Amanda L. Creech

Reduced-representation phosphosignatures measured by quantitative targeted MS capture cellular states and enable large-scale comparison of drug-induced phenotypes

Profiling post-translational modifications represents an alternative dimension to gene expression data in characterizing cellular processes. Many cellular responses to drugs are mediated by changes in cellular phosphosignaling. We sought to develop a common platform on which phosphosignaling responses could be profiled across thousands of samples, and created a targeted MS assay that profiles a reduced-representation set of phosphopeptides that we show to be strong indicators of responses to chemical perturbagens. To develop the assay, we investigated the coordinate regulation of phosphosites in samples derived from three cell lines treated with 26 different bioactive small molecules. Phosphopeptide analytes were selected from these discovery studies by clustering and picking 1 to 2 proxy members from each cluster. A quantitative, targeted parallel reaction monitoring assay was developed to directly measure 96 reduced-representation probes. Sample processing for proteolytic digestion, protein quantification, peptide desalting, and phosphopeptide enrichment have been fully automated, making possible the simultaneous processing of 96 samples in only 3 days, with a plate phosphopeptide enrichment variance of 12%. This highly reproducible process allowed ∼95% of the reduced-representation phosphopeptide probes to be detected in ∼200 samples. The performance of the assay was evaluated by measuring the probes in new samples generated under treatment conditions from discovery experiments, recapitulating the observations of deeper experiments using a fraction of the analytical effort. We measured these probes in new experiments varying the treatments, cell types, and timepoints to demonstrate generalizability. We demonstrated that the assay is sensitive to disruptions in common signaling pathways (e.g. MAPK, PI3K/mTOR, and CDK). The high-throughput, reduced-representation phosphoproteomics assay provides a platform for the comparison of perturbations across a range of biological conditions, suitable for profiling thousands of samples. We believe the assay will prove highly useful for classification of known and novel drug and genetic mechanisms through comparison of phosphoproteomic signatures.

Building the connectivity map of epigenetics: Chromatin profiling by quantitative targeted mass spectrometry

Epigenetic control of genome function is an important regulatory mechanism in diverse processes such as lineage commitment and environmental sensing, and in disease etiologies ranging from neuropsychiatric disorders to cancer. Here we report a robust, high-throughput targeted, quantitative mass spectrometry (MS) method to rapidly profile modifications of the core histones of chromatin that compose the epigenetic landscape, enabling comparisons among cells with differing genetic backgrounds, genomic perturbations, and drug treatments.

Functional Proteomic Analysis of Repressive Histone Methyltransferase Complexes Reveals ZNF518B as a G9A Regulator

Cell-type specific gene silencing by histone H3 lysine 27 and lysine 9 methyltransferase complexes PRC2 and G9A-GLP is crucial both during development and to maintain cell identity. Although studying their interaction partners has yielded valuable insight into their functions, how these factors are regulated on a network level remains incompletely understood. Here, we present a new approach that combines quantitative interaction proteomics with global chromatin profiling to functionally characterize repressive chromatin modifying protein complexes in embryonic stem cells. We define binding stoichiometries of 9 new and 12 known interaction partners of PRC2 and 10 known and 29 new interaction partners of G9A-GLP, respectively. We demonstrate that PRC2 and G9A-GLP interact physically and share several interaction partners, including the zinc finger proteins ZNF518A and ZNF518B. Using global chromatin profiling by targeted mass spectrometry, we discover that even sub-stoichiometric binding partners such as ZNF518B can positively regulate global H3K9me2 levels. Biochemical analysis reveals that ZNF518B directly interacts with EZH2 and G9A. Our systematic analysis suggests that ZNF518B may mediate the structural association between PRC2 and G9A-GLP histone methyltransferases and additionally regulates the activity of G9A-GLP.

A Library of Phosphoproteomic and Chromatin Signatures for Characterizing Cellular Responses to Drug Perturbations

SUMMARY Although the value of proteomics has been demonstrated, cost and scale are typically prohibitive, and gene expression profiling remains dominant for characterizing cellular responses to perturbations. However, high-throughput sentinel assays provide an opportunity for proteomics to contribute at a meaningful scale. We present a systematic library resource (90 drugs 3 6 cell lines) of proteomic signatures that measure changes in the reduced-representation phosphoproteome (P100) and changes in epigenetic marks on histones (GCP). A majority of these drugs elicited reproducible signatures, but notable cell line- and assay-specific differences were observed. Using the “connectivity” framework, we compared signatures across cell types and integrated data across assays, including a transcriptional assay (L1000). Consistent connectivity among cell types revealed cellular responses that transcended lineage, and consistent connectivity among assays revealed unexpected associations between drugs. We further leveraged the resource against public data to formulate hypotheses for treatment of multiple myeloma and acute lymphocytic leukemia. This resource is publicly available at https://clue.io/proteomics.

