ABSTRACT
Members of the PP2A family of serine/threonine phosphatases are important human tumor suppressor genes. Unlike most tumor suppressors, they are rarely mutated/deleted, but rather are impaired by "inhibitor proteins." Two papers in this issue of Cell show how some phenothiazine derivatives reactivate specific PP2A isozymes with potential benefit in cancer and other diseases.
Subject(s)
Neoplasms , Protein Phosphatase 2 , Genes, Tumor Suppressor , Humans , Neoplasms/drug therapy , Neoplasms/genetics , Protein Phosphatase 2/geneticsABSTRACT
Loss-of-function mutations in TET2 occur frequently in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and are associated with a DNA hypermethylation phenotype. To determine the role of TET2 deficiency in leukemia stem cell maintenance, we generated a reversible transgenic RNAi mouse to model restoration of endogenous Tet2 expression. Tet2 restoration reverses aberrant hematopoietic stem and progenitor cell (HSPC) self-renewal in vitro and in vivo. Treatment with vitamin C, a co-factor of Fe2+ and α-KG-dependent dioxygenases, mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in Tet2-deficient mouse HSPCs and suppresses human leukemic colony formation and leukemia progression of primary human leukemia PDXs. Vitamin C also drives DNA hypomethylation and expression of a TET2-dependent gene signature in human leukemia cell lines. Furthermore, TET-mediated DNA oxidation induced by vitamin C treatment in leukemia cells enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to selectively target TET deficiency in cancer. PAPERCLIP.
Subject(s)
Ascorbic Acid/pharmacology , DNA-Binding Proteins/metabolism , Leukemia, Myeloid, Acute/drug therapy , Myelodysplastic Syndromes/drug therapy , Proto-Oncogene Proteins/metabolism , Vitamins/pharmacology , Animals , Ascorbic Acid/administration & dosage , Cell Death , Cell Line, Tumor , DNA Methylation , DNA-Binding Proteins/genetics , Dioxygenases , Gene Knockdown Techniques , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Myelodysplastic Syndromes/genetics , Neoplasm Transplantation , Poly (ADP-Ribose) Polymerase-1/genetics , Proto-Oncogene Proteins/genetics , Transcription, Genetic , Transplantation, Heterologous , Vitamins/administration & dosageABSTRACT
Large-scale genomic studies have identified multiple somatic aberrations in breast cancer, including copy number alterations and point mutations. Still, identifying causal variants and emergent vulnerabilities that arise as a consequence of genetic alterations remain major challenges. We performed whole-genome small hairpin RNA (shRNA) "dropout screens" on 77 breast cancer cell lines. Using a hierarchical linear regression algorithm to score our screen results and integrate them with accompanying detailed genetic and proteomic information, we identify vulnerabilities in breast cancer, including candidate "drivers," and reveal general functional genomic properties of cancer cells. Comparisons of gene essentiality with drug sensitivity data suggest potential resistance mechanisms, effects of existing anti-cancer drugs, and opportunities for combination therapy. Finally, we demonstrate the utility of this large dataset by identifying BRD4 as a potential target in luminal breast cancer and PIK3CA mutations as a resistance determinant for BET-inhibitors.
Subject(s)
Algorithms , Breast Neoplasms/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Cycle Proteins , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases , Cluster Analysis , Drug Resistance, Neoplasm , Gene Dosage , Gene Expression Profiling , Genome-Wide Association Study , Humans , Linear Models , Nuclear Proteins/genetics , Phosphatidylinositol 3-Kinases , Transcription Factors/geneticsABSTRACT
The primary task of white adipose tissue (WAT) is the storage of lipids. However, "beige" adipocytes also exist in WAT. Beige adipocytes burn fat and dissipate the energy as heat, but their abundance is diminished in obesity. Stimulating beige adipocyte development, or WAT browning, increases energy expenditure and holds potential for combating metabolic disease and obesity. Here, we report that insulin and leptin act together on hypothalamic neurons to promote WAT browning and weight loss. Deletion of the phosphatases PTP1B and TCPTP enhanced insulin and leptin signaling in proopiomelanocortin neurons and prevented diet-induced obesity by increasing WAT browning and energy expenditure. The coinfusion of insulin plus leptin into the CNS or the activation of proopiomelanocortin neurons also increased WAT browning and decreased adiposity. Our findings identify a homeostatic mechanism for coordinating the status of energy stores, as relayed by insulin and leptin, with the central control of WAT browning.
