The human CDK8 subcomplex is a molecular switch that controls Mediator coactivator function.

The human CDK8 subcomplex (CDK8, cyclin C, Med12, and Med13) negatively regulates transcription in ways not completely defined; past studies suggested CDK8 kinase activity was required for its repressive function. Using a reconstituted transcription system together with recombinant or endogenous CDK8 subcomplexes, we demonstrate that, in fact, Med12 and Med13 are critical for subcomplex-dependent repression, whereas CDK8 kinase activity is not. A hallmark of activated transcription is efficient reinitiation from promoter-bound scaffold complexes that recruit a series of pol II enzymes to the gene. Notably, the CDK8 submodule strongly represses even reinitiation events, suggesting a means to fine tune transcript levels. Structural and biochemical studies confirm the CDK8 submodule binds the Mediator leg/tail domain via the Med13 subunit, and this submodule-Mediator association precludes pol II recruitment. Collectively, these results reveal the CDK8 subcomplex functions as a simple switch that controls the Mediator-pol II interaction to help regulate transcription initiation and reinitiation events. As Mediator is generally required for expression of protein-coding genes, this may reflect a common mechanism by which activated transcription is shut down in human cells.

Cooperative activity of cdk8 and GCN5L within Mediator directs tandem phosphoacetylation of histone H3.

The human Mediator complex is generally required for expression of protein-coding genes. Here, we show that the GCN5L acetyltransferase stably associates with Mediator together with the TRRAP polypeptide. Yet, contrary to expectations, TRRAP/GCN5L does not associate with the transcriptionally active core Mediator but rather with Mediator that contains the cdk8 subcomplex. Consequently, this derivative 'T/G-Mediator' complex does not directly activate transcription in a reconstituted human transcription system. However, within T/G-Mediator, cdk8 phosphorylates serine-10 on histone H3, which in turn stimulates H3K14 acetylation by GCN5L within the complex. Tandem phosphoacetylation of H3 correlates with transcriptional activation, and ChIP assays demonstrate co-occupancy of T/G-Mediator components at several activated genes in vivo. Moreover, cdk8 knockdown causes substantial reduction of global H3 phosphoacetylation, suggesting that T/G-Mediator is a major regulator of this H3 mark. Cooperative H3 modification provides a mechanistic basis for GCN5L association with cdk8-Mediator and also identifies a biochemical means by which cdk8 can indirectly activate gene expression. Indeed our results suggest that T/G-Mediator directs early events-such as modification of chromatin templates-in transcriptional activation.

Detection of Hepatocellular Carcinoma in a High-Risk Population by a Mass Spectrometry-Based Test.

Hepatocellular carcinoma (HCC) is one of the fastest growing causes of cancer-related death. Guidelines recommend obtaining a screening ultrasound with or without alpha-fetoprotein (AFP) every 6 months in at-risk adults. AFP as a screening biomarker is plagued by low sensitivity/specificity, prompting interest in discovering alternatives. Mass spectrometry-based techniques are promising in their ability to identify potential biomarkers. This study aimed to use machine learning utilizing spectral data and AFP to create a model for early detection. Serum samples were collected from three separate cohorts, and data were compiled to make Development, Internal Validation, and Independent Validation sets. AFP levels were measured, and Deep MALDI® analysis was used to generate mass spectra. Spectral data were input into the VeriStrat® classification algorithm. Machine learning techniques then classified each sample as "Cancer" or "No Cancer". Sensitivity and specificity of the test were >80% to detect HCC. High specificity of the test was independent of cause and severity of underlying disease. When compared to AFP, there was improved cancer detection for all tumor sizes, especially small lesions. Overall, a machine learning algorithm incorporating mass spectral data and AFP values from serum samples offers a novel approach to diagnose HCC. Given the small sample size of the Independent Validation set, a further independent, prospective study is warranted.

A proposal for score assignment to characterize biological processes from mass spectral analysis of serum.

