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ATF4 induction through an atypical integrated stress response to ONC201 triggers p53-independent apoptosis in hematological malignancies

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Science Signaling  16 Feb 2016:
Vol. 9, Issue 415, pp. ra17
DOI: 10.1126/scisignal.aac4380
  • Fig. 1 ONC201 induces apoptosis in MCL and AML cells independent of TP53 mutation status.

    (A) Annexin V–positive cells were counted by flow cytometry after a 72-hour treatment with ONC201. HL-60 and THP-1 cells were treated with the same concentrations for 72 and 120 hours (n = 3 experiments for each cell line). (B) Annexin V/propidium iodide (PI) double-negative cells were counted as live cells and normalized by the live cell count in untreated control samples (n = 3 experiments for each cell line). (C) Immunoblot of p53 in Z-138 and JVM-2 cells transduced with lentivirus expressing control (C) short hairpin RNA (shRNA) or shRNA specific to TP53. (D) Specific apoptosis (see Materials and Methods) induced by ONC201 treatment for 72 hours in TP53 knockdown or intact Z-138 and JVM-2 cells (n = 3 experiments for each isogenic line).

  • Fig. 2 Effects of ONC201 on primary MCL and AML cells and normal bone marrow cells.

    (A and B) Specific apoptosis induced by a 72-hour in vitro treatment with ONC201 (5 μM) in normal BMMCs (n = 9 samples), primary MCL cells (n = 8 samples), and AML cells (n = 18 samples) (A) or in stem/progenitor (CD45+CD34+CD38) populations in normal bone marrow samples and primary AML cells (B). (C to E) Comparison of effects of ONC201 treatment (5 μM for 72 hours) on live cell numbers of primary AML cells according to FLT3 mutation (mut) status (C), TP53 mutation status (D), or complex/noncomplex karyotype (E). (F) Specific apoptosis by ONC201 or Nutlin-3a treatment (72 hours) in two primary MCL samples with TP53 mutation. Representative of two technical replicates. (G and H) Absolute number of human CD45-positive cells in peripheral blood (PB) (per milliliter, n = 10 mice for each group) (G) and in bone marrow (per tibia, n = 3 for each group) (H) at 4 weeks after transplantation into NSG mice of viable AML cells after treatment with (5 μM for 48 hours, then washed) or without ONC201 in vitro into NSG mice. (I) Survival curves of mice (n = 10 mice for each group) after transplantation of AML cells as in (G) and (H). **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 3 ONC201-induced apoptosis is independent of the extrinsic pathway of apoptosis.

    (A) Real-time quantitative polymerase chain reaction (PCR) for DR5 and TRAIL mRNA after exposure to ONC201 (5 μM) of Z-138, MINO, JeKo-1, and OCI-AML3 cells. Relative quantity (RQ) values of mRNA expression are calculated as the fold change relative to mRNA expression at time 0 (normalized to GAPDH) (n = 3 experiments). (B) Specific apoptosis of MCL cell lines treated for 72 hours with ONC201 (5 μM), with or without Z-IETD-FMK (25 μM). JeKo-1 cells were also treated with TRAIL (10 nM), with or without Z-IETD-FMK, for 72 hours (n = 3 experiments). (C) Specific apoptosis and number of live cells (PI and annexin V double-negative cells) in parental Jurkat and caspase-8–deficient Jurkat I9.2 cells after treatment with ONC201 (5 μM for 72 hours) (n = 3 experiments). (D) Specific apoptosis induced by ONC201 with or without the pan-caspase inhibitor Z-VAD-FMK (50 μM) in JeKo-1 and Jurkat I9.2 cells (n = 3 experiments). ***P < 0.001, ****P < 0.0001.

  • Fig. 4 Gene expression profiling of JeKo-1 cells treated with ONC201.

    (A) Mean-centered heat map of variant genes from a time course treatment of JeKo-1 cells with ONC201 (5 μM for 12, 24, and 36 hours) (n = 3 for each time point). (B) Distribution of slope and Pearson’s r values of correlation between gene expression and time. Boxes indicate probes with extreme values of slope and r (data files S1 and S2). (C) Enrichment plot of target genes of CHOP. False discovery rate (FDR) q = 0.016. (D) Enrichment plot of genes that positively correlate with mTOR inhibition. FDR q = 0.000. (E) Subtracted heat map of expression changes (absolute log2 mean-centered value >1 in at least 1 of 12 replicates) in genes of JeKo-1 cells treated with ONC201 (5 μM), rapamycin (10 nM), or tunicamycin (1 μM) for the times indicated (12, 24, or 36 hours) (n ≥ 2 for each condition). (F) Real-time PCR analysis of DDIT3, GADD34, DR5, and ERO1L mRNA expression in JeKo-1 cells during treatment with ONC201 relative to that of GAPDH (n = 3 experiments).

  • Fig. 5 Effects of ONC201 on eIF2α kinases, eIF2α, ATF4, and mTORC1.

