Massively parallel identification of sequence motifs triggering ribosome-associated mRNA quality control.

Decay of mRNAs can be triggered by ribosome slowdown at stretches of rare codons or positively charged amino acids. However, the full diversity of sequences that trigger co-translational mRNA decay is poorly understood. To comprehensively identify sequence motifs that trigger mRNA decay, we use a massively parallel reporter assay to measure the effect of all possible combinations of codon pairs on mRNA levels in S. cerevisiae. In addition to known mRNA-destabilizing sequences, we identify several dipeptide repeats whose translation reduces mRNA levels. These include combinations of positively charged and bulky residues, as well as proline-glycine and proline-aspartate dipeptide repeats. Genetic deletion of the ribosome collision sensor Hel2 rescues the mRNA effects of these motifs, suggesting that they trigger ribosome slowdown and activate the ribosome-associated quality control (RQC) pathway. Deep mutational scanning of an mRNA-destabilizing dipeptide repeat reveals a complex interplay between the charge, bulkiness, and location of amino acid residues in conferring mRNA instability. Finally, we show that the mRNA effects of codon pairs are predictive of the effects of endogenous sequences. Our work highlights the complexity of sequence motifs driving co-translational mRNA decay in eukaryotes, and presents a high throughput approach to dissect their requirements at the codon level.

Massively parallel identification of sequence motifs triggering ribosome-associated mRNA quality control.

Decay of mRNAs can be triggered by ribosome slowdown at stretches of rare codons or positively charged amino acids. However, the full diversity of sequences that trigger co-translational mRNA decay is poorly understood. To comprehensively identify sequence motifs that trigger mRNA decay, we use a massively parallel reporter assay to measure the effect of all possible combinations of codon pairs on mRNA levels in S. cerevisiae. In addition to known mRNA-destabilizing sequences, we identify several dipeptide repeats whose translation reduces mRNA levels. These include combinations of positively charged and bulky residues, as well as proline-glycine and proline-aspartate dipeptide repeats. Genetic deletion of the ribosome collision sensor Hel2 rescues the mRNA effects of these motifs, suggesting that they trigger ribosome slowdown and activate the ribosome-associated quality control (RQC) pathway. Deep mutational scanning of an mRNA-destabilizing dipeptide repeat reveals a complex interplay between the charge, bulkiness, and location of amino acid residues in conferring mRNA instability. Finally, we show that the mRNA effects of codon pairs are predictive of the effects of endogenous sequences. Our work highlights the complexity of sequence motifs driving co-translational mRNA decay in eukaryotes, and presents a high throughput approach to dissect their requirements at the codon level.

Heparan Sulfate Synthesized by Ext1 Regulates Receptor Tyrosine Kinase Signaling and Promotes Resistance to EGFR Inhibitors in GBM.

Signaling from multiple receptor tyrosine kinases (RTK) contributes to therapeutic resistance in glioblastoma (GBM). Heparan sulfate (HS), present on cell surfaces and in the extracellular matrix, regulates cell signaling via several mechanisms. To investigate the role for HS in promoting RTK signaling in GBM, we generated neural progenitor cells deficient for HS by knockout of the essential HS-biosynthetic enzyme Ext1, and studied tumor initiation and progression. HS-null cells had decreased proliferation, invasion, and reduced activation of multiple RTKs compared with control. In vivo tumor establishment was significantly decreased, and rate of tumor growth reduced with HS-deficient cells implanted in an HS-poor microenvironment. To investigate if HS regulates RTK activation through platelet-derived growth factor receptor α (PDGFRα) signaling, we removed cell surface HS in patient-derived GBM lines and identified reduced cell surface PDGF-BB ligand. Reduced ligand levels were associated with decreased phosphorylation of PDGFRα, suggesting HS promotes ligand-receptor interaction. Using human GBM tumorspheres and a murine GBM model, we show that ligand-mediated signaling can partially rescue cells from targeted RTK inhibition and that this effect is regulated by HS. Indeed, tumor cells deficient for HS had increased sensitivity to EGFR inhibition in vitro and in vivo. IMPLICATIONS: Our study shows that HS expressed on tumor cells and in the tumor microenvironment regulates ligand-mediated signaling, promoting tumor cell proliferation and invasion, and these factors contribute to decreased tumor cell response to targeted RTK inhibition.

Mechanisms of Resistance to EGFR Inhibition Reveal Metabolic Vulnerabilities in Human GBM.

Amplification of the epidermal growth factor receptor gene (EGFR) represents one of the most commonly observed genetic lesions in glioblastoma (GBM); however, therapies targeting this signaling pathway have failed clinically. Here, using human tumors, primary patient-derived xenografts (PDX), and a murine model for GBM, we demonstrate that EGFR inhibition leads to increased invasion of tumor cells. Further, EGFR inhibitor-treated GBM demonstrates altered oxidative stress, with increased lipid peroxidation, and generation of toxic lipid peroxidation products. A tumor cell subpopulation with elevated aldehyde dehydrogenase (ALDH) levels was determined to comprise a significant proportion of the invasive cells observed in EGFR inhibitor-treated GBM. Our analysis of the ALDH1A1 protein in newly diagnosed GBM revealed detectable ALDH1A1 expression in 69% (35/51) of the cases, but in relatively low percentages of tumor cells. Analysis of paired human GBM before and after EGFR inhibitor therapy showed an increase in ALDH1A1 expression in EGFR-amplified tumors (P < 0.05, n = 13 tumor pairs), and in murine GBM ALDH1A1-high clones were more resistant to EGFR inhibition than ALDH1A1-low clones. Our data identify ALDH levels as a biomarker of GBM cells with high invasive potential, altered oxidative stress, and resistance to EGFR inhibition, and reveal a therapeutic target whose inhibition should limit GBM invasion.