Supplementary Figure 1. Mutation panel (OncoCarta panel v.1.0) tested in phase 1 expansion study (19 oncogenes and 225 mutations)

Time of day variation in metabolites in healthy volunteers and in patients treated with the MEK inhibitor.

Supplementary Figure 2. Case study: 64-year old female with BRAF V600-mutant melanoma (A) Immunhistochemistry for Ki67 expression showing a reduction between baseline and cycle1/day15 in lymph node biopsies (B) Immunhistochemistry for pERK expression showing a reduction between baseline and cycle5/day1 in lymph node biopsies (C) FDG-PET uptake at baseline (D) FDG-PET metabolic partial response at day 15 of cycle 1 (E) CT scan images at baseline (F) CT scan images at day 1 of cycle 5 show a 80% reduction in target lesion size, indicating partial response

Applied MEK signature to the PI3K treated patient cohort Heat map illustrating the changes relative to baseline treatment in 21 metabolites (MEK signature) in Pictilisib Phase I cohort. Dose range 45 - 450 mg.

Abstract MAPK pathway activation is frequently observed in human malignancies, including melanoma, and is associated with sensitivity to MEK inhibition and changes in cellular metabolism. Using quantitative mass spectrometry–based metabolomics, we identified in preclinical models 21 plasma metabolites including amino acids, propionylcarnitine, phosphatidylcholines, and sphingomyelins that were significantly altered in two B-RAF–mutant melanoma xenografts and that were reversed following a single dose of the potent and selective MEK inhibitor RO4987655. Treatment of non–tumor-bearing animals and mice bearing the PTEN-null U87MG human glioblastoma xenograft elicited plasma changes only in amino acids and propionylcarnitine. In patients with advanced melanoma treated with RO4987655, on-treatment changes of amino acids were observed in patients with disease progression and not in responders. In contrast, changes in phosphatidylcholines and sphingomyelins were observed in responders. Furthermore, pretreatment levels of seven lipids identified in the preclinical screen were statistically significantly able to predict objective responses to RO4987655. The RO4987655 treatment–related changes were greater than baseline physiological variability in nontreated individuals. This study provides evidence of a translational exo-metabolomic plasma readout predictive of clinical efficacy together with pharmacodynamic utility following treatment with a signal transduction inhibitor. Mol Cancer Ther; 16(10); 2315–23. ©2017 AACR.

The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website.

Inhibition of mitogen-activated protein kinase (MEK, also known as MAPK2, MAPKK), a key molecule of the Ras/MAPK (mitogen-activated protein kinase) pathway, has shown promising effects on B-raf-mutated and some RAS (rat sarcoma)-activated tumors in clinical trials. The objective of this study is to examine the efficacy of a novel allosteric MEK inhibitor RO4987655 in K-ras-mutated human tumor xenograft models using [18F] FDG-PET imaging and proteomics technology.

Abstract Purpose: This phase I expansion study assessed safety, pharmacodynamic effects, and antitumor activity of RO4987655, a pure MEK inhibitor, in selected patients with advanced solid tumor. Experimental Design: We undertook a multicenter phase I two-part study (dose escalation and cohort expansion). Here, we present the part 2 expansion that included melanoma, non–small cell lung cancer (NSCLC), and colorectal cancer with oral RO4987655 administered continuously at recommended doses of 8.5 mg twice daily until progressive disease (PD). Sequential tumor sampling investigated multiple markers of pathway activation/tumor effects, including ERK phosphorylation and Ki-67 expression. BRAF and KRAS testing were implemented as selection criteria and broader tumor mutational analysis added. Results: Ninety-five patients received RO4987655, including 18 BRAF-mutant melanoma, 23 BRAF wild-type melanoma, 24 KRAS-mutant NSCLC, and 30 KRAS-mutant colorectal cancer. Most frequent adverse events were rash, acneiform dermatitis, and gastrointestinal disorders, mostly grade 1/2. Four (24%) of 17 BRAF-mutated melanoma had partial response as did four (20%) of 20 BRAF wild-type melanoma and two (11%) of 18 KRAS-mutant NSCLC. All KRAS-mutant colorectal cancer developed PD. Paired tumor biopsies demonstrated reduced ERK phosphorylation among all cohorts but significant differences among cohorts in Ki-67 modulation. Sixty-nine percent showed a decrease in fluorodeoxyglucose uptake between baseline and day 15. Detailed mutational profiling confirmed RAS/RAF screening and identified additional aberrations (NRAS/non-BRAF melanomas; PIK3CA/KRAS colorectal cancer) without therapeutic implications. Conclusions: Safety profile of RO4987655 was comparable with other MEK inhibitors. Single-agent activity was observed in all entities except colorectal cancer. Evidence of target modulation and early biologic activity was shown among all indications independent of mutational status. Clin Cancer Res; 20(16); 4251–61. ©2014 AACR.

Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.

Predictive biomarkers are discovered and used in oncology research to formulate hypotheses aimed at the identification of patients benefiting from specific therapeutic intervention(s). They pave the way to the development of companion diagnostic tests which are tools readily implemented in the clinic and serve to qualify a patient for treatment with a particular targeted drug or the continued use of a particular drug, thus maximizing the benefit to risk ratio of the medical intervention to the patient. Predictive biomarkers are defined by biological characteristics of the patient's or tumor status that can be measured objectively and correlated with clinical outcome: these can be molecular, cellular or biochemical features. Predictive markers need extensive analytical validation - specific for the tool utilized for their assessment - as well as rigorous clinical qualification in the context of the drug treatment for which they define clinical utility. The process of companion diagnostic development is a highly interdisciplinary and complex one, driven by key crucial milestones and accompanying the same and typical process of a whole drug discovery and development continuum, from marker discovery and validation, assay development, clinical qualification until test approval and commercialization.

Theraptosis S.A., Romainville,France.Tumor necrosis factor a (TNFa), a pro-inflammatory cytokine, is associatedwith major cardiomyopathy. In the heart, TNFa binding to the TNF receptor1 (TNFR1) has been implicated in TNFa mediating negative inotropic effectsaswellasapoptosis.TNFa-TNFR1activatescaspase-8whichleadstocaspase-3 activation either directly or following mitochondrial disruption. Here we in-vestigated whether caspase-8-induced mitochondrial dysfunction could lead toTNFa-induced alterations of Ca

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Protein-kinase inhibitors are among the most advanced compounds in development using the new drug discovery paradigm of developing small-molecule drugs against specific molecular targets in cancer. After treatment with a cyclin dependent kinase CDK2 inhibitor in monkey, histopathological analysis of the eye showed specific cellular damage in the photoreceptor layer. Since this CDK2 inhibitor showed activity also on other CDKs, in order to investigate the mechanism of toxicity of this compound, we isolated cones and rods from the retina of normal monkey and humans by Laser Capture Microdissection. Using Real-Time PCR we first measured the expression of cyclin dependent protein-kinases (CDK)1, 2, 4, 5, Glycogen synthase kinase 3β (GSK3β) and microtubule associated protein TAU. We additionally verified the presence of these proteins in monkey eye sections by immunohistochemistry and immunofluorescence analysis and afterwards quantified GSK3β, phospho-GSK3β and TAU by Reverse Phase Protein Microarrays. With this work we demonstrate how complementary gene expression and protein-based technologies constitute a powerful tool for the understanding of the molecular mechanism of a CDK2 inhibitor induced toxicity. Moreover, this investigative approach is helpful to better understand and characterize the mechanism of species-specific toxicities and further support a rational, molecular mechanism-based safety assessment in humans.

