Ronald C. Hendrickson

Constitutive Activation of Signal Transducers and Activators of Transcription Predicts Vorinostat Resistance in Cutaneous T-Cell Lymphoma

Vorinostat is a histone deacetylase inhibitor that induces differentiation, growth arrest, and/or apoptosis of malignant cells both in vitro and in vivo and has shown clinical responses in approximately 30% of patients with advanced mycosis fungoides and Sézary syndrome cutaneous T-cell lymphoma (CTCL). The purpose of this study was to identify biomarkers predictive of vorinostat response in CTCL using preclinical model systems and to assess these biomarkers in clinical samples. The signal transducer and activator of transcription (STAT) signaling pathway was evaluated. The data indicate that persistent activation of STAT1, STAT3, and STAT5 correlate with resistance to vorinostat in lymphoma cell lines. Simultaneous treatment with a pan-Janus-activated kinase inhibitor resulted in synergistic antiproliferative effect and down-regulation of the expression of several antiapoptotic genes. Immunohistochemical analysis of STAT1 and phosphorylated tyrosine STAT3 (pSTAT3) in skin biopsies obtained from CTCL patients enrolled in the vorinostat phase IIb trial showed that nuclear accumulation of STAT1 and high levels of nuclear pSTAT3 in malignant T cells correlate with a lack of clinical response. These results suggest that deregulation of STAT activity plays a role in vorinostat resistance in CTCL, and strategies that block this pathway may improve vorinostat response. Furthermore, these findings may be of prognostic value in predicting the response of CTCL patients to vorinostat.

Pathway-Based Identification of Biomarkers for Targeted Therapeutics: Personalized Oncology with PI3K Pathway Inhibitors

Phosphorylation sites on proteins in the phosphatidylinositol 3-kinase pathway that are regulated by candidate drugs can serve as useful biomarkers to predict tumor sensitivity to AKT inhibitors. Toward Customizing Tumor Treatment Just as our view of Earth has become increasingly global, cells are now seen as complex networks of interacting and intersecting signaling pathways rather than a collection of regulated genes. This new view applies to cancer cells as well, which we now know have entire dysregulated pathways and not just dysregulated genes. Andersen and colleagues have identified phosphoprotein biomarkers for a pathway often altered in cancer—the phosphatidylinositol 3-kinase (PI3K) pathway—and have shown that one of these predicts the sensitivity of cancer cells to a promising class of cancer drugs: inhibitors of AKT, a kinase that promotes growth and inhibits cell death. To find useful markers of the PI3K pathway, the authors focused on a vital biochemical event—the addition of phosphate groups to serines and threonines in cellular proteins. Cells use this simple covalent modification over and over again to regulate protein-protein binding and activity of key enzymes. Measurement of this modification in specific proteins reveals their activation. The authors monitored 375 phosphorylation sites in the PI3K pathway after treating prostate cancer cells with three different PI3K pathway inhibitors, potentially useful drugs. They found that each drug modulated a specific array of phosphoproteins, with some overlap, many of them within proteins that participate in cytoskeletal remodeling, vesicle transport, and protein translation. In theory, each phosphopeptide that decreased in abundance after drug treatment could, if elevated in cancer cells, serve as a biomarker of sensitivity to that drug. To show that this was the case, the authors chose one of the phosphorylated sites (the threonine at position 246 of the cytoplasmic protein PRAS40) and generated a high-quality antibody to it. The amount of phosphorylation at Thr246 correlated with activation of the PI3K pathway in human cancer cell lines, in a mouse prostate tumor, and in triple-negative breast tumors. Of potentially even more utility, Thr246 phosphorylation predicted the sensitivity of these cells to AKT inhibitors. Cancers are extremely heterogeneous, even within tissues, and for optimal effectiveness, treatments need to be customized accordingly. As this work shows, phosphorylated amino acids can serve as biomarkers for activated pathways in cancer and, because specific antibodies can easily be made to these phosphorylated peptides, can be readily measured. These results point to a way, after further development of more biomarkers, to routinely characterize the activated pathways in patients’ cancers. A tumor characterized in this way can then be treated with the appropriate pathway-specific drugs, optimizing the chances of eradicating the tumor. Although we have made great progress in understanding the complex genetic alterations that underlie human cancer, it has proven difficult to identify which molecularly targeted therapeutics will benefit which patients. Drug-specific modulation of oncogenic signaling pathways in specific patient subpopulations can predict responsiveness to targeted therapy. Here, we report a pathway-based phosphoprofiling approach to identify and quantify clinically relevant, drug-specific biomarkers for phosphatidylinositol 3-kinase (PI3K) pathway inhibitors that target AKT, phosphoinositide-dependent kinase 1 (PDK1), and PI3K–mammalian target of rapamycin (mTOR). We quantified 375 nonredundant PI3K pathway–relevant phosphopeptides, all containing AKT, PDK1, or mitogen-activated protein kinase substrate recognition motifs. Of these phosphopeptides, 71 were drug-regulated, 11 of them by all three inhibitors. Drug-modulated phosphoproteins were enriched for involvement in cytoskeletal reorganization (filamin, stathmin, dynamin, PAK4, and PTPN14), vesicle transport (LARP1, VPS13D, and SLC20A1), and protein translation (S6RP and PRAS40). We then generated phosphospecific antibodies against selected, drug-regulated phosphorylation sites that would be suitable as biomarker tools for PI3K pathway inhibitors. As proof of concept, we show clinical translation feasibility for an antibody against phospho-PRAS40Thr246. Evaluation of binding of this antibody in human cancer cell lines, a PTEN (phosphatase and tensin homolog deleted from chromosome 10)–deficient mouse prostate tumor model, and triple-negative breast tumor tissues showed that phospho-PRAS40Thr246 positively correlates with PI3K pathway activation and predicts AKT inhibitor sensitivity. In contrast to phosphorylation of AKTThr308, the phospho-PRAS40Thr246 epitope is highly stable in tissue samples and thus is ideal for immunohistochemistry. In summary, our study illustrates a rational approach for discovery of drug-specific biomarkers toward development of patient-tailored treatments.