    (A) Representative immunoblot analysis (from three experiments) of the abundance and phosphorylation (p) of eIF2α kinases, eIF2α, and ATF4 in OCI-AML3 and JeKo-1 cells from a time course of treatment with ONC201 (5 μM for 36 hours). (B) Representative immunoblot analysis (from three experiments) of GCN2, eIF2α, and ATF4 in HL-60, MOLM-13, JVM-2, and MINO cells treated with control or ONC201 (5 μM) for 24 hours. (C) Representative immunoblot analysis (from three experiments) of mTORC1 target proteins in JeKo-1 cells from a time course of treatment with ONC201 (5 μM for 36 hours).

  • Fig. 6 ATF4 induction by ONC201 and its role in apoptosis.

    (A) Immunoblot analysis of total and phosphorylated eIF2α in OCI-AML3 cells transfected with control shRNA and shRNAs against eIF2α (eIF2α_1 and eIF2α_2) and effects of eIF2α knockdown on apoptosis of cells treated with ONC201 (5 μM) for 72 hours. (B) Representative immunoblots (from three experiments) of ATF4 and eIF2α in OCI-AML3 cells transfected with control or eIF2α-targeting shRNA and treated with ONC201 (5 μM for 24 hours) or tunicamycin (Tm; 1 μM for 6 hours). (C) Green fluorescent protein (GFP) reporter assay of ATF4 translation. 4′,6-Diamidino-2-phenylindole (DAPI)–negative and GFP-positive cells were counted by flow cytometry after treatment with dimethyl sulfoxide (ctrl), ONC201 (5 μM for 27 hours), or thapsigargin (Tg; 1 μM for 1 or 6 hours) (n = 3 experiments). (D) Immunoblot verification of ATF4 knockdown in JeKo-1 (shATF4_1) and OCI-AML3 (shATF4_2) cells. (E) Specific apoptosis induced by ONC201 in control and ATF4 knockdown JeKo-1 (shATF_1) and OCI-AML3 (shATF_2) cells (n = 3 experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 7 Effects of antiapoptotic BCL-2 family proteins on ONC201-induced apoptosis.

    (A) Representative immunoblots (from three experiments) of BCL-XL, BCL-2, and MCL-1 in JeKo-1 cells during treatment with ONC201 (5 μM). (B) Immunoblots of BCL-XL, BCL-2, and MCL-1 in BCL-XL– or BCL-2–overexpressing HL-60 cells. HL-60 cells transfected with vector control were used as control. (C) Specific apoptosis of BCL-2– or BCL-XL–overexpressing HL-60 cells after a 72-hour ONC201 treatment (2.5 and 5 μM) (n = 3 experiments). (D) Immunoblots of BCL-XL, BCL-2, and MCL-1 after MCL-1 knockdown in OCI-AML3 cells. (E) Specific apoptosis after MCL-1 knockdown in OCI-AML3 cells after a 72-hour ONC201 treatment (2.5 and 5 μM) (n = 3 experiments). (F) Representative immunoblots (from three experiments) of BCL-XL, BCL-2, and MCL-1 in MCL or AML cell lines. (G) Synergistic effects on specific apoptosis of OCI-AML3, THP-1, and JVM-2 cells by combination of ABT-199 and ONC201 (n = 3 experiments). *P < 0.05, ***P < 0.001, ****P < 0.0001.

  • Fig. 8 Schematic of atypical ISR induced by ONC201 leading to ATF4-mediated apoptosis, and comparison to other causes of ATF4 induction.

    ONC201 appears to up-regulate ATF4 translation through a mechanism involving μORFs of the ATF4 gene that partially resembles that used by the classical ISR and UPR. However, ONC201-induced ATF4 up-regulation does not require an increase in the phosphorylation of eIF2a, indicating a mechanism of action of ONC201 that indirectly activates this pathway. ONC201-induced ATF4 serves as an apoptosis-promoting factor that also inhibits mTORC1 activity. ONC201 also reduces MCL-1 protein abundance, causing synergistic effects with the BCL-2 inhibitor ABT-199. AA, amino acids; dsRNA, double-stranded RNA.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/415/ra17/dc1

    Fig. S1. Cell cycle changes and DNA damage induced by ONC201.

    Fig. S2. Effect of ONC201 on primary MCL and AML cells and normal bone marrow cells.

    Fig. S3. Efficacy of ONC201 in bortezomib-resistant MM cells and combination therapy with ibrutinib in primary MCL cells.

    Fig. S4. Apoptosis induced by ONC201 in caspase-8– or Bax/Bak-deficient cells.

    Fig. S5. Changes in the AKT/ERK/FOXO3a pathway with ONC201 treatment.

    Fig. S6. Immunoblot and PCR analysis of ER stress–related molecules.

    Fig. S7. Quantification of the immunoblots in Fig. 5 (A and B).

    Fig. S8. Effects of ER stress inducers on GCN2, eIF2α, ATF4, and mTORC1.

    Fig. S9. Immunoblot analysis of ISR-related molecules after amino acid or glucose deprivation in OCI-AML3 and JeKo-1 cells.

    Fig. S10. ATF4 induction by ONC201 or tunicamycin in OCI-AML3 cells with eIF2α knockdown.

    Fig. S11. ATF4 expression over time in response to ONC201 in OCI-AML3 and JeKo-1 cells.