INTRODUCTION: Most diseases involve a complex interplay between multiple biological processes at the cellular, tissue, organ, and systemic levels. Clinical tests and biomarkers based on the measurement of a single or few analytes may not be able to capture the complexity of a patient's disease. Novel approaches for comprehensively assessing biological processes from easily obtained samples could help in the monitoring, treatment, and understanding of many conditions. OBJECTIVES: We propose a method of creating scores associated with specific biological processes from mass spectral analysis of serum samples. METHODS: A score for a process of interest is created by: (i) identifying mass spectral features associated with the process using set enrichment analysis methods, and (ii) combining these features into a score using a principal component analysis-based approach. We investigate the creation of scores using cohorts of patients with non-small cell lung cancer, melanoma, and ovarian cancer. Since the circulating proteome is amenable to the study of immune responses, which play a critical role in cancer development and progression, we focus on functions related to the host response to disease. RESULTS: We demonstrate the feasibility of generating scores, their reproducibility, and their associations with clinical outcomes. Once the scores are constructed, only 3 µL of serum is required for the assessment of multiple biological functions from the circulating proteome. CONCLUSION: These mass spectrometry-based scores could be useful for future multivariate biomarker or test development studies for informing treatment, disease monitoring and improving understanding of the roles of various biological functions in multiple disease settings.

Extending the information content of the MALDI analysis of biological fluids via multi-million shot analysis.

INTRODUCTION: Reliable measurements of the protein content of biological fluids like serum or plasma can provide valuable input for the development of personalized medicine tests. Standard MALDI analysis typically only shows high abundance proteins, which limits its utility for test development. It also exhibits reproducibility issues with respect to quantitative measurements. In this paper we show how the sensitivity of MALDI profiling of intact proteins in unfractionated human serum can be substantially increased by exposing a sample to many more laser shots than are commonly used. Analytical reproducibility is also improved. METHODS: To assess what is theoretically achievable we utilized spectra from the same samples obtained over many years and combined them to generate MALDI spectral averages of up to 100,000,000 shots for a single sample, and up to 8,000,000 shots for a set of 40 different serum samples. Spectral attributes, such as number of peaks and spectral noise of such averaged spectra were investigated together with analytical reproducibility as a function of the number of shots. We confirmed that results were similar on MALDI instruments from different manufacturers. RESULTS: We observed an expected decrease of noise, roughly proportional to the square root of the number of shots, over the whole investigated range of the number of shots (5 orders of magnitude), resulting in an increase in the number of reliably detected peaks. The reproducibility of the amplitude of these peaks, measured by CV and concordance analysis also improves with very similar dependence on shot number, reaching median CVs below 2% for shot numbers > 4 million. Measures of analytical information content and association with biological processes increase with increasing number of shots. CONCLUSIONS: We demonstrate that substantially increasing the number of laser shots in a MALDI-TOF analysis leads to more informative and reliable data on the protein content of unfractionated serum. This approach has already been used in the development of clinical tests in oncology.

Proteomic test for anti-PD-1 checkpoint blockade treatment of metastatic melanoma with and without BRAF mutations.

The therapeutic landscape in metastatic melanoma has changed dramatically in the last decade, with the success of immune checkpoint inhibitors resulting in durable responses for a large number of patients. For patients with BRAF mutations, combinations of BRAF and MEK inhibitors demonstrated response rates and benefit comparable to those from immune checkpoint inhibitors, providing the rationale for sequential treatment with targeted and immunotherapies and raising the question of optimal treatment sequencing.Biomarkers for the selection of anti-PD-1 therapy in BRAF wild type (BRAF WT) and in BRAF mutated (BRAF MUT) patients help development of alternative treatments for patients unlikely to benefit, and might lead to better understanding of the interaction of checkpoint inhibition and targeted therapy. In this paper we evaluate the performance of a previously developed serum proteomic test, BDX008, in metastatic melanoma patients treated with anti-PD-1 agents and investigate the role of BRAF mutation status. BDX008, a pre-treatment proteomic test associated with acute phase reactants, wound healing and complement activation, stratifies patients into two groups, BDX008+ and BDX008-, with better and worse outcomes on immunotherapy.Serum samples were available from 71 patients treated with anti-PD1 inhibitors; 25 patients had BRAF mutations, 39 were wild type. Overall, BDX008+ patients had significantly better overall survival (OS) (HR = 0.50, P = 0.016) and a trend for better progression-free survival (PFS) (HR = 0.61, P = 0.060) than BDX008- patients. BDX008 classification was statistically significant in the analyses adjusted for mutation status, LDH, and line of treatment (P = 0.009 for OS and 0.031 for PFS). BRAF WT BDX008+ patients had markedly long median OS of 32.5 months and 53% landmark 2 years survival, with statistically significantly superior OS as compared to BDX008- patients (HR = 0.41, P = 0.032). The difference between BDX008+ and BDX008- in PFS in BRAF WT patients and in OS and PFS in BRAF MUT patients did not reach statistical significance, though numerically was consistent with overall results. The test demonstrated significant interaction with neutrophil-to-lymphocyte ratio (NLR) (PFS P = 0.041, OS P = 0.004). BDX008 as a biomarker selecting for benefit from immune checkpoint blockade, especially in patients with wild type BRAF and in subgroups with low NLR, warrants further evaluation.

