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Synthetic lethality by targeting the RUVBL1/2-TTT complex in mTORC1-hyperactive cancer cells

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Science Advances  31 Jul 2020:
Vol. 6, no. 31, eaay9131
DOI: 10.1126/sciadv.aay9131
  • Fig. 1 High-throughput screening for compounds with synthetic lethality against mTORC1-high cancer cells.

    (A) Scheme of screening. Chemical library containing 1576 FDA-approved drugs and bioactive compounds were treated to SW480 shControl (WT) and SW480 shTSC2 (KD) cells. shControl or shTSC2 viral vectors were infected to SW480 cells and cells were selected with puromycin. Capability to induce cell death was measured to rule out cytostatic compounds. Hits were validated and further confirmed in the cells. (B) Cytotoxicity in WT and KD cells were measured after compounds were treated for 36 hours in 96-well plates in duplicate. Fluorescence-based cytotoxicity assay (CellTox Green) was used and to determine the selectivity, and the level of cell death induced in WT cells was subtracted from the level of cell death induced in KD cells. Higher values indicate higher selective cytotoxicity against mTORC1-high cells. RFU, relative fluorescence unit. (C) Increase in mTORC1 activity enhances PL-mediated cell death. SW480 shControl and SW480 shTSC2 cells were treated with PL (10 μM) for 48 hours, and cell viability was measured. (D) Analysis of mTORC1 signaling in SW480 shControl and SW480 shTSC2 cells. OVCAR-8 (OV8) cells were used as a control for comparison. (E) Correlation between PL-induced cell death and mTORC1 levels. Cell death was measured in various normal and cancer cells after 48 hours of PL (10 μM) treatment. Normal or nonmalignant immortalized cells, 184B5, human dermal fibroblast (HDF), CCD-18Co, and BJ-hTERT (BJ); low mTORC1 cancer cells, LS174T and SW480; high mTORC1 cancer cells, TOV-112D, IGROV-1, FAMPAC, OVCAR-8, T24, and BJ-ELR. Immunoblot results show increased mTORC1 activity in PL-sensitive cells. (F) Inhibition of mTORC1 activity reduces PL-mediated cell death. Dimethyl sulfoxide (DMSO) or 100 nM rapamycin was pretreated for 16 hours, and then PL 10 μM was treated to T24 cells. (G) PL selectively kills cancer cells with high mTORC1 activity. All data are presented as means ± SD. Significant differences were calculated by one-way analysis of variance (ANOVA) compared with shControl group or rapamycin-untreated group (***P < 0.001).

  • Fig. 2 PL inhibits mTORC1-high tumor growth in vivo.

    (A) The effect of PL on OVCAR-8 xenograft. PL (7 mg/kg) was administered intraperitoneally everyday to nude mice injected with OVCAR-8 cells. Eight mice per group were used. (B) The effect of PL on SW480 xenograft. PL (7 mg/kg) was administered intraperitoneally everyday to nude mice injected with SW480 shControl and SW480 shTSC2 cells. Eight mice per group were used. (C) The effect of PL on colon cancer PDX. PL (7 mg/kg) was administered intraperitoneally everyday to female SCID mice implanted with HJG152, HJG172, or HJG78 tumors. Seven mice per group for HJG152 and HJG78 and six mice per group for HJG172 were used. (D) The effect of PL on lung cancer PDX. PL (7 mg/kg) was administered intraperitoneally everyday to female severe combined immunodeficient (SCID) mice implanted with LG70, LG43, or LG55 tumors. Six mice per group for LG70 and seven mice per group for LG43 and LG55 were used. (E) The effect of PL on esophageal cancer PDX. PL (7 mg/kg) was administered intraperitoneally everyday to female SCID mice implanted with LEG139, LEG8, or LEG110 tumors. Seven mice per group for LEG139 and LEG8 and five mice per group for LEG110 were used. The volume and weight were measured as described in Materials and Methods. All data are presented as means ± SD (one-way ANOVA; *P < 0.05, **P < 0.01, significant difference compared to the vehicle group.)

  • Fig. 3 PL targets the RUVBL1/2-TTT pathway.

    (A) Target identification using mass spectrometry. Cell lysates were mixed with or without PL, and biotin-labeled PL was used to detect PL-binding proteins. Avidin beads were used to pull down PL-biotin adduct and was subjected to mass spectrometry analysis. (B) PL-biotin binds to RUVBL1 and RUVBL2 and free PL competes with this binding. Glyceraldehyde-3-phosphate dehydrogenase was used as a negative control. (C) The effect of PL on RUVBL1/2-TTT complex formation. T24 cells and T24 cells overexpressing Flag-Tel2 were used for immunoprecipitation with anti-Flag antibody and affinity agarose beads. PL (10 μM) was treated for 2 and 4 hours. (D) The effect of PL on RUVBL1/2-TTT proteins. T24 and OVCAR-8 cells were treated with PL (10 μM) for 1, 3, 6, and 12 hours and examined for RUVBL1/2-TTT proteins and its downstream PIKK pathway by immunoblotting. (E) PL suppresses doxorubicin (DOX)– and camptothecin (CPT)–induced DNA damage response signaling in T24 cells. PL was pretreated for 40 min before the addition of DOX (2 μM) and CPT (1 μM) for 1 hour. pChk1, pChk2, and pH2AX were examined by immunoblotting. (F) PL inhibits ionizing radiation (IR)–induced 53BP1, BRCA1, and Rad51 foci formation. PL was pretreated for 1 hour before IR at 4 gray for 3 hours.

