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Genetic and epigenetic inactivation of SESTRIN1 controls mTORC1 and response to EZH2 inhibition in follicular lymphoma

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Science Translational Medicine  28 Jun 2017:
Vol. 9, Issue 396, eaak9969
DOI: 10.1126/scitranslmed.aak9969
  • Fig. 1. Genomic analyses and a genetic screen identify SESTRIN1 as a functional target of Chr. 6q deletions in FL.

    (A) Integrative Genome View of Chr. 6q deletions occurring in 34% (89 of 260) of FL tumors. (B) Analysis of recurrent deletions in Chr. 6 in FL using GISTIC algorithm. (C) Frequency of homozygous and heterozygous deletions in Chr. 6q. (D) Integrative Genome View visualization of 10 of 260 cases with focal deletions (<5 Mb) on Chr. 6q. Representative FL case with focal deletion affecting SESTRIN1 and TNFAIP3 locus is shown in detail. (E) Flow cytometry validation of individual shRNAs linked to green fluorescent protein (GFP) showing percentages of GFP-positive cells before and after IL-3 withdrawal in pro–B cells transduced with the indicated constructs.

  • Fig. 2. Loss of Sestrin1 promotes FL development in vivo.

    (A) Graphic representation of chimeric mouse model based on the transplantation of vavBcl2 transgenic hematopoietic progenitor cells. WT, wild type. (B) Analysis of tumor latency in the vavBcl2 chimeric mouse model with sh-1 Sestrin1 (blue; n = 10), sh-2 Sestrin1 (red; n = 12), and vector (black; n = 24). Significant difference in survival was measured by log-rank test between animals expressing empty vector and shRNAs targeting Sestrin1. (C and D) Characterization of the shRNAs against Sestrin1. (C) Sestrin1, Sestrin2, and Sestrin3 mRNA expression in tumors expressing vector or shRNA for Sestrin1. P values were calculated using two-tailed t test. n.s., nonsignificant (P < 0.05). Data are shown as a bar graph corresponding to the mean ± SD. (D) Immunoblot analysis in pro–B cells expressing shRNA for Sestrin1 or vector. The antibody recognized two Sestrin1 isoforms, and they were both targeted by shRNA. (E) Representative images of immunohistochemistry and histopathology analyses of indolent vavBcl2 tumors expressing shRNAs against Sestrin1. Ki67 quantification was performed on three different slides for each tumor type using ImageJ. Scale bars, 100 μm. H&E, hematoxylin and eosin; PNA, peanut agglutinin; BCL6, B cell lymphoma 6. (F) Representative immunohistochemistry analyses of phospho-S6 and phospho-4EBP1 in vavBcl2 tumors expressing vector or shRNA #2 for Sestrin1. Quantification was performed on two different slides for each tumor type using ImageJ. Scale bars, 100 μm. (G) Analysis of phosphorylation of S6 and 4EBP1 by Western blot in B cells expressing vector, short hairpin for Sestrin1, or overexpressing Akt as control.

  • Fig. 3. SESTRIN1 is a direct target of EZH2Y641X silencing in lymphomas.

