Unbiased Proteomic Profiling Uncovers a Targetable GNAS/PKA/PP2A Axis in Small Cell Lung Cancer Stem Cells.

Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.

Phosphoregulation of the oncogenic protein regulator of cytokinesis 1 (PRC1) by the atypical CDK16/CCNY complex.

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that forms an active complex with cyclin Y (CCNY). Although both proteins have been recently implicated in cancer pathogenesis, it is still unclear how the CDK16/CCNY complex exerts its biological activity. To understand the CDK16/CCNY network, we used complementary proteomic approaches to identify potential substrates of this complex. We identified several candidates implicating the CDK16/CCNY complex in cytoskeletal dynamics, and we focused on the microtubule-associated protein regulator of cytokinesis (PRC1), an essential protein for cell division that organizes antiparallel microtubules and whose deregulation may drive genomic instability in cancer. Using analog-sensitive (AS) CDK16 generated by CRISPR-Cas9 mutagenesis in 293T cells, we found that specific inhibition of CDK16 induces PRC1 dephosphorylation at Thr481 and delocalization to the nucleus during interphase. The observation that CDK16 inhibition and PRC1 downregulation exhibit epistatic effects on cell viability confirms that these proteins can act through a single pathway. In conclusion, we identified PRC1 as the first substrate of the CDK16/CCNY complex and demonstrated that the proliferative function of CDK16 is mediated by PRC1 phosphorylation. As CDK16 is emerging as a critical node in cancer, our study reveals novel potential therapeutic targets.

Kinome rewiring reveals AURKA limits PI3K-pathway inhibitor efficacy in breast cancer.

Dysregulation of the PI3K-AKT-mTOR signaling network is a prominent feature of breast cancers. However, clinical responses to drugs targeting this pathway have been modest, possibly because of dynamic changes in cellular signaling that drive resistance and limit drug efficacy. Using a quantitative chemoproteomics approach, we mapped kinome dynamics in response to inhibitors of this pathway and identified signaling changes that correlate with drug sensitivity. Maintenance of AURKA after drug treatment was associated with resistance in breast cancer models. Incomplete inhibition of AURKA was a common source of therapy failure, and combinations of PI3K, AKT or mTOR inhibitors with the AURKA inhibitor MLN8237 were highly synergistic and durably suppressed mTOR signaling, resulting in apoptosis and tumor regression in vivo. This signaling map identifies survival factors whose presence limits the efficacy of targeted therapies and reveals new drug combinations that may unlock the full potential of PI3K-AKT-mTOR pathway inhibitors in breast cancer.

Discovery of new substrates of the elongation factor-2 kinase suggests a broader role in the cellular nutrient response.

Elongation Factor-2 Kinase (eEF2K) in an unusual mammalian enzyme that has one known substrate, elongation factor-2. It belongs to a class of kinases, called alpha kinases, that has little sequence identity to the >500 conventional protein kinases, but performs the same reaction and has similar catalytic residues. The phosphorylation of eEF2 blocks translation elongation, which is thought to be critical to regulating cellular energy usage. Here we report a system for discovering new substrates of alpha kinases and identify the first new substrates of eEF2K including AMPK and alpha4, and determine a sequence motif for the kinase that shows a requirement for threonine residues as the target of phosphorylation. These new substrates suggest that eEF2K has a more diverse role in regulating cellular energy usage that involves multiple pathways and regulatory feedback.

An Optimized Chromatographic Strategy for Multiplexing In Parallel Reaction Monitoring Mass Spectrometry: Insights from Quantitation of Activated Kinases.

