorly with NC cells but, upon comigration into the head, differentiate into neurons and exhibit decreased survival. Furthermore, we demonstrate that this in vivo differentiation requires retinoic acid and brain-derived neurotrophic factor signaling from the microenvironment, as well as cell-autonomous intersectin-1-dependent phosphoinositide 3-kinase-mediated signaling, likely via Akt kinase activation. Our findings suggest a microenvironment-driven explanation for NB's trunk-biased localization and highlight the potential for induced differentiation to promote NB resolution in vivo.


Assuntos
Diferenciação Celular/fisiologia , Neuroblastoma/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Humanos , Masculino , Camundongos , Crista Neural/metabolismo , Neurônios/citologia , Neurônios/fisiologia , Transdução de Sinais , Transplante Heterólogo/métodos , Tretinoína/metabolismo , Tretinoína/farmacologia , Microambiente Tumoral , Peixe-Zebra/metabolismo
16.
Nat Commun ; 12(1): 3091, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035268

RESUMO

VEGFR2 (KDR/Flk1) signaling in endothelial cells (ECs) plays a central role in angiogenesis. The P-type ATPase transporter ATP7A regulates copper homeostasis, and its role in VEGFR2 signaling and angiogenesis is entirely unknown. Here, we describe the unexpected crosstalk between the Copper transporter ATP7A, autophagy, and VEGFR2 degradation. The functional significance of this Copper transporter was demonstrated by the finding that inducible EC-specific ATP7A deficient mice or ATP7A-dysfunctional ATP7Amut mice showed impaired post-ischemic neovascularization. In ECs, loss of ATP7A inhibited VEGF-induced VEGFR2 signaling and angiogenic responses, in part by promoting ligand-induced VEGFR2 protein degradation. Mechanistically, VEGF stimulated ATP7A translocation from the trans-Golgi network to the plasma membrane where it bound to VEGFR2, which prevented autophagy-mediated lysosomal VEGFR2 degradation by inhibiting autophagic cargo/adapter p62/SQSTM1 binding to ubiquitinated VEGFR2. Enhanced autophagy flux due to ATP7A dysfunction in vivo was confirmed by autophagy reporter CAG-ATP7Amut -RFP-EGFP-LC3 transgenic mice. In summary, our study uncovers a novel function of ATP7A to limit autophagy-mediated degradation of VEGFR2, thereby promoting VEGFR2 signaling and angiogenesis, which restores perfusion recovery and neovascularization. Thus, endothelial ATP7A is identified as a potential therapeutic target for treatment of ischemic cardiovascular diseases.


Assuntos
Autofagia/genética , Vasos Sanguíneos/metabolismo , ATPases Transportadoras de Cobre/genética , ATPases do Tipo-P/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/fisiologia , Células COS , Células Cultivadas , Chlorocebus aethiops , ATPases Transportadoras de Cobre/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , ATPases do Tipo-P/metabolismo , Interferência de RNA , Transdução de Sinais/genética , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
17.
Nat Commun ; 12(1): 2656, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976200

RESUMO

Activating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors. Here, we report a monobody, termed 12VC1, that recognizes the active state of both KRAS(G12V) and KRAS(G12C) up to 400-times more tightly than wild-type KRAS. The crystal structures reveal that 12VC1 recognizes the mutations through a shallow pocket, and 12VC1 competes against RAS-effector interaction. When expressed intracellularly, 12VC1 potently inhibits ERK activation and the proliferation of RAS-driven cancer cell lines in vitro and in mouse xenograft models. 12VC1 fused to VHL selectively degrades the KRAS mutants and provides more extended suppression of mutant RAS activity than inhibition by 12VC1 alone. These results demonstrate the feasibility of selective targeting and degradation of KRAS mutants in the active state with noncovalent reagents and provide a starting point for designing noncovalent therapeutics against oncogenic RAS mutants.


Assuntos
Anticorpos Monoclonais/farmacologia , Proteínas Mutantes/antagonistas & inibidores , Mutação , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Camundongos Nus , Proteínas Mutantes/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
18.
Methods Mol Biol ; 2262: 281-302, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977484

RESUMO

RAS is frequently mutated in human cancers with nearly 20% of all cancers harboring mutations in one of three RAS isoforms (KRAS, HRAS, or NRAS). Furthermore, RAS proteins are critical oncogenic drivers of tumorigenesis. As such, RAS has been a prime focus for development of targeted cancer therapeutics. Although RAS is viewed by many as undruggable, the recent development of allele-specific covalent inhibitors to KRAS(G12C) has provided significant hope for the eventual pharmacological inhibition of RAS (Ostrem et al., Nature 503(7477):548-551, 2013; Patricelli et al., Cancer Discov 6(3):316-329, 2016; Janes et al., Cell 172(3):578-589.e17, 2018; Canon et al., Nature 575(7781):217-223, 2019; Hallin et al., Cancer Discov 10(1):54-71, 2020). Indeed, these (G12C)-specific inhibitors have elicited promising responses in early phase clinical trials (Canon et al., Nature 575(7781):217-223, 2019; Hallin et al., Cancer Discov 10(1):54-71, 2020). Despite this success in pharmacologically targeting KRAS(G12C), the remaining RAS mutants lack readily tractable chemistries for development of covalent inhibitors. Thus, alternative approaches are needed to develop broadly efficacious RAS inhibitors. We have utilized Monobody (Mb) technology to identify vulnerabilities in RAS that can potentially be exploited for development of novel RAS inhibitors. Here, we describe the methods used to isolate RAS-specific Mbs and to define their inhibitory activity.


