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. 2013 Mar;3(3):350-62.
doi: 10.1158/2159-8290.CD-12-0470. Epub 2013 Jan 3.

A genome-scale RNA interference screen implicates NF1 loss in resistance to RAF inhibition (VSports app下载)

Steven R Whittaker  1 , Jean-Philippe Theurillat, Eliezer Van Allen, Nikhil Wagle, Jessica Hsiao, Glenn S Cowley, Dirk Schadendorf, David E Root, Levi A Garraway
Affiliations

A genome-scale RNA interference screen implicates NF1 loss in resistance to RAF inhibition

VSports注册入口 - Steven R Whittaker et al. Cancer Discov. 2013 Mar.

Abstract

RAF inhibitors such as vemurafenib and dabrafenib block BRAF-mediated cell proliferation and achieve meaningful clinical benefit in the vast majority of patients with BRAF(V600E)-mutant melanoma. However, some patients do not respond to this regimen, and nearly all progress to therapeutic resistance. We used a pooled RNA interference screen targeting more than 16,500 genes to discover loss-of-function events that could drive resistance to RAF inhibition. The highest ranking gene was NF1, which encodes neurofibromin, a tumor suppressor that inhibits RAS activity. NF1 loss mediates resistance to RAF and mitogen-activated protein kinase (MAPK) kinase kinase (MEK) inhibitors through sustained MAPK pathway activation. However, cells lacking NF1 retained sensitivity to the irreversible RAF inhibitor AZ628 and an ERK inhibitor. NF1 mutations were observed in BRAF-mutant tumor cells that are intrinsically resistant to RAF inhibition and in melanoma tumors obtained from patients exhibiting resistance to vemurafenib, thus showing the clinical potential for NF1-driven resistance to RAF/MEK-targeted therapies VSports手机版. .

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Conflict of interest statement

Conflicts of interest: L. A. G. is a consultant for Foundation Medicine, Novartis, and Millennium/Takeda, and an equity holder in Foundation Medicine V体育安卓版.

Figures (VSports最新版本)