Prognostic role of the VeriStrat test in first line patients with non-small cell lung cancer treated with platinum-based chemotherapy.

OBJECTIVES: VeriStrat® is a blood-based test that utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI ToF) mass spectrometry to assign a binary classification of VeriStrat Good or VeriStrat Poor that is associated with treatment outcomes in cancer patients. A number of other studies have shown an association between VeriStrat status and clinical outcomes in second and subsequent lines of therapy. The prognostic properties of VeriStrat were demonstrated in the placebo arms of two randomized studies in non-small cell lung cancer (NSCLC): TOPICAL and BR.21; the predictive properties of the test were shown in a prospective randomized phase III study PROSE in the second line treatment of NSCLC with erlotinib versus chemotherapy. Motivated by these observations, we sought to extend the clinical utility of VeriStrat to standard first line chemotherapy and evaluated the performance of the test in a number of clinical studies of patients treated with platinum-based regimens. MATERIALS AND METHODS: We examine the performance of VeriStrat in three independent clinical trials where the test classification was acquired for prospectively collected baseline samples from 481 patients treated with platinum-based chemotherapy in first line. RESULTS: Across these trials, 66-70% of patients were classified as VeriStrat Good; patients classified as VeriStrat Good had significantly longer progression-free survival and overall survival than VeriStrat Poor patients, with hazard ratios ranging from 0.36 to 0.72 and 0.26 to 0.51, respectively. These results demonstrated that VeriStrat is a strong prognostic test in NSCLC patients treated with platinum-based regimens in the first line. CONCLUSION: VeriStrat provides valuable clinical information that may be used to support patient-physician conversations regarding prognosis and treatment options, and to identify a subset of patients who might benefit from other treatment strategies, possibly in the framework of clinical trials.

A Serum Protein Signature Associated with Outcome after Anti-PD-1 Therapy in Metastatic Melanoma.

A mass spectrometry analysis was performed using serum from patients receiving checkpoint inhibitors to define baseline protein signatures associated with outcome in metastatic melanoma. Pretreatment serum was obtained from a development set of 119 melanoma patients on a trial of nivolumab with or without a multipeptide vaccine and from patients receiving pembrolizumab, nivolumab, ipilimumab, or both nivolumab and ipilimumab. Spectra were obtained using matrix-assisted laser desorption/ionization time of flight mass spectrometry. These data combined with clinical data identified patients with better or worse outcomes. The test was applied to five independent patient cohorts treated with checkpoint inhibitors and its biology investigated using enrichment analyses. A signature consisting of 209 proteins or peptides was associated with progression-free and overall survival in a multivariate analysis. The test performance across validation cohorts was consistent with the development set results. A pooled analysis, stratified by set, demonstrated a significantly better overall survival for "sensitive" relative to "resistant" patients, HR = 0.15 (95% confidence interval: 0.06-0.40, P < 0.001). The test was also associated with survival in a cohort of ipilimumab-treated patients. Test classification was found to be associated with acute phase reactant, complement, and wound healing pathways. We conclude that a pretreatment signature of proteins, defined by mass spectrometry analysis and machine learning, predicted survival in patients receiving PD-1 blocking antibodies. This signature of proteins was associated with acute phase reactants and elements of wound healing and the complement cascade. This signature merits further study to determine if it identifies patients who would benefit from PD-1 blockade. Cancer Immunol Res; 6(1); 79-86. ©2017 AACR.