    (A) Differential SESTRIN1 expression in 57 primary FL samples with and without EZH2Y641X mutation. The P value was calculated by Wilcoxon test. Data are shown using the R boxplot function with whiskers extending to minimum and maximum values. FPKM, fragments per kilobase million. (B) Differential SESTRIN1 expression in 38 germinal center B cell–DLBCL (GCB-DLBCL) samples, including 8 samples with the EZH2Y641X gain-of-function mutation. The P value was calculated by Wilcoxon test. Data are shown using the R boxplot function with whiskers extending to minimum and maximum values. (C) ChIP for EZH2 and immunoglobulin G (IgG) in SU-DHL-4 lymphoma cell lines, followed by qPCR (ChIP-qPCR) using three primers spanning SESTRIN1 promoter (P1, P2, and P3), two negative control primers mapping on Chr. 6 upstream of FRK and TNFAIP3 coding sequence (CDS), and two other negative controls mapping on Chr. 7 and Chr. 3. Data are shown as a bar graph corresponding to the mean ± SEM. (D) H3K27me3 ChiP sequencing (ChIP-seq) signal spanning SESTRIN1 genomic locus (Chr. 6: 109,305,000 to 109,420,000) in OCI-LY7 EZH2WT cells (light blue) and Karpas-422 (dark red) and SU-DHL-6 (red) EZH2Y641X mutant cells. Bottom: Zoom-in in SESTRIN1 promoter and coding start site region (Chr. 6: 109,410,000 to 109,420,000). (E) Differential expression analysis focused on genes within the Chr. 6q region in EZH2WT lymphoma cell lines (n = 2), EZH2Y641X-mutated (n = 6), and mutated lines treated with the EZH2 inhibitor GSK126 for 72 hours (n = 6 EZH2Y641X + GSK126). Each cell line has been analyzed in duplicate. SESTRIN1 is significantly differentially expressed between the three groups (FDR < 0.01). (F) Western blot analysis of SESTRIN1 and SESTRIN2 in EZH2Y641X (SU-DHL-6) and EZH2WT (OCI-LY19) lymphoma cell lines treated with the EZH2 inhibitor (1 μM GSK126 for 72 hours). Sestrin1 antibody detects two SESTRIN1 predicted isoforms. Quantification was performed using Image Studio Lite, and the average of the signal intensity was normalized to the control. (G) SESTRIN1 expression in the indicated EZH2WT and EZH2Y641X-mutated lymphoma cell lines treated for 72 hours with dimethyl sulfoxide (DMSO) or 1 μM GSK126. Data are shown as a bar graph corresponding to the mean ± SEM. (H) ChIP for H3K27me3 and histone-3 in SU-DHL-6 lymphoma cell line with and without GSK126 (1 μM for 72 hours), showing loss of SESTRIN1 promoter binding upon EZH2 inhibition. Data are shown as a bar graph corresponding to the mean ± SEM.

  • Fig. 4. SESTRIN1 determines the effects of EZH2 inhibition in lymphoma.

    (A) SESTRIN1 and SESTRIN2 expression in OCI-LY19 and SU-DHL-5 lymphoma cells (EZH2WT/p53WT) and SU-DHL-10 and VAL cells (EZH2Y641X/p53WT) treated with DXR or vehicle for 6 hours. Two-tailed t test was used to define significant (P < 0.05) or nonsignificant (P > 0.05) difference between EZH2WT and EZH2Y641X mutant cells. Data are shown as a bar graph corresponding to the mean ± SEM. Each experiment had two or three technical replicates. (B) Immunoblot with the indicated antibodies in OCI-LY19 and SU-DHL-5 lymphoma cells (EZH2WT/p53WT) and SU-DHL-10 and VAL cells (EZH2Y641X/p53WT) treated with DXR or vehicle for 6 hours. (C) Analysis of S6 and 4EBP1 phosphorylation in EZH2Y641X-mutated lymphoma cell lines SU-DHL-10 and SU-DHL-6 treated for 72 hours with DMSO (vehicle), or 1 or 2 μM GSK126. (D) Immunoblot to assess S6 and 4EBP1 phosphorylation in SU-DHL-10 cells with mutations in SESTRIN1 genomic locus (CRISPR-SESTRIN1) or expressing shRNA targeting SESTRIN1 (sh-SESTRIN1) treated with vehicle (DMSO) or GSK126 (1 or 2 μM for 72 hours). (E) Flow cytometry analysis to measure methionine analog (AHA) incorporation in lymphoma cells treated with the EZH2 inhibitor (GSK126) or rapamycin with or without SESTRIN1. (F) Quantification of the methionine synthetic analog incorporation. Data are shown as a bar graph corresponding to the mean ± SD. (G and H) Tumor growth curves of xenografted SU-DHL-10 expressing vector (G) or CRISPR-SESTRIN1 (H), treated for 14 days with vehicle or GSK126. The mean tumor volumes ± SD are expressed in cubic millimeter. P values were calculated by two-tailed t test. n.s., nonsignificant difference (P > 0.05). (I) Tumor weight in animals xenografted with SU-DHL-10 cells expressing vector or single-guide RNA (sgRNA) targeting SESTRIN1 (CRISPR-SESTRIN1) and treated with the EZH2 inhibitor (75 mg/kg ip) for 14 days. P value was calculated by paired t test. Data are shown as single values with mean ± SD. (J) Representative images of immunohistochemistry and histopathology analyses of xenografted tumors expressing vector or sgRNA targeting Sestrin1 (CRISPR-SESTRIN1) and stained for cleaved caspase3, phospho-S6, and phospho-4EBP1. Quantification was performed with ImageJ. Scale bars, 200 μm. (K) Graphic representation of RapaLink-1 molecular structure and the FAT (FRAP-ATM-TTRAP), FRB, and kinase mTOR domains targeted by the inhibitor. (L) Percentage of viable cells in EZH2Y641X mutant and EZH2WT cells treated with the mTOR inhibitor (12.5 nM RapaLink-1) for 48 hours. Each cell line was analyzed in triplicate, and two-tailed t test was used to assess the significance of the difference in response between EZH2Y641X mutant and EZH2WT cells. Data are shown as a bar graph corresponding to the mean ± SD.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/396/eaak9969/DC1

