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HIF activation causes synthetic lethality between the VHL tumor suppressor and the EZH1 histone methyltransferase

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Science Translational Medicine  12 Jul 2017:
Vol. 9, Issue 398, eaal5272
DOI: 10.1126/scitranslmed.aal5272
  • Fig. 1. VHL−/− ccRCC cells exhibit altered H3K27 modifications and increased dependence on EZH1.

    (A) Quantification of the indicated histone modifications, as measured by mass spectrometry analysis (described in fig. S1), in renal tumor samples annotated as ccRCCs, typical pRCCs (type 1), atypical pRCCs, and chRCCs. P values were calculated using Student’s t test, and P < 0.05 was considered significant. NS, not significant. (B) Short hairpin RNA (shRNA) screen schema. (C) Genes whose shRNAs exhibited the greatest depletion or enrichment in the VHL−/− ccRCC relative to isogenic pVHL-proficient cells, as determined by RIGER. (D) Immunoblots of UMRC-2 cells that were lentivirally infected to produce pVHL and then stably infected to produce the indicated EZH1 shRNAs or an shRNA against firefly luciferase (Luc). The shRNAs in the screen are indicated in red. (E) Crystal violet staining of UMRC-2 that were lentivirally infected to produce pVHL (VHL) or infected with an empty lentivirus (VEC) and then stably infected to produce the indicated EZH1 shRNAs or an shRNA against firefly luciferase (Luc). (F) Photomicrographs of CAKI-2 cells that were infected to produce exogenous wild-type (WT) EZH1, catalytic-dead EZH1 (ΔSET), or with the empty vector (VEC) and then with a lentivirus encoding an EZH1 shRNA (10708) or control shRNA. The EZH1 WT vector contained synonymous mutations in the sequence recognized by the 10708 shRNA. The ΔSET mutation also eliminates the 10708 recognition sequence.

  • Fig. 2. CRISPR/Cas9-based ablation of EZH1 inhibits VHL−/− RCC cells.

    (A) Immunoblots of UMRC-2 ccRCC cells that were lentivirally infected to produce pVHL and then stably infected to express sgRNAs targeting EZH1 or a nontargeting control, as indicated. (B) Viable cell counts, as determined by Vi-CELL, of UMRC-2 cells that were lentivirally infected to produce pVHL (VHL) or with the empty vector (VEC) and then transduced to express sgRNAs targeting either EZH1 or a nontargeting control. Cell counts of both VEC and VHL cells were determined 18 days after selection and are presented as fold change after normalization to the number of viable sgControl expressing cells (n = 3, means ± SD). P values were calculated using Student’s t test. *P < 0.05 (significant). (C to E) Immunoblots (C), viable cell counts determined by Vi-CELL (D) (n = 3, means ± SD), and photomicrographs (E) of UMRC-2 cells infected with a DOX-inducible EZH1 expression vector and then transduced to express sgRNAs designed to inactivate the endogenous EZH1 locus (INEX2) or, as a control, an ineffective sgRNA. Analysis in (C) was done after expressing the indicated sgRNA for 10 days. DOX was present for the first 7 days and then maintained (+) or withdrawn (−) for 3 days. (D) and (E) were done 18 days after DOX withdrawal. P values were calculated using Student’s t test. *P < 0.05 (significant).

  • Fig. 3. JQ-EZ-05 is a specific and reversible EZH1/2 inhibitor.

    (A) JQ-EZ-05 structure. (B) Inhibition of recombinant EZH1 and EZH2 by JQ-EZ-05 in vitro. (C) Immunoblots of CAKI-2 cells treated with 1 μM JQ-EZ-05 for the indicated number of days or treated for 5 days followed by removal (washout) for 1 to 3 days. (D) Heatmap displaying gain (red and green) or loss (blue and black) of histone marks in two biological replicates (labeled 1 and 2) of CAKI-2 cells treated with the indicated concentrations of JQ-EZ-05 or dimethyl sulfoxide (DMSO) for either 24 hours (D1) or 1 week (D7).

  • Fig. 4. JQ-EZ-05 treatment preferentially inhibits VHL−/− ccRCC.

