Research ArticleCANCER TREATMENT

Targeting the kinase activities of ATR and ATM exhibits antitumoral activity in mouse models of MLL-rearranged AML

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Science Signaling  13 Sep 2016:
Vol. 9, Issue 445, pp. ra91
DOI: 10.1126/scisignal.aad8243
  • Fig. 1 p53-independent toxicity of ATRi in AMLMLL cells in culture.

    (A) Fluorescence activated cell sorting (FACS) analysis showing the percentage of viable AMLMLL cells [identified by size and 4′,6-diamidino-2-phenylindole (DAPI) exclusion] either untreated or exposed to ATRi (10 μM, 24 hours); FSC-A, forward scattered light. Data are representative of two independent experiments. (B) FACS analysis of DNA content [propidium iodide (PI)] from the cultures used in (A) illustrating the depletion of G2 cells observed in response to ATRi. Data are representative of two independent experiments. (C) Western blot of SMC1, KAP1, and γH2AX phosphorylation and poly(ADP-ribose) polymerase 1 (PARP1) cleavage products in AMLMLL cells exposed to ATRi (5 μM, 6 hours). Data are representative of two independent experiments. (D) Western blot confirming the depletion of p53 in AMLMLL cells after infection with retroviruses expressing a p53-targeting shRNA. β-Actin abundance is shown as a loading control. (E) FACS analysis showing the percentage of viable AMLMLL cells (identified by size and DAPI exclusion) from control or p53 shRNA–infected AMLMLL cells either untreated or exposed to ATRi (10 μM, 24 hours). Data are representative of two independent experiments. (F) FACS analysis of DNA content (PI) from the cultures used in (E) illustrating the depletion of G2 cells observed in response to ATRi. Data are representative of two independent experiments.

  • Fig. 2 Increased levels of ATRi-induced replication stress in AMLMLL cells.

    (A) Western blot of CHK1, RPA, and γH2AX phosphorylation in AMLMLL cells exposed to HU (2 mM, 2 hours). Data are representative of two independent experiments. (B) FACS analysis showing the percentage of viable AMLMLL and AMLETO cells (identified by size and DAPI exclusion) either untreated or exposed to ATRi (3 μM, 16 hours). Data are representative of two independent experiments. (C) FACS analysis of DNA content (PI) and H2AX phosphorylation in AMLMLL and AMLETO cells exposed to ATRi (10 μM, 5 hours). Data are representative of two independent experiments. (D) Fork rates were measured in stretched DNA fibers prepared from AMLMLL and AMLETO cells exposed (or not) to ATRi (10 μM, 5 hours). At least 200 tracks were measured per condition. ***P < 0.001 by two-tailed t test.

  • Fig. 3 In vivo responses of AMLMLL to ATRi.

    (A) Kaplan-Meier curves of AMLMLL transplanted mice that were either treated with vehicle (n = 9), ATRi from day 1 (ATRiPr; n = 7), or ATRi from day 13 (ATRiTh; n = 7). Treatment on the prevention group stopped at day 40. P value was calculated with the Mantel-Cox log-rank test. ***P < 0.001. (B) Representative IVIS of the luciferase signals observed on mice from the groups indicated on (A) on day 18. (C) Representative examples of the luciferase signal observed by IVIS on isolated organs from the indicated groups at day 23. (D) Picture of the spleen sizes observed at day 23 of the in vivo treatment experiment. Scale bar, 1 cm. (E) Representative images of γH2AX immunohistochemistry on spleens of AMLMLL transplanted mice treated with vehicle or ATRi (60 mg/kg, 11 days). Scale bar (black), 50 μm. Numbers indicate the percentage of γH2AX-positive cells in each case (means ± SD). (F) FACS analysis from the bone marrow collected from mice at day 23 of the treatment experiment indicated in (A). GFP (x axis) is used to monitor the presence of AMLMLL cells. y axis indicates FSC-A. The percentage of live GFP+ cells detected in each case is indicated. Data are representative of two independent groups. (G) Effect of ATRi as monotherapy on the growth of xenografts from the human MV4:11 cell line of MLL-driven AML. Treatment started when tumors became palpable, and eight animals were used per group. ***P < 0.001 by two-way analysis of variance.

  • Fig. 4 In vivo responses of AMLMLL to ATMi.

