Student Recital (December 12, 2012)

What Good Would the Moon Be / Kurt Weill Jordan Ennis, soprano Concerto / Nikolai Rimsky-Korsakov Andante Cantabile Sage Lewis, trombone Du bist wie eine Blume, Op. 25, No. 24 / Robert Schumann Long Time Ago / Aaron Copland Richard Moran, tenor Estudios Sencillos / Leo Brouwer Five Studies / Fredric Hand Nolan Driscoll, guitar Study No. 7 in A minor / Matteo Carcassi Jeremy Place, guitar Concerto no. 3 in G, K. 216 / Wolfgang Amadeus Mozart Allegro Carla Mason, violin Etude No. 14 in D Major / M. Carcassi Etude No. 1 in E minor / Heitor Villa-Lobos James Davidson, guitar Prelude No. 4 / H. Villa-Lobos Mark Gavin, guitar Bois Epais / Jean-Baptiste Lully The Call / Ralph Vaughan Williams Justine Smigel, mezzo-soprano Allegro, Op. 20 / Joseph Hector Fiocco Gail Colombo, violin Improvisation II et III pour Saxophone Alto Seul / Ryo Noda III Chelsea Fisk, alto saxophonehttps://vc.bridgew.edu/student_concerts/1034/thumbnail.jp

Systematic genomic and translational efficiency studies of uveal melanoma

To further our understanding of the somatic genetic basis of uveal melanoma, we sequenced the protein-coding regions of 52 primary tumors and 3 liver metastases together with paired normal DNA. Known recurrent mutations were identified in GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. The role of mutated EIF1AX was tested using loss of function approaches including viability and translational efficiency assays. Knockdown of both wild type and mutant EIF1AX was lethal to uveal melanoma cells. We probed the function of N-terminal tail EIF1AX mutations by performing RNA sequencing of polysome-associated transcripts in cells expressing endogenous wild type or mutant EIF1AX. Ribosome occupancy of the global translational apparatus was sensitive to suppression of wild type but not mutant EIF1AX. Together, these studies suggest that cells expressing mutant EIF1AX may exhibit aberrant translational regulation, which may provide clonal selective advantage in the subset of uveal melanoma that harbors this mutation

Genomic and Functional Studies of Uveal Melanoma

Uveal melanoma (UM) is a rare form of melanoma that is lethal once metastatic. Primary tumors in the iris, ciliary body, and choroid of the eye metastasize in 50% of patients, despite effective treatment of the initial tumor. The majority of uveal melanomas harbor activating mutations in GNAQ or GNA11, which relay signaling to downstream effectors including protein kinase C (PKC) and the mitogen-activated protein kinase (MAPK) signaling pathway (RAF-MEK-ERK). Both PKC and MEK inhibitors are currently in clinical trials, however, MEK inhibition alone is insufficient to improve overall survival. These observations highlight a need to identify new drug targets for the design of novel therapies including combinations. To uncover novel UM biology and nominate strategies for combination therapy, genomic and functional genomic approaches were applied. Whole exome sequencing of primary and metastatic tumors identified somatic genetic alterations that drive tumorigenesis. Recurrent mutations in GNAQ, GNA11, BAP1, SF3B1, and EIF1AX were confirmed. Mutations in potential drivers of metastasis, SMARCA4 and IQGAP1, were also identified. Furthermore, the function of N-terminal tail mutations in the translation initiation factor EIF1AX was probed using loss of function studies to assess both viability and mRNA regulation at the level of translation. Upon EIF1AX knockdown, the efficiency of ribosomal protein translation was reduced in wild type, but not mutant cells. Deregulated translation may play an important role in UM tumorigenesis. To identify putative co-targets of MEK and PKC inhibitors, genome-scale RNA interference drug enhancer screens were performed. These screens nominated several novel genes and pathways for further study in UM. In particular, the mitochondrial folate pathway enzyme MTHFD2 was identified as a novel PKC inhibitor sensitizer. The strongest MEK inhibitor enhancer was the MAPK pathway member, BRAF. Indeed, targeting multiple nodes of the MAPK signaling pathway achieved stronger pathway suppression and synergistic effects. Co-inhibition of RAF/MEK or MEK/ERK may warrant clinical investigation in patients. Overall, these studies provide a foundation for our understanding of UM genomics and combination therapy opportunities. Several novel avenues for future study of UM biology and co-dependencies are uncovered. Translation of these findings into clinical studies will be of the utmost importance.Medical Science

The genetic landscape of clinical resistance to RAF inhibition in metastatic melanoma

Most patients with BRAF(V600)-mutant metastatic melanoma develop resistance to selective RAF kinase inhibitors. The spectrum of clinical genetic resistance mechanisms to RAF inhibitors and options for salvage therapy are incompletely understood. We performed whole-exome sequencing on formalin-fixed, paraffin-embedded tumors from 45 patients with BRAF(V600)-mutant metastatic melanoma who received vemurafenib or dabrafenib monotherapy. Genetic alterations in known or putative RAF inhibitor resistance genes were observed in 23 of 45 patients (51%). Besides previously characterized alterations, we discovered a "long tail" of new mitogen-activated protein kinase (MAPK) pathway alterations (MAP2K2, MITF) that confer RAF inhibitor resistance. In three cases, multiple resistance gene alterations were observed within the same tumor biopsy. Overall, RAF inhibitor therapy leads to diverse clinical genetic resistance mechanisms, mostly involving MAPK pathway reactivation. Novel therapeutic combinations may be needed to achieve durable clinical control of BRAF(V600)-mutant melanoma. Integrating clinical genomics with preclinical screens may model subsequent resistance studies

