Modulation of Plasma Metabolite Biomarkers of the MAPK Pathway with MEK Inhibitor RO4987655: Pharmacodynamic and Predictive Potential in Metastatic Melanoma.

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.

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues.

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.

Phase I expansion and pharmacodynamic study of the oral MEK inhibitor RO4987655 (CH4987655) in selected patients with advanced cancer with RAS-RAF mutations.

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.

Evaluation of efficacy of a new MEK inhibitor, RO4987655, in human tumor xenografts by [(18)F] FDG-PET imaging combined with proteomic approaches.

BACKGROUND: 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 [(18)F] FDG-PET imaging and proteomics technology. METHODS: [(18)F] FDG uptake was studied in human lung carcinoma xenografts from day 0 to day 9 of RO4987655 therapy using microPET Focus 120 (CTI Concorde Microsystems, Knoxville, TN, USA). The expression levels of GLUT1 and hexokinase 1 were examined using semi-quantitative fluorescent immunohistochemistry (fIHC). The in vivo effects of RO4987655 on MAPK/PI3K pathway components were assessed by reverse phase protein arrays (RPPA). RESULTS: We have observed modest metabolic decreases in tumor [(18)F] FDG uptake after MEK inhibition by RO4987655 as early as 2 h post-treatment. The greatest [(18)F] FDG decreases were found on day 1, followed by a rebound in [(18)F] FDG uptake on day 3 in parallel with decreasing tumor volumes. Molecular analysis of the tumors by fIHC did not reveal statistically significant correlations of GLUT1 and hexokinase 1 expressions with the [(18)F] FDG changes. RPPA signaling response profiling revealed not only down-regulation of pERK1/2, pMKK4, and pmTOR on day 1 after RO4987655 treatment but also significant up-regulation of pMEK1/2, pMEK2, pC-RAF, and pAKT on day 3. The up-regulation of these markers is interpreted to be indicative of a reactivation of the MAPK and activation of the compensatory PI3K pathway, which can also explain the rebound in [(18)F] FDG uptake following MEK inhibition with RO4987655 in the K-ras-mutated human tumor xenografts. CONCLUSIONS: We have performed the first preclinical evaluation of a new MEK inhibitor, RO4987655, using a combination of [(18)F] FDG-PET imaging and molecular proteomics. These results provide support for using preclinical [(18)F] FDG-PET imaging in early, non-invasive monitoring of the effects of MEK and perhaps other Ras/MAPK signaling pathway inhibitors, which should facilitate a wider implementation of clinical [(18)F] FDG-PET to optimize their clinical use.

Multiple protein analysis of formalin-fixed and paraffin-embedded tissue samples with reverse phase protein arrays.

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 markers in early research and companion diagnostic developments in oncology.

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.

Expression of serine/threonine protein-kinases and related factors in normal monkey and human retinas: the mechanistic understanding of a CDK2 inhibitor induced retinal toxicity.

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 3beta (GSK3beta) and microtubule associated protein TAU. We additionally verified the presence of these proteins in monkey eye sections by immuno-histochemistry and immunofluorescence analysis and afterwards quantified GSK3beta, phospho-GSK3beta 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.

Assessing and managing toxicities induced by kinase inhibitors.

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.

Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH.

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.

Structural basis of tyrosine sulfation and VH-gene usage in antibodies that recognize the HIV type 1 coreceptor-binding site on gp120.

The conserved surface of the HIV-1 gp120 envelope glycoprotein that binds to the HIV-1 coreceptor is protected from humoral recognition by multiple layers of camouflage. Here we present sequence and genomic analyses for 12 antibodies that pierce these defenses and determine the crystal structures of 5. The data reveal mechanisms and atomic-level details for three unusual immune features: posttranslational mimicry of coreceptor by tyrosine sulfation of antibody, an alternative molecular mechanism controlling such sulfation, and highly selective V(H)-gene usage. When confronted by extraordinary viral defenses, the immune system unveils novel adaptive capabilities, with tyrosine sulfation enhancing the vocabulary of antigen recognition.

Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1.