Assuntos
Antineoplásicos/farmacologia , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Engenharia de Proteínas/métodos , Anticorpos de Domínio Único/imunologia , Proteínas ras/antagonistas & inibidores , Proteínas ras/imunologia , Humanos , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia
19.
Biochem Soc Trans ; 48(5): 1831-1841, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-32869838

RESUMO

RAS is a membrane localized small GTPase frequently mutated in human cancer. As such, RAS has been a focal target for developing cancer therapeutics since its discovery nearly four decades ago. However, efforts to directly target RAS have been challenging due to the apparent lack of readily discernable deep pockets for binding small molecule inhibitors leading many to consider RAS as undruggable. An important milestone in direct RAS inhibition was achieved recently with the groundbreaking discovery of covalent inhibitors that target the mutant Cys residue in KRAS(G12C). Surprisingly, these G12C-reactive compounds only target mutant RAS in the GDP-bound state thereby locking it in the inactive conformation and blocking its ability to couple with downstream effector pathways. Building on this success, several groups have developed similar compounds that selectively target KRAS(G12C), with AMG510 and MRTX849 the first to advance to clinical trials. Both have shown early promising results. Though the success with these compounds has reignited the possibility of direct pharmacological inhibition of RAS, these covalent inhibitors are limited to treating KRAS(G12C) tumors which account for <15% of all RAS mutants in human tumors. Thus, there remains an unmet need to identify more broadly efficacious RAS inhibitors. Here, we will discuss the current state of RAS(G12C) inhibitors and the potential for inhibiting additional RAS mutants through targeting RAS dimerization which has emerged as an important step in the allosteric regulation of RAS function.


Assuntos
Antineoplásicos/farmacologia , Mutação , Neoplasias/terapia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas ras/antagonistas & inibidores , Acetonitrilas/farmacologia , Regulação Alostérica , Sítio Alostérico , Animais , Domínio Catalítico , Membrana Celular/metabolismo , Dimerização , Desenho de Fármacos , GTP Fosfo-Hidrolases/metabolismo , Humanos , Metabolismo , Camundongos , Conformação Molecular , Transplante de Neoplasias , Neoplasias/metabolismo , Piperazinas/farmacologia , Conformação Proteica , Piridinas/farmacologia , Piridinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/metabolismo
20.
Am J Physiol Cell Physiol ; 319(5): C933-C944, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32936699

RESUMO

Caveolin-1 (Cav-1) is a scaffolding protein and a major component of caveolae/lipid rafts. Previous reports have shown that endothelial dysfunction in Cav-1-deficient (Cav-1-/-) mice is mediated by elevated oxidative stress through endothelial nitric oxide synthase (eNOS) uncoupling and increased NADPH oxidase. Oxidant stress is the net balance of oxidant generation and scavenging, and the role of Cav-1 as a regulator of antioxidant enzymes in vascular tissue is poorly understood. Extracellular SOD (SOD3) is a copper (Cu)-containing enzyme that is secreted from vascular smooth muscle cells/fibroblasts and subsequently binds to the endothelial cells surface, where it scavenges extracellular [Formula: see text] and preserves endothelial function. SOD3 activity is dependent on Cu, supplied by the Cu transporter ATP7A, but whether Cav-1 regulates the ATP7A-SOD3 axis and its role in oxidative stress-mediated vascular dysfunction has not been studied. Here we show that the activity of SOD3, but not SOD1, was significantly decreased in Cav-1-/- vessels, which was rescued by re-expression of Cav-1 or Cu supplementation. Loss of Cav-1 reduced ATP7A protein, but not mRNA, and this was mediated by ubiquitination of ATP7A and proteasomal degradation. ATP7A bound to Cav-1 and was colocalized with SOD3 in caveolae/lipid rafts or perinucleus in vascular tissues or cells. Impaired endothelium-dependent vasorelaxation in Cav-1-/- mice was rescued by gene transfer of SOD3 or by ATP7A-overexpressing transgenic mice. These data reveal an unexpected role of Cav-1 in stabilizing ATP7A protein expression by preventing its ubiquitination and proteasomal degradation, thereby increasing SOD3 activity, which in turn protects against vascular oxidative stress-mediated endothelial dysfunction.


Assuntos
Caveolina 1/genética , ATPases Transportadoras de Cobre/genética , Células Endoteliais/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase/genética , Animais , Aorta/citologia , Aorta/metabolismo , Caveolina 1/deficiência , Cobre/farmacologia , Proteínas de Transporte de Cobre/genética , Proteínas de Transporte de Cobre/metabolismo , ATPases Transportadoras de Cobre/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Masculino , Artérias Mesentéricas/citologia , Artérias Mesentéricas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Estresse Oxidativo , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Transdução de Sinais , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/metabolismo , Ubiquitinação/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos
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