Reliable quantitation of protein abundances in defined sets of cellular proteins is critical to numerous biological applications. Traditional immunodetection-based methods are limited by the quality and availability of specific antibodies, especially for site-specific post-translational modifications. Targeted proteomic methods, including the recently developed parallel reaction monitoring (PRM) mass spectrometry, have enabled accurate quantitative measurements of up to a few hundred specific target peptides. However, the degree of practical multiplexing in label-free PRM workflows remains a significant limitation for the technique. Here we present a strategy for significantly increasing multiplexing in label-free PRM that takes advantage of the superior separation characteristics and retention time stability of meter-scale monolithic silica-C18 column-based chromatography. We show the utility of the approach in quantifying kinase abundances downstream of previously developed active kinase enrichment methodology based on multidrug inhibitor beads. We examine kinase activation dynamics in response to three different MAP kinase inhibitors in colorectal carcinoma cells and demonstrate reliable quantitation of over 800 target peptides from over 150 kinases in a single label-free PRM run. The kinase activity profiles obtained from these analyses reveal compensatory activation of TGF-ß family receptors as a response to MAPK blockade. The gains achieved using this label-free PRM multiplexing strategy will benefit a wide array of biological applications.

Innate immunity kinase TAK1 phosphorylates Rab1 on a hotspot for posttranslational modifications by host and pathogen.

TGF-ß activated kinase 1 (TAK1) is a critical signaling hub responsible for translating antigen binding signals to immune receptors for the activation of the AP-1 and NF-κB master transcriptional programs. Despite its importance, known substrates of TAK1 are limited to kinases of the MAPK and IKK families and include no direct effectors of biochemical processes. Here, we identify over 200 substrates of TAK1 using a chemical genetic kinase strategy. We validate phosphorylation of the dynamic switch II region of GTPase Rab1, a mediator of endoplasmic reticulum to Golgi vesicular transport, at T75 to be regulated by TAK1 in vivo. TAK1 preferentially phosphorylates the inactive (GDP-bound) state of Rab1. Phosphorylation of Rab1 disrupts interaction with GDP dissociation inhibitor 1 (GDI1), but not guanine exchange factor (GEF) or GTPase-activating protein (GAP) enzymes, and is exclusive to membrane-localized Rab1, suggesting phosphorylation may stimulate Rab1 membrane association. Furthermore, we found phosphorylation of Rab1 at T75 to be essential for Rab1 function. Previous studies established that the pathogen Legionella pneumophila is capable of hijacking Rab1 function through posttranslational modifications of the switch II region. Here, we present evidence that Rab1 is regulated by the host in a similar fashion, and that the innate immunity kinase TAK1 and Legionella effectors compete to regulate Rab1 by switch II modifications during infection.

Synthetic Lethal Targeting of ARID1A-Mutant Ovarian Clear Cell Tumors with Dasatinib.

New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G1-S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1A-mutant OCCC. Mol Cancer Ther; 15(7); 1472-84. ©2016 AACR.

Isocitrate Dehydrogenase Mutations Confer Dasatinib Hypersensitivity and SRC Dependence in Intrahepatic Cholangiocarcinoma.

UNLABELLED: Intrahepatic cholangiocarcinoma (ICC) is an aggressive liver bile duct malignancy exhibiting frequent isocitrate dehydrogenase (IDH1/IDH2) mutations. Through a high-throughput drug screen of a large panel of cancer cell lines, including 17 biliary tract cancers, we found that IDH mutant (IDHm) ICC cells demonstrate a striking response to the multikinase inhibitor dasatinib, with the highest sensitivity among 682 solid tumor cell lines. Using unbiased proteomics to capture the activated kinome and CRISPR/Cas9-based genome editing to introduce dasatinib-resistant "gatekeeper" mutant kinases, we identified SRC as a critical dasatinib target in IDHm ICC. Importantly, dasatinib-treated IDHm xenografts exhibited pronounced apoptosis and tumor regression. Our results show that IDHm ICC cells have a unique dependency on SRC and suggest that dasatinib may have therapeutic benefit against IDHm ICC. Moreover, these proteomic and genome-editing strategies provide a systematic and broadly applicable approach to define targets of kinase inhibitors underlying drug responsiveness. SIGNIFICANCE: IDH mutations define a distinct subtype of ICC, a malignancy that is largely refractory to current therapies. Our work demonstrates that IDHm ICC cells are hypersensitive to dasatinib and critically dependent on SRC activity for survival and proliferation, pointing to new therapeutic strategies against these cancers. Cancer Discov; 6(7); 727-39. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 681.