  • Fig. 4 Cancer cells with high mTORC1 activity have increased dependency on RUVBL1/2 for survival.

    (A) The effect of RUVBL1/2 depletion on cell viability. Cell death was measured 4 days (T24) or 6 days (CCD-18co, HDF, SW480, IGROV-1, and OVCAR-8) after siRUVBL1 (si#1, si#2) or siRUVBL2 (si#1, si#2) transfection. (B) Increase in mTORC1 activity enhances sensitivity to RUVBL1/2 silencing–induced cell death. Cell death was measured 6 days after siRUVBL2 transfection to SW480 shControl and SW480 shTSC2 cells. (C) Reduction in mTOR activity prevents cell death induced by RUVBL1 or RUVBL2 knockdown. To reduce mTOR activity in T24 cells, rapamycin or Torin1 (100 nM) was pretreated to T24 cells 16 hours before siRUVBL1/2 transfection. Cell death was measured 4 days (T24) or 6 days (OVCAR8) after transfection. (D) The effect of RUVBL1/2 knockdown on TTT and PIKK family proteins. Protein levels of RUVBL1/2, TTT, and PIKK family members were examined 48 hours after knockdown of RUVBL1-#2 and RUVBL2-#1. (E) RUVBL2 is overexpressed in human cancer tissues. Various human cancer and corresponding normal tissues were stained with RUVBL2 (green) and phospho-S6 (red), and RUVBL2 expression levels were quantified using ImageJ. Representative picture of RUVBL2 (green) and phospho-S6 (red) staining are shown from esophagus cancer and corresponding normal tissue. (**P < 0.01, significant difference of expression between normal and cancer tissues). (F) mTORC1 activity (p-S6) and RUVBL2 expression show positive correlation in human cancer tissues. (Pearson correlation coefficient = 0.70, P < 0.001). (G) Cancer cells with high mTORC1 activity require RUVBL1/2 for survival. All data are presented as means ± SD. Significant differences were calculated by one-way ANOVA compared with DMSO-treated group for each siRNA treatment (***P < 0.001).

  • Fig. 5 Hyperactive mTORC1 causes overreliance on RUVBL1/2 for maintenance of DNA damage stress.

    (A) Differential DNA damage induction by RUVBL2 knockdown (siRUVBL2-#1). DNA damage was measured using comet assay. DNA damage was quantified on the basis of the olive tail movement (OTM) value, automatically calculated using a computer program, CometScore. OTM is computed as the summation of each tail intensity integral value, multiplied by its relative distance from the center of the head, and divided by the total comet intensity. A minimum of 80 cells or more were analyzed per group. (B) Differential DNA damage induction by PL. Cells were treated with PL for 14 hours before the occurrence of evident cell death and collected for DNA damage measurement using comet assay. A minimum of 70 cells or more were analyzed per group. DNA damage was quantified on the basis of the OTM value. (C) mTORC1 activity affects DNA damage levels. SW480 cells stably expressing shControl or shTSC2 were analyzed for phospho-histone H2AX (Ser139). Histone H3 and β-actin were used as loading control. (D) T24 cells were harvested 24 hours after Torin1 treatment (100 nM). (E) Depletion of c-Myc reduces RUVBL1/2 silencing–mediated cell death in SW480 cells. Cells were transfected with either siControl or siMyc (si pool) at seeding and were subsequently transfected with siRUVBL1-#2 or siRUVBL2-#1 24 hours after seeding. Cell viability was measured 6 days after transfection. Significant differences between shControl and shTSC2 cells transfected with siRUVBL1/2 (***P < 0.001) and significant differences between shTSC2 cells transfected with siRUVBL1/2-only and siRUVBL1/2 and siMyc (###P < 0.001). (F) SW480 cells infected with shControl or shTSC2 were transfected with siMyc. Cells were lysed 48 hours after transfection, and proteins were analyzed by immunoblotting. (G) Depletion of c-Myc reduces RUVBL1/2 silencing–mediated cell death in T24 cells. Cells were transfected with either siControl or siMyc (si pool) at seeding and were subsequently transfected with siRUVBL1-#2 or siRUVBL2-#1 24 hours after seeding. Cell viability was measured 4 days after transfection. (H) T24 cells were analyzed for immunoblot after siMyc transfection (48 hours). (I) Model for synthetic lethality of RUVBL1/2 inhibition in cancer cells with mTORC1 hyperactivation. Cancer cells with high mTORC1 activity have increased DNA damage stress, which is partially through c-Myc. Proper functioning of RUVBL1/2 is critical in mitigating the stress. Blockage of RUVBL1/2 selectively kills cancer cells with high mTORC1 activity. All data are presented as means ± SD. Significant differences were calculated by one-way ANOVA compared with DMSO-treated group for each siRNA treatment (***P < 0.001).

Supplementary Materials

  • Supplementary Materials

    Synthetic lethality by targeting the RUVBL1/2-TTT complex in mTORC1-hyperactive cancer cells

    Seung Ho Shin, Ji Su Lee, Jia-Min Zhang, Sungbin Choi, Zarko V. Boskovic, Ran Zhao, Mengqiu Song, Rui Wang, Jie Tian, Mee-Hyun Lee, Jae Hwan Kim, Minju Jeong, Jung Hyun Lee, Michael Petukhov, Sam W. Lee, Sang Gyun Kim, Lee Zou, Sanguine Byun

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