Anti-human immunodeficiency virus type 1 (HIV-1) antibodies whose binding to gp120 is enhanced by CD4 binding (CD4i antibodies) are generally considered nonneutralizing for primary HIV-1 isolates. However, a novel CD4i-specific Fab fragment, X5, has recently been found to neutralize a wide range of primary isolates. To investigate the precise nature of the extraordinary neutralizing ability of Fab X5, we evaluated the abilities of different forms (immunoglobulin G [IgG], Fab, and single-chain Fv) of X5 and other CD4i monoclonal antibodies to neutralize a range of primary HIV-1 isolates. Our results show that, for a number of isolates, the size of the neutralizing agent is inversely correlated with its ability to neutralize. Thus, the poor ability of CD4i-specific antibodies to neutralize primary isolates is due, at least in part, to steric factors that limit antibody access to the gp120 epitopes. Studies of temperature-regulated neutralization or fusion-arrested intermediates suggest that the steric effects are important in limiting the binding of IgG to the viral envelope glycoproteins after HIV-1 has engaged CD4 on the target cell membrane. The results identify hurdles in using CD4i epitopes as targets for antibody-mediated neutralization in vaccine design but also indicate that the CD4i regions could be efficiently targeted by small molecule entry inhibitors.

Tyrosine sulfation of human antibodies contributes to recognition of the CCR5 binding region of HIV-1 gp120.

Sulfated tyrosines at the amino terminus of the principal HIV-1 coreceptor CCR5 play a critical role in its ability to bind the HIV-1 envelope glycoprotein gp120 and mediate HIV-1 infection. Here, we show that a number of human antibodies directed against gp120 are tyrosine sulfated at their antigen binding sites. Like that of CCR5, antibody association with gp120 is dependent on sulfate moieties, enhanced by CD4, and inhibited by sulfated CCR5-derived peptides. Most of these antibodies preferentially associate with gp120 molecules of CCR5-utilizing (R5) isolates and neutralize primary R5 isolates more efficiently than laboratory-adapted isolates. These studies identify a distinct subset of CD4-induced HIV-1 neutralizing antibodies that closely emulate CCR5 and demonstrate that tyrosine sulfation can contribute to the potency and diversity of the human humoral response.

Monoclonal antibodies for the structural analysis of the Na+/H+ antiporter NhaA from Escherichia coli.

Since their advent some 25 years ago, monoclonal antibodies have developed into powerful tools for structural and functional analysis of their cognate antigens. Together with the respective antigen binding fragments, antibodies offer exclusive capacities in detection, characterization, purification and functional assays for every given ligand. Antibody-fragment mediated crystallization represents a major advance in determining the three-dimensional structure of membrane-bound protein complexes. In this review, we focus on the methods used to generate monoclonal antibodies against the NhaA antiporter from Escherichia coli as a paradigm of secondary transporters. We describe examples on how antibodies are helpful in understanding structure and function relationships for this important class of integral membrane proteins. The generated conformation-specific antibody fragments are highly valuable reagents for co-crystallization attempts and structure determination of the antiporter.

CD4 binding site antibodies inhibit human immunodeficiency virus gp120 envelope glycoprotein interaction with CCR5.

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior glycoprotein is conformationally flexible. Upon binding the host cell receptor, CD4, gp120 assumes a conformation that is able to bind the chemokine receptors CCR5 or CXCR4, which act as coreceptors for the virus. CD4-binding-site (CD4BS) antibodies are neutralizing antibodies elicited during natural infection that are directed against gp120 epitopes that overlap the binding site for CD4. Recent studies (S. H. Xiang et al., J. Virol. 76:9888-9899, 2002) suggest that CD4BS antibodies recognize conformations of gp120 distinct from the CD4-bound conformation. This predicts that the binding of CD4BS antibodies will inhibit chemokine receptor binding. Here, we show that Fab fragments and complete immunoglobulin molecules of CD4BS antibodies inhibit CD4-independent gp120 binding to CCR5 and cell-cell fusion mediated by CD4-independent HIV-1 envelope glycoproteins. These results are consistent with a model in which the binding of CD4BS antibodies limits the ability of gp120 to assume a conformation required for coreceptor binding.

HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites.

