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1.
Blood Adv ; 5(20): 4003-4016, 2021 10 26.
Article in English | MEDLINE | ID: mdl-34474469

ABSTRACT

Tolinapant (ASTX660) is a potent, nonpeptidomimetic antagonist of cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2) and X-linked IAP, which is currently being evaluated in a phase 2 study in T-cell lymphoma (TCL) patients. Tolinapant has demonstrated evidence of single-agent clinical activity in relapsed/refractory peripheral TCL and cutaneous TCL. To investigate the mechanism of action underlying the single-agent activity observed in the clinic, we have used a comprehensive translational approach integrating in vitro and in vivo models of TCL confirmed by data from human tumor biopsies. Here, we show that tolinapant acts as an efficacious immunomodulatory molecule capable of inducing complete tumor regression in a syngeneic model of TCL exclusively in the presence of an intact immune system. These findings were confirmed in samples from our ongoing clinical study showing that tolinapant treatment can induce changes in gene expression and cytokine profile consistent with immune modulation. Mechanistically, we show that tolinapant can activate both the adaptive and the innate arms of the immune system through the induction of immunogenic forms of cell death. In summary, we describe a novel role for IAP antagonists as immunomodulatory molecules capable of promoting a robust antitumor immune response in TCL.


Subject(s)
Lymphoma, T-Cell , Skin Neoplasms , Apoptosis , Humans , Immunity , Morpholines , Neoplasm Recurrence, Local , Piperazines , Pyrroles
3.
Front Cell Dev Biol ; 6: 148, 2018.
Article in English | MEDLINE | ID: mdl-30443548

ABSTRACT

Autophagy protease ATG4B is a key regulator of the LC3/GABARAP conjugation system required for autophagosome formation, maturation and closure. Members of the ATG4 and the LC3/GABARAP family have been implicated in various diseases including cancer, and targeting the ATG4B protease has been suggested as a potential therapeutic anti-cancer strategy. Recently, it has been demonstrated that ATG4B is regulated by multiple post-translational modifications, including phosphorylation and de-phosphorylation. In order to identify regulators of ATG4B activity, we optimized a cell-based luciferase assay based on ATG4B-dependent release of Gaussia luciferase. We applied this assay in a proof-of-concept small molecule compound screen and identified activating compounds that increase cellular ATG4B activity. Next, we performed a high-throughput screen to identify kinases and phosphatases that regulate cellular ATG4B activity using siRNA mediated knockdown and cDNA overexpression. Of these, we provide preliminary evidence that the kinase AKT2 enhances ATG4B activity in cells. We provide all raw and processed data from the screens as a resource for further analysis. Overall, our findings provide novel insights into the regulation of ATG4B and highlight the importance of post-translational modifications of ATG4B.

4.
Mol Biol Cell ; 27(25): 4033-4042, 2016 12 15.
Article in English | MEDLINE | ID: mdl-27798239

ABSTRACT

The type II phosphatidylinositol 4-kinase (PI4KII) enzymes synthesize the lipid phosphatidylinositol 4-phosphate (PI(4)P), which has been detected at the Golgi complex and endosomal compartments and recruits clathrin adaptors. Despite common mechanistic similarities between the isoforms, the extent of their redundancy is unclear. We found that depletion of PI4KIIα and PI4KIIß using small interfering RNA led to actin remodeling. Depletion of PI4KIIß also induced the formation of invadopodia containing membrane type I matrix metalloproteinase (MT1-MMP). Depletion of PI4KII isoforms also differentially affected trans-Golgi network (TGN) pools of PI(4)P and post-TGN traffic. PI4KIIß depletion caused increased MT1-MMP trafficking to invasive structures at the plasma membrane and was accompanied by reduced colocalization of MT1-MMP with membranes containing the endosomal markers Rab5 and Rab7 but increased localization with the exocytic Rab8. Depletion of PI4KIIß was sufficient to confer an aggressive invasive phenotype on minimally invasive HeLa and MCF-7 cell lines. Mining oncogenomic databases revealed that loss of the PI4K2B allele and underexpression of PI4KIIß mRNA are associated with human cancers. This finding supports the cell data and suggests that PI4KIIß may be a clinically significant suppressor of invasion. We propose that PI4KIIß synthesizes a pool of PI(4)P that maintains MT1-MMP traffic in the degradative pathway and suppresses the formation of invadopodia.


