Inhibition of PERK-Nrf2 signaling sensitizes de-differentiated cells to chemotherapy.

<p>(a) Schematic of treatment timing and dosage for cell survival experiments described in (b–f). (b) Fraction of cells surviving 2 nM Tax or 30 nM Dox following pretreatment with 1 µM PERKi or DMSO, normalized to individual vehicle-treated controls. (c and d) Fraction of cells surviving 30 nM Dox (c) or 2 nM Tax (d) following pretreatment with 1 µM PERKi or DMSO and rescue with 3 mM NAC, normalized to individual vehicle-treated controls. (e and f) Fraction of cells surviving 30 nM Dox (e) or 2 nM Tax (f) following pretreatment with 1 µM PERKi or DMSO and rescue with 25 µM Oltipraz, normalized to individual vehicle-treated controls. (g) Quantitative RT-PCR analysis of Nrf2 gene expression in cells stably expressing control (shLuc) of Nrf2-specific shRNA (shNrf2 1 and shNrf2 2). Expression is shown normalized to the HMLE-Twist shLuc sample. (h and i) Fraction of control or Nrf2 knockdown cells surviving 30 nM Dox (h) or 2 nM Tax (i) following pretreatment with 1 µM PERKi or DMSO and rescue with 25 µM Oltipraz, normalized to individual vehicle-treated controls. *<i>p</i><0.05. Data are presented as mean ± SEM.</p

De-differentiated cells activate MDR and Nrf2 in the absence of oxidative or chemotherapy stress.

<p>(a) Fraction of HMLE-shGFP or HMLE-Twist cells surviving 3-d treatment with 2 nM Tax or 30 nM Dox, normalized to individual vehicle-treated controls. (b) Flow cytometry quantification of MDR1-mediated efflux ability. HMLE-shGFP or HMLE-Twist cells were loaded with cell-permeable DiOC2(3)-dye and efflux ability measured by loss of fluorescent signal after 1.5 h (efflux) compared to the loading control. (c) Fluorescent microscopy images of relative cellular ROS levels using the mitochondrial superoxide (MitoSOX) probe (red channel) or general oxidative stress (CellROX) probe (red channel) and cell nuclei labeled with DAPI (blue channel). (d) Flow cytometry quantification of MitoSOX fluorescence relative to individual vehicle-treated controls. Cells were treated with 40 µM menadione, 1 µM Dox, 1 µM Tax, or DMSO for 2 h prior to analysis. (e) Fluorescent microscopy images of relative lipid peroxidation levels (green channel) and cell nuclei labeled with DAPI (blue channel). Indicated cells were treated with 100 µM cumene hydroperoxide or DMSO for 2 h prior to analysis. (f) Relative amounts of reduced (GSH) to oxidized (GSSG) glutathione measured by luminescence-based assay. Indicated cells were treated with 40 µM menadione or DMSO for 2 h prior to analysis. Each sample is normalized to HMLE-shGFP DMSO control. (g) Western blot analysis of SOD1, CAT, and β-actin. (h) Overlap of genes >2-fold up-regulated in differentiated cells treated with 40 µM menadione (ROS) for 2 h compared to corresponding DMSO control (blue circle), and de-differentiated cells compared to differentiated cells in the absence of treatment (purple circle). (i) Immunofluorescence microscopy images of Nrf2 localization upon treatment with 40 µM menadione or DMSO for 2 h. Nrf2 protein was indirectly labeled with a secondary Alexa Fluor 488 antibody (green channel) and cell nuclei labeled with DAPI (blue channel). *<i>p</i><0.05. Data are presented as mean ± SEM.</p

Nrf2 is constitutively activated by PERK in de-differentiated cells.