The ability of human immunodeficiency virus (HIV-1) to persist and cause AIDS is dependent on its avoidance of antibody-mediated neutralization. The virus elicits abundant, envelope-directed antibodies that have little neutralization capacity. This lack of neutralization is paradoxical, given the functional conservation and exposure of receptor-binding sites on the gp120 envelope glycoprotein, which are larger than the typical antibody footprint and should therefore be accessible for antibody binding. Because gp120-receptor interactions involve conformational reorganization, we measured the entropies of binding for 20 gp120-reactive antibodies. Here we show that recognition by receptor-binding-site antibodies induces conformational change. Correlation with neutralization potency and analysis of receptor-antibody thermodynamic cycles suggested a receptor-binding-site 'conformational masking' mechanism of neutralization escape. To understand how such an escape mechanism would be compatible with virus-receptor interactions, we tested a soluble dodecameric receptor molecule and found that it neutralized primary HIV-1 isolates with great potency, showing that simultaneous binding of viral envelope glycoproteins by multiple receptors creates sufficient avidity to compensate for such masking. Because this solution is available for cell-surface receptors but not for most antibodies, conformational masking enables HIV-1 to maintain receptor binding and simultaneously to resist neutralization.

The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120.

We have analyzed the unique epitope for the broadly neutralizing human monoclonal antibody (MAb) 2G12 on the gp120 surface glycoprotein of human immunodeficiency virus type 1 (HIV-1). Sequence analysis, focusing on the conservation of relevant residues across multiple HIV-1 isolates, refined the epitope that was defined previously by substitutional mutagenesis (A. Trkola, M. Purtscher, T. Muster, C. Ballaun, A. Buchacher, N. Sullivan, K. Srinivasan, J. Sodroski, J. P. Moore, and H. Katinger, J. Virol. 70:1100-1108, 1996). In a biochemical study, we digested recombinant gp120 with various glycosidase enzymes of known specificities and showed that the 2G12 epitope is lost when gp120 is treated with mannosidases. Computational analyses were used to position the epitope in the context of the virion-associated envelope glycoprotein complex, to determine the variability of the surrounding surface, and to calculate the surface accessibility of possible glycan- and polypeptide-epitope components. Together, these analyses suggest that the 2G12 epitope is centered on the high-mannose and/or hybrid glycans of residues 295, 332, and 392, with peripheral glycans from 386 and 448 on either flank. The epitope is mannose dependent and composed primarily of carbohydrate, with probably no direct involvement of the gp120 polypeptide surface. It resides on a face orthogonal to the CD4 binding face, on a surface proximal to, but distinct from, that implicated in coreceptor binding. Its conservation amidst an otherwise highly variable gp120 surface suggests a functional role for the 2G12 binding site, perhaps related to the mannose-dependent attachment of HIV-1 to DC-SIGN or related lectins that facilitate virus entry into susceptible target cells.

High level production of functional antibody Fab fragments in an oxidizing bacterial cytoplasm.

The antigen-binding fragments (Fab) of antibodies are powerful tools in clinical therapy, molecular diagnostics and basic research. However, their principal applications require pure recombinant molecules in large amounts, which are challenging to obtain. Severe limitations in yield, folding and functionality are commonly encountered in bacterial production of Fab fragments. Secretion into the oxidizing periplasm generally results in low yield, whereas expression in the reducing cytoplasmic environment produces unfolded or non-functional protein. We hypothesized that an impaired reducing environment of the cytoplasm would permit correctly folded, functional cytoplasmic expression of Fabs with high yield. We used the Escherichia coli strain FA113, which has no activity of both thioredoxin and glutathione reductase, and thus has an oxidizing cytoplasmic environment. With the newly constructed vector pFAB1 we tested the cytoplasmic expression of two Fab fragments, which recognize the integral membrane protein NhaA, a bacterial Na(+)/H(+) antiporter. These antibodies differ in terms of DNA sequence and stability. Both antibody fragments were produced to very high yields (10-30 mg/l from bacterial cultures at an A(600 nm)=1.2-1.3). This is a factor 50-250 times higher than any other reported over-expression strategy for Fab fragments and currently represents the highest production rate ever been reported for antibody Fab fragments in bacteria grown to similar cell densities. The fragments are fully functional and can be efficiently purified by His-tag chromatography. Expression of active Fab fragments in the bacterial cytoplasm unlocks the possibility of using antibody specific targeting in an intracellular environment. Such a capacity opens new perspectives for investigating metabolic and regulatory pathways in vivo and also provides a powerful selection system for functional genomics.