ABSTRACT
Investigating therapeutic "outliers" that show exceptional responses to anti-cancer treatment can uncover biomarkers of drug sensitivity. We performed preclinical trials investigating primary murine acute myeloid leukemias (AMLs) generated by retroviral insertional mutagenesis in KrasG12D "knockin" mice with the MEK inhibitor PD0325901 (PD901). One outlier AML responded and exhibited intrinsic drug resistance at relapse. Loss of wild-type (WT) Kras enhanced the fitness of the dominant clone and rendered it sensitive to MEK inhibition. Similarly, human colorectal cancer cell lines with increased KRAS mutant allele frequency were more sensitive to MAP kinase inhibition, and CRISPR-Cas9-mediated replacement of WT KRAS with a mutant allele sensitized heterozygous mutant HCT116 cells to treatment. In a prospectively characterized cohort of patients with advanced cancer, 642 of 1,168 (55%) with KRAS mutations exhibited allelic imbalance. These studies demonstrate that serial genetic changes at the Kras/KRAS locus are frequent in cancer and modulate competitive fitness and MEK dependency.
Subject(s)
Antineoplastic Agents/therapeutic use , Benzamides/therapeutic use , Colorectal Neoplasms/genetics , Diphenylamine/analogs & derivatives , MAP Kinase Signaling System , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Cell Line, Tumor , Clonal Evolution , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Diphenylamine/pharmacology , Diphenylamine/therapeutic use , Drug Resistance, Neoplasm , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , MAP Kinase Signaling System/drug effects , Mice , Mutation , RetroviridaeABSTRACT
Recent advances in targeted covalent inhibitors have aroused significant interest for their potential in drug development for difficult therapeutic targets. Proteome-wide profiling of functional residues is an integral step of covalent drug discovery aimed at defining actionable sites and evaluating compound selectivity in cells. A classical workflow for this purpose is called IsoTOP-ABPP, which employs an activity-based probe and two isotopically labeled azide-TEV-biotin tags to mark, enrich, and quantify proteome from two samples. Here we report a novel isobaric 11plex-AzidoTMT reagent and a new workflow, named AT-MAPP, that significantly expands multiplexing power as compared to the original isoTOP-ABPP. We demonstrate its application in identifying cysteine on- and off-targets using a KRAS G12C covalent inhibitor ARS-1620. However, changes in some of these hits can be explained by modulation at the protein and post-translational levels. Thus, it would be crucial to interrogate site-level bona fide changes in concurrence to proteome-level changes for corroboration. In addition, we perform a multiplexed covalent fragment screening using four acrylamide-based compounds as a proof-of-concept. This study identifies a diverse set of liganded cysteine residues in a compound-dependent manner with an average hit rate of 0.07% in intact cell. Lastly, we screened 20 sulfonyl fluoride-based compounds to demonstrate that the AT-MAPP assay is flexible for noncysteine functional residues such as tyrosine and lysine. Overall, we envision that 11plex-AzidoTMT will be a useful addition to the current toolbox for activity-based protein profiling and covalent drug development.
Subject(s)
Cysteine , Proteome , Proteome/metabolism , Cysteine/metabolism , Proteomics , Protein Processing, Post-Translational , Drug DiscoveryABSTRACT
Imprinted genes are expressed from only one parental allele and heterozygous loss involving the expressed allele is sufficient to produce complete loss of protein expression. Genetic alterations are common in tumorigenesis but the role of imprinted genes in this process is not well understood. In earlier work we mutagenized mice heterozygous for the Neurofibromatosis I tumor suppressor gene (NF1) to model radiotherapy-associated second malignant neoplasms that arise in irradiated NF1 patients. Expression analysis of tumor cell lines established from our mouse models identified Grb10 expression as widely absent. Grb10 is an imprinted gene and polymorphism analysis of cell lines and primary tumors demonstrates that the expressed allele is commonly lost in diverse Nf1 mutant tumors arising in our mouse models. We performed functional studies to test whether Grb10 restoration or loss alter fundamental features of the tumor growth. Restoring Grb10 in Nf1 mutant tumors decreases proliferation, decreases soft agar colony formation and downregulates Ras signaling. Conversely, Grb10 silencing in untransformed mouse embryo fibroblasts significantly increased cell proliferation and increased Ras-GTP levels. Expression of a constitutively activated MEK rescued tumor cells from Grb10-mediated reduction in colony formation. These studies reveal that Grb10 loss can occur during in vivo tumorigenesis, with a functional consequence in untransformed primary cells. In tumors, Grb10 loss independently promotes Ras pathway hyperactivation, which promotes hyperproliferation, an early feature of tumor development. In the context of a robust Nf1 mutant mouse model of cancer this work identifies a novel role for an imprinted gene in tumorigenesis.