Subject(s)
Minor Histocompatibility Antigens/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Podosomes/enzymology , Cell Line, Tumor , Cell Membrane/metabolism , Cell Movement/physiology , Endosomes/metabolism , Extracellular Matrix/metabolism , HeLa Cells , Humans , Isoenzymes , MCF-7 Cells , Matrix Metalloproteinase 14/metabolism , Minor Histocompatibility Antigens/genetics , Neoplasms/enzymology , Neoplasms/pathology , Phosphatidylinositol Phosphates/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Protein Transport/physiology , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , trans-Golgi Network/metabolism
5.
Oncotarget ; 7(27): 41203-41216, 2016 Jul 05.
Article in English | MEDLINE | ID: mdl-27256984

ABSTRACT

The cellular stress response autophagy has been implicated in various diseases including neuro-degeneration and cancer. The role of autophagy in cancer is not clearly understood and both tumour promoting and tumour suppressive effects of autophagy have been reported, which complicates the design of therapeutic strategies based on targeting the autophagy pathway. Here, we have systematically analyzed gene expression data for 47 autophagy genes for deletions, amplifications and mutations in various cancers. We found that several cancer types have frequent autophagy gene amplifications, whereas deletions are more frequent in prostate adenocarcinomas. Other cancer types such as glioblastoma and thyroid carcinoma show very few alterations in any of the 47 autophagy genes. Overall, individual autophagy core genes are altered at low frequency in cancer, suggesting that cancer cells require functional autophagy. Some autophagy genes show frequent single base mutations, such as members of the ULK family of protein kinases. Furthermore, we found hotspot mutations in the arginine-rich stretch in MAP1LC3A resulting in reduced cleavage of MAP1LC3A by ATG4B both in vitro and in vivo, suggesting a functional implication of this gene mutation in cancer development.


Subject(s)
Autophagy-Related Proteins/genetics , Autophagy/genetics , Microtubule-Associated Proteins/genetics , Mutation , Neoplasms/genetics , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Microarray Analysis , Mutant Proteins/genetics
6.
Methods Mol Biol ; 1376: 55-75, 2016.
Article in English | MEDLINE | ID: mdl-26552675

ABSTRACT

Phosphoinositides compromise a family of eight membrane lipids which play important roles in many cellular signaling pathways. Signaling through phosphoinositides has been shown in a variety of cellular functions such cell proliferation, cell growth, apoptosis, and vesicle trafficking. Phospholipid phosphatases regulate cell signaling by modifying the concentration of phosphoinositides and their dephosphorylated products. To understand the role of individual lipid phosphatases in phosphoinositide turnover and functional signaling, it is crucial to determine the substrate specificity of the lipid phosphatase of interest. In this chapter we describe how the substrate specificity of an individual lipid phosphatase can be qualitatively and quantitatively measured in an in vitro radiometric assay. In addition, we specify the different expression systems and purification methods required to produce the necessary yield and functionality in order to further characterize these enzymes. The outstanding versatility and sensitivity of this assay system are yet unmatched and are therefore currently considered the standard of the field.


Subject(s)
Enzyme Assays/methods , Phosphatidylinositols/metabolism , Phosphoric Monoester Hydrolases/metabolism , Animals , Cell Line , Gene Expression , In Vitro Techniques , Lipids/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Staining and Labeling , Substrate Specificity
7.
Nat Struct Mol Biol ; 22(12): 983-90, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26551075

ABSTRACT

Aberrant activation of the EGF receptor (EGFR) contributes to many human cancers by activating the Ras-MAPK pathway and other pathways. EGFR signaling is augmented by Src-family kinases, but the mechanism is poorly understood. Here, we show that human EGFR preferentially phosphorylates peptide substrates that are primed by a prior phosphorylation. Using peptides based on the sequence of the adaptor protein Shc1, we show that Src mediates the priming phosphorylation, thus promoting subsequent phosphorylation by EGFR. Importantly, the doubly phosphorylated Shc1 peptide binds more tightly than singly phosphorylated peptide to the Ras activator Grb2; this binding is a key step in activating the Ras-MAPK pathway. Finally, a crystal structure of EGFR in complex with a primed Shc1 peptide reveals the structural basis for EGFR substrate specificity. These results provide a molecular explanation for the integration of Src and EGFR signaling with downstream effectors such as Ras.


Subject(s)
ErbB Receptors/drug effects , ErbB Receptors/metabolism , Peptides/metabolism , Phosphotyrosine/metabolism , Shc Signaling Adaptor Proteins/metabolism , Crystallography, X-Ray , ErbB Receptors/chemistry , GRB2 Adaptor Protein/metabolism , Humans , Phosphorylation , Protein Binding , Protein Conformation , Protein Processing, Post-Translational , Sensitivity and Specificity , Shc Signaling Adaptor Proteins/chemistry , Signal Transduction , Src Homology 2 Domain-Containing, Transforming Protein 1 , Substrate Specificity
8.
Oncotarget ; 6(12): 10548-62, 2015 Apr 30.
Article in English | MEDLINE | ID: mdl-25868852