    Fig. S12. Effects of CHOP knockdown on ONC201-induced apoptosis.

    Fig. S13. Effects of ONC201 on IRE1α.

    Fig. S14. The role of ATF4 in mTORC1 inhibition by ONC201.

    Fig. S15. Quantification of the immunoblots in Fig. 7A.

    Table S1. Clinical information for the primary AML samples.

    Table S2. Summary of changes in the GCN2/eIF2α/ATF4 axis and mTORC1 targets by tunicamycin, amino acid or glucose deprivation, and ONC201 in AML and MCL cells.

    Table S3. Combination index of each cell line treated with ONC201 and ABT-199.

    Table S4. shRNA sequences used.

    Table S5. Antibodies for immunoblot analysis.

    Table S6. Primers used for PCR.

    Data file S1. Genes with most positive slope and r in Fig. 4B.

    Data file S2. Genes with most negative slope and r in Fig. 4B.

    Data file S3. Common up-regulated genes in ONC201-sensitive MCL cell lines.

  • Supplementary Materials for:

    ATF4 induction through an atypical integrated stress response to ONC201 triggers p53-independent apoptosis in hematological malignancies

    Jo Ishizawa, Kensuke Kojima, Dhruv Chachad, Peter Ruvolo, Vivian Ruvolo, Rodrigo O. Jacamo, Gautam Borthakur, Hong Mu, Zhihong Zeng, Yoko Tabe, Joshua E. Allen, Zhiqiang Wang, Wencai Ma, Hans C. Lee, Robert Orlowski, Dos D. Sarbassov, Philip L. Lorenzi, Xuelin Huang, Sattva S. Neelapu, Timothy McDonnell, Roberto N. Miranda, Michael Wang, Hagop Kantarjian, Marina Konopleva, R. Eric. Davis,* Michael Andreeff*

    *Corresponding author. E-mail: mandreef{at}mdanderson.org (M.A.) redavis1{at}mdanderson.org (R.E.D.)

    This PDF file includes:

    • Fig. S1. Cell cycle changes and DNA damage induced by ONC201.
    • Fig. S2. Effect of ONC201 on primary MCL and AML cells and normal bone marrow cells.
    • Fig. S3. Efficacy of ONC201 in bortezomib-resistant MM cells and combination therapy with ibrutinib in primary MCL cells.
    • Fig. S4. Apoptosis induced by ONC201 in caspase-8– or Bax/Bak-deficient cells.
    • Fig. S5. Changes in the AKT/ERK/FOXO3a pathway with ONC201 treatment.
    • Fig. S6. Immunoblot and PCR analysis of ER stress–related molecules.
    • Fig. S7. Quantification of the immunoblots in Fig. 5 (A and B).
    • Fig. S8. Effects of ER stress inducers on GCN2, eIF2α, ATF4, and mTORC1.
    • Fig. S9. Immunoblot analysis of ISR-related molecules after amino acid or glucose deprivation in OCI-AML3 and JeKo-1 cells.
    • Fig. S10. ATF4 induction by ONC201 or tunicamycin in OCI-AML3 cells with eIF2α knockdown.
    • Fig. S11. ATF4 expression over time in response to ONC201 in OCI-AML3 and JeKo-1 cells.
    • Fig. S12. Effects of CHOP knockdown on ONC201-induced apoptosis.
    • Fig. S13. Effects of ONC201 on IRE1α.
    • Fig. S14. The role of ATF4 in mTORC1 inhibition by ONC201.
    • Fig. S15. Quantification of the immunoblots in Fig. 7A.
    • Table S1. Clinical information for the primary AML samples.
    • Table S2. Summary of changes in the GCN2/eIF2α/ATF4 axis and mTORC1 targets by tunicamycin, amino acid or glucose deprivation, and ONC201 in AML and MCL cells.
    • Table S3. Combination index of each cell line treated with ONC201 and ABT-199.
    • Table S4. shRNA sequences used.
    • Table S5. Antibodies for immunoblot analysis.
    • Table S6. Primers used for PCR.
    • Legends for data files S1 to S3

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 2.60 MB

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Genes with most positive slope and r in Fig. 4B.
    • Data file S2 (Microsoft Excel format). Genes with most negative slope and r in Fig. 4B.
    • Data file S3 (Microsoft Excel format). Common up-regulated genes in ONC201-sensitive MCL cell lines.

    [Download Data Files S1 to S3]


    Citation: J. Ishizawa, K. Kojima, D. Chachad, P. Ruvolo, V. Ruvolo, R. O. Jacamo, G. Borthakur, H. Mu, Z. Zeng, Y. Tabe, J. E. Allen, Z. Wang, W. Ma, H. C. Lee, R. Orlowski, D. D. Sarbassov, P. L. Lorenzi, X. Huang, S. S. Neelapu, T. McDonnell, R. N. Miranda, M. Wang, H. Kantarjian, M. Konopleva, R. E. Davis, M. Andreeff, ATF4 induction through an atypical integrated stress response to ONC201 triggers p53- independent apoptosis in hematological malignancies. Sci. Signal. 9, ra17 (2016).

    © 2016 American Association for the Advancement of Science

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