Discovery and characterization of the mTOR-p73 signaling axis in human cancer

To my parents, and to San iii ACKNOWLEDGEMENTS I am grateful to all those who have supported my dissertation research through helpful discussions and advice over the past four years. First and foremost, I have innumerable thanks for my mentor, Jennifer Pietenpol. Without her renowned energy and commitment none of this would have been possible. I am thankful that such a brilliant scientist took the time to teach me both the details of data generation, and about finding the big picture in the fight against cancer. Thanks also to all the members of the Pietenpol lab, past and present, who have made my training experience both fruitful and entertaining. I‟ve enjoyed hanging out by the lab drawer, our „office water cooler‟, and coffee machine in both its old and new incarnations. These were the shoulders I cried on, as my lab ornament went from second to last, to last place, to triaged year after year

ISG20L1 is a p53 family target gene that modulates genotoxic stress-induced autophagy

<p>Abstract</p> <p>Background</p> <p>Autophagy is characterized by the sequestration of cytoplasm and organelles into multimembrane vesicles and subsequent degradation by the cell's lysosomal system. It is linked to many physiological functions in human cells including stress response, protein degradation, organelle turnover, caspase-independent cell death and tumor suppression. Malignant transformation is frequently associated with deregulation of autophagy and several tumor suppressors can modulate autophagic processes. The tumor suppressor p53 can induce autophagy after metabolic or genotoxic stress through transcriptionally-dependent and -independent mechanisms. In this study we expand on the former mechanism by functionally characterizing a p53 family target gene, ISG20L1 under conditions of genotoxic stress.</p> <p>Results</p> <p>We identified a p53 target gene, ISG20L1, and show that transcription of the gene can be regulated by all three p53 family members (p53, p63, and p73). We generated an antibody to ISG20L1 and found that it localizes to the nucleolar and perinucleolar regions of the nucleus and its protein levels increase in a p53- and p73-dependent manner after various forms of genotoxic stress. When ectopically expressed in epithelial cancer-derived cell lines, ISG20L1 expression decreased clonogenic survival without a concomitant elevation in apoptosis and this effect was partially rescued in cells that were ATG5 deficient. Knockdown of ISG20L1 did not alter 5-FU induced apoptosis as assessed by PARP and caspase-3 cleavage, sub-G₁content, and DNA laddering. Thus, we investigated the role of ISG20L1 in autophagy, a process commonly associated with type II cell death, and found that ISG20L1 knockdown decreased levels of autophagic vacuoles and LC3-II after genotoxic stress as assessed by electron microscopy, biochemical, and immunohistochemical measurements of LC3-II.</p> <p>Conclusions</p> <p>Our identification of ISG20L1 as a p53 family target and discovery that modulation of this target can regulate autophagic processes further strengthens the connection between p53 signaling and autophagy. Given the keen interest in targeting autophagy as an anticancer therapeutic approach in tumor cells that are defective in apoptosis, investigation of genes and signaling pathways involved in cell death associated with autophagy is critical.</p

A large peptidome dataset improves HLA class I epitope prediction across most of the human population

Published in final edited form as: Nat Biotechnol. 2020 February ; 38(2): 199–209. doi:10.1038/s41587-019-0322-9.Prediction of HLA epitopes is important for the development of cancer immunotherapies and vaccines. However, current prediction algorithms have limited predictive power, in part because they were not trained on high-quality epitope datasets covering a broad range of HLA alleles. To enable prediction of endogenous HLA class I-associated peptides across a large fraction of the human population, we used mass spectrometry to profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines. We identified canonical peptide motifs per HLA allele, unique and shared binding submotifs across alleles and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, we developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumor cell lines.P01 CA229092 - NCI NIH HHS; P50 CA101942 - NCI NIH HHS; T32 HG002295 - NHGRI NIH HHS; T32 CA009172 - NCI NIH HHS; U24 CA224331 - NCI NIH HHS; R21 CA216772 - NCI NIH HHS; R01 CA155010 - NCI NIH HHS; U01 CA214125 - NCI NIH HHS; T32 CA207021 - NCI NIH HHS; R01 HL103532 - NHLBI NIH HHS; U24 CA210986 - NCI NIH HHSAccepted manuscrip

A Gene Signature-Based Approach Identifies mTOR as a Regulator of p73▿

Although genomic technologies have advanced the characterization of gene regulatory networks downstream of transcription factors, the identification of pathways upstream of these transcription factors has been more challenging. In this study we present a gene signature-based approach for connecting signaling pathways to transcription factors, as exemplified by p73. We generated a p73 gene signature by integrating whole-genome chromatin immunoprecipitation and expression profiling. The p73 signature was linked to corresponding signatures produced by drug candidates, using the in silico Connectivity Map resource, to identify drugs that would induce p73 activity. Of the pharmaceutical agents identified, there was enrichment for direct or indirect inhibitors of mammalian Target of Rapamycin (mTOR) signaling. Treatment of both primary cells and cancer cell lines with rapamycin, metformin, and pyrvinium resulted in an increase in p73 levels, as did RNA interference-mediated knockdown of mTOR. Further, a subset of genes associated with insulin response or autophagy exhibited mTOR-mediated, p73-dependent expression. Thus, downstream gene signatures can be used to identify upstream regulators of transcription factor activity, and in doing so, we identified a new link between mTOR, p73, and p73-regulated genes associated with autophagy and metabolic pathways

p73 Is Required for Multiciliogenesis and Regulates the Foxj1-Associated Gene Network

We report that p73 is expressed in multiciliated cells (MCCs), is required for MCC differentiation, and directly regulates transcriptional modulators of multiciliogenesis. Loss of ciliary biogenesis provides a unifying mechanism for many phenotypes observed in p73 knockout mice including hydrocephalus; hippocampal dysgenesis; sterility; and chronic inflammation/infection of lung, middle ear, and sinus. Through p73 and p63 ChIP-seq using murine tracheal cells, we identified over 100 putative p73 target genes that regulate MCC differentiation and homeostasis. We validated Foxj1, a transcriptional regulator of multiciliogenesis, and many other cilia-associated genes as direct target genes of p73 and p63. We show p73 and p63 are co-expressed in a subset of basal cells and suggest that p73 marks these cells for MCC differentiation. In summary, p73 is essential for MCC differentiation, functions as a critical regulator of a transcriptome required for MCC differentiation, and, like p63, has an essential role in development of tissues