P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates.

The transcription cycle of RNA polymerase II (Pol II) is regulated at discrete transition points by cyclin-dependent kinases (CDKs). Positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1, promotes release of paused Pol II into elongation, but the precise mechanisms and targets of Cdk9 action remain largely unknown. Here, by a chemical genetic strategy, we identified ∼ 100 putative substrates of human P-TEFb, which were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors phosphorylated by Cdk9 was the 5'-to-3' "torpedo" exoribonuclease Xrn2, required in transcription termination by Pol II, which we validated as a bona fide P-TEFb substrate in vivo and in vitro. Phosphorylation by Cdk9 or phosphomimetic substitution of its target residue, Thr439, enhanced enzymatic activity of Xrn2 on synthetic substrates in vitro. Conversely, inhibition or depletion of Cdk9 or mutation of Xrn2-Thr439 to a nonphosphorylatable Ala residue caused phenotypes consistent with inefficient termination in human cells: impaired Xrn2 chromatin localization and increased readthrough transcription of endogenous genes. Therefore, in addition to its role in elongation, P-TEFb regulates termination by promoting chromatin recruitment and activation of a cotranscriptional RNA processing enzyme, Xrn2.

Identification of AMPK Phosphorylation Sites Reveals a Network of Proteins Involved in Cell Invasion and Facilitates Large-Scale Substrate Prediction.

AMP-activated protein kinase (AMPK) is a central energy gauge that regulates metabolism and has been increasingly involved in non-metabolic processes and diseases. However, AMPK's direct substrates in non-metabolic contexts are largely unknown. To better understand the AMPK network, we use a chemical genetics screen coupled to a peptide capture approach in whole cells, resulting in identification of direct AMPK phosphorylation sites. Interestingly, the high-confidence AMPK substrates contain many proteins involved in cell motility, adhesion, and invasion. AMPK phosphorylation of the RHOA guanine nucleotide exchange factor NET1A inhibits extracellular matrix degradation, an early step in cell invasion. The identification of direct AMPK phosphorylation sites also facilitates large-scale prediction of AMPK substrates. We provide an AMPK motif matrix and a pipeline to predict additional AMPK substrates from quantitative phosphoproteomics datasets. As AMPK is emerging as a critical node in aging and pathological processes, our study identifies potential targets for therapeutic strategies.

Linking tumor mutations to drug responses via a quantitative chemical-genetic interaction map.

UNLABELLED: There is an urgent need in oncology to link molecular aberrations in tumors with therapeutics that can be administered in a personalized fashion. One approach identifies synthetic-lethal genetic interactions or dependencies that cancer cells acquire in the presence of specific mutations. Using engineered isogenic cells, we generated a systematic and quantitative chemical-genetic interaction map that charts the influence of 51 aberrant cancer genes on 90 drug responses. The dataset strongly predicts drug responses found in cancer cell line collections, indicating that isogenic cells can model complex cellular contexts. Applying this dataset to triple-negative breast cancer, we report clinically actionable interactions with the MYC oncogene, including resistance to AKT-PI3K pathway inhibitors and an unexpected sensitivity to dasatinib through LYN inhibition in a synthetic lethal manner, providing new drug and biomarker pairs for clinical investigation. This scalable approach enables the prediction of drug responses from patient data and can accelerate the development of new genotype-directed therapies. SIGNIFICANCE: Determining how the plethora of genomic abnormalities that exist within a given tumor cell affects drug responses remains a major challenge in oncology. Here, we develop a new mapping approach to connect cancer genotypes to drug responses using engineered isogenic cell lines and demonstrate how the resulting dataset can guide clinical interrogation.