Subject(s)
Alleles , GRB10 Adaptor Protein/genetics , Genomic Imprinting , Neurofibromatosis 1/genetics , Animals , Cell Line, Tumor , Cell Proliferation , Disease Models, Animal , Down-Regulation , Fibroblasts/metabolism , GRB10 Adaptor Protein/metabolism , Gene Knockdown Techniques , Gene Silencing , Genes, Neurofibromatosis 1 , Loss of Heterozygosity , Mice , Mice, Knockout , Phosphorylation , Proto-Oncogene Proteins p21(ras) , Radiation , Signal Transduction , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Although targeting cancer metabolism is a promising therapeutic strategy, clinical success will depend on an accurate diagnostic identification of tumor subtypes with specific metabolic requirements. Through broad metabolite profiling, we successfully identified three highly distinct metabolic subtypes in pancreatic ductal adenocarcinoma (PDAC). One subtype was defined by reduced proliferative capacity, whereas the other two subtypes (glycolytic and lipogenic) showed distinct metabolite levels associated with glycolysis, lipogenesis, and redox pathways, confirmed at the transcriptional level. The glycolytic and lipogenic subtypes showed striking differences in glucose and glutamine utilization, as well as mitochondrial function, and corresponded to differences in cell sensitivity to inhibitors of glycolysis, glutamine metabolism, lipid synthesis, and redox balance. In PDAC clinical samples, the lipogenic subtype associated with the epithelial (classical) subtype, whereas the glycolytic subtype strongly associated with the mesenchymal (QM-PDA) subtype, suggesting functional relevance in disease progression. Pharmacogenomic screening of an additional â¼ 200 non-PDAC cell lines validated the association between mesenchymal status and metabolic drug response in other tumor indications. Our findings highlight the utility of broad metabolite profiling to predict sensitivity of tumors to a variety of metabolic inhibitors.
Subject(s)
Adenocarcinoma/classification , Adenocarcinoma/metabolism , Carcinoma, Pancreatic Ductal/classification , Carcinoma, Pancreatic Ductal/metabolism , Metabolome , Metabolomics , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Biomarkers, Tumor/metabolism , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Proliferation , Glucose/metabolism , Glutamine/metabolism , Glycolysis/genetics , Humans , Inhibitory Concentration 50 , Lipogenesis/genetics , Mesoderm/metabolism , Mesoderm/pathology , Metabolome/genetics , Reproducibility of Results , Transcription, GeneticABSTRACT
Intercellular communication is essential for glandular functions and tissue homeostasis. Gap junctions couple cells homotypically and heterotypically and co-ordinate reciprocal responses between the different cell types. Connexins (Cxs) are the main mammalian gap junction proteins, and the distribution of some Cx subtypes in the heterotypic gap junctions is not symmetrical; in the murine mammary gland, Cx26, Cx30 and Cx32 are expressed only in the luminal epithelial cells and Cx43 is expressed only in myoepithelial cells. Expression of all four Cxs peaks during late pregnancy and throughout lactation suggesting essential roles for these proteins in the functional secretory activity of the gland. Transgenic (Tg) mice over-expressing Cx26 driven by keratin 5 promoter had an unexpected mammary phenotype: the mothers were unable to feed their pups to weaning age leading to litter starvation and demise in early to mid-lactation. The mammary gland of K5-Cx26 female mice developed normally and produced normal levels of milk protein, suggesting a defect in delivery rather than milk production. Because the mammary gland of K5-Cx26 mothers contained excessive milk, we hypothesized that the defect may be in an inability to eject the milk. Using ex vivo three-dimensional mammary organoid cultures, we showed that tissues isolated from wild-type FVB females contracted upon treatment with oxytocin, whereas, organoids from Tg mice failed to do so. Unexpectedly, we found that ectopic expression of Cx26 in myoepithelial cells altered the expression of endogenous Cx43 resulting in impaired gap junction communication, demonstrated by defective dye coupling in mammary epithelial cells of Tg mice. Inhibition of gap junction communication or knock-down of Cx43 in organoids from wild-type mice impaired contraction in response to oxytocin, recapitulating the observations from the mammary glands of Tg mice. We conclude that Cx26 acts as a trans-dominant negative for Cx43 function in myoepithelial cells, highlighting the importance of cell type-specific expression of Cxs for optimal contractile function of the mammary myoepithelium.