The Genetic Landscape of Clinical Resistance to RAF Inhibition in Metastatic Melanoma

Most patients with BRAF(V600)-mutant metastatic melanoma develop resistance to selective RAF kinase inhibitors. The spectrum of clinical genetic resistance mechanisms to RAF inhibitors and options for salvage therapy are incompletely understood. We performed whole-exome sequencing on formalin-fixed, paraffin-embedded tumors from 45 patients with BRAF(V600)-mutant metastatic melanoma who received vemurafenib or dabrafenib monotherapy. Genetic alterations in known or putative RAF inhibitor resistance genes were observed in 23 of 45 patients (51%). Besides previously characterized alterations, we discovered a “long tail” of new mitogen-activated protein kinase (MAPK) pathway alterations (MAP2K2, MITF) that confer RAF inhibitor resistance. In three cases, multiple resistance gene alterations were observed within the same tumor biopsy. Overall, RAF inhibitor therapy leads to diverse clinical genetic resistance mechanisms, mostly involving MAPK pathway reactivation. Novel therapeutic combinations may be needed to achieve durable clinical control of BRAF(V600)-mutant melanoma. Integrating clinical genomics with preclinical screens may model subsequent resistance studies. SIGNIFICANCE: The use of RAF inhibitors for BRAF(V600)-mutant metastatic melanoma improves patient outcomes, but most patients demonstrate early or acquired resistance to this targeted therapy. We reveal the genetic landscape of clinical resistance mechanisms to RAF inhibitors from patients using whole-exome sequencing, and experimentally assess new observed mechanisms to define potential subsequent treatment strategies. (C)2013 AACR

EIF1AX-regulated growth and translation in uveal melanoma.

<p><b>(A)</b> Distribution of <i>EIF1AX</i> mutations observed in cohort of 52 uveal melanomas in comparison to other cancer types (as reported by <a href="http://www.tumorportal.org" target="_blank">http://www.tumorportal.org</a>). <b>(B)</b> <i>EIF1AX</i> wild type (WT) or mutant (MUT) uveal melanoma cells were infected with <i>EIF1AX</i> or control shRNAs and cell viability was determined after 6 days using MTS. Percent growth is relative to shLuc-expressing cells. Error bars represent SD of mean from 3 independent experiments. <b>(C)</b> Immunoblot analysis of EIF1AX protein levels in shRNA-expressing cells. <b>(D)</b> Polysome profiles of cell lines expressing shRNAs against <i>EIF1AX</i> and <i>Luciferase</i>.</p

Somatic mutations in primary and metastatic uveal melanoma.

<p><b>(A)</b> The number of synonymous and nonsynonymous mutations per megabase of DNA sequence for 52 samples, arranged in columns. <b>(B)</b> Mutations in recurrently mutated genes are color-coded and ordered by significance. <b>(C)</b> Boxplots represent the distributions of allelic fractions observed per sample where the thick line represents 25-75^th percentile, and thin line 5-95^th. <b>(D)</b> The percentage of tumor cells (CCF) harboring a given mutation in the primary tumor in comparison to a metastatic liver sample from the same patient (UM45). <b>(E)</b> As in (D), but comparing a pre-treatment metastatic tumor sample to a post-treatment metastasis (Trio 2).</p

Decreased EIF1AX expression impairs translation of protein synthesis machinery in wildtype, but not mutated setting.

<p><b>(A)</b> Principal component analysis depicts 4 color-coded clusters of 141 genes. <b>(B)</b> The trend in translational efficiency is depicted for each cluster in cells expressing control shRNAs (CN) or <i>EIF1AX</i> shRNAs (KD). Each line represents a different gene. Ribosomal protein genes are highlighted in red. Translational efficiency was calculated as polysome CPM / total CPM. <b>(C)</b> Boxplots demonstrate the distribution of the translational efficiencies of 78 ribosomal proteins in cells as in (B).</p

A Landscape of Driver Mutations in Melanoma

Despite recent insights into melanoma genetics, systematic surveys for driver mutations are challenged by an abundance of passenger mutations caused by carcinogenic UV light exposure. We developed a permutation-based framework to address this challenge, employing mutation data from intronic sequences to control for passenger mutational load on a per gene basis. Analysis of large-scale melanoma exome data by this approach discovered six novel melanoma genes (PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2), three of which—RAC1, PPP6C, and STK19—harbored recurrent and potentially targetable mutations. Integration with chromosomal copy number data contextualized the landscape of driver mutations, providing oncogenic insights in BRAF- and NRAS-driven melanoma as well as those without known NRAS/BRAF mutations. The landscape also clarified a mutational basis for RB and p53 pathway deregulation in this malignancy. Finally, the spectrum of driver mutations provided unequivocal genomic evidence for a direct mutagenic role of UV light in melanoma pathogenesis.National Human Genome Research Institute (U.S.) (Large Scale Sequencing Program Grant U54 HG003067)Melanoma Research AllianceNational Cancer Institute (U.S.) (Support Grant CA-16672