Oncogene mimicry as a mechanism of primary resistance to BRAF inhibitors.

Despite the development of potent RAF/mitogen-activated protein kinase (MAPK) pathway inhibitors, only a fraction of BRAF-mutant patients benefit from treatment with these drugs. Using a combined chemogenomics and chemoproteomics approach, we identify drug-induced RAS-RAF-MEK complex formation in a subset of BRAF-mutant cancer cells characterized by primary resistance to vemurafenib. In these cells, autocrine interleukin-6 (IL-6) secretion may contribute to the primary resistance phenotype via induction of JAK/STAT3 and MAPK signaling. In a subset of cell lines, combined IL-6/MAPK inhibition is able to overcome primary resistance to BRAF-targeted therapy. Overall, we show that the signaling plasticity exerted by primary resistant BRAF-mutant cells is achieved by their ability to mimic signaling features of oncogenic RAS, a strategy that we term "oncogene mimicry." This model may guide future strategies for overcoming primary resistance observed in these tumors.

Global turnover of histone post-translational modifications and variants in human cells.

BACKGROUND: Post-translational modifications (PTMs) on the N-terminal tails of histones and histone variants regulate distinct transcriptional states and nuclear events. Whereas the functional effects of specific PTMs are the current subject of intense investigation, most studies characterize histone PTMs/variants in a non-temporal fashion and very few studies have reported kinetic information about these histone forms. Previous studies have used radiolabeling, fluorescence microscopy and chromatin immunoprecipitation to determine rates of histone turnover, and have found interesting correlations between increased turnover and increased gene expression. Therefore, histone turnover is an understudied yet potentially important parameter that may contribute to epigenetic regulation. Understanding turnover in the context of histone modifications and sequence variants could provide valuable additional insight into the function of histone replacement. RESULTS: In this study, we measured the metabolic rate of labeled isotope incorporation into the histone proteins of HeLa cells by combining stable isotope labeling of amino acids in cell culture (SILAC) pulse experiments with quantitative mass spectrometry-based proteomics. In general, we found that most core histones have similar turnover rates, with the exception of the H2A variants, which exhibit a wider range of rates, potentially consistent with their epigenetic function. In addition, acetylated histones have a significantly faster turnover compared with general histone protein and methylated histones, although these rates vary considerably, depending on the site and overall degree of methylation. Histones containing transcriptionally active marks have been consistently found to have faster turnover rates than histones containing silent marks. Interestingly, the presence of both active and silent marks on the same peptide resulted in a slower turnover rate than either mark alone on that same peptide. Lastly, we observed little difference in the turnover between nearly all modified forms of the H3.1, H3.2 and H3.3 variants, with the notable exception that H3.2K36me2 has a faster turnover than this mark on the other H3 variants. CONCLUSIONS: Quantitative proteomics provides complementary insight to previous work aimed at quantitatively measuring histone turnover, and our results suggest that turnover rates are dependent upon site-specific post-translational modifications and sequence variants.

In vivo residue-specific histone methylation dynamics.

Methylation of specific histone residues is capable of both gene activation and silencing. Despite vast work on the function of methylation, most studies either present a static snapshot of methylation or fail to assign kinetic information to specific residues. Using liquid chromatography-tandem mass spectrometry on a high-resolution mass spectrometer and heavy methyl-SILAC labeling, we studied site-specific histone lysine and arginine methylation dynamics. The detection of labeled intermediates within a methylation state revealed that mono-, di-, and trimethylated residues generally have progressively slower rates of formation. Furthermore, methylations associated with active genes have faster rates than methylations associated with silent genes. Finally, the presence of both an active and silencing mark on the same peptide results in a slower rate of methylation than the presence of either mark alone. Here we show that quantitative proteomic approaches such as this can determine the dynamics of multiple methylated residues, an understudied portion of histone biology.