ABSTRACT

Treatment options for ovarian cancer patients remain limited and overall survival is less than 50% despite recent clinical advances. The lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B) has been described as a tumor suppressor in the PI3K/Akt pathway with loss of expression found most pronounced in breast, ovarian cancer and melanoma. Using microarray technology we identified a DNA repair defect in INPP4B-deficient cells, which we further characterized by comet assays and quantification of γH2AX, RAD51 and 53BP1 foci formation. INPP4B loss resulted in significantly increased sensitivity towards PARP inhibition, comparable to loss of BRCA1 in two- and three-dimensional in vitro models, as well as in in vivo xenograft models. Mechanistically, we discovered that INPP4B forms a protein complex with the key players of DNA repair, ATR and BRCA1, in GST pulldown and 293T overexpression assays, and INPP4B loss affects BRCA1, ATM and ATR protein stability resulting in the observed DNA repair defect. Given that INPP4B loss has been found in 40% of ovarian cancer patients, this study provides the rationale for establishing INPP4B as a biomarker of PARP inhibitor response, and consequently offers novel therapeutic options for a significant subset of patients. Loss of the tumor suppressor inositol polyphosphate 4-phosphatase type II (INPP4B) results in a DNA repair defect due to concomitant loss of BRCA1, ATR and ATM and can be therapeutically targeted with PARP inhibitors.


Subject(s)
Breast Neoplasms/genetics , DNA Repair , Ovarian Neoplasms/genetics , Phosphoric Monoester Hydrolases/deficiency , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Animals , Ataxia Telangiectasia Mutated Proteins/deficiency , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , BRCA1 Protein/deficiency , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Female , Genes, Tumor Suppressor , HEK293 Cells , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Transcriptome , Transfection , Xenograft Model Antitumor Assays
9.
Mol Cell ; 52(5): 679-92, 2013 Dec 12.
Article in English | MEDLINE | ID: mdl-24211266

ABSTRACT

SHOC2 is mutated in Noonan syndrome and plays a key role in the activation of the ERK-MAPK pathway, which is upregulated in the majority of human cancers. SHOC2 functions as a PP1-regulatory protein and as an effector of MRAS. Here we show that SHOC2 and MRAS form a complex with SCRIB, a polarity protein with tumor suppressor properties. SCRIB functions as a PP1-regulatory protein and antagonizes SHOC2-mediated RAF dephosphorylation through a mechanism involving competition for PP1 molecules within the same macromolecular complex. SHOC2 function is selectively required for the malignant properties of tumor cells with mutant RAS, and both MRAS and SHOC2 play a key role in polarized migration. We propose that MRAS, through its ability to recruit a complex with paradoxical components, coordinates ERK pathway spatiotemporal dynamics with polarity and that this complex plays a key role during tumorigenic growth.


Subject(s)
Intracellular Signaling Peptides and Proteins/genetics , MAP Kinase Signaling System/genetics , Membrane Proteins/genetics , Tumor Suppressor Proteins/genetics , ras Proteins/genetics , Carcinogenesis/genetics , Carcinogenesis/metabolism , Cell Line , Cell Movement/genetics , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Macromolecular Substances/metabolism , Membrane Proteins/metabolism , Phosphorylation , Receptors, Neuropeptide Y/genetics , Receptors, Neuropeptide Y/metabolism , Tumor Suppressor Proteins/metabolism , raf Kinases/genetics , raf Kinases/metabolism , ras Proteins/metabolism
10.
Sci Signal ; 6(277): ra40, 2013 May 28.
Article in English | MEDLINE | ID: mdl-23716717

ABSTRACT

The nonreceptor tyrosine kinase BMX (bone marrow tyrosine kinase gene on chromosome X) is abundant in various cell types and activated downstream of phosphatidylinositol-3 kinase (PI3K) and the kinase Src, but its substrates are unknown. Positional scanning peptide library screening revealed a marked preference for a priming phosphorylated tyrosine (pY) in the -1 position, indicating that BMX substrates may include multiple tyrosine kinases that are fully activated by pYpY sites in the kinase domain. BMX phosphorylated focal adhesion kinase (FAK) at Tyr577 subsequent to its Src-mediated phosphorylation at Tyr576. Loss of BMX by RNA interference or by genetic deletion in mouse embryonic fibroblasts (MEFs) markedly impaired FAK activity. Phosphorylation of the insulin receptor in the kinase domain at Tyr¹¹89 and Tyr¹¹9°, as well as Tyr¹¹85, and downstream phosphorylation of the kinase AKT at Thr³°8 were similarly impaired by BMX deficiency. However, insulin-induced phosphorylation of AKT at Ser47³ was not impaired in Bmx knockout MEFs or liver tissue from Bmx knockout mice, which also showed increased insulin-stimulated glucose uptake, possibly because of decreased abundance of the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase). Thus, by identifying the pYpY motif as a substrate for BMX, our findings suggest that BMX functions as a central regulator among multiple signaling pathways mediated by tyrosine kinases.