Figure 1
Figure 1. A genome-wide RNAi pooled screen identifies NF1 as a key determinant of B-RAF inhibitor sensitivity in melanoma cells
(A) Outline of the pooled screening strategy employed in B-RAFV600E A375 melanoma cells. A 90,000 shRNA pooled lentiviral library targeting approximately 16,600 genes was introduced into the cells, selection of infected cells was achieved using 0.5 μg/ml puromycin for 3 d. Cells were then treated with either DMSO or 3 μM PLX4720 for up to 14 population doublings. shRNA sequences were amplified by PCR and the relative abundance of each shRNA determined by Illumina sequencing. (B) Growth of A375 cells infected with approximately 90,000 shRNAs and cultured in the presence of either DMSO or 3 μM PLX4720 for up to 14 population doublings. (C) The number of reads per shRNA was normalized and log2 transformed and shRNA data for 2 DMSO controls and 6 PLX4720-treated replicas was analyzed using a ‘2nd best shRNA’, 2-class comparison of log-fold change (LFC) in RIGER to generate a ranked list of genes that were enriched in the PLX4720-treated cells. The screening hits are visualized by plotting the function y=1/normalized enrichment score (NES). The top 5-ranking candidate genes are indicated. (D) Heat map of shRNA representation across early time point, DMSO-and PLX4720-treated replicas for the screen. shRNAs 39714 and 39717 were enriched across all drug-treated replicates. (E) Validation of shRNA-mediated knockdown of NF1 in A375 cells. A375 cells were infected with shRNAs against luciferase or NF1, after selection in puromycin, cell lysates were made and levels of NF1 protein determined by Western blotting. (F) A375 cells infected with either shLuc or shNF1 were cultured in the presence or absence of 3 μM PLX4720 for 2 weeks. Cells were fixed, then stained with crystal violet and photographed.
Figure 2
Figure 2. Knockdown of NF1 drives resistance to selective B-RAF inhibitors through reactivation of the MAPK pathway
(A) A375, SKMEL28 and UACC62 cells are resistant to PLX4720 following knockdown of NF1. shRNA-infected cells were treated with a 10-point concentration response of the inhibitor for 4 d and cell proliferation determined using cell-titer glo. Cells were also infected with LacZ and KRASG12V-expressing lentivirus to act as negative and positive controls respectively. (B) Loss of NF1 is permissive for MAPK-signaling in the presence of B-RAF inhibitors. shRNA-infected A375, SKMEL28 and UACC62 cells were treated with the indicated concentrations of the inhibitors for 16 h, cell lysates were made and analyzed by Western blotting for the indicated proteins. Cells were also infected with LacZ and KRASG12V-expressing lentivirus to act as negative and positive controls respectively.
Figure 3
Figure 3. Activation of RAS and C-RAF drives resistance to PLX4720
(A) A375 cells were depleted of NF1 using shRNA and RAS-GTP levels in A375 cells were determined by a RAS-GTP affinity pull-down, followed by Western blotting for the indicated proteins. (B) Combinatorial knockdown of NF1 and C-RAF abrogates NF1-mediated resistance to B-RAF inhibition at the level of ERK phosphorylation. A375 cells were infected with NF1 shRNA and treated with either DMSO or PLX4720 for 16 h. Cell lysates were analyzed for the indicated proteins. (C) Combinatorial knockdown of NF1 and C-RAF abrogates NF1-mediated resistance to RAF inhibition. Quantitative analysis of the Western blots from Figure 3B for phospho-ERK normalized to ERK2 (red) and for cyclin D1 normalized to vinculin (green). Data from 3 independent experiments is presented. (D) Combinatorial knockdown of NF1 and C-RAF abrogates NF1-mediated resistance to RAF inhibition. shRNA-infected cells were treated with a 10-point concentration response of the inhibitors for 4 d and cell proliferation determined using cell-titer glo.
Figure 4
Figure 4. Both MEK inhibition and combined RAF/MEK inhibition does not completely reverse NF1-mediated resistance
(A) A375 cells infected with shRNAs targeting NF1 were treated with a 10-point concentration response of AZD6244 for 4 d and cell proliferation determined using cell-titer glo. Cells were also infected with LacZ and KRASG12V-expressing lentivirus to act as negative and positive controls respectively. (B) In parallel to the above, cells were treated with 0.2 μM AZD6244 for 16 h and cell lysates analyzed by Western blotting for the indicated proteins. (C) Combinatorial inhibition of B-RAF and MEK using PLX4720 and AZD6244 does not fully overcome NF1-mediated resistance. A375 cells were infected with the stated shRNAs/ORFs and then exposed to a 10-point concentration response of PLX4720 alone or PLX4720 plus 0.2 μM AZD6244 for 4 d. Cell proliferation was determined by cell-titer glo. (D) Combined B-RAF/MEK inhibition does not fully block NF1-mediated resistance at the level of ERK phosphorylation. A375 cells were infected with shRNAs/ORFs as above and then treated with either DMSO (D), 1 μM PLX4720 (P), 0.2 μM AZD6244 (A) or a combination of PLX4720 and AZD6244 (C) for 16 h. Cell lysates were analyzed by Western blotting for the indicated proteins.
Figure 5
Figure 5. Cells lacking NF1 expression are sensitive to sustained B-RAF/C-RAF inhibition and to ERK inhibition
(A) A375 cells infected with the indicated shRNAs/ORFs were treated with a 10-point concentration response of AZ628 for 4 d and cell proliferation determined using cell-titer glo. (B) In parallel to the above, cells were also treated with 0.2 μM AZ628 for 16 h, cell lysates were analyzed by Western blotting for the indicated proteins. (C) A375 cells infected with the indicated shRNAs/ORFs were treated with a 10-point concentration response of VTX-11e for 4 d and cell proliferation determined using cell-titer glo. (D) In parallel to the above, cells were also treated with 1μM VTX-11e for 16 h, cell lysates were analyzed by Western blotting for the indicated proteins.
Figure 6
Figure 6. Loss of NF1 is observed in some B-RAF-mutant human melanoma and colorectal cell lines
(A) Melanoma and colorectal cancer cell lines from the Broad Cancer Cell Line Encyclopedia were assessed for expression of B-RAFV600E and damaging mutations in NF1. 3 such lines were identified (HS695T, LOXIMVI and RKO, colored red) and analyzed alongside a panel of B-RAFV600E/NF1WT cells (colored blue) for expression of NF1 protein by quantitative Western blotting. A two-tailed t-test was performed to determine if the expression of NF1 protein was significantly different between cells expressing either wild-type or mutant protein. Incomplete suppression of ERK phosphorylation by PLX4720 in NF1-mutant cell lines. Cells were treated with the indicated concentrations of PLX4720 for 16 h, cells were analyzed for ERK2 and phospho-ERK1/2 by In-cell Western. Cells with wild-type NF1 are labeled blue, those with mutant NF1 are labeled red. (B) Reduced expression of NF1 protein is associated with resistance to PLX4720. NF1-wild-type and mutant cell lines were treated with a 10-point titration of PLX4720 for 16 h and ERK phosphorylation was assessed using an in-cell Western assay. Cells with wild-type NF1 are labeled blue, those with mutant NF1 are labeled red. (C) Reduced expression of NF1 protein is associated with resistance to PLX4720. NF1-wild-type and mutant cell lines were treated with a 10-point titration of PLX4720 for 4 d and cell proliferation was assessed using cell-titer glo. Cells with wild-type NF1 are labeled blue, those with mutant NF1 are labeled red. (D) Human B-RAF- and NF1-mutant melanoma and colorectal cancer cell lines display only modest resistance to inhibition of ERK. Cells were exposed to a 10-point titration of the ERK inhibitor VTX-11e for 4 d and cell proliferation was assessed using cell-titer glo. Cells with wild-type NF1 are labeled blue, those with mutant NF1 are labeled red.
Figure 7
Figure 7. Whole-exome sequencing identifies NF1 mutations in melanoma patient tumors exhibiting resistance to vemurafenib
(A) Melanoma patients who progressed on treatment with vemurafenib were biopsied pre- and post-treatment. Whole-exome sequencing was performed and NF1 alterations assessed. The NF1 mutations are listed alongside the functional impact on the expressed protein. Silent mutations were assessed for their potential effects on splicing sites using the human splicing finder. The splicing motif affected by the mutation (in red) is indicated and ‘site broken’ indicates potentially damaging effects on splice site function. (B) Progression free survival data was extrapolated from Chapman et al (2) and the number of patients who progressed per month is in blue, the cumulative number of progressing patients is in gray. NF1 mutations observed in our cohort of patients are overlaid at their respective PFS times. Patients with a PFS of less than three months were nominated for de novo resistance, while those with a PFS of greater than or equal to three months demonstrated acquired resistance. Three NF1 alterations are observed in pre-treatment B-RAF-mutant melanoma patient samples, and all three patients demonstrated de novo resistance. A fourth patient with a PFS of five months exhibited acquired resistance. (C) Integrative Genomics Viewer (48) plot showing an NF1 silent mutation observed in the post-relapse biopsy sample only (c.4023G>A) in a patient with a PFS of five months.
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