Trang T. Pham

Sporadic human mismatch repair (MMR)-deficient colorectal cancers account for approximately 12.5% of all cases of colorectal cancer. MMR-deficient colorectal cancers are classically characterized by right-sided location, multifocality, mucinous histology, and lymphocytic infiltration. However, tumors in germ-line MMR-deficient mouse models lack these histopathologic features. Mice lacking the heterotrimeric G protein alpha subunit Gialpha2 develop chronic colitis and multifocal, right-sided cancers with mucinous histopathology, similar to human MMR-deficient colorectal cancer. Young Gialpha2-/- colonic epithelium has normal MMR expression but selectively loses MLH1 and consequently PMS2 expression following inflammation. Gialpha2-/- cancers have microsatellite instability. Mlh1 is epigenetically silenced not by promoter hypermethylation but by decreased histone acetylation. Chronically inflamed Gialpha2-/- colonic mucosa contains patchy hypoxia, with increased crypt expression of the hypoxia markers DEC-1 and BNIP3. Chromatin immunoprecipitation identified increased binding of the transcriptional repressor DEC-1 to the proximal Mlh1 promoter in hypoxic YAMC cells and colitic Gialpha2-/- crypts. Treating Gialpha2-/- mice with the histone deacetylase inhibitor suberoylanilide hydroxamic acid significantly decreased colitis activity and rescued MLH1 expression in crypt epithelial cells, which was associated with increased acetyl histone H3 levels and decreased DEC-1 binding at the proximal Mlh1 promoter, consistent with a histone deacetylase-dependent mechanism. These data link chronic hypoxic inflammation, epigenetic MMR protein down-regulation, development of MMR-deficient colorectal cancer, and the firstmouse model of somatically acquired MMR-deficient colorectal cancer.

// Kyla A.L. Collins 1, * , Timothy J. Stuhlmiller 2, 3, * , Jon S. Zawistowski 2, 3 , Michael P. East 2, 3 , Trang T. Pham 2, 3 , Claire R. Hall 4 , Daniel R. Goulet 2, 3 , Samantha M. Bevill 2, 3 , Steven P. Angus 2, 3 , Sara H. Velarde 2, 3 , Noah Sciaky 2 , Tudor I. Oprea 5, 6 , Lee M. Graves 2, 3 , Gary L. Johnson 2, 3 and Shawn M. Gomez 1, 2, 4 1 Curriculum in Bioinformatics and Computational Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA 2 Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA 3 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA 4 Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC 27514, USA 5 Translational Informatics Division, School of Medicine, University of New Mexico, Albuquerque, NM 87106, USA 6 UNM Comprehensive Cancer Center, University of New Mexico, Albuquerque, NM 87131, USA * These authors contributed equally to this work Correspondence to: Gary L. Johnson, email: gary_johnson@med.unc.edu Shawn M. Gomez, email: smgomez@unc.edu Keywords: cancer biology; proteomics; kinase signaling Received: September 27, 2017      Accepted: January 19, 2018      Epub: January 29, 2018      Published: March 20, 2018 ABSTRACT Multiplexed small molecule inhibitors covalently bound to Sepharose beads (MIBs) were used to capture functional kinases in luminal, HER2-enriched and triple negative (basal-like and claudin-low) breast cancer cell lines and tumors. Kinase MIB-binding profiles at baseline without perturbation proteomically distinguished the four breast cancer subtypes. Understudied kinases, whose disease associations and pharmacology are generally unexplored, were highly represented in MIB-binding taxonomies and are integrated into signaling subnetworks with kinases that have been previously well characterized in breast cancer. Computationally it was possible to define subtypes using profiles of less than 50 of the more than 300 kinases bound to MIBs that included understudied as well as metabolic and lipid kinases. Furthermore, analysis of MIB-binding profiles established potential functional annotations for these understudied kinases. Thus, comprehensive MIBs-based capture of kinases provides a unique proteomics-based method for integration of poorly characterized kinases of the understudied kinome into functional subnetworks in breast cancer cells and tumors that is not possible using genomic strategies. The MIB-binding profiles readily defined subtype-selective differential adaptive kinome reprogramming in response to targeted kinase inhibition, demonstrating how MIB profiles can be used in determining dynamic kinome changes that result in subtype selective phenotypic state changes.

Eukaryotic cells form stress granules under a variety of stresses, however the signaling pathways regulating their formation remain largely unknown. We have determined that the Saccharomyces cerevisiae lysine acetyltransferase complex NuA4 is required for stress granule formation upon glucose deprivation but not heat stress. Further, the Tip60 complex, the human homolog of the NuA4 complex, is required for stress granule formation in cancer cell lines. Surprisingly, the impact of NuA4 on glucose-deprived stress granule formation is partially mediated through regulation of acetyl-CoA levels, which are elevated in NuA4 mutants. While elevated acetyl-CoA levels suppress the formation of glucose-deprived stress granules, decreased acetyl-CoA levels enhance stress granule formation upon glucose deprivation. Further our work suggests that NuA4 regulates acetyl-CoA levels through the Acetyl-CoA carboxylase Acc1. Altogether this work establishes both NuA4 and the metabolite acetyl-CoA as critical signaling pathways regulating the formation of glucose-deprived stress granules.