Phase II trial of sorafenib and erlotinib in advanced pancreatic cancer.

This trial was designed to assess efficacy and safety of erlotinib with sorafenib in the treatment of patients with advanced pancreatic adenocarcinoma. An exploratory correlative study analyzing pretreatment serum samples using a multivariate protein mass spectrometry-based test (VeriStrat®), previously shown to correlate with outcomes in lung cancer patients treated with erlotinib, was performed. Patients received sorafenib 400 mg daily along with erlotinib 150 mg daily with a primary endpoint of 8-week progression free survival (PFS) rate. Pretreatment serum sample analysis by VeriStrat was done blinded to clinical and outcome data; the endpoints were PFS and overall survival (OS). Difference between groups (by VeriStrat classification) was assessed using log-rank P values; hazard ratios (HR) were obtained from Cox proportional hazards model. Thirty-six patients received study drug and were included in the survival analysis. Eight-week PFS rate of 46% (95% confidence interval (CI): 0.32-0.67) did not meet the primary endpoint of a rate ≥70%. Thirty-two patients were included in the correlative analysis, and VeriStrat "Good" patients had superior PFS (HR = 0.18, 95% CI: 0.06-0.57; P = 0.001) and OS (HR = 0.31 95% CI: 0.13-0.77, P = 0.008) compared to VeriStrat "Poor" patients. Grade 3 toxicities of this regimen included fever, anemia, diarrhea, dehydration, rash, and altered liver function. This study did not meet the primary endpoint, and this combination will not be further pursued. In this small retrospective analysis, the proteomic classification was significantly associated with clinical outcomes and is being further evaluated in ongoing studies.

p53 activates transcription by directing structural shifts in Mediator.

It is not well understood how the human Mediator complex, transcription factor IIH and RNA polymerase II (Pol II) work together with activators to initiate transcription. Activator binding alters Mediator structure, yet the functional consequences of such structural shifts remain unknown. The p53 C terminus and its activation domain interact with different Mediator subunits, and we find that each interaction differentially affects Mediator structure; strikingly, distinct p53-Mediator structures differentially affect Pol II activity. Only the p53 activation domain induces the formation of a large pocket domain at the Mediator-Pol II interaction site, and this correlates with activation of stalled Pol II to a productively elongating state. Moreover, we define a Mediator requirement for TFIIH-dependent Pol II C-terminal domain phosphorylation and identify substantial differences in Pol II C-terminal domain processing that correspond to distinct p53-Mediator structural states. Our results define a fundamental mechanism by which p53 activates transcription and suggest that Mediator structural shifts trigger activation of stalled Pol II complexes.

The human CDK8 subcomplex is a histone kinase that requires Med12 for activity and can function independently of mediator.

The four proteins CDK8, cyclin C, Med12, and Med13 can associate with Mediator and are presumed to form a stable "CDK8 subcomplex" in cells. We describe here the isolation and enzymatic activity of the 600-kDa CDK8 subcomplex purified directly from human cells and also via recombinant expression in insect cells. Biochemical analysis of the recombinant CDK8 subcomplex identifies predicted (TFIIH and RNA polymerase II C-terminal domain [Pol II CTD]) and novel (histone H3, Med13, and CDK8 itself) substrates for the CDK8 kinase. Notably, these novel substrates appear to be metazoan-specific. Such diverse targets imply strict regulation of CDK8 kinase activity. Along these lines, we observe that Mediator itself enables CDK8 kinase activity on chromatin, and we identify Med12--but not Med13--to be essential for activating the CDK8 kinase. Moreover, mass spectrometry analysis of the endogenous CDK8 subcomplex reveals several associated factors, including GCN1L1 and the TRiC chaperonin, that may help control its biological function. In support of this, electron microscopy analysis suggests TRiC sequesters the CDK8 subcomplex and kinase assays reveal the endogenous CDK8 subcomplex--unlike the recombinant submodule--is unable to phosphorylate the Pol II CTD.