Subject(s)
Adipose Tissue, Brown/metabolism , Adipose Tissue, White/metabolism , Insulin/metabolism , Leptin/metabolism , Pro-Opiomelanocortin/metabolism , Adiposity , Animals , Body Temperature Regulation , Mice, Inbred C57BL , Mice, Knockout , Neurons/metabolism , Obesity/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolismABSTRACT
Knowledge of fundamental differences between breast cancer subtypes has driven therapeutic advances; however, basal-like breast cancer (BLBC) remains clinically intractable. Because BLBC exhibits alterations in DNA repair enzymes and cell-cycle checkpoints, elucidation of factors enabling the genomic instability present in this subtype has the potential to reveal novel anti-cancer strategies. Here, we demonstrate that BLBC is especially sensitive to suppression of iron-sulfur cluster (ISC) biosynthesis and identify DNA polymerase epsilon (POLE) as an ISC-containing protein that underlies this phenotype. In BLBC cells, POLE suppression leads to replication fork stalling, DNA damage, and a senescence-like state or cell death. In contrast, luminal breast cancer and non-transformed mammary cells maintain viability upon POLE suppression but become dependent upon an ATR/CHK1/CDC25A/CDK2 DNA damage response axis. We find that CDK1/2 targets exhibit hyperphosphorylation selectively in BLBC tumors, indicating that CDK2 hyperactivity is a genome integrity vulnerability exploitable by targeting POLE.
Subject(s)
Breast Neoplasms/pathology , Carcinoma, Basal Cell/pathology , Cyclin-Dependent Kinase 2/metabolism , DNA Polymerase II/metabolism , Genomic Instability , Poly-ADP-Ribose Binding Proteins/metabolism , Animals , Apoptosis , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Carcinoma, Basal Cell/genetics , Carcinoma, Basal Cell/metabolism , Cell Cycle , Cell Proliferation , Cyclin-Dependent Kinase 2/genetics , DNA Damage , DNA Polymerase II/genetics , Female , Humans , Mice , Mice, Inbred NOD , Phosphorylation , Poly-ADP-Ribose Binding Proteins/genetics , Signal Transduction , Tumor Cells, CulturedABSTRACT
The HapImmuneTM platform exploits covalent inhibitors as haptens for creating major histocompatibility complex (MHC)-presented tumor-specific neoantigens by design, combining targeted therapies with immunotherapy for the treatment of drug-resistant cancers. A HapImmune antibody, R023, recognizes multiple sotorasib-conjugated KRAS(G12C) peptides presented by different human leukocyte antigens (HLAs). This high specificity to sotorasib, coupled with broad HLA-binding capability, enables such antibodies, when reformatted as T cell engagers, to potently and selectively kill sotorasib-resistant KRAS(G12C) cancer cells expressing different HLAs upon sotorasib treatment. The loosening of HLA restriction could increase the patient population that can benefit from this therapeutic approach. To understand the molecular basis for its unconventional binding capability, we used single-particle cryogenic electron microscopy to determine the structures of R023 bound to multiple sotorasib-peptide conjugates presented by different HLAs. R023 forms a pocket for sotorasib between the VH and VL domains, binds HLAs in an unconventional, angled way, with VL making most contacts with them, and makes few contacts with the peptide moieties. This binding mode enables the antibody to accommodate different hapten-peptide conjugates and to adjust its conformation to different HLAs presenting hapten-peptides. Deep mutational scanning validated the structures and revealed distinct levels of mutation tolerance by sotorasib- and HLA-binding residues. Together, our structural information and sequence landscape analysis reveal key features for achieving MHC-restricted recognition of multiple hapten-peptide antigens, which will inform the development of next-generation therapeutic antibodies.