    Materials and Methods

    Fig. S1. Genomic analysis of frequently altered regions in 260 FLs.

    Fig. S2. Characterization of vavBcl2 lymphoma mouse model.

    Fig. S3. Characterization of epigenetics and expression of SESTRIN1, SESTRIN2, and SESTRIN3 in FL and DLBCL.

    Fig. S4. Regulation of mTOR signaling by SESTRIN1 in lymphoma.

    Table S1. Copy number analysis of 260 indolent FL samples.

    Table S2. Quantification of homozygous and heterozygous deletions on Chr. 6q in 260 FL.

    Table S3. Analysis of focal deletions on Chr. 6q.

    Table S4. shRNA screen analysis.

    Table S5. Sestrin1 expression in EZH2WT or EZH2Y641X GCB-DLBCL patient samples.

    Table S6. Differential expression analysis of genes on Chr. 6q in EZH2WT or EZH2Y641X cell lines.

    Table S7. Differential expression analysis of cell lines treated with the EZH2 inhibitor (GSK126) or DMSO.

    Table S8. List of lymphoma cell lines with relevant genomic lesions.

    References (3743)

  • Supplementary Material for:

    Genetic and epigenetic inactivation of SESTRIN1 controls mTORC1 and response to EZH2 inhibition in follicular lymphoma

    Elisa Oricchio,* Natalya Katanayeva, Maria Christine Donaldson, Stephanie Sungalee, Joyce P. Pasion, Wendy Béguelin, Elena Battistello, Viraj R. Sanghvi, Man Jiang, Yanwen Jiang, Matt Teater, Anita Parmigiani, Andrei V. Budanov, Fong Chun Chan, Sohrab P. Shah, Robert Kridel, Ari M. Melnick, Giovanni Ciriello, Hans-Guido Wendel

    *Corresponding author. Email: elisa.oricchio{at}epfl.ch

    Published 28 June 2017, Sci. Transl. Med. 9, eaak9969 (2017)
    DOI: 10.1126/scitranslmed.aak9969

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Genomic analysis of frequently altered regions in 260 FLs.
    • Fig. S2. Characterization of vavBcl2 lymphoma mouse model.
    • Fig. S3. Characterization of epigenetics and expression of SESTRIN1, SESTRIN2, and SESTRIN3 in FL and DLBCL.
    • Fig. S4. Regulation of mTOR signaling by SESTRIN1 in lymphoma.
    • References (3743)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Copy number analysis of 260 indolent FL samples.
    • Table S2 (Microsoft Excel format). Quantification of homozygous and heterozygous deletions on Chr. 6q in 260 FL.
    • Table S3 (Microsoft Excel format). Analysis of focal deletions on Chr. 6q.
    • Table S4 (Microsoft Excel format). shRNA screen analysis.
    • Table S5 (Microsoft Excel format). Sestrin1 expression in EZH2WT or EZH2Y641X GCB-DLBCL patient samples.
    • Table S6 (Microsoft Excel format). Differential expression analysis of genes on Chr. 6q in EZH2WT or EZH2Y641X cell lines.
    • Table S7 (Microsoft Excel format). Differential expression analysis of cell lines treated with the EZH2 inhibitor (GSK126) or DMSO.
    • Table S8 (Microsoft Excel format). List of lymphoma cell lines with relevant genomic lesions.

    [Download Tables S1 to S8]

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