<p>(a) Immunofluorescence microscopy images of phospho-PERK (pPERK) upon treatment with 40 nM thapsigargin (Tg) for 2 h or 1 µM PERK inhibitor (PERKi) for 2 d. pPERK protein was indirectly labeled with a secondary Alexa Fluor 488 antibody (green channel) and cell nuclei labeled with DAPI (blue channel). (b) Immunofluorescence microscopy images of Nrf2 localization upon treatment with 1 µM PERKi or DMSO for 2 d, followed by 40 µM menadione or DMSO for 2 h. Nrf2 protein was indirectly labeled with a secondary Alexa Fluor 488 antibody (green channel) and cell nuclei labeled with DAPI (blue channel). (c) Quantitative RT-PCR analysis of PERK gene expression in cells stably expressing control (shLuc) or PERK-specific shRNA (shPERK 1 and shPERK 2). Expression is shown normalized to the HMLE-Twist shLuc sample. (d) Immunofluorescence microscopy images of Nrf2 localization in cell lines with stable knockdown of PERK compared to control knockdown cells. Nrf2 protein was indirectly labeled with a secondary Alexa Fluor 488 antibody (green channel) and cell nuclei labeled with DAPI (blue channel). Quantification of the number of cells with nuclear versus cytoplasmic Nrf2 localization is shown below. One hundred cells were analyzed per group, and the resulting ratio is normalized to the HMLE-shGFP shLuc group. (e) Western blot analysis of HMOX-1 and β-actin. HMLE-shGFP or HMLE-Twist cells were treated with 1 µM PERKi or DMSO for 2 d, followed by 40 µM menadione or DMSO for 2 h prior to cell lysis. (f) PERK or Nrf2-bound proteins were immunoprecipitated from HMLE-Twist cells, followed by immunoblotting for Nrf2. (g) Nrf2 immunoprecipitation followed by Western blot analysis of pan-phosphorylation. HMLE-Twist cells were treated with 1 µM PERKi or DMSO for 2 d prior to analysis.</p

PERK activates MDR mechanisms in de-differentiated cells.

<p>(a) Fluorescent microscopy images of cellular ROS levels using the MitoSOX probe (red channel) and cell nuclei labeled with DAPI (blue channel). HMLE-shGFP or HMLE-Twist cells were treated with 1 µM PERKi or DMSO for 2 d prior to imaging. Quantification per cell is shown on the right. Each group is normalized to the HMLE-Twist DMSO group. (b) Fluorescent microscopy images of relative lipid peroxidation levels (green channel) and cell nuclei labeled with DAPI (blue channel). HMLE-shGFP or HMLE-Twist cells were treated with 1 µM PERKi or DMSO for 2 d prior to imaging. Quantification per cell is shown on the right. Each group is normalized to the HMLE-shGFP DMSO group. (c) Flow cytometry quantification of ROS buffering by measuring MitoSOX fluorescence relative to individual vehicle-treated controls. HMLE-shGFP or HMLE-Twist cells were treated with 1 µM PERKi or DMSO for 2 d, followed by 40 µM menadione, 1 µM Dox, or DMSO for 2 h prior to analysis. (d) Western blot analysis of SOD1, CAT, and β-actin. HMLE-shGFP or HMLE-Twist cells were treated with 1 µM PERKi or DMSO for 2 d prior to cell lysis. (e) Flow cytometry was utilized to quantitate efflux ability of HMLE-shGFP or HMLE-Twist cells treated with 1 µM PERKi or DMSO for 5 d. Results are shown as percentage of cells with the ability to efflux >50% of loaded DiOC2(3)-dye. (f) Fluorescent microscopy images of cellular ROS levels in luminal and basal breast cancer cell lines using the MitoSOX probe as in (a). Each group is normalized to the MDA.MB.231 DMSO group. (g) Relative amounts of reduced (GSH) to oxidized (GSSG) glutathione. Luminal and basal breast cancer cells were treated with 1 µM PERKi or DMSO for 2 d prior to analysis. Each sample is normalized to the MCF7 DMSO group. *<i>p</i><0.05. Data are presented as mean ± SEM.</p

Breakpoint Analysis of Transcriptional and Genomic Profiles Uncovers Novel Gene Fusions Spanning Multiple Human Cancer Types

