Transcriptional response to the HSP70 inhibitor MAL3-101 in parental rhabdomyosarcoma cells and isogenic acquired-resistance lines.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Amit Sabnis
PROVIDER: E-GEOD-80525 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:We demonstrate that inhibition of HSP70 with MAL3-101 induces activation of the unfolded protein response, and that cells with acquired resistance upregulate a cytosolic HSP70 at the level of mRNA.
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Project description:Molecular chaperones prevent the accumulation of toxic proteins in the cell, but when chaperones are overwhelmed, the rapid modulation of proteostasis pathways provide corrective responses. One molecular chaperone in particular, Hsp70, plays a central role in both cellular protection and the response to proteotoxic insults. This is perhaps most evident in cancer cells, some of which overexpress Hsp70 and thrive even when harboring high levels of misfolded, aggregated, and/or unassembled protein complexes. In an effort to define how cells compensate for compromised proteostasis, we examined the adaptive response in breast cancer cells after challenge with a specific Hsp70 inhibitor, MAL3-101 and further define the pathways involved in the differential MAL3-101 response between sensitive and resistant cells.
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Project description:Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first map proteasome-associated genetic co-dependencies. We identify cytosolic heat shock protein 70 (HSP70) chaperones as a potential target, consistent with proposed mechanisms of myeloma tumor cells overcoming PI-induced stress. These results led us to explore allosteric HSP70 inhibitors as myeloma therapeutics. We show these compounds exhibit increased efficacy against both acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Surprisingly, shotgun and pulsed-SILAC proteomics reveal that JG’s overcome PI resistance not via the expected mechanism of inhibiting cytosolic HSP70s, but instead through mitochondrial-localized HSP70, HSPA9, destabilizing the 55S mitoribosome. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize dynamics of myeloma proteostasis networks under therapeutic pressure while further motivating investigation of HSPA9 as a specific target in PI-resistant disease.
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Project description:NRAS-mutant melanoma is currently a challenge to treat. This is due to an absence of inhibitors directed against NRAS, along with acquired and adaptive resistance of this tumor type to inhibitors in the MAPK pathway. Inhibitors to MEK (mitogen-activated protein kinase kinase) have shown some promise for this tumor type. In this work we explored the use of MEK inhibitors for NRAS-mutant melanoma, and at the same time investigated the impact of the brain micro-environment, specifically astrocytes, on the response of a melanoma brain metastatic cell line to MEK inhibition. This led to the surprising finding that astrocytes enhance the sensitivity of melanoma tumors to MEK inhibitors (MEKi). We show that MEKi cause an upregulation of the transcription factor ID3, but this is blocked by conditioned media from astrocytes. We show that silencing ID3 enhances the sensitivity of melanoma to MEK inhibitors, thus mimicking the effect of the brain microenvironment. Moreover, we report that ID3 is a client protein of the chaperone HSP70, and that HSP70 inhibition causes ID3 to misfold and accumulate in a detergent-insoluble fraction in cells. We show that HSP70 inhibitors synergize with MEK inhibitors against NRAS-mutant melanoma, and that this combination significantly enhances the survival of mice in two different models of NRAS-mutant melanoma. These studies highlight ID3 as a mediator of adaptive resistance, and support the combined use of MEK and HSP70 inhibitors for the therapy of NRAS-mutant melanoma.
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Project description:MTI-101 is a first-in-class novel cyclic peptide shown to have anti-tumor activity in both multiple myeloma and castrate-resistant prostate in vivo cancer models. These data suggest the potential for a broad spectrum of anti-cancer activity for this class of compounds. To further delineate determinants of sensitivity and resistance that were not dependent on oncogenic drivers, two isogenic drug resistant cell lines were generated with chronic exposure to MTI-101 in a non-small cell lung cancer (NSCLC) PC-9 (EGFR driven) and a small cell lung cancer (SCLC) H446 (PTEN deleted and c-MYC amplified). Using the isogenic cell line systems coupled with RNA-SEQ, the top enriched GSEA hallmark was downregulation of genes that contribute to epithelial to mesenchymal transition (EMT), a finding that was consistent in both acquired MTI-101 selected resistant cell lines. The RNA-SEQ data correlated with predicted changes in the phenotype, including significant decreased invasion in Matrigel and changes of EMT markers (E-Cadherin, Vimentin and Twist) at the protein level. Furthermore, in the EGFR driven PC-9 cell line, selection for resistance towards MTI-101 resulted in collateral sensitivity toward EGFR inhibitors. To determine the stability of the MET phenotype, cells were removed from drug and tested for invasion and drug resistance every month. These data indicated that the mesenchymal to epithelial transition (MET) phenotype and genotype was stable for a minimum of six months. MTI-101 treatment in PC-9 and H446 cells showed synergistic activity with the standard of care agents Erlotinib, Osimertinib, and Cisplatin when used in combination in PC-9 and H446 cells, respectively. Finally, in vivo data indicates that MTI-101 treatment selects for increased E-Cadherin and decreased Vimentin in H446. In addition, treatment with MTI-101 decreased incidence of bone metastasis in the PC-9 in vivo model. Together, these data indicate that MTI-101 treatment could be used as a tool to delay the emergence of metastatic disease for the treatment of lung cancer.
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Project description:Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first mapped proteasome-associated genetic co-dependencies. We identified cytosolic heat shock protein 70 (HSP70) chaperones as potential targets, consistent with proposed mechanisms of myeloma tumor cells overcoming PI-induced stress. These results led us to explore allosteric HSP70 inhibitors (JG compounds) as myeloma therapeutics. We showed these compounds exhibit increased efficacy against acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Surprisingly, shotgun and pulsed-SILAC mass spectrometry found that JGs have the most pronounced effect on the proteome not through inhibiting cytosolic HSP70s but instead through mitochondrial-localized HSP70, HSPA9/mortalin, destabilizing the 55S mitoribosome. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize myeloma proteostasis networks under therapeutic pressure while motivating further investigation of HSPA9 as a specific vulnerability in PI-resistant disease.