Subject(s)
Peptides , Humans , Peptides/immunology , Peptides/chemistry , HLA Antigens/immunology , HLA Antigens/metabolism , Major Histocompatibility Complex/immunology , Haptens/immunology , Protein Binding , Cryoelectron MicroscopyABSTRACT
Protein-tyrosine phosphatases (PTPs), along with protein-tyrosine kinases, play key roles in cellular signaling. All Class I PTPs contain an essential active site cysteinyl residue, which executes a nucleophilic attack on substrate phosphotyrosyl residues. The high reactivity of the catalytic cysteine also predisposes PTPs to oxidation by reactive oxygen species, such as H(2)O(2). Reversible PTP oxidation is emerging as an important cellular regulatory mechanism and might contribute to diseases such as cancer. We exploited these unique features of PTP enzymology to develop proteomic methods, broadly applicable to cell and tissue samples, that enable the comprehensive identification and quantification of expressed classical PTPs (PTPome) and the oxidized subset of the PTPome (oxPTPome). We find that mouse and human cells and tissues, including cancer cells, display distinctive PTPomes and oxPTPomes, revealing additional levels of complexity in the regulation of protein-tyrosine phosphorylation in normal and malignant cells.
Subject(s)
Protein Tyrosine Phosphatases/analysis , Proteomics/methods , Animals , Cell Line , Humans , Mice , Mice, Inbred C57BL , Neoplasms/metabolism , Oxidation-Reduction , RatsABSTRACT
Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the membrane yeast two-hybrid (MYTH) and the mammalian membrane two-hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provides a broad and deep view of RTK signaling.
Subject(s)
ErbB Receptors/metabolism , Protein Interaction Maps , Signal Transduction , src-Family Kinases/metabolism , Animals , Enzyme Activation , Epidermal Growth Factor/pharmacology , ErbB Receptors/agonists , ErbB Receptors/genetics , HEK293 Cells , Humans , Mice , Mutation , Phosphorylation , Protein Interaction Mapping , Receptor-Like Protein Tyrosine Phosphatases, Class 3/genetics , Receptor-Like Protein Tyrosine Phosphatases, Class 3/metabolism , Receptor-Like Protein Tyrosine Phosphatases, Class 4/genetics , Receptor-Like Protein Tyrosine Phosphatases, Class 4/metabolism , Reproducibility of Results , Signal Transduction/drug effects , Transfection , Two-Hybrid System Techniques , src-Family Kinases/geneticsABSTRACT
Treatment and relevant targets for breast cancer (BC) remain limited, especially for triple-negative BC (TNBC). We identified 6091 proteins of 76 human BC cell lines using data-independent acquisition (DIA). Integrating our proteomic findings with prior multi-omics datasets, we found that including proteomics data improved drug sensitivity predictions and provided insights into the mechanisms of action. We subsequently profiled the proteomic changes in nine cell lines (five TNBC and four non-TNBC) treated with EGFR/AKT/mTOR inhibitors. In TNBC, metabolism pathways were dysregulated after EGFR/mTOR inhibitor treatment, while RNA modification and cell cycle pathways were affected by AKT inhibitor. This systematic multi-omics and in-depth analysis of the proteome of BC cells can help prioritize potential therapeutic targets and provide insights into adaptive resistance in TNBC.
Subject(s)
Signal Transduction , Triple Negative Breast Neoplasms , Humans , Proto-Oncogene Proteins c-akt/metabolism , Proteomics , Cell Proliferation , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Triple Negative Breast Neoplasms/metabolism , ErbB Receptors/metabolismABSTRACT
High-grade serous tubo-ovarian carcinoma (HGSC) is a major cause of cancer-related death. Treatment is not uniform, with some patients undergoing primary debulking surgery followed by chemotherapy (PDS) and others being treated directly with chemotherapy and only having surgery after three to four cycles (NACT). Which strategy is optimal remains controversial. We developed a mathematical framework that simulates hierarchical or stochastic models of tumor initiation and reproduces the clinical course of HGSC. After estimating parameter values, we infer that most patients harbor chemoresistant HGSC cells at diagnosis and that, if the tumor burden is not too large and complete debulking can be achieved, PDS is superior to NACT due to better depletion of resistant cells. We further predict that earlier diagnosis of primary HGSC, followed by complete debulking, could improve survival, but its benefit in relapsed patients is likely to be limited. These predictions are supported by primary clinical data from multiple cohorts. Our results have clear implications for these key issues in HGSC management.