Sensitive multiplexed analysis of kinase activities and activity-based kinase identification

Constitutive activation of one or more kinase signaling pathways is a hallmark of many cancers. Here we extend the previously described mass spectrometry–based KAYAK approach by monitoring kinase activities from multiple signaling pathways simultaneously. This improved single-reaction strategy, which quantifies the phosphorylation of 90 synthetic peptides in a single mass spectrometry run, is compatible with nanogram to microgram amounts of cell lysate. Furthermore, the approach enhances kinase monospecificity through substrate competition effects, faithfully reporting the signatures of many signaling pathways after mitogen stimulation or of basal pathway activation differences across a panel of well-studied cancer cell lines. Hierarchical clustering of activities from related experiments groups peptides phosphorylated by similar kinases together and, when combined with pathway alteration using pharmacological inhibitors, distinguishes underlying differences in potency, off-target effects and genetic backgrounds. Finally, we introduce a strategy to identify the kinase, and even associated protein complex members, responsible for phosphorylation events of interest.

Quantitative analysis of intact apolipoproteins in human HDL by top-down differential mass spectrometry

Top-down mass spectrometry holds tremendous potential for the characterization and quantification of intact proteins, including individual protein isoforms and specific posttranslationally modified forms. This technique does not require antibody reagents and thus offers a rapid path for assay development with increased specificity based on the amino acid sequence. Top-down MS is efficient whereby intact protein mass measurement, purification by mass separation, dissociation, and measurement of product ions with ppm mass accuracy occurs on the seconds to minutes time scale. Moreover, as the analysis is based on the accurate measurement of an intact protein, top-down mass spectrometry opens a research paradigm to perform quantitative analysis of “unknown” proteins that differ in accurate mass. As a proof of concept, we have applied differential mass spectrometry (dMS) to the top-down analysis of apolipoproteins isolated from human HDL3. The protein species at 9415.45 Da demonstrates an average fold change of 4.7 (p-value 0.017) and was identified as an O-glycosylated form of apolipoprotein C-III [NANA-(2 → 3)-Gal-β(1 → 3)-GalNAc, +656.2037 Da], a protein associated with coronary artery disease. This work demonstrates the utility of top-down dMS for quantitative analysis of intact protein mixtures and holds potential for facilitating a better understanding of HDL biology and complex biological systems at the protein level.

Application of an End-to-End Biomarker Discovery Platform to Identify Target Engagement Markers in Cerebrospinal Fluid by High Resolution Differential Mass Spectrometry