Subject(s)
Connexin 43/metabolism , Connexins/metabolism , Epithelial Cells/metabolism , Mammary Glands, Animal/metabolism , Animals , Blotting, Western , Cell Differentiation/genetics , Cells, Cultured , Connexin 43/genetics , Connexins/genetics , Epithelial Cells/drug effects , Epithelial Cells/physiology , Female , Gap Junctions/metabolism , Gap Junctions/physiology , Gene Expression , Lactation/genetics , Lactation/physiology , Male , Mammary Glands, Animal/cytology , Mammary Glands, Animal/physiology , Mice, Transgenic , Microscopy, Confocal , Muscle Contraction/drug effects , Muscle Contraction/genetics , Muscle Contraction/physiology , Organ Culture Techniques , Oxytocin/pharmacology , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Videotape RecordingABSTRACT
Glandular tissues form ducts (tubes) and acini (spheres) in multicellular organisms. This process is best demonstrated in the organization of the ductal tree of the mammary gland and in 3D models of morphogenesis in culture. Here, we asked a fundamental question: How do single adult epithelial cells generate polarized acini when placed in a surrogate basement membrane 3D gel? Using human breast epithelial cells from either reduction mammoplasty or nonmalignant breast cell lines, we observed a unique cellular movement where single cells undergo multiple rotations and then maintain it cohesively as they divide to assemble into acini. This coherent angular motion (CAMo) was observed in both primary cells and breast cell lines. If CAMo was disrupted, the final geometry was not a sphere. The malignant counterparts of the human breast cell lines in 3D were randomly motile, did not display CAMo, and did not form spheres. Upon "phenotypic reversion" of malignant cells, both CAMo and spherical architecture were restored. We show that cell-cell adhesion and tissue polarity are essential for the formation of acini and link the functional relevance of CAMo to the establishment of spherical architecture rather than to multicellular aggregation or growth. We propose that CAMo is an integral step in the formation of the tissue architecture and that its disruption is involved in malignant transformation.
Subject(s)
Breast/cytology , Cell Movement , Morphogenesis , Acinar Cells/cytology , Cell Adhesion , Cell Line , Cell Polarity , Epithelial Cells/cytology , Female , Humans , RotationABSTRACT
Purpose: This study explores the potential of preclinical in vitro cell line response data and computational modeling in identifying optimal dosage requirements of pan-RAF (Belvarafenib) and MEK (Cobimetinib) inhibitors in melanoma treatment. Our research is motivated by the critical role of drug combinations in enhancing anti-cancer responses and the need to close the knowledge gap around selecting effective dosing strategies to maximize their potential. Results: In a drug combination screen of 43 melanoma cell lines, we identified unique dosage landscapes of panRAF and MEK inhibitors for NRAS vs BRAF mutant melanomas. Both experienced benefits, but with a notably more synergistic and narrow dosage range for NRAS mutant melanoma. Computational modeling and molecular experiments attributed the difference to a mechanism of adaptive resistance by negative feedback. We validated in vivo translatability of in vitro dose-response maps by accurately predicting tumor growth in xenografts. Then, we analyzed pharmacokinetic and tumor growth data from Phase 1 clinical trials of Belvarafenib with Cobimetinib to show that the synergy requirement imposes stricter precision dose constraints in NRAS mutant melanoma patients. Conclusion: Leveraging pre-clinical data and computational modeling, our approach proposes dosage strategies that can optimize synergy in drug combinations, while also bringing forth the real-world challenges of staying within a precise dose range. Simple Summary: Combining drugs is crucial for enhancing anti-cancer responses. However, the potential of pre-clinical data in identifying suitable combinations and dosage is often underutilized. In this study, we leverage preclinical in vitro cell line drug response data and computational modeling of signal transduction and of pharmacokinetics to elucidate distinct dose requirements for the combination of pan-RAF and MEK inhibitors in melanoma. Our findings reveal a more synergistic, but narrower dosing landscape in NRAS vs BRAF mutant melanoma, which we linked to a mechanism of adaptive resistance through negative feedback. Further, our analysis suggests the importance of drug dosing strategies to optimize synergy based on mutational context, yet highlights the real-world challenges of maintaining a narrow dose range. This approach establishes a framework for translational investigation of drug responses in the refinement of combination therapy, balancing the potential for synergy and practical feasibility in cancer treatment planning.