    (A) Kaplan-Meier curves of mice that were transplanted with ATM wild-type (WT) (n = 6) and ATM−/− (n = 8) bone marrow hematopoietic progenitors that had been infected with retroviruses expressing MLL-AF9-IRES-neo and N-RASG12D-IRES-GFP. P value was calculated with the Mantel-Cox log-rank test. (B) Chemical structure of the ATM inhibitor AZD0156 (ATMi). (C) Representative IVIS of the luciferase signals observed on AMLMLL transplanted mice that were either treated with vehicle, ATMi from day 1 (ATMiPr), or ATMi from day 8 (ATMiTh). IVIS imaging was conducted on day 22 of the experiment. (D) Image of the spleen sizes observed at day 22 of the in vivo treatment experiment explained in (C). Scale bar, 1 cm. (E) Representative examples of the luciferase signal observed by IVIS on isolated organs from the indicated groups at day 22. (F) Kaplan-Meier curves of AMLMLL transplanted mice that were either treated with vehicle (n = 9), ATMi from day 1 (ATMiPr; n = 9), or ATMi from day 8 (ATMiTh; n = 9). Treatment on both groups stopped at day 40. P value was calculated with the Mantel-Cox log-rank test. ***P < 0.001.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/445/ra91/DC1

    Fig. S1. CHEK1 mRNA expression is highest in lymphomas and leukemias.

    Fig. S2. Toxicity, replication stress, and DNA breakage induced by two distinct ATR inhibitors in AMLMLL cells.

    Movie S1. Control mice for the ATRi experiment with animals injected with AMLMLL cells after treatment with vehicle.

    Movie S2. Mice injected with AMLMLL cells and treated with ATRi on the prevention protocol.

    Movie S3. Mice injected with AMLMLL cells and treated with ATRi on the therapy protocol.

    Movie S4. Control mice for the ATMi experiment with animals injected with AMLMLL cells after treatment with vehicle.

    Movie S5. Mice injected with AMLMLL cells and treated with ATMi on the prevention protocol.

    Movie S6. Mice injected with AMLMLL and treated with ATMi on the therapy protocol.

  • Supplementary Materials for:

    Targeting the kinase activities of ATR and ATM exhibits antitumoral activity in mouse models of MLL-rearranged AML

    Isabel Morgado-Palacin, Amanda Day, Matilde Murga, Vanesa Lafarga, Marta Elena Anton, Anthony Tubbs, Hua-Tang Chen, Aysegul Ergan, Rhonda Anderson, Avinash Bhandoola, Kurt G. Pike, Bernard Barlaam, Elaine Cadogan, Xi Wang, Andrew J. Pierce, Chad Hubbard, Scott A. Armstrong, André Nussenzweig,* Oscar Fernandez-Capetillo*

    *Corresponding author. Email: ofernandez{at}cnio.es (O.F.-C.); nussenza{at}mail.nih.gov (A.N.)

    This PDF file includes:

    • Fig. S1. CHEK1 mRNA expression is highest in lymphomas and leukemias.
    • Fig. S2. Toxicity, replication stress, and DNA breakage induced by two distinct ATR inhibitors in AMLMLL cells.
    • Legends for movies S1 to S6

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 247 KB

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Control mice for the ATRi experiment with animals injected with AMLMLL cells after treatment with vehicle.
    • Movie S2 (.mov format). Mice injected with AMLMLL cells and treated with ATRi on the prevention protocol.
    • Movie S3 (.mov format). Mice injected with AMLMLL cells and treated with ATRi on the therapy protocol.
    • Movie S4 (.mov format). Control mice for the ATMi experiment with animals injected with AMLMLL cells after treatment with vehicle.
    • Movie S5 (.mov format). Mice injected with AMLMLL cells and treated with ATMi on the prevention protocol.
    • Movie S6 (.mov format). Mice injected with AMLMLL and treated with ATMi on the therapy protocol.

    Citation: I. Morgado-Palacin, A. Day, M. Murga, V. Lafarga, M. E. Anton, A. Tubbs, H.-T. Chen, A. Ergan, R. Anderson, A. Bhandoola, K. G. Pike, B. Barlaam, E. Cadogan, X. Wang, A. J. Pierce, C. Hubbard, S. A. Armstrong, A. Nussenzweig, O. Fernandez-Capetillo, Targeting the kinase activities of ATR and ATM exhibits antitumoral activity in mouse models of MLL-rearranged AML. Sci. Signal. 9, ra91 (2016).

    © 2016 American Association for the Advancement of Science

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