    (A and B) Immunoblots (A) and cell survival (XTT assay) (n = 3, means ± SD) (B) of CAKI-2 cells stably expressing pVHL (VHL) or empty vector (VEC) and treated with the indicated concentrations of JQ-EZ-05 for 3 days (A) or 10 days (B). In (B), P values were calculated using Student’s t test. *P < 0.05 (significant). (C to E) Viable cell counts (Vi-CELL) (C) and soft agar assays (D and E) of UMRC-2 cells stably expressing pVHL (VHL) or empty vector (VEC) treated with JQ-EZ-05 for 10 days (C) or 3 weeks (D to E). In (D), the top medium was changed every 4 days. In (E), three representative fields were quantified from each biological replicate. In (C) and (E) (n = 3, means ± SD), P values were calculated using Student’s t test, and P < 0.05 considered significant. (F and G) Apoptosis, as measured by annexin V staining (y axis) (F) and resulting quantification (G), of UMRC-2 cells infected with a lentivirus encoding pVHL (VHL) or empty vector (VEC) that were treated with the indicated concentrations of JQ-EZ-05 for 8 days. P values were calculated using Student’s t test, and P < 0.05 was considered significant. (H and I) Bioluminescence imaging (BLI) of nonobese diabetic/severe combined immunodeficient mice orthotopically injected with UMRC-2 cells engineered to produce firefly luciferase. Once tumors were established, as determined by serial BLI, mice were randomized (day 0) to JQ-EZ-05 (75 mg/kg, intraperitoneally, three times per week) or vehicle. Representative images (H) and quantification (I) of change in tumor burden on day 45 relative to day 0, as determined by BLI. In (I), P values were calculated by using the nonparametric Mann-Whitney U test, and P < 0.05 was considered significant. (J) Representative images of kidneys excised from tumor-bearing mice at necropsy after treatment as described in (H) and (I) or from an age- and sex-matched control mouse without any orthotopic tumors (“Neg.”). (K) Body weights of mice (n = 3, means ± SD) after the indicated duration of treatment with either vehicle or JQ-EZ-05 (75 mg/kg, intraperitoneally, three times per week).

  • Fig. 5. Antiproliferative effects of JQ-EZ-05 on VHL−/− ccRCC are on-target.

    (A) Sequence alignment of EZH1 and EZH2 catalytic SET domains. (B and C) Immunoblots (B) and crystal violet staining (C) of 786-O cells infected to exogenously produce WT or ΔPSET EZH1 and treated with the indicated amounts of JQ-EZ-05 for 3 and 7 days, respectively. (D and E) Photomicrographs (D) and immunoblots (E) of 786-O cells infected with a lentivirus encoding GFP, WT EZH1, or randomly mutagenized EZH1 (XL-Red) and treated with the indicated concentrations of JQ-EZ-05 for 3 days (E) or with either 4 μM JQ-EZ-05 or DMSO for 21 days (D). (F) Crystal violet staining of UMRC-2 cells infected to exogenously produce the indicated EZH1 variants and treated with the indicated concentrations of JQ-EZ-05 for 10 days. (G) Structural model of JQ-EZ-05 bound to WT EZH1. (H) Immunoblots of UMRC-2 cells expressing EZH1 variants as in (F) treated with the indicated concentrations of JQ-EZ-05 for 3 days.

  • Fig. 6. Sensitivity of VHL−/− ccRCC cells to EZH1 loss is HIF-dependent.

    (A) Schema describing DOX treatment of cells (described in Fig. 2, C to E) and transduction of these cells to either express sgRNA-targeting ARNT1 (sgARNT) or nontargeting control (sgCon). (B to D) Viable cell counts (n = 3, means ± SD) (B), immunoblot analysis (C), and representative photomicrographs (D) of cells [described in (A)] expressing the indicated sgRNA. In (B), “Days” indicates time after withdrawal of DOX. Images in (D) were recorded 15 days after DOX withdrawal. In (B), P values were calculated using Student’s t test. *P < 0.05 (significant). (E and F) Crystal violet staining (E) and immunoblots (F) of 786-O cells infected to produce the indicated ARNT1 shRNAs or a control shRNA (Con). (E) Cells were treated with the indicated amounts of JQ-EZ-05 for 10 days. SE, short exposure; LE, long exposure. (G and H) Crystal violet staining (G) and immunoblots (H) of 786-O cells infected with lentiviruses encoding either ARNT1 sgRNA or a control sgRNA. In (G), cells were treated with the indicated amounts of JQ-EZ-05 for 10 days. NDRG1 was included in (C), (F), and (H) as an HIF2α-responsive gene product.