ABSTRACT
The Hippo pathway is a key growth control pathway that is conserved across species. The downstream effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), are frequently activated in cancers to drive proliferation and survival. Based on the premise that sustained interactions between YAP/TAZ and TEADs (transcriptional enhanced associate domain) are central to their transcriptional activities, we discovered a potent small-molecule inhibitor (SMI), GNE-7883, that allosterically blocks the interactions between YAP/TAZ and all human TEAD paralogs through binding to the TEAD lipid pocket. GNE-7883 effectively reduces chromatin accessibility specifically at TEAD motifs, suppresses cell proliferation in a variety of cell line models and achieves strong antitumor efficacy in vivo. Furthermore, we uncovered that GNE-7883 effectively overcomes both intrinsic and acquired resistance to KRAS (Kirsten rat sarcoma viral oncogene homolog) G12C inhibitors in diverse preclinical models through the inhibition of YAP/TAZ activation. Taken together, this work demonstrates the activities of TEAD SMIs in YAP/TAZ-dependent cancers and highlights their potential broad applications in precision oncology and therapy resistance.
Subject(s)
Neoplasms , Proto-Oncogene Proteins p21(ras) , Humans , Proto-Oncogene Proteins p21(ras)/genetics , Precision Medicine , Transcription Factors/metabolism , Signal TransductionABSTRACT
In the mammary gland, epithelial cells are embedded in a 'soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for beta-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of beta-casein expression required both laminin signalling and a 'soft' extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of beta-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues' unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.
Subject(s)
Biomimetic Materials/metabolism , Cell Differentiation , Epithelial Cells/cytology , Extracellular Matrix/metabolism , Laminin/metabolism , Mammary Glands, Animal/cytology , Actins/metabolism , Animals , Caseins/metabolism , Cell Movement , Cell Shape , Down-Regulation , Elasticity , Epithelial Cells/metabolism , Female , Mice , Myosin Type II/metabolism , Organoids/cytology , Organoids/metabolism , Pregnancy , Receptors, Laminin/metabolism , Signal TransductionABSTRACT
Small molecules that stabilize inactive protein conformations are an underutilized strategy for drugging dynamic or otherwise intractable proteins. To facilitate the discovery and characterization of such inhibitors, we created a screening platform to identify conformation-locking antibodies for molecular probes (CLAMPs) that distinguish and induce rare protein conformational states. Applying the approach to KRAS, we discovered CLAMPs that recognize the open conformation of KRASG12C stabilized by covalent inhibitors. One CLAMP enables the visualization of KRASG12C covalent modification in vivo and can be used to investigate response heterogeneity to KRASG12C inhibitors in patient tumors. A second CLAMP enhances the affinity of weak ligands binding to the KRASG12C switch II region (SWII) by stabilizing a specific conformation of KRASG12C, thereby enabling the discovery of such ligands that could serve as leads for the development of drugs in a high-throughput screen. We show that combining the complementary properties of antibodies and small molecules facilitates the study and drugging of dynamic proteins.