Functional proteomic analysis of repressive histone methyltransferase complexes PRC2 and G9A reveals ZNF518B as a G9A regulator

Cell-type specific gene silencing by histone H3 lysine 27 and lysine 9 methyltransferase complexes PRC2 and G9A-GLP is crucial both during development and to maintain cell identity. Although studying their interaction partners has yielded valuable insight into their functions, how these factors are regulated on a network level remains incompletely understood. Here, we present a new approach that combines quantitative interaction proteomics with global chromatin profiling to functionally characterize repressive chromatin modifying protein complexes in embryonic stem cells. We define binding stoichiometries of 9 new and 12 known interaction partners of PRC2 and 10 known and 29 new interaction partners of G9A-GLP, respectively. We demonstrate that PRC2 and G9A-GLP interact physically and share several interaction partners, including the zinc finger proteins ZNF518A and ZNF518B. Using global chromatin profiling by targeted mass spectrometry, we discover that even sub-stoichiometric binding partners such as ZNF518B can positively regulate global H3K9me2 levels. Biochemical analysis reveals that ZNF518B directly interacts with EZH2 and G9A. Our systematic analysis suggests that ZNF518B may mediate the structural association between PRC2 and G9A-GLP histone methyltransferases and additionally regulates the activity of G9A-GLP.

The Role of Mass Spectrometry and Proteogenomics in the Advancement of HLA Epitope Prediction

A challenge in developing personalized cancer immunotherapies is the prediction of putative cancer‐specific antigens. Currently, predictive algorithms are used to infer binding of peptides to human leukocyte antigen (HLA) heterodimers to aid in the selection of putative epitope targets. One drawback of current epitope prediction algorithms is that they are trained on datasets containing biochemical HLA‐peptide binding data that may not completely capture the rules associated with endogenous processing and presentation. The field of MS has made great improvements in instrumentation speed and sensitivity, chromatographic resolution, and proteogenomic database search strategies to facilitate the identification of HLA‐ligands from a variety of cell types and tumor tissues. As such, these advances have enabled MS profiling of HLA‐binding peptides to be a tractable, orthogonal approach to lower throughput biochemical assays for generating comprehensive datasets to train epitope prediction algorithms. In this review, we will highlight the progress made in the field of HLA‐ligand profiling enabled by MS and its impact on current and future epitope prediction strategies.

Abstract 2674: High fat diet accelerates MYC-driven prostate cancer through metabolic and epigenomic rewiring

Introduction: The mechanisms underlying the association between high dietary fat intake and prostate cancer (PCa) are unknown. Using a MYC-driven PCa mouse model, we sought to identify metabolic and epigenomic alterations driven by high fat diet (HFD) that facilitate PCa progression. Additionally, we investigated whether these alterations were relevant to PCa progression and lethality in humans. Material and Methods: Wild-type (WT) and transgenic Hi-MYC (MYC) mice were assigned either a HFD or control diet and were sacrificed at 12, 24, and 36 weeks of age for histologic and phenotypic characterization. Metabolic and epigenomic analyses were carried on the ventral prostates of 12-week old mice. Human PCa gene expression profiling data were obtained from 319 men with PCa and well-annotated post-diagnostic saturated fat intake (SFI) data from the Physicians’ Health Study and Health Professionals Follow-up Study prospective cohorts. Results: HFD does not affect the incidence of MYC-induced murine prostate intraepithelial neoplasia (mPIN) at 12 weeks, but increases mPIN proliferative index (Ki-67) at 24 weeks and tumor burden at 36 weeks. MYC overexpression, as expected, induces a significant metabolic reprogramming and HFD further enhances this rewiring to provide additional anabolic metabolites to sustain the increased proliferation of MYC prostate while having little effect on the WT prostate. Moreover, MYC altered key metabolites of the methionine cycle in a direction suggestive of a global hypomethylation, again amplified by HFD. Targeted quantitative histone mass spectrometry revealed a robust MYC-driven signature, including a global demethylation of H3K27 and H4K20 marks, the latter enhanced by HFD. Moreover, ChIP-seq revealed an intricate crosstalk between MYC and the H4K20me1 demethylase PHF8, resulting in enhanced genomic instability in the context of HFD. Finally, RNA-seq and ATAC-seq analyses showed that HFD rewires MYC-driven PCa through the alteration of genes implicated in chromatin function and remodeling. In humans, SFI was associated with enrichment in genes associated with increased MYC transcriptional activity in the prostate. Furthermore, this MYC transcriptional signature was associated with PCa lethality overall (OR = 3.21; 95% CI = 1.47, 7.35 comparing extreme score tertiles), and the association was stronger among men with high post-diagnostic SFI (OR = 1.32; 95%CI = 1.11, 1.66) than those with low SFI (OR = 1.05; 95%CI = 0.98, 1.12). Conclusions: HFD supports a coordinated metabolomic and epigenomic rewiring to increase epigenomic plasticity and MYC transcriptional activity prior to the appearance of phenotypic alterations in the prostate. Importantly, HFD requires MYC-mediated transformation to trigger its deleterious effects. In humans, SFI also enhances MYC transcriptional activity, which is associated with increased PCa lethality. Citation Format: David P. Labbe’, Giorgia Zadra, Meng Yang, Charles Y. Lin, Jaime M. Reyes, Stefano Cacciatore, Ericka M. Ebot, Maura B. Cotter, Amanda L. Creech, Jacob D. Jaffe, Philip W. Kantoff, James E. Bradner, Lorelei A. Mucci, Jorge E. Chavarro, Massimo Loda, Myles Brown. High fat diet accelerates MYC-driven prostate cancer through metabolic and epigenomic rewiring. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2674.