The rapid identification of protein biomarkers in biofluids is important to drug discovery and development. Here, we describe a general proteomic approach for the discovery and identification of proteins that exhibit a statistically significant difference in abundance in cerebrospinal fluid (CSF) before and after pharmacological intervention. This approach, differential mass spectrometry (dMS), is based on the analysis of full scan mass spectrometry data. The dMS workflow does not require complex mixing and pooling strategies, or isotope labeling techniques. Accordingly, clinical samples can be analyzed individually, allowing the use of longitudinal designs and within-subject data analysis in which each subject acts as its own control. As a proof of concept, we performed multifactorial dMS analyses on CSF samples drawn at 6 time points from n = 6 cisterna magna ported (CMP) rhesus monkeys treated with 2 potent gamma secretase inhibitors (GSI) or comparable vehicle in a 3-way crossover study that included a total of 108 individual CSF samples. Using analysis of variance and statistical filtering on the aligned and normalized LC-MS data sets, we detected 26 features that were significantly altered in CSF by drug treatment. Of those 26 features, which belong to 10 distinct isotopic distributions, 20 were identified by MS/MS as 7 peptides from CD99, a cell surface protein. Six features from the remaining 3 isotopic distributions were not identified. A subsequent analysis showed that the relative abundance of these 26 features showed the same temporal profile as the ELISA measured levels of CSF A beta 42 peptide, a known pharmacodynamic marker for gamma-secretase inhibition. These data demonstrate that dMS is a promising approach for the discovery, quantification, and identification of candidate target engagement biomarkers in CSF.

PI3K pathway regulates ER-dependent transcription in breast cancer through the epigenetic regulator KMT2D

Tumor cells develop resistance to a drug used to treat breast cancer through a chromatin remodeling mechanism. Chromatin state dictates drug response Drugs inhibiting the phosphoinositide-(3)-kinase (PI3K) signaling pathway are effective in a subset of breast cancer patients. Tumors become resistant to these drugs, however, and this transition is often accompanied by increased transcription of genes regulated by the estrogen receptor. A better understanding of the mechanism linking PI3K signaling and estrogen receptor activity could potentially suggest strategies to prevent drug resistance. Toska et al. found that PI3K inhibition activates a specific epigenetic regulator, the histone methyltransferase KMT2D. The protein modifications catalyzed by KMT2D create a more open chromatin state, which unleashes estrogen receptor–dependent transcription. Thus, combination therapies consisting of PI3K inhibitors and KMT2D inhibitors may be more effective than PI3K inhibitors alone. Science, this issue p. 1324 Activating mutations in PIK3CA, the gene encoding phosphoinositide-(3)-kinase α (PI3Kα), are frequently found in estrogen receptor (ER)–positive breast cancer. PI3Kα inhibitors, now in late-stage clinical development, elicit a robust compensatory increase in ER-dependent transcription that limits therapeutic efficacy. We investigated the chromatin-based mechanisms leading to the activation of ER upon PI3Kα inhibition. We found that PI3Kα inhibition mediates an open chromatin state at the ER target loci in breast cancer models and clinical samples. KMT2D, a histone H3 lysine 4 methyltransferase, is required for FOXA1, PBX1, and ER recruitment and activation. AKT binds and phosphorylates KMT2D, attenuating methyltransferase activity and ER function, whereas PI3Kα inhibition enhances KMT2D activity. These findings uncover a mechanism that controls the activation of ER by the posttranslational modification of epigenetic regulators, providing a rationale for epigenetic therapy in ER-positive breast cancer.

Nutrient-regulated Phosphorylation of ATG13 Inhibits Starvation-induced Autophagy

Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser-224 and Ser-258. mTOR directly phosphorylates ATG13 on Ser-258 while Ser-224 is modulated by the AMPK pathway. In ATG13 knock-out cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ATG13 and ULK1 translocation. These events culminated in a more rapid starvation-induced autophagy response. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation.

Proteome-wide analysis of mutant p53 targets in breast cancer identifies new levels of gain-of-function that influence PARP, PCNA, and MCM4