Subject(s)
Computer Simulation , Early Detection of Cancer , Ovarian Neoplasms/diagnosis , Ovarian Neoplasms/therapy , Aged , Cohort Studies , Cystadenocarcinoma, Serous/diagnosis , Cystadenocarcinoma, Serous/pathology , Cystadenocarcinoma, Serous/therapy , Cytoreduction Surgical Procedures , Female , Humans , Middle Aged , Models, Biological , Neoadjuvant Therapy , Neoplasm Grading , Ovarian Neoplasms/pathology , Ovarian Neoplasms/surgery , Survival Analysis , Treatment Outcome , Tumor BurdenABSTRACT
The motheaten mouse has long served as a paradigm for complex autoimmune and inflammatory disease. Null mutations in Ptpn6, which encodes the nonreceptor protein-tyrosine phosphatase Shp1, cause the motheaten phenotype. However, Shp1 regulates multiple signaling pathways in different hematopoietic cell types, so the cellular and molecular mechanism of autoimmunity and inflammation in the motheaten mouse has remained unclear. By using floxed Ptpn6 mice, we dissected the contribution of innate immune cells to the motheaten phenotype. Ptpn6 deletion in neutrophils resulted in cutaneous inflammation, but not autoimmunity, providing an animal model of human neutrophilic dermatoses. By contrast, dendritic cell deletion caused severe autoimmunity, without inflammation. Genetic and biochemical analysis showed that inflammation was caused by enhanced neutrophil integrin signaling through Src-family and Syk kinases, whereas autoimmunity resulted from exaggerated MyD88-dependent signaling in dendritic cells. Our data demonstrate that disruption of distinct Shp1-regulated pathways in different cell types combine to cause motheaten disease.
Subject(s)
Autoimmunity/immunology , Dendritic Cells/immunology , Inflammation/immunology , Neutrophils/immunology , Animals , Autoimmunity/genetics , CD11c Antigen/genetics , CD11c Antigen/immunology , CD11c Antigen/metabolism , Calgranulin A/genetics , Calgranulin A/immunology , Calgranulin A/metabolism , Cell Line, Tumor , Cells, Cultured , Dendritic Cells/metabolism , Dermis/immunology , Dermis/metabolism , Dermis/pathology , Female , Flow Cytometry , Humans , Immunoblotting , Inflammation/genetics , Inflammation/metabolism , Intracellular Signaling Peptides and Proteins/immunology , Intracellular Signaling Peptides and Proteins/metabolism , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Mutation , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/immunology , Myeloid Differentiation Factor 88/metabolism , Neutrophils/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 6/immunology , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Protein-Tyrosine Kinases/immunology , Protein-Tyrosine Kinases/metabolism , Syk Kinase , src-Family Kinases/immunology , src-Family Kinases/metabolismABSTRACT
Oncogene-induced senescence (OIS) provides an important, but incompletely understood, barrier to tumorigenesis. In this issue, Yang et al. (2014) surprisingly report that inactivation of PTP1B by reactive oxygen species is essential for OIS, via effects on AGO2 and microRNA maturation.