  • Fig. 7. Sensitivity of VHL−/− ccRCC to EZH1 loss is driven by HIF-dependent induction of H3K27 demethylases.

    (A) GSEA of RNA sequencing (RNA-seq) data comparing gene expression in 786-O empty vector control cells versus 786-O cells in which ARNT1 was inactivated with either shRNA (3819; used in Fig. 6, E and F) or sgRNA (as in Fig. 6, G and H). Gene sets scoring with a false discovery rate <0.25, and nominal P < 0.05, were considered significant. NES, normalized enrichment score. (B) Immunoblots of purified histones incubated for the indicated time periods with lysates from UMRC-2 cells infected to produce WT pVHL (+VHL) or with an empty lentivirus (VHL−/−). (C and D) Immunoblots (C) and percent viable cells relative to DMSO-treated control (D) (n = 3, means ± SD) of UMRC-2 cells transduced to express sgRNAs targeting HIF2α (sgH2a4 and sgH2a6) or a nontargeting control. In (D), cells were treated with 3 μM JQ-EZ-05 or DMSO for 8 days. P values were calculated using Student’s t test. *P < 0.05 (significant). (E) Left: Crystal violet staining of 786-O cells expressing either shRNA against KDM6B (JMJD3) (sh6B#1 and sh6B#2) or control shRNA (shCON) and treated with the indicated concentrations of JQ-EZ-05 for 8 days. Right: Quantification (n = 3, means ± SD) of crystal violet stain in cells treated with 4.5 μM EZ-05 relative to cells treated with DMSO.

  • Fig. 8. Transcriptional response to EZH1 loss is influenced by pVHL status.

    (A and B) Schema for RNA-seq analysis to measure the transcriptional response to EZH1 inactivation, either using pharmacological inhibition (A) or genetic loss (B). In (A), 786-O cells stably expressing WT EZH1 (WT) or the Y727F (YF) mutant were treated with 4 μM JQ-EZ-05 or DMSO for 3 days. In (B), RNA was prepared from UMRC-2 cells expressing the indicated sgRNA for 7 days. (C) Venn diagram showing GSEA gene sets up-regulated by either pharmacological or genetic inactivation of EZH1. Right: Representative gene sets that were induced under both conditions. (D) Heatmap depicting mRNA expression, as measured by RNA-seq, of the indicated genes under conditions described in (A) and (B). (E) Abundance of the indicated mRNAs (relative to actin mRNA), as determined by real-time quantitative polymerase chain reaction, in UMRC-2 cells expressing either pVHL (VHL) or empty vector control (VEC) that were superinfected to express the indicated sgRNAs. Values (n = 3, means ± SD) are expressed relative to sgCon cells. RNA was prepared from cells expressing the indicated sgRNA for 7 days, before the onset of overt toxicity linked to EZH1 loss. P values were calculated using Student’s t test. *P < 0.05 (significant).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/398/eaal5272/DC1

    Materials and Methods

    Fig. S1. Analysis of histone modification patterns in renal tumors.

    Fig. S2. Histopathological characterization of representative tumor samples.

    Fig. S3. Genotypic characterization of representative tumor samples.

    Fig. S4. Molecular characterization of ccRCC lines used in the shRNA minipool screen.

    Fig. S5. Effects of genetic inactivation of EZH1 or EZH2 on ccRCC.

    Fig. S6. Characterizing the on-target effects of EZH1 shRNA.

    Fig. S7. Effects of pharmacological EZH inhibition using JQ-EZ-05 on lymphoid cells.

    Fig. S8. Effects of pharmacological EZH inhibition using JQ-EZ-05 and GSK126 on renal cells.

    Fig. S9. Validation of candidate JQ-EZ-05–resistant EZH1 mutants.

    Fig. S10. Effects of JQ-EZ-05 on VHL−/− cells lacking ARNT1.