Significance Mutant p53 (mtp53) is a driver oncogene of breast cancer. Here, for the first time, to our knowledge, using an inducible endogenous knockdown system, we explore the mtp53 driven proteome. We report this key data set that highlights mtp53-driven proteome diversity at the level of protein localization, as well as changes in protein levels without corresponding changes in transcription. We validated two protein pathways that include increased chromatin association of poly(ADP ribose) polymerase 1, and the increase of nuclear replication proteins minichromosome maintenance 4 and proliferating cell nuclear antigen. The addition of mtp53 proteomic targets to the previously identified transcriptional targets suggests that effective treatment of mtp53-driven breast cancers may be facilitated by new combination protocols blocking proteins of the metabolic pathways of cholesterol biosynthesis, DNA replication, and DNA repair. The gain-of-function mutant p53 (mtp53) transcriptome has been studied, but, to date, no detailed analysis of the mtp53-associated proteome has been described. We coupled cell fractionation with stable isotope labeling with amino acids in cell culture (SILAC) and inducible knockdown of endogenous mtp53 to determine the mtp53-driven proteome. Our fractionation data highlight the underappreciated biology that missense mtp53 proteins R273H, R280K, and L194F are tightly associated with chromatin. Using SILAC coupled to tandem MS, we identified that R273H mtp53 expression in MDA-MB-468 breast cancer cells up- and down-regulated multiple proteins and metabolic pathways. Here we provide the data set obtained from sequencing 73,154 peptide pairs that then corresponded to 3,010 proteins detected under reciprocal labeling conditions. Importantly, the high impact regulated targets included the previously identified transcriptionally regulated mevalonate pathway proteins but also identified two new levels of mtp53 protein regulation for nontranscriptional targets. Interestingly, mtp53 depletion profoundly influenced poly(ADP ribose) polymerase 1 (PARP1) localization, with increased cytoplasmic and decreased chromatin-associated protein. An enzymatic PARP shift occurred with high mtp53 expression, resulting in increased poly-ADP-ribosylated proteins in the nucleus. Mtp53 increased the level of proliferating cell nuclear antigen (PCNA) and minichromosome maintenance 4 (MCM4) proteins without changing the amount of pcna and mcm4 transcripts. Pathway enrichment analysis ranked the DNA replication pathway above the cholesterol biosynthesis pathway as a R273H mtp53 activated proteomic target. Knowledge of the proteome diversity driven by mtp53 suggests that DNA replication and repair pathways are major targets of mtp53 and highlights consideration of combination chemotherapeutic strategies targeting cholesterol biosynthesis and PARP inhibition.

Identification of direct target engagement biomarkers for kinase-targeted therapeutics.

Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is often unknown. By definition, proximal PD biomarkers aim to measure the interaction of a drug with its biological target. For kinase drug discovery, protein substrate phosphorylation sites represent candidate PD biomarkers. However, substrate phosphorylation is often controlled by input from multiple converging pathways complicating assessment of how potently a small molecule drug hits its target based on substrate phoshorylation measurements alone. Here, we report the use of quantitative, differential mass-spectrometry to identify and monitor novel drug-regulated phosphorylation sites on target kinases. Autophosphorylation sites constitute clinically validated biomarkers for select protein tyrosine kinase inhibitors. The present study extends this principle to phosphorylation sites in serine/threonine kinases looking beyond the T-loop autophosphorylation site. Specifically, for the 3′-phosphoinositide-dependent protein kinase 1 (PDK1), two phospho-residues p-PDK1Ser410 and p-PDK1Thr513 are modulated by small-molecule PDK1 inhibitors, and their degree of dephosphorylation correlates with inhibitor potency. We note that classical, ATP-competitive PDK1 inhibitors do not modulate PDK1 T-loop phosphorylation (p-PDK1Ser241), highlighting the value of an unbiased approach to identify drug target-regulated phosphorylation sites as these are complementary to pathway PD biomarkers. Finally, we extend our analysis to another protein Ser/Thr kinase, highlighting a broader utility of our approach for identification of kinase drug-target engagement biomarkers.

Measurement of Fractional Synthetic Rates of Multiple Protein Analytes by Triple Quadrupole Mass Spectrometry

BACKGROUND Current approaches to measure protein turnover that use stable isotope-labeled tracers via GC-MS are limited to a small number of relatively abundant proteins. We developed a multiplexed liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM) assay to measure protein turnover and compared the fractional synthetic rates (FSRs) for 2 proteins, VLDL apolipoprotein B100 (VLDL apoB100) and HDL apoA-I, measured by both methods. We applied this technique to other proteins for which kinetics are not readily measured with GC-MS. METHODS Subjects were given a primed-constant infusion of [5,5,5-D(3)]-leucine (D(3)-leucine) for 15 h with blood samples collected at selected time points. Apolipoproteins isolated by SDS-PAGE from lipoprotein fractions were analyzed by GC-MS or an LC-SRM assay designed to measure the M+3/M+0 ratio at >1% D(3)-leucine incorporation. We calculated the FSR for each apolipoprotein by curve fitting the tracer incorporation data from each subject. RESULTS The LC-SRM method was linear over the range of tracer enrichment values tested and highly correlated with GC-MS (R(2) > 0.9). The FSRs determined from both methods were similar for HDL apoA-I and VLDL apoB100. We were able to apply the LC-SRM approach to determine the tracer enrichment of multiple proteins from a single sample as well as proteins isolated from plasma after immunoprecipitation. CONCLUSIONS The LC-SRM method provides a new technique for measuring the enrichment of proteins labeled with stable isotopes. LC-SRM is amenable to a multiplexed format to provide a relatively rapid and inexpensive means to measure turnover of multiple proteins simultaneously.