Subject(s)
Antibodies , Neoplasms , Proto-Oncogene Proteins p21(ras) , Antibodies/chemistry , Humans , Ligands , Mutation , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitorsABSTRACT
Connexins constitute a large family of trans-membrane proteins that allow intercellular communication and the transfer of ions and small signaling molecules between cells. Recent studies have revealed complex translational and post-translational mechanisms that regulate connexin synthesis, maturation, membrane transport and degradation that in turn modulate gap junction intercellular communication. With the growing myriad of connexin interacting proteins, including cytoskeletal elements, junctional proteins, and enzymes, gap junctions are now perceived, not only as channels between neighboring cells, but as signaling complexes that regulate cell function and transformation. Connexins have also been shown to form functional hemichannels and have roles altogether independent of channel functions, where they exert their effects on proliferation and other aspects of life and death of the cell through mostly-undefined mechanisms. This review provides an updated overview of current knowledge of connexins and their interacting proteins, and it describes connexin modulation in disease and tumorigenesis.
ABSTRACT
Gap junctions (GJ) are required for mammary epithelial differentiation. Using epithelial (SCp2) and myoepithelial-like (SCg6) mouse-derived mammary cells, the role of heterocellular interaction in assembly of GJ complexes and functional differentiation (beta-casein expression) was evaluated. Heterocellular interaction is critical for beta-casein expression, independent of exogenous basement membrane or cell anchoring substrata. Functional differentiation of SCp2, co-cultured with SCg6, is more sensitive to GJ inhibition relative to homocellular SCp2 cultures differentiated by exogenous basement membrane. Connexin (Cx)32 and Cx43 levels were not regulated across culture conditions; however, GJ functionality was enhanced under differentiation-permissive conditions. Immunoprecipitation studies demonstrated association of junctional complex components (alpha-catenin, beta-catenin and ZO-2) with Cx32 and Cx43, in differentiation conditions, and additionally with Cx30 in heterocellular cultures. Although beta-catenin did not shuttle between cadherin and GJ complexes, increased association between connexins and beta-catenin in heterocellular cultures was observed. This was concomitant with reduced nuclear beta-catenin, suggesting that differentiation in heterocellular cultures involves sequestration of beta-catenin in GJ complexes.
Subject(s)
Cell Communication/physiology , Epithelial Cells/physiology , Gap Junctions/physiology , Membrane Proteins/metabolism , alpha Catenin/metabolism , beta Catenin/metabolism , Animals , Basement Membrane/metabolism , Cell Adhesion/physiology , Cell Cycle Proteins/metabolism , Cell Differentiation/physiology , Cell Membrane Permeability/physiology , Connexin 30 , Connexin 43/metabolism , Connexins/metabolism , Epithelial Cells/cytology , Female , Mammary Glands, Animal/cytology , Mice , Zonula Occludens-2 Protein , Gap Junction beta-1 ProteinABSTRACT
The mammary gland is an ideal "model organism" for studying tissue specificity and gene expression in mammals: it is one of the few organs that develop after birth and it undergoes multiple cycles of growth, differentiation and regression during the animal's lifetime in preparation for the important function of lactation. The basic "functional differentiation" unit in the gland is the mammary acinus made up of a layer of polarized epithelial cells specialized for milk production surrounded by myoepithelial contractile cells, and the two-layered structure is surrounded by basement membrane. Much knowledge about the regulation of mammary gland development has been acquired from studying the physiology of the gland and of lactation in rodents. Culture studies, however, were hampered by the inability to maintain functional differentiation on conventional tissue culture plastic. We now know that the microenvironment, including the extracellular matrix and tissue architecture, plays a crucial role in directing functional differentiation of organs. Thus, in order for culture systems to be effective experimental models, they need to recapitulate the basic unit of differentiated function in the tissue or organ and to maintain its three-dimensional (3D) structure. Mouse mammary culture models evolved from basic monolayers of cells to an array of complex 3D systems that observe the importance of the microenvironment in dictating proper tissue function and structure. In this chapter, we focus on how 3D mouse mammary epithelial cultures have enabled investigators to gain a better understanding of the organization, development and function of the acinus, and to identify key molecular, structural, and mechanical cues important for maintaining mammary function and architecture. The accompanying chapter of Vidi et al. describes 3D models developed for human cells. Here, we describe how mouse primary epithelial cells and cell lines--essentially those we use in our laboratory--are cultured in relevant 3D microenvironments. We focus on the design of functional assays that enable us to understand the intricate signaling events underlying mammary gland biology, and address the advantages and limitations of the different culture settings. Finally we also discuss how advances in bioengineering tools may help towards the ultimate goal of building tissues and organs in culture for basic research and clinical studies.