    Fig. S11. Effects of pVHL expression on H3K27 methylation.

    Fig. S12. Effects of JQ-EZ-05 on H3K27 histone demethylase–deficient VHL−/− ccRCC.

    Fig. S13. Oncomine analysis of H3K27 demethylases and methyltransferases in renal tumors.

    Fig. S14. Identification of biomarkers to define EZH1-VHL synthetic lethality.

    Table S1. Annotated list of pLKO-based shRNAs used in the minipool screen.

    Table S2. RIGER analysis to identify genes corresponding to shRNAs that were enriched or depleted selectively in the VHL−/− cells.

    Table S3. RNA-seq data of transcriptional changes observed upon ARNT loss in 786-O cells.

    Table S4. GSEA of 786-O cells with high (VEC) or low (sh/sgARNT) HIF activity.

    Table S5. GSEA of ccRCC cells upon EZH1 inactivation using either pharmacological or genetic tools.

    Table S6. GSEA comparing ccRCC cells that either do (present) or do not (absent) display EZH1-VHL synthetic lethality.

    Table S7. Primer sequences.

    References (5260)

  • Supplementary Material for:

    HIF activation causes synthetic lethality between the VHL tumor suppressor and the EZH1 histone methyltransferase

    Abhishek A. Chakraborty, Eijiro Nakamura, Jun Qi, Amanda Creech, Jacob D. Jaffe, Joshiawa Paulk, Jesse S. Novak, Kshithija Nagulapalli, Samuel K. McBrayer, Glenn S. Cowley, Javier Pineda, Jiaxi Song, Yaoyu E. Wang, Steven A. Carr, David E. Root, Sabina Signoretti, James E. Bradner, William G. Kaelin Jr.*

    *Corresponding author. Email: william_kaelin{at}dfci.harvard.edu

    Published 12 July 2017, Sci. Transl. Med. 9, eaal5272 (2017)
    DOI: 10.1126/scitranslmed.aal5272

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Analysis of histone modification patterns in renal tumors.
    • Fig. S2. Histopathological characterization of representative tumor samples.
    • Fig. S3. Genotypic characterization of representative tumor samples.
    • Fig. S4. Molecular characterization of ccRCC lines used in the shRNA minipool screen.
    • Fig. S5. Effects of genetic inactivation of EZH1 or EZH2 on ccRCC.
    • Fig. S6. Characterizing the on-target effects of EZH1 shRNA.
    • Fig. S7. Effects of pharmacological EZH inhibition using JQ-EZ-05 on lymphoid cells.
    • Fig. S8. Effects of pharmacological EZH inhibition using JQ-EZ-05 and GSK126 on renal cells.
    • Fig. S9. Validation of candidate JQ-EZ-05–resistant EZH1 mutants.
    • Fig. S10. Effects of JQ-EZ-05 on VHL−/− cells lacking ARNT1.
    • Fig. S11. Effects of pVHL expression on H3K27 methylation.
    • Fig. S12. Effects of JQ-EZ-05 on H3K27 histone demethylase–deficient VHL−/− ccRCC.
    • Fig. S13. Oncomine analysis of H3K27 demethylases and methyltransferases in renal tumors.
    • Fig. S14. Identification of biomarkers to define EZH1-VHL synthetic lethality.
    • Legends for tables S1 to S7
    • References (5260)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Annotated list of pLKO-based shRNAs used in the minipool screen.
    • Table S2 (Microsoft Excel format). RIGER analysis to identify genes corresponding to shRNAs that were enriched or depleted selectively in the VHL−/− cells.
    • Table S3 (Microsoft Excel format). RNA-seq data of transcriptional changes observed upon ARNT loss in 786-O cells.
    • Table S4 (Microsoft Excel format). GSEA of 786-O cells with high (VEC) or low (sh/sgARNT) HIF activity.
    • Table S5 (Microsoft Excel format). GSEA of ccRCC cells upon EZH1 inactivation using either pharmacological or genetic tools.
    • Table S6 (Microsoft Excel format). GSEA comparing ccRCC cells that either do (present) or do not (absent) display EZH1-VHL synthetic lethality.
    • Table S7 (Microsoft Excel format). Primer sequences.

    [Download Tables S1 to S7]