Currently, several protein kinase-modulating compounds have received market approval across a range of diverse therapeutic indications. Furthermore, a large number of chemical and biological protein kinase-modulating compounds are undergoing testing at the preclinical and clinical level. Protein kinases are both major pharmacological targets and diagnostically useful. Progression of kinase modulators toward clinically viable therapies is aided by a reversible mechanism of action, short treatment durations and patient-compliant administration routes. However, the physiological role and essential functional activity of protein kinases in many organs and tissues complicates, to different extents, the development of useful, highly potent protein kinase modulators. In this review, we will highlight common problems in the development of these compounds and lessons learned from the extensive preclinical and clinical characterization of some key protein kinase modulators, some of which have either entered and successfully completed clinical trials or have been abandoned as a consequence of unacceptable toxicity issues. We will ultimately explore how molecular profiling tools combined with histopathological endpoints can be adopted to address and further understand these toxicities in humans and understand their relevance and characterization when identified during early animal experiments.

Control by Na/H antiporters of sodium/proton concentration and cell volume is crucial for the viability of all cells. Adaptation to high salinity and/or extreme pH in plants and bacteria or in human heart muscles requires the action of Na/H antiporters. Their activity is tightly controlled by pH. We determined the first crystal structure of a member of this family of secondary transporters, namely the structure of NhaA [1]. The latter is the main antiporter of Escherichia coli and many enterobacteria. An iterative process of optimizing protein and crystal quality was pursued to permit structure determination. The protein has been crystallized at low pH, i.e. NhaA is present in its pH down-regulated conformation. A negatively charged ion funnel opens to the cytoplasm and ends in the middle of themembrane at the putative ion-binding site. There, a unique assembly of two pairs of short helices connected by crossed, extended chains creates a balanced electrostatic environment. Based on the structure and on previous biochemical and genetic data, a working model for the pH-regulated ion translocation has been suggested. Binding of charged substrates causes electric imbalance inducingmovements, which allow for a rapid alternating access mechanism. This ion exchange machinery is regulated by a conformational change elicited by a pH signal perceived at the cytoplasmic funnel entry.

The Na+/H+ antiporter NhaA is the main Na+ extrusion system in E. coli. Using direct current measurements combined with a solid supported membrane (SSM), we obtained electrical data of the function of NhaA purified and reconstituted in liposomes. These measurements demonstrate NhaA's electrogenicity, its specificity for Li+ and Na+ and its pronounced pH dependence in the range pH 6.5-8.5. The mutant G338S, in contrast, presents a pH independent profile, as reported previously. A complete right-side-out orientation of the NhaA antiporter within the proteoliposomal membrane was determined using a NhaA-specific antibody based ELISA assay. This allowed for the first time the investigation of NhaA in the passive downhill uptake mode corresponding to the transport of Na+ from the periplasmic to the cytoplasmic side of the membrane. In this mode, the transporter has kinetic properties differing significantly from those of the previously investigated efflux mode. The apparent Km values were 11 mM for Na+ and 7.3 mM for Li+ at basic pH and 180 mM for Na+ and 50 mM for Li+ at neutral pH. The data demonstrate that in the passive downhill uptake mode pH regulation of the carrier affects both apparent Km as well as turnover (Vmax).

The control by Na + /H + antiporters of sodium/proton concentration and cell volume is crucial for the viability of all cells. Adaptation to high salinity and/or extreme pH in plants and bacteria or in human heart muscles requires the action of Na + /H + antiporters. Their activity is tightly controlled by pH. Here we present the crystal structure of pH-downregulated NhaA, the main antiporter of Escherichia coli and many enterobacteria. A negatively charged ion funnel opens to the cytoplasm and ends in the middle of the membrane at the putative ion-binding site. There, a unique assembly of two pairs of short helices connected by crossed, extended chains creates a balanced electrostatic environment. We propose that the binding of charged substrates causes an electric imbalance, inducing movements, that permit a rapid alternating-access mechanism. This ion-exchange machinery is regulated by a conformational change elicited by a pH signal perceived at the entry to the cytoplasmic funnel.