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Project description:Glycosylation is an abundant post-translational modification of both intracellular and extracellular proteins [1]. The majority of glycans are classified as N-linked chains, where the carbohydrate moiety is attached to asparagine residues, or O-linked chains, most commonly linked to a serine or threonine. N-linked glycosylation is initiated by the oligosaccharyltransferase complex with only two paralogs of the catalytic subunit, whereas O-glycan initiation is more complex. There are several types of O-linked glycosylation, but among the most diverse is the mucin or GalNAc type (hereafter referred to as O-glycosylation). O-glycosylation is initiated by 20 evolutionarily conserved polypeptide GalNAc-transferases (GalNAc-Ts), which catalyze the first step in the O-glycosylation of proteins by adding GalNAc residues to threonine, serine, and tyrosine amino acids (Fig 1A). Each of the GalNAc-Ts are differentially expressed in various tissues and have both distinct and overlapping peptide substrate specificities [2-12]. Thus, the repertoire of GalNAc-Ts expressed in a given cell determines the subset and O-glycosite pattern of glycosylated proteins [13]. Substantial efforts have been made to characterize and predict the substrate specificities of GalNAc-Ts in vitro, but understanding of the in vivo specificities of the individual GalNAc-Ts or their biological functions is limited [13-15]. This lack of insight prevents an understanding of how site-specific O-linked glycosylation affects diseases, such as metabolic disorders, cardiovascular disease, and various malignancies, that have been associated with GalNAc-Ts through genome-wide association studies and other linkage studies [16-26]. Therefore, it is imperative that we establish how O-glycosylation at specific sites in proteins affects protein function. A major task in achieving this goal is to identify the non-redundant biological functions of site-specific O-glycosylation. We and others recently developed new strategies for identifying specific sites on proteins that undergo O-glycosylation in different cell types and tissues [27-31]. Characterization of the O-glycoproteomic landscape in isolated human cells and multiple human cell lines suggests that more than 80 % of all proteins that traffic through the secretory pathway are O-glycoproteins [28, 30]. Probing the non-redundant contributions of individual GalNAc-Ts in cells with and without specific GalNAc-Ts [32-34] has revealed broad substrate specificities for some of the individual isoforms, whereas others seem to have very restricted substrate specificities [33-35]. Assessing all of the mapped O-glycosylation sites to identify associations between O-glycosites and protein annotations, we recently found that O-glycans are over-represented close to tandem repeat regions, protease cleavage sites, within propeptides, and on a select group of protein domains [28, 30, 36]. Although such general associations between the location of O-glycans and protein functions may direct future investigations, the strategy does not define the function of site-specific glycosylation. Further progress in discovering and defining novel functions of site-specific glycosylation events requires direct quantitative analysis of potential biological responses induced by the loss of distinct GalNAc-T isoforms, and such biological responses are not easily observed in single cell culture systems. Instead, more complex model systems can be used to examine and dissect the molecular mechanisms underlying the important biological functions of site-specific glycosylation. We previously used an organotypic tissue model equipped with genetically engineered cells to decipher the function of elongated O-glycans [29]. In the present study, we use the model combined with quantitative O-glycoproteomics and phosphoproteomics to perform open-ended discovery of the biological functions of site-specific glycosylation governed by GalNAc-Ts (Fig 1B). With this combinatorial strategy, we demonstrate that loss of individual GalNAc-T isoforms has distinct phenotypic consequences through their effect on distinct biological pathways, suggesting specific roles during epithelial formation.
2020-05-22 | PXD016618 | Pride
Project description:Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating this resistance, we mapped genetic co-dependencies associated with the proteasome. These studies identified cytosolic heat shock protein 70 (HSP70) chaperones as a potential target, mirroring recent studies that have shown mechanism of overcoming PI-induced stress. Here, we first underscore this relationship by mapping genetic co-dependencies in cancer proteostasis. These results lead us to explore HSP70 inhibitors as potential therapeutics. We show these compounds exhibit increased efficacy against both acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Surprisingly, shotgun and pulsed-SILAC proteomics reveal that JG’s overcome PI resistance not via the expected mechanism of inhibiting cytosolic HSP70s, but instead through mitochondrial-localized HSP70, HSPA9, destabilizing the 55S mitoribosome. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on response to PI. Our results characterize dynamics of myeloma proteostasis networks under therapeutic pressure while further motivating investigation of HSPA9 as a specific target in PI-resistant disease. This dataset corresponds to experiment outlined in figure 4a.
2020-04-24 | PXD018617 | Pride
Project description:Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating this resistance, we mapped genetic co-dependencies associated with the proteasome. These studies identified cytosolic heat shock protein 70 (HSP70) chaperones as a potential target, mirroring recent studies that have shown mechanism of overcoming PI-induced stress. Here, we first underscore this relationship by mapping genetic co-dependencies in cancer proteostasis. These results lead us to explore HSP70 inhibitors as potential therapeutics. We show these compounds exhibit increased efficacy against both acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Surprisingly, shotgun and pulsed-SILAC proteomics reveal that JG’s overcome PI resistance not via the expected mechanism of inhibiting cytosolic HSP70s, but instead through mitochondrial-localized HSP70, HSPA9, destabilizing the 55S mitoribosome. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on response to PI. Our results characterize dynamics of myeloma proteostasis networks under therapeutic pressure while further motivating investigation of HSPA9 as a specific target in PI-resistant disease. This dataset corresponds to figure 5, experiment outlined in figure 5a.
2020-04-24 | PXD018387 | Pride