Subject(s)
Argonaute Proteins/metabolism , Gene Silencing , Protein Tyrosine Phosphatase, Non-Receptor Type 1/physiology , Tyrosine/metabolism , ras Proteins/physiology , HumansABSTRACT
RASopathies are a family of related syndromes caused by mutations in regulators of the RAS/Extracellular Regulated Kinase 1/2 (ERK1/2) signaling cascade that often result in neurological deficits. RASopathy mutations in upstream regulatory components, such as NF1, PTPN11/SHP2, and RAS have been well-characterized, but mutation-specific differences in the pathogenesis of nervous system abnormalities remain poorly understood, especially those involving mutations downstream of RAS. Here, we assessed cellular and behavioral phenotypes in mice expressing a Raf1L613V gain-of-function mutation associated with the RASopathy, Noonan Syndrome. We report that Raf1L613V/wt mutants do not exhibit a significantly altered number of excitatory or inhibitory neurons in the cortex. However, we observed a significant increase in the number of specific glial subtypes in the forebrain. The density of GFAP+ astrocytes was significantly increased in the adult Raf1L613V/wt cortex and hippocampus relative to controls. OLIG2+ oligodendrocyte progenitor cells were also increased in number in mutant cortices, but we detected no significant change in myelination. Behavioral analyses revealed no significant changes in voluntary locomotor activity, anxiety-like behavior, or sociability. Surprisingly, Raf1L613V/wt mice performed better than controls in select aspects of the water radial-arm maze, Morris water maze, and cued fear conditioning tasks. Overall, these data show that increased astrocyte and oligodendrocyte progenitor cell (OPC) density in the cortex coincides with enhanced cognition in Raf1L613V/wt mutants and further highlight the distinct effects of RASopathy mutations on nervous system development and function.
Subject(s)
Cerebral Cortex/metabolism , Learning , Mutation , Neuroglia/metabolism , Noonan Syndrome/genetics , Noonan Syndrome/psychology , Proto-Oncogene Proteins c-raf/genetics , Animals , Biomarkers , Glial Fibrillary Acidic Protein/metabolism , Immunohistochemistry , MAP Kinase Signaling System , Maze Learning , Memory , Mice , Mice, Transgenic , Neurons/metabolism , Noonan Syndrome/metabolism , Oligodendroglia/metabolism , Proto-Oncogene Proteins c-raf/metabolismABSTRACT
Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell-mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell-mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1-triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1-targeting therapeutic approaches.
Subject(s)
Cell Adhesion , Immunity, Cellular/immunology , Phosphoproteins/metabolism , Programmed Cell Death 1 Receptor/metabolism , Proteome/analysis , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , Cytokines/metabolism , Humans , Ligands , Lymphocyte Activation , Phosphorylation , Signal Transduction , T-Lymphocytes/metabolism , Transcriptional ActivationABSTRACT
Conditional knockout (KO) mouse models are invaluable for elucidating the physiological roles of platelets. The Platelet factor 4-Cre recombinase (Pf4-Cre) transgenic mouse is the current model of choice for generating megakaryocyte/platelet-specific KO mice. Platelets and leukocytes work closely together in a wide range of disease settings, yet the specific contribution of platelets to these processes remains unclear. This is partially a result of the Pf4-Cre transgene being expressed in a variety of leukocyte populations. To overcome this issue, we developed a Gp1ba-Cre transgenic mouse strain in which Cre expression is driven by the endogenous Gp1ba locus. By crossing Gp1ba-Cre and Pf4-Cre mice to the mT/mG dual-fluorescence reporter mouse and performing a head-to-head comparison, we demonstrate more stringent megakaryocyte lineage-specific expression of the Gp1ba-Cre transgene. Broader tissue expression was observed with the Pf4-Cre transgene, leading to recombination in many hematopoietic lineages, including monocytes, macrophages, granulocytes, and dendritic and B and T cells. Direct comparison of phenotypes of Csk, Shp1, or CD148 conditional KO mice generated using either the Gp1ba-Cre or Pf4-Cre strains revealed similar platelet phenotypes. However, additional inflammatory and immunological anomalies were observed in Pf4-Cre-generated KO mice as a result of nonspecific deletion in other hematopoietic lineages. By excluding leukocyte contributions to phenotypes, the Gp1ba-Cre mouse will advance our understanding of the role of platelets in inflammation and other pathophysiological processes in which platelet-leukocyte interactions are involved.
