Routine Multiplex Mutational Profiling of Melanomas Enables Enrollment in Genotype-Driven Therapeutic Trials

Purpose: Knowledge of tumor mutation status is becoming increasingly important for the treatment of cancer, as mutation-specific inhibitors are being developed for clinical use that target only sub-populations of patients with particular tumor genotypes. Melanoma provides a recent example of this paradigm. We report here development, validation, and implementation of an assay designed to simultaneously detect 43 common somatic point mutations in 6 genes (BRAF, NRAS, KIT, GNAQ, GNA11, and CTNNB1) potentially relevant to existing and emerging targeted therapies specifically in melanoma. Methods: The test utilizes the SNaPshot method (multiplex PCR, multiplex primer extension, and capillary electrophoresis) and can be performed rapidly with high sensitivity (requiring 5–10% mutant allele frequency) and minimal amounts of DNA (10–20 nanograms). The assay was validated using cell lines, fresh-frozen tissue, and formalin-fixed paraffin embedded tissue. Clinical characteristics and the impact on clinical trial enrollment were then assessed for the first 150 melanoma patients whose tumors were genotyped in the Vanderbilt molecular diagnostics lab. Results: Directing this test to a single disease, 90 of 150 (60%) melanomas from sites throughout the body harbored a mutation tested, including 57, 23, 6, 3, and 2 mutations in BRAF, NRAS, GNAQ, KIT, and CTNNB1, respectively. Among BRAF V600 mutations, 79%, 12%, 5%, and 4% were V600E, V600K, V600R, and V600M, respectively. 23 of 54 (43%) patients with mutation harboring metastatic disease were subsequently enrolled in genotype-driven trials. Conclusion: We present development of a simple mutational profiling screen for clinically relevant mutations in melanoma. Adoption of this genetically-informed approach to the treatment of melanoma has already had an impact on clinical trial enrollment and prioritization of therapy for patients with the disease

IQGAP1 is a novel CXCR2-interacting protein and essential component of the "chemosynapse".

Chemotaxis is essential for a number of physiological processes including leukocyte recruitment. Chemokines initiate intracellular signaling pathways necessary for chemotaxis through binding seven transmembrane G protein-couple receptors. Little is known about the proteins that interact with the intracellular domains of chemokine receptors to initiate cellular signaling upon ligand binding. CXCR2 is a major chemokine receptor expressed on several cell types, including endothelial cells and neutrophils. We hypothesize that multiple proteins interact with the intracellular domains of CXCR2 upon ligand stimulation and these interactions comprise a "chemosynapse", and play important roles in transducing CXCR2 mediated signaling processes.In an effort to define the complex of proteins that assemble upon CXCR2 activation to relay signals from activated chemokine receptors, a proteomics approach was employed to identify proteins that co-associate with CXCR2 with or without ligand stimulation. The components of the CXCR2 "chemosynapse" are involved in processes ranging from intracellular trafficking to cytoskeletal modification. IQ motif containing GTPase activating protein 1 (IQGAP1) was among the novel proteins identified to interact directly with CXCR2. Herein, we demonstrate that CXCR2 co-localizes with IQGAP1 at the leading edge of polarized human neutrophils and CXCR2 expressing differentiated HL-60 cells. Moreover, amino acids 1-160 of IQGAP1 directly interact with the carboxyl-terminal domain of CXCR2 and stimulation with CXCL8 enhances IQGAP1 association with Cdc42.Our studies indicate that IQGAP1 is a novel essential component of the CXCR2 "chemosynapse"

VASP is a CXCR2-interacting protein that regulates CXCR2-mediated polarization and chemotaxis

Chemotaxis regulates the recruitment of leukocytes, which is integral for a number of biological processes and is mediated through the interaction of chemokines with seven transmembrane G-protein-coupled receptors. Several studies have indicated that chemotactic signaling pathways might be activated via G-protein-independent mechanisms, perhaps through novel receptor-interacting proteins. CXCR2 is a major chemokine receptor expressed on neutrophils. We used a proteomics approach to identify unique ligand-dependent CXCR2-interacting proteins in differentiated neutrophil-like HL-60 cells. Using this approach, vasodilator-stimulated phosphoprotein (VASP) was identified as a CXCR2-interacting protein. The interaction between CXCR2 and VASP is direct and enhanced by CXCL8 stimulation, which triggers VASP phosphorylation via PKA- and PKCδ-mediated pathways. The interaction between CXCR2 and VASP requires free F-actin barbed ends to recruit VASP to the leading edge. Finally, knockdown of VASP in HL-60 cells results in severely impaired CXCR2-mediated chemotaxis and polarization. These data provide the first demonstration that direct interaction of VASP with CXCR2 is essential for proper CXCR2 function and demonstrate a crucial role for VASP in mediating chemotaxis in leukocytes

The Lymphotoxin-β Receptor Is an Upstream Activator of NF-κB-mediated Transcription in Melanoma Cells*

The pleiotropic transcription factor nuclear factor-κB (NF-κB (p50/p65)) regulates the transcription of genes involved in the modulation of cell proliferation, apoptosis, and oncogenesis. Furthermore, a host of solid and hematopoietic tumor types exhibit constitutive activation of NF-κB (Basseres, D. S., and Baldwin, A. S. (2006) 25, 6817-6830). However, the mechanism for this constitutive activation of NF-κB has not been elucidated in the tumors. We have previously shown that NF-κB-inducing kinase (NIK) protein and its association with Inhibitor of κB kinase αβ are elevated in melanoma cells compared with their normal counterpart, leading to constitutive activation of NF-κB. Moreover, expression of dominant negative NIK blocked this base-line NF-κB activity in melanoma cells. Of the three receptors that require NIK for activation of NF-κB, only the lymphotoxin-β receptor (LTβ-R) is expressed in melanoma. We show in this manuscript that for melanoma there is a strong relationship between expression of the LTβ-R and constitutive NF-κB transcriptional activity. Moreover, we show that activation of the LTβ-R can drive NF-κB activity to regulate gene expression that leads to enhanced cell growth. The inhibition by LTβ-R shRNA resulted in decreased NF-κB promoter activity, decreased growth, and decreased invasiveness as compared with control. These results indicate that the LTβ-R constitutively induces NF-κB activation, and this event may be associated with autonomous growth of melanoma cells

Melanoma SNaPshot screen (v1.0).

<p>A, five multiplexed panels can detect the mutational status of twenty gene loci. Each peak color represents a particular nucleotide at that locus. The gene name, amino acid, and nucleotide are labeled above each peak. An “(R)” after the nucleotide denotes a reverse extension primer. B, pan-positive control for melanoma SNaPshot screen. Peaks are labeled as described in A. C, SNaPshot sensitivity measurement using cell line DNA carrying known mutations. Numbers indicate the arbitrary fluorescence units of WT (panel 1: green, panels 2, 3: blue) and mutant (panel 1: blue, panels 2, 3: green) peaks. Solid arrows indicate mutant peaks and dotted arrows show background peaks. Background peaks in the negative controls (far right panel) are indicated by their peak height and a star (*).</p

Spectrum of mutations in the first 150 melanomas genotyped in the molecular diagnostic lab.

*<p>CSD – chronic sun damage.</p>#<p>This CTNNB1 mutation (CTNNB1 S45P) occurred concurrently with an NRAS Q61L mutation.</p

The SNaPshot melanoma screen can detect 43 point mutations in 6 genes relevant to targeted therapy in melanoma.

*<p>SNaPshot assays in bold text were previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035309#pone.0035309-DiasSantagata1" target="_blank">[23]</a>.</p