<div><p>Gene fusions, like <i>BCR/ABL1</i> in chronic myelogenous leukemia, have long been recognized in hematologic and mesenchymal malignancies. The recent finding of gene fusions in prostate and lung cancers has motivated the search for pathogenic gene fusions in other malignancies. Here, we developed a “breakpoint analysis” pipeline to discover candidate gene fusions by tell-tale transcript level or genomic DNA copy number transitions occurring within genes. Mining data from 974 diverse cancer samples, we identified 198 candidate fusions involving annotated cancer genes. From these, we validated and further characterized novel gene fusions involving <i>ROS1</i> tyrosine kinase in angiosarcoma (<i>CEP85L/ROS1</i>), <i>SLC1A2</i> glutamate transporter in colon cancer (<i>APIP/SLC1A2</i>), <i>RAF1</i> kinase in pancreatic cancer (<i>ATG7/RAF1</i>) and anaplastic astrocytoma (<i>BCL6/RAF1</i>), <i>EWSR1</i> in melanoma (<i>EWSR1/CREM</i>), <i>CDK6</i> kinase in T-cell acute lymphoblastic leukemia (<i>FAM133B/CDK6</i>), and <i>CLTC</i> in breast cancer (<i>CLTC/VMP1</i>). Notably, while these fusions involved known cancer genes, all occurred with novel fusion partners and in previously unreported cancer types. Moreover, several constituted druggable targets (including kinases), with therapeutic implications for their respective malignancies. Lastly, breakpoint analysis identified new cell line models for known rearrangements, including <i>EGFRvIII</i> and <i>FIP1L1/PDGFRA</i>. Taken together, we provide a robust approach for gene fusion discovery, and our results highlight a more widespread role of fusion genes in cancer pathogenesis.</p></div

Discovery and characterization of <i>EWSR1/CREM</i> in melanoma.

<p>(<i>A</i>) Array CGH heatmap displaying intragenic <i>EWSR1</i> breakpoints identified in the SH-4 and CHL-1 melanoma cell lines. (<i>B</i>) Paired-end RNA-seq identification of <i>EWSR1/CREM</i> in CHL-1. Paired-end reads supporting the rearrangement are depicted along with the predicted gene fusion structure. CREM contributes a basic leucine zipper motif (ZIP), while EWSR1 contributes the EWS Activation Domain (EAD). (<i>C</i>) RT-PCR verification of <i>EWSR1/CREM</i> in CHL-1. (<i>D</i>) Quantitative RT-PCR using primers flanking the gene fusion junction verifies <i>EWSR1/CREM</i> knockdown following transfection of an siRNA pool targeting the 3′ end of <i>CREM</i>. (<i>E</i>, <i>F</i>, <i>G</i>) Transfection of CHL-1 with <i>CREM</i>-targeting siRNA pool results in (<i>E</i>) decreased cell proliferation, (<i>F</i>) decreased invasion, and (<i>G</i>) a higher fraction of senescent cells, compared to non-targeting control (NTC). **<i>P</i><0.01 (two-sided Student's t-test).</p

Discovery of new cell line models for the known rearrangements, <i>EGFRvIII</i> and <i>FIP1L1/PDGFRA</i>.

<p>(<i>A</i>) Heatmap depicting genomic breakpoints within <i>EGFR</i> in the glioblastoma cell lines, CAS-1 and DKMG. (<i>B</i>) Identification of <i>EGFRvIII</i> in DKMG cells by paired-end RNA-seq. Paired-end reads supporting the rearrangement are depicted. (<i>C</i>) Verification of <i>EGFRvIII</i> expression by RT-PCR (top panel) and Western blotting (bottom panel) in DKMG. RT-PCR was done using primers flanking the exon 1/exon 8 junction of <i>EGFRvIII</i>, and Western blotting was done using an antibody specific to the EGFRvIII isoform. Control samples include U87 glioblastoma cells without <i>EGFR</i> rearrangement, U87-vIII cells engineered to express exogenous <i>EGFRvIII</i>, and A431 epidermoid carcinoma cells with <i>EGFR</i> amplification. (<i>D</i>) RBA identification of expression-level breakpoint within <i>PDGFRA</i> in SUPT13 T-ALL cells. ***<i>P<10⁻¹¹</i> (Student's t-test). (<i>E</i>) RNA-seq identification of <i>FIP1L1/PDGFRA</i>. (<i>F</i>) RT-PCR validation of <i>FIP1L1/PDGFRA</i> expression in SUPT13. (<i>G</i>) SUPT13 cells are sensitive to imatinib (IC₅₀ = 0.036 µM). K562 is a positive control CML cell line harboring BCR/ABL1 with known sensitivity to imatinib (IC₅₀ = 0.18 µM).</p

Breakpoint analysis for discovering novel cancer gene rearrangements.

