EZH2 modifies sunitinib resistance in renal cell carcinoma by kinome reprogramming

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represent a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft (PDX) model that is intrinsically resistant to the RTKI sunitinib but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its anti-angiogenic and anti-metastatic activity but lost its direct anti-tumor effects due to kinome reprogramming, which resulted in suppression of pro- apoptotic and cell cycle regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTK, restoring the anti-tumor effects of sunitnib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease.This research was funded by Roswell Park Cancer Institute’s Cancer Center Support Grant from National Cancer Institute, NIH P30CA016056 (RP) and a generous donation by Richard and Deidre Turner (RP). This investigation was conducted in-part in a facility constructed with support from Research Facilities Improvement Program Grant Number C06 RR020128-01 from the National Center for Research Resources, National Institutes of Health

Development of Proteomic Strategies to Elucidate Disease Processes in Cancer and Neurological Disorders

Advances in instrumentation and data analysis software have allowed mass spectrometry to become perhaps the most important tool in large scale proteomic studies. Using optimized workflows, it is possible to identify thousands of proteins and protein post-translational modifications in a single mass spectrometry (MS) run from a variety of sample sources, including immortalized cell lines, tissues, and biofluids. However, protein phosphorylation still presents a challenge for analysis because its low abundance and dynamic nature necessitates additional sample preparation steps and special considerations that must be taken into account when quantifying phosphorylation events. Even more difficult is dissection of intricate phosphorylation signaling pathways, which are involved in healthy cellular processes as well as disease pathogenesis. Protein post-translational modifications aside, identification of low abundance proteins from certain sample types can also be problematic. A good example of this is the so-called secretome, or the proteins that are secreted by cells in response to various stimuli. Secreted proteins are released in small amounts into large volumes of complex biofluids or cell culture media, making it difficult to recover these proteins for MS analysis This dissertation discusses strategies for identifying segments of both the phosphoproteome and the secretome. Chapter one is an overview of current phosphoproteomic techniques while chapters two and three highlight the work that has been done to identify the direct substrates of the ABL and CDKL5 kinases, whose dysregulation leads to aberrant phosphorylation signaling and the development of cancer or severe infantile epilepsy, respectively. Chapter four details the development of a novel nanopolymer-based reagent for capture of the secretome from Helicobacter pylori, a pathogen that has been linked to peptic ulcer formation and several types of cancer

Viral proteogenomic and expression profiling during productive replication of a skin-tropic herpesvirus in the natural host.

Efficient transmission of herpesviruses is essential for dissemination in host populations; however, little is known about the viral genes that mediate transmission, mostly due to a lack of natural virus-host model systems. Marek's disease is a devastating herpesviral disease of chickens caused by Marek's disease virus (MDV) and an excellent natural model to study skin-tropic herpesviruses and transmission. Like varicella zoster virus that causes chicken pox in humans, the only site where infectious cell-free MD virions are efficiently produced is in epithelial skin cells, a requirement for host-to-host transmission. Here, we enriched for heavily infected feather follicle epithelial skin cells of live chickens to measure both viral transcription and protein expression using combined short- and long-read RNA sequencing and LC/MS-MS bottom-up proteomics. Enrichment produced a previously unseen breadth and depth of viral peptide sequencing. We confirmed protein translation for 84 viral genes at high confidence (1% FDR) and correlated relative protein abundance with RNA expression levels. Using a proteogenomic approach, we confirmed translation of most well-characterized spliced viral transcripts and identified a novel, abundant isoform of the 14 kDa transcript family via IsoSeq transcripts, short-read intron-spanning sequencing reads, and a high-quality junction-spanning peptide identification. We identified peptides representing alternative start codon usage in several genes and putative novel microORFs at the 5' ends of two core herpesviral genes, pUL47 and ICP4, along with strong evidence of independent transcription and translation of the capsid scaffold protein pUL26.5. Using a natural animal host model system to examine viral gene expression provides a robust, efficient, and meaningful way of validating results gathered from cell culture systems

Identification of Upstream Kinases by Fluorescence Complementation Mass Spectrometry

Protein kinases and their substrates comprise extensive signaling networks that regulate many diverse cellular functions. However, methods and techniques to systematically identify kinases directly responsible for specific phosphorylation events have remained elusive. Here we describe a novel proteomic strategy termed fluorescence complementation mass spectrometry (FCMS) to identify kinase–substrate pairs in high throughput. The FCMS strategy employs a specific substrate and a kinase library, both of which are fused with fluorescence complemented protein fragments. Transient and weak kinase–substrate interactions in living cells are stabilized by the association of fluorescence protein fragments. These kinase–substrate pairs are then isolated with high specificity and are identified and quantified by LC–MS. FCMS was applied to the identification of both known and novel kinases of the transcription factor, cAMP response element-binding protein (CREB). Novel CREB kinases were validated by <i>in vitro</i> kinase assays, and the phosphorylation sites were unambiguously located. These results uncovered possible new roles for CREB in multiple important signaling pathways and demonstrated the great potential of this new proteomic strategy

Osteogenesis on nanoparticulate mineralized collagen scaffolds via autogenous activation of the canonical BMP receptor signaling pathway

Skeletal regenerative medicine frequently incorporates deliverable growth factors to stimulate osteogenesis. However, the cost and side effects secondary to supraphysiologic dosages of growth factors warrant investigation of alternative methods of stimulating osteogenesis for clinical utilization. In this work, we describe growth factor independent osteogenic induction of human mesenchymal stem cells (hMSCs) on a novel nanoparticulate mineralized collagen glycosaminoglycan scaffold (MC-GAG). hMSCs demonstrated elevated osteogenic gene expression and mineralization on MC-GAG with minimal to no effect upon addition of BMP-2 when compared to non-mineralized scaffolds (Col-GAG). To investigate the intracellular pathways responsible for the increase in osteogenesis, we examined the canonical and non-canonical pathways downstream from BMP receptor activation. Constitutive Smad1/5 phosphorylation with nuclear translocation occurred on MC-GAG independent of BMP-2, whereas Smad1/5 phosphorylation depended on BMP-2 stimulation on Col-GAG. When non-canonical BMPR signaling molecules were examined, ERK1/2 phosphorylation was found to be decreased in MC-GAG but elevated in Col-GAG. No differences in Smad2/3 or p38 activation were detected. Collectively, these results demonstrated that MC-GAG scaffolds induce osteogenesis without exogenous BMP-2 addition via endogenous activation of the canonical BMP receptor signaling pathway