Subject(s)
Blood Platelets/metabolism , Integrases/metabolism , Leukocytes/metabolism , Platelet Glycoprotein GPIb-IX Complex/metabolism , Agglutination , Animals , Bone Marrow Cells/cytology , CSK Tyrosine-Protein Kinase , Cell Lineage , Cell Size , Gene Targeting , Homeostasis , Lymphocyte Count , Megakaryocytes/cytology , Megakaryocytes/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Models, Animal , Phenotype , Platelet Aggregation , Platelet Factor 4/metabolism , Receptor-Like Protein Tyrosine Phosphatases, Class 3/metabolism , Recombination, Genetic/genetics , Spleen/cytology , src-Family Kinases/metabolismABSTRACT
Src homology 2 domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a ubiquitous protein tyrosine phosphatase that is a critical regulator of signal transduction. Germ line mutations in the PTPN11 gene responsible for catalytic gain or loss of function of SHP2 cause 2 disorders with multiple organ defects: Noonan syndrome (NS) and NS with multiple lentigines (NSML), respectively. Bleeding anomalies have been frequently reported in NS, but causes remain unclear. This study investigates platelet activation in patients with NS and NSML and in 2 mouse models carrying PTPN11 mutations responsible for these 2 syndromes. Platelets from NS mice and patients displayed a significant reduction in aggregation induced by low concentrations of GPVI and CLEC-2 agonists and a decrease in thrombus growth on a collagen surface under arterial shear stress. This was associated with deficiencies in GPVI and αIIbß3 integrin signaling, platelet secretion, and thromboxane A2 generation. Similarly, arterial thrombus formation was significantly reduced in response to a local carotid injury in NS mice, associated with a significant increase in tail bleeding time. In contrast, NSML mouse platelets exhibited increased platelet activation after GPVI and CLEC-2 stimulation and enhanced platelet thrombotic phenotype on collagen matrix under shear stress. Blood samples from NSML patients also showed a shear stress-dependent elevation of platelet responses on collagen matrix. This study brings new insights into the understanding of SHP2 function in platelets, points to new thrombopathies linked to platelet signaling defects, and provides important information for the medical care of patients with NS in situations involving risk of bleeding.
Subject(s)
Blood Platelets/enzymology , Germ-Line Mutation , Noonan Syndrome/enzymology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Signal Transduction , Animals , Blood Platelets/pathology , Humans , Mice , Mice, Mutant Strains , Noonan Syndrome/pathology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/geneticsABSTRACT
MYC (also known as c-MYC) overexpression or hyperactivation is one of the most common drivers of human cancer. Despite intensive study, the MYC oncogene remains recalcitrant to therapeutic inhibition. MYC is a transcription factor, and many of its pro-tumorigenic functions have been attributed to its ability to regulate gene expression programs. Notably, oncogenic MYC activation has also been shown to increase total RNA and protein production in many tissue and disease contexts. While such increases in RNA and protein production may endow cancer cells with pro-tumour hallmarks, this increase in synthesis may also generate new or heightened burden on MYC-driven cancer cells to process these macromolecules properly. Here we discover that the spliceosome is a new target of oncogenic stress in MYC-driven cancers. We identify BUD31 as a MYC-synthetic lethal gene in human mammary epithelial cells, and demonstrate that BUD31 is a component of the core spliceosome required for its assembly and catalytic activity. Core spliceosomal factors (such as SF3B1 and U2AF1) associated with BUD31 are also required to tolerate oncogenic MYC. Notably, MYC hyperactivation induces an increase in total precursor messenger RNA synthesis, suggesting an increased burden on the core spliceosome to process pre-mRNA. In contrast to normal cells, partial inhibition of the spliceosome in MYC-hyperactivated cells leads to global intron retention, widespread defects in pre-mRNA maturation, and deregulation of many essential cell processes. Notably, genetic or pharmacological inhibition of the spliceosome in vivo impairs survival, tumorigenicity and metastatic proclivity of MYC-dependent breast cancers. Collectively, these data suggest that oncogenic MYC confers a collateral stress on splicing, and that components of the spliceosome may be therapeutic entry points for aggressive MYC-driven cancers.
Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Genes, myc/genetics , Spliceosomes/drug effects , Spliceosomes/metabolism , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Survival/drug effects , Cell Transformation, Neoplastic/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , HeLa Cells , Humans , Introns/genetics , Mice , Mice, Nude , Neoplasm Metastasis/drug therapy , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA Precursors/biosynthesis , RNA Precursors/genetics , RNA Splicing/drug effects , RNA Splicing Factors , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Ribonucleoprotein, U2 Small Nuclear/metabolism , Ribonucleoproteins/metabolism , Splicing Factor U2AF , Xenograft Model Antitumor AssaysABSTRACT
Programmed cell death-1 (PD-1) is an essential inhibitory receptor in T cells. Antibodies targeting PD-1 elicit durable clinical responses in patients with multiple tumor indications. Nevertheless, a significant proportion of patients do not respond to anti-PD-1 treatment, and a better understanding of the signaling pathways downstream of PD-1 could provide biomarkers for those whose tumors respond and new therapeutic approaches for those whose tumors do not. We used affinity purification mass spectrometry to uncover multiple proteins associated with PD-1. Among these proteins, signaling lymphocytic activation molecule-associated protein (SAP) was functionally and mechanistically analyzed for its contribution to PD-1 inhibitory responses. Silencing of SAP augmented and overexpression blocked PD-1 function. T cells from patients with X-linked lymphoproliferative disease (XLP), who lack functional SAP, were hyperresponsive to PD-1 signaling, confirming its inhibitory role downstream of PD-1. Strikingly, signaling downstream of PD-1 in purified T cell subsets did not correlate with PD-1 surface expression but was inversely correlated with intracellular SAP levels. Mechanistically, SAP opposed PD-1 function by acting as a molecular shield of key tyrosine residues that are targets for the tyrosine phosphatase SHP2, which mediates PD-1 inhibitory properties. Our results identify SAP as an inhibitor of PD-1 function and SHP2 as a potential therapeutic target in patients with XLP.
Subject(s)
Cell Cycle Checkpoints/physiology , Mass Spectrometry/methods , Programmed Cell Death 1 Receptor/metabolism , Signaling Lymphocytic Activation Molecule Family/metabolism , T-Lymphocytes/metabolism , Animals , Biomarkers, Tumor , Cell Proliferation/physiology , Cytokines/genetics , Cytokines/metabolism , Gene Expression Regulation, Enzymologic , Gene Silencing , HEK293 Cells , Humans , Jurkat Cells , Male , Mice , Mice, Knockout , Programmed Cell Death 1 Receptor/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Signaling Lymphocytic Activation Molecule Family/geneticsABSTRACT
Growth retardation is a constant feature of Noonan syndrome (NS) but its physiopathology remains poorly understood. We previously reported that hyperactive NS-causing SHP2 mutants impair the systemic production of insulin-like growth factor 1 (IGF1) through hyperactivation of the RAS/extracellular signal-regulated kinases (ERK) signalling pathway. Besides endocrine defects, a direct effect of these mutants on growth plate has not been explored, although recent studies have revealed an important physiological role for SHP2 in endochondral bone growth. We demonstrated that growth plate length was reduced in NS mice, mostly due to a shortening of the hypertrophic zone and to a lesser extent of the proliferating zone. These histological features were correlated with decreased expression of early chondrocyte differentiation markers, and with reduced alkaline phosphatase staining and activity, in NS murine primary chondrocytes. Although IGF1 treatment improved growth of NS mice, it did not fully reverse growth plate abnormalities, notably the decreased hypertrophic zone. In contrast, we documented a role of RAS/ERK hyperactivation at the growth plate level since 1) NS-causing SHP2 mutants enhance RAS/ERK activation in chondrocytes in vivo (NS mice) and in vitro (ATDC5 cells) and 2) inhibition of RAS/ERK hyperactivation by U0126 treatment alleviated growth plate abnormalities and enhanced chondrocyte differentiation. Similar effects were obtained by chronic treatment of NS mice with statins. In conclusion, we demonstrated that hyperactive NS-causing SHP2 mutants impair chondrocyte differentiation during endochondral bone growth through a local hyperactivation of the RAS/ERK signalling pathway, and that statin treatment may be a possible therapeutic approach in NS.