<p>Schematic depiction of the approach and workflow, demonstrated by example of the rediscovery of a known gene fusion, <i>SET/NUP214</i>, in the T-ALL cell line LOUCY. Various publicly-available and in-house exon microarray and high-density CGH/SNP array experiments were analyzed. RNA breakpoint analysis (RBA) identifies significant transitions in exon expression level, which may reflect elevated expression of exons distal (3′ partner) or proximal (5′ partner) to a gene fusion junction. To identify such transitions a “walking” Student's t-test was applied, comparing expression levels of proximal and distal exons. Candidate rearrangements were subsequently filtered for those disrupting genes of the Cancer Gene Census, with directional orientation (i.e. being the 5′ or 3′ partner) consistent with known rearrangements of that gene. RBA candidates were further filtered using a Bonferroni correction to adjust for multiple t-tests. DNA breakpoint analysis (DBA) screens for intragenic DNA copy number transitions, which may reflect unbalanced chromosomal rearrangements leading to the formation of gene fusions. The fused lasso method (FDR 1%) followed by a copy number smoothing algorithm was applied to identify CNAs. Copy number transitions were filtered for those disrupting any annotated gene and then further filtered for those disrupting genes of the Cancer Gene Census. We included only candidate breakpoints where the directional orientation of the copy number transition was consistent with known rearrangements of that gene. Several candidates were then validated using molecular and cytogenetic approaches. The average numbers of candidate rearrangements per cancer sample are depicted along the left and right panels at various stages of the workflow.</p

Identification and characterization of novel <i>RAF1</i> gene fusions in pancreatic cancer and anaplastic astrocytoma.

<p>(<i>A</i>) Array CGH heatmaps displaying intragenic <i>RAF1</i> genomic breakpoints identified in the PL5 pancreatic cancer cell line (<i>left panel</i>) and the D-538MG anaplastic astrocytoma cell line (<i>right panel</i>). Unsmoothed log₂ ratios are displayed. (<i>B</i>) Identification of <i>ATG7/RAF1</i> (left) and <i>BCL6/RAF1</i> (right) in PL5 and D-538MG cells, respectively, by paired-end RNA-seq. A subset of the paired-end reads supporting each gene fusion is displayed. Both gene fusions are in-frame and the <i>RAF1</i> serine threonine kinase domain (STK) is retained in both fusions. (<i>C</i>) Experimental validation of gene fusions by RT-PCR, using primers flanking the respective gene fusion junction. (<i>D</i>) Western blotting verifies knockdown of ATG7/RAF1 in PL5 following transfection of a <i>RAF1</i>-targeting siRNA pool. ATG7/RAF1 protein levels were monitored using an anti-<i>RAF1</i> antibody, with anti-<i>GAPDH</i> providing a loading control. (<i>E</i>) Decreased cell proliferation and (<i>F</i>) invasion rates of PL5 following transfection of a <i>RAF1</i>-targeting siRNA pool, compared to transfection of a non-targeting control (NTC) siRNA pool. ** <i>P</i><0.01 (two-sided Student's t-test). (<i>G</i>) Break-apart FISH demonstrates rearrangement of <i>BRAF</i> in a pancreatic cancer case from the TMA, as evidenced by physical separation of the red and green probes (arrows) flanking <i>BRAF</i> (single interphase nucleus shown).</p

DBA discovery of recurrent rearrangements of <i>CLTC</i> and <i>VMP1</i> across diverse cancer types.

<p>(<i>A</i>) Heatmap depicting focal deletions between <i>CLTC</i> and <i>VMP1</i> in the breast cancer cell lines BT-549 and HCC1954. (<i>B</i>) Discovery of the recurrent <i>CLTC/VMP1</i> rearrangement in BT-549 (<i>left</i> panel) and HCC1954 (<i>right</i> panel) by paired-end RNA-seq. (<i>C</i>) RT-PCR verification of <i>CLTC/VMP1</i> fusion in BT-549 and HCC1954. (<i>D</i>) Heatmap depicting focal deletions disrupting <i>CLTC</i>, <i>PTRH2</i> and/or <i>VMP1</i> in various cancer types (see legend). (<i>E</i>) A renal cell carcinoma line, RXF393, was also profiled by exon microarray where an expression breakpoint was evident within <i>CLTC</i>. ***<i>P<10⁻⁹</i> (Student's t-test).</p