A phosphoproteomic signature in endothelial cells predicts vascular toxicity of tyrosine kinase inhibitors used in CML

Key Points Newer CML kinase inhibitors increase ischemia risk and are toxic to endothelial cells where they produce a proteomic toxicity signature. This phosphoproteomic EC toxicity signature predicts bosutinib to be safe, providing a potential screening tool for safer drug development.

Abstract A10: High-fat diet enhances MYC-driven prostate cancer through epigenomic and metabolomic rewiring

Diet is hypothesized to be a critical environmental risk factor for prostate cancer (PCa) development, and progression; however, the mechanisms underlying these associations remain elusive. In a MYC-driven PCa mouse model we find that a high fat diet significantly alters the transcription of genes implicated in chromatin function and remodeling in prostatic tumor tissues but not in the normal prostate. Importantly, this chromatin associated gene expression signature was observed well before the appearance of a high fat diet-driven phenotype that was characterized by greater cell proliferation and increased tumor burden. Consistent with this finding, high-throughput targeted quantitative histone mass spectrometry revealed a robust MYC-driven signature affecting more than half of the 68 histone marks profiled. Surprisingly, high fat diet further enhanced the MYC-induced epigenetic signature while it was unable to affect the normal murine prostate. Epigenetic remodeling relies on substrates and cofactors that are obtained from the diet. Untargeted metabolomic analyses revealed that MYC overexpression, as expected, impacted glutamine uptake. In addition, high fat diet leads to additional carbohydrates, amino acids, lipids and nucleotides necessary to sustain an increased cellular proliferation in MYC-driven cancers while it had little influence on the normal prostate. Moreover, the pool of metabolites altered by high fat diet in the context of MYC overexpression is highly suggestive of a global methylation defect. Finally, using the genome-wide mRNA profiles of tumor (N=402) and adjacent normal (N=200) prostate tissues from the Health Professionals Follow-up Study and the Physicians9 Health Study cohorts, we have discovered an enrichment in genes implicated in chromatin function and remodeling in tumor tissues from overweight/obese men, but not in normal adjacent tissues, consistent with the high fat diet signature observed in mice. Strikingly, men whose tumors had high expression of this chromatin signature had worse clinical characteristics and were more likely to die from prostate cancer (OR = 5.01; 95% CI = 2.31, 11.38 comparing extreme score quartiles). Taken together, these results demonstrate that a high fat diet does not drive significant epigenomic and metabolomic alterations in the normal prostate while it leads to important alterations in MYC-driven PCa that results in increased aggressiveness. Our results suggest that the impact of diet on PCa risk may be to augment the growth of already established subclinical disease. In addition, as MYC is one of the most commonly amplified genes in PCa, the ability of a high fat diet to augment MYC-driven cancers in this pre-clinical model suggest that a healthy diet may slow the progression of the disease. Citation Format: David P. Labbe, Giorgia Zadra, Ericka M. Ebot, Charles Y. Lin, Jaime M. Reyes, Stefano Cacciatore, Maura Cotter, Amanda L. Creech, Jacob D. Jaffe, Philip W. Kantoff, James E. Bradner, Lorelei A. Mucci, Massimo Loda, Myles Brown. High-fat diet enhances MYC-driven prostate cancer through epigenomic and metabolomic rewiring. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A10.

Functional proteomics defines a PRC2-G9A interaction network and reveals ZNF518B as a G9A regulator

Cell-type specific gene silencing by histone H3 lysine 27 and lysine 9 methyltransferase complexes PRC2 and G9A-GLP is crucial both during development and to maintain cell identity. Although studying their interaction partners has yielded valuable insight into their functions, how these factors are regulated on a network level remains incompletely understood. Here, we present a new approach that combines quantitative interaction proteomics with global chromatin profiling to functionally characterize repressive chromatin modifying protein complexes in embryonic stem cells. We define binding stoichiometries of 9 new and 12 known interaction partners of PRC2 and 10 known and 29 new interaction partners of G9A-GLP, respectively. We demonstrate that PRC2 and G9A-GLP interact physically and share several interaction partners, including the zinc finger proteins ZNF518A and ZNF518B. Using global chromatin profiling by targeted mass spectrometry, we discover that even sub-stoichiometric binding partners such as ZNF518B can positively regulate global H3K9me2 levels. Biochemical analysis reveals that ZNF518B directly interacts with EZH2 and G9A. Our systematic analysis suggests that ZNF518B may mediate the structural association between PRC2 and G9A-GLP histone methyltransferases and additionally regulates the activity of G9A-GLP.