Subject(s)
Enzyme Activation/physiology , Phosphotyrosine/metabolism , Protein-Tyrosine Kinases/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction/physiology , Animals , Chromatography, Liquid , Fluorescent Antibody Technique , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Immunoblotting , Mice , Mice, Knockout , Peptide Library , Phosphorylation , Protein-Tyrosine Kinases/genetics , RNA Interference , Receptor, Insulin/metabolism , Tandem Mass Spectrometry
11.
Cancer Cell ; 16(2): 115-25, 2009 Aug 04.
Article in English | MEDLINE | ID: mdl-19647222

ABSTRACT

We report that knocking down the expression of inositol polyphosphate 4-phosphatase type II (INPP4B) in human epithelial cells, like knockdown of PTEN, resulted in enhanced Akt activation and anchorage-independent growth and enhanced overall motility. In xenograft experiments, overexpression of INPP4B resulted in reduced tumor growth. INPP4B preferentially hydrolyzes phosphatidylinositol-3,4-bisphosphate (PI(3,4)P(2)) with no effect on phosphatidylinositol-3.4.5-triphosphate (PI(3,4,5)P(3)), suggesting that PI(3,4)P(2) and PI(3,4,5)P(3) may cooperate in Akt activation and cell transformation. Dual knockdown of INPP4B and PTEN resulted in cellular senescence. Finally, we found loss of heterozygosity (LOH) at the INPP4B locus in a majority of basal-like breast cancers, as well as in a significant fraction of ovarian cancers, which correlated with lower overall patient survival, suggesting that INPP4B is a tumor suppressor.


Subject(s)
Phosphatidylinositol 3-Kinases/metabolism , Phosphoric Monoester Hydrolases/physiology , Signal Transduction , Tumor Suppressor Proteins/physiology , Breast Neoplasms/genetics , Cell Movement/genetics , Cells, Cultured , Cellular Senescence/genetics , Female , Humans , Insulin/pharmacology , Loss of Heterozygosity , Ovarian Neoplasms/genetics , Ovarian Neoplasms/mortality , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/physiology , Phosphoric Monoester Hydrolases/genetics , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Substrate Specificity
12.
J Biol Chem ; 279(5): 3563-72, 2004 Jan 30.
Article in English | MEDLINE | ID: mdl-14597631

ABSTRACT

The signal transducer and activator of transcription (Stat) gene family comprises seven members with similarities in their domain structure and a common mode of activation. Members of this gene family mediate interferon induction of gene transcription and the response to a large number of growth factors and hormones. Extracellular ligand binding to transmembrane receptors causes the intracellular activation of associated tyrosine kinases, phosphorylation of Stat molecules, dimerization, and translocation to the nucleus. Prolactin-induced phosphorylation of Stat5 is a key event in the development and differentiation of mammary epithelial cells. In addition to the crucial phosphorylation at tyrosine 694, we have identified an O-linked N-acetylglucosamine (O-GlcNAc) as another secondary modification essential for the transcriptional induction by Stat5. This modification was only found on nuclear Stat5 after cytokine activation. Similar observations were made with Stat1, Stat3, and Stat6. Glycosylation of Stat5, however, does not seem to be a prerequisite for nuclear translocation. Mass spectrometric analysis revealed a glycosylated peptide in the N-terminal region of Stat5. Replacement of threonine 92 by an alanine residue (Stat5a-T92A) strongly reduced the prolactin induction of Stat5a glycosylation and abolished transactivation of a target gene promoter. Only the glycosylated form of Stat5 was able to bind the coactivator of transcription CBP, an essential interaction for Stat5-mediated gene transcription.


Subject(s)
DNA-Binding Proteins/physiology , Milk Proteins , Nuclear Proteins/metabolism , Trans-Activators/metabolism , Trans-Activators/physiology , Acetylglucosamine/pharmacology , Active Transport, Cell Nucleus , Alanine/chemistry , Animals , CREB-Binding Protein , Cell Differentiation , Cell Division , Cell Line , Cell Nucleus/metabolism , Cytoplasm/metabolism , DNA-Binding Proteins/metabolism , Dimerization , Epithelial Cells/cytology , Galactosyltransferases/metabolism , Glycosylation , Growth Substances/metabolism , Humans , Insecta , Interferons/metabolism , Lectins/metabolism , Ligands , Luciferases/metabolism , Mass Spectrometry , Microscopy, Fluorescence , Mutation , Nuclear Proteins/chemistry , Phosphorylation , Prolactin/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , STAT5 Transcription Factor , Serine/chemistry , Threonine/chemistry , Trans-Activators/chemistry , Transcription, Genetic , Transfection , Tumor Suppressor Proteins , Tyrosine/chemistry
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