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.
Inhibitor of DNA binding 4 (ID4) is a helix-loop-helix protein that heterodimerizes with basic helix-loop-helix transcription factors inhibiting their function. ID4 expression is important for ...adipogenic differentiation of the 3T3-L1 cell line, and inhibition of ID4 is associated with a concomitant decrease in CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ mRNA and protein expression. Mice with a homozygous deletion of Id4 (Id4−/−) have reduced body fat and gain much less weight compared with wild-type littermates when placed on diets with high fat content. Mouse embryonic fibroblasts (MEFs) isolated from Id4−/− mice have reduced adipogenic potential when compared with wild-type MEFs. In agreement with changes in morphological differentiation, the levels of CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ were also reduced in MEFs from Id4−/− mice. Our results demonstrate the importance of ID4 in adipocyte differentiation and the implications of this regulation for adipose tissue formation.
Abstract
Uveal melanoma (UM) is the most common form of ocular cancer in adults and metastatic disease currently has no effective therapy. Over 80% of UM tumors harbor activating mutations in the ...heterotrimeric G protein α subunits GNAQ or GNA11. These mutations induce protein kinase C (PKC) and the MAP kinase pathway, which signals through RAF, MEK, and ERK. Clinical trials of MEK and PKC inhibitors are underway to treat UM, however, novel therapeutic strategies may be required for effective treatment. To identify rational combination therapies in the context of MEK or PKC inhibition, we performed a pooled, genome-wide synthetic lethal RNA interference screen. Candidate genes whose silencing sensitizes GNAQ/11-mutant UM cells to MEK or PKC inhibition were nominated and further validated across a panel of cell lines using a custom pool of shRNAs. Several genes scored well across all cell lines. The top MEK inhibitor sensitizer was BRAF, thereby highlighting the importance of MAPK signaling in UM. Several non-MAPK pathway genes were also nominated and may represent novel pathways for the development of combination therapies.
Citation Format: Chelsea S. Place, Glenn S. Cowley, David E. Root, Levi A. Garraway. Synthetic lethal RNAi screens to nominate potential therapeutic combinations for uveal melanoma. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3429. doi:10.1158/1538-7445.AM2014-3429
Abstract
Uveal melanoma (UM) is a rare type of melanoma that occurs in the iris, ciliary body, and choroid of the eye. Metastatic disease is typically found in the liver and has no effective ...therapeutic options. To improve our understanding of the genetic drivers of UM, we sequenced the exome of 61 primary tumors and 3 liver metastases, each with matched normal DNA. Consistent with prior studies, the majority of UM tumors harbored mutually exclusive mutations in GNAQ and GNA11. Co-occurring with GNAQ and GNA11 mutations were inactivating mutations throughout the BAP1 coding region as well as recurrent mutations in the splicing factor SF3B1 and the translation initiation factor EIF1AX. Somatic mutations found only in metastatic tumor samples were also identified. The function of mutant EIF1AX was probed using loss of function experiments. Reduction of EIF1AX expression impaired the growth of both wild type and mutant cells. To identify transcripts regulated by EIF1AX at the level of translation, RNA sequencing of polysome-associated mRNAs was performed. Knockdown of wild type, but not mutant EIF1AX reduced the efficiency of ribosomal protein translation. Cancer cells expressing mutant EIF1AX may harbor changes in protein translation, which may be important for tumorigenesis.
Citation Format: Chelsea S. Place, Ivana K. Kim, Bita Esmaeli, Ali Amin-Mansour, Daniel J. Treacy, Scott L. Carter, Eran Hodis, Nikhil Wagle, Sara Seepo, Xiaoxing Yu, Francisca Vazquez, Elizabeth Nickerson, Kristian Cibulskis, Aaron McKenna, Stacey B. Gabriel, Gad Getz, Eliezer M. Van Allen, Levi A. Garraway, Scott E. Woodman. Systematic genomic characterization of uveal melanoma. abstract. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-15.
The declining trend in vehicle emissions has underscored the growing significance of volatile organic compound (VOC) emissions from volatile chemical products (VCPs). However, accurately representing ...VOC chemistry in simplified chemical mechanisms remains challenging due to its chemical complexity including speciation and reactivity. Previous studies have predominantly focused on VOCs from fossil fuel sources, leading to an underrepresentation of VOC chemistry from VCP sources. We developed an integrated chemical mechanism, RACM2B-VCP, that is compatible with WRF-Chem and is aimed at enhancing the representation of VOC chemistry, particularly from VCP sources, within the present urban environment. Evaluation against the Air Quality System (AQS) network data demonstrates that our model configured with RACM2B-VCP reproduces both the magnitude and spatial variability of O3 and PM2.5 in Los Angeles. Furthermore, evaluation against comprehensive measurements of O3 and PM2.5 precursors from the Reevaluating the Chemistry of Air Pollutants in California (RECAP-CA) airborne campaign and the Southwest Urban NOx and VOC Experiment (SUNVEx) ground site and mobile laboratory campaign confirm the model's accuracy in representing NOx and many VOCs and highlight remaining biases. Although there exists an underprediction in the total VOC reactivity of observed VOC species, our model with RACM2B-VCP exhibits good agreement for VOC markers emitted from different sectors, including biogenic, fossil fuel, and VCP sources. Through sensitivity analyses, we probe the contributions of VCP and fossil fuel emissions to total VOC reactivity and O3. Our results reveal that 52 % of the VOC reactivity and 35 % of the local enhancement of MDA8 O3 arise from anthropogenic VOC emissions in Los Angeles. Significantly, over 50 % of this anthropogenic fraction of either VOC reactivity or O3 is attributed to VCP emissions. The RACM2B-VCP mechanism created, described, and evaluated in this work is ideally suited for accurately representing ozone for the right reasons in the present urban environment where mobile, biogenic, and VCP VOCs are all important contributors to ozone formation.
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.
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► Landscape of driver mutations by exon sequencing of 121 melanoma tumor/normal pairs ► Method for detecting genes with driver mutations in high-mutation-rate setting ► PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2 are significantly mutated melanoma genes ► Signature spectrum of UV mutagenesis accounts for 46% of driver mutations found
A statistical approach for analyzing exome sequencing data differentiates between driver mutations and the abundant passenger mutations found in melanoma due to UV light exposure. Analysis of whole-exome sequence data from 121 tumors identifies six new melanoma genes and defines a landscape of driver mutations in this challenging malignancy.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Abstract
Melanoma is an aggressive skin cancer of melanocytic origin characterized by high metastatic potential and mutation rate. Affording a survey of the wide breadth of genomic lesions found in ...melanoma, we present here an analysis of the somatic mutations discovered in the sequenced exomes of 121 melanoma tumor-normal pairs. We identify frequent genomic alterations both in genes previously implicated in melanoma (BRAF, NRAS, TP53, CDKN2A, PTEN) as well as in several genes whose role in melanoma tumorigenesis has not yet been established and thus are of particular interest. To do so we implement a novel method to increase the identification of genes that are significantly recurrently mutated in melanoma in the setting of its exceptionally high mutation rate. A preponderance of C>T transitions (∼85%) in the observed mutational profile reflects a history of DNA damage due to UV radiation, though the majority of somatic mutations in known melanoma genes are not C>T events. Our study broadens understanding of the genomic lesions involved in melanoma tumorigenesis, and we expect our analysis approach to inform future genomic studies of cancer lineages with similarly high mutation rates.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5056. doi:1538-7445.AM2012-5056
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 Sciences
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