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. 2012 Jan 12;119(2):476-87.
doi: 10.1182/blood-2011-04-346601. Epub 2011 Nov 11.

Dual mTORC1/mTORC2 inhibition diminishes Akt activation and induces Puma-dependent apoptosis in lymphoid malignancies

Mamta Gupta  1 , Andrea E Wahner Hendrickson, Seong Seok Yun, Jing Jing Han, Paula A Schneider, Brian D Koh, Mary J Stenson, Linda E Wellik, Jennifer C Shing, Kevin L Peterson, Karen S Flatten, Allan D Hess, B Douglas Smith, Judith E Karp, Sharon Barr, Thomas E Witzig, Scott H Kaufmann
Affiliations

Dual mTORC1/mTORC2 inhibition diminishes Akt activation and induces Puma-dependent apoptosis in lymphoid malignancies

Mamta Gupta et al. Blood. .

Abstract

The mammalian target of rapamycin (mTOR) plays crucial roles in proliferative and antiapoptotic signaling in lymphoid malignancies. Rapamycin analogs, which are allosteric mTOR complex 1 (mTORC1) inhibitors, are active in mantle cell lymphoma and other lymphoid neoplasms, but responses are usually partial and short-lived. In the present study we compared the effects of rapamycin with the dual mTORC1/mTORC2 inhibitor OSI-027 in cell lines and clinical samples representing divers lymphoid malignancies. In contrast to rapamycin, OSI-027 markedly diminished proliferation and induced apoptosis in a variety of lymphoid cell lines and clinical samples, including specimens of B-cell acute lymphocytic leukemia (ALL), mantle cell lymphoma, marginal zone lymphoma and Sezary syndrome. Additional analysis demonstrated that OSI-027-induced apoptosis depended on transcriptional activation of the PUMA and BIM genes. Overexpression of Bcl-2, which neutralizes Puma and Bim, or loss of procaspase 9 diminished OSI-027-induced apoptosis in vitro. Moreover, OSI-027 inhibited phosphorylation of mTORC1 and mTORC2 substrates, up-regulated Puma, and induced regressions in Jeko xenografts. Collectively, these results not only identify a pathway that is critical for the cytotoxicity of dual mTORC1/mTORC2 inhibitors, but also suggest that simultaneously targeting mTORC1 and mTORC2 might be an effective anti-lymphoma strategy in vivo.

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Figures

Figure 1
Figure 1
Activation of mTORC1 and mTORC2 signaling in clinical lymphoma samples. MCL samples (n = 10) were stained using antibodies to rictor, raptor, and autophosphorylated mTOR as well as phosphorylation site-specific antibodies for targets downstream of mTORC1 (phospho-Thr37/46-4EBP1, phospho-Ser235/236-ribosomal S6 protein) and mTORC2 (phospho-Ser473-Akt). Similar results were observed in all 10 specimens.
Figure 2
Figure 2
Effects of OSI-027 and rapamycin on phosphorylation of mTORC1 and mTORC2 substrates in malignant human lymphoid lines. (A,D) The MCL line Jeko (left) and T-cell ALL line Jurkat (right) were treated for 8 hours with diluent (lanes 1,8); OSI-127 at 1.25, 2.5, 5, 10 or 20μM (lanes 2-6 and 9-13, respectively); or 10nM rapamycin (lanes 7,14). Whole cell lysates were then subjected to SDS-PAGE followed by immunoblotting with antibody that recognizes the indicated antigen. (B,E) Jeko cells were treated for 24 hours with diluent (lanes 1,5); OSI-127 at 0.5, 2, or 5μM (lanes 2-4); or rapamycin at 1, 10 and 100nM (lanes 6-8). Cell lysates were then subjected to SDS-PAGE followed by immunoblotting with antibody that recognizes the indicated antigen. (C,F) Samples from 3 MCL patients were treated for 24 hours with diluent or OSI-127 at 2 and 5μM. After SDS-PAGE, cell lysates were probed as indicated. (G) After Jeko cells were treated with the indicated OSI-027 concentration for 24 hours, mTOR immunoprecipitates (top) or cell lysates (bottom) were probed as indicated.
Figure 3
Figure 3
Effects of OSI-027 in Akt Thr308 phosphorylation and Akt-associated phosphatases. (A) Jeko (left) and Jurkat (right) cells were treated for 8 hours with diluent (lanes 1,8); OSI-127 at 1.25, 2.5, 5, 10 or 20μM (lanes 2-6 and 9-13, respectively); or 10nM rapamycin (lanes 7,14). Whole cell lysates were then subjected to SDS-PAGE followed by immunoblotting with antibody that recognizes the indicated antigen. (B) Jeko cells were treated for 24 hours with diluent (lanes 1,5); OSI-127 at 0.5, 2, or 5μM (lanes 2-4); or rapamycin at 1, 10 and 100nM (lanes 6-8). Cell lysates were then subjected to SDS-PAGE followed by immunoblotting with antibody that recognizes the indicated antigen. (C) After a 24-hour treatment with OSI-027, Akt immunoprecipitates (top) or cell lysates (bottom) from Jeko cells were probed with antibodies to PP1 and PP2A as indicated. (D, E) After a 24-hour treatment with OSI-027, immunoprecipitates of Akt2 (D) or Akt1 (E) from Jeko cells were probed for PHLPP isoforms as indicated. Additional experiments (not shown) indicated that PHLPP1 failed to immunoprecipitate with Akt1 and PHLPP2 failed to immunoprecipitate with Akt2.
Figure 4
Figure 4
OSI-027 inhibits 3H-thymidine incorporation and diminishes viable cell mass in cultures of malignant lymphoid cells. (A-B) After lymphoid cell lines were treated for 48 hours with OSI-027 or rapamycin as indicated, 3H-thymidine incorporation into DNA was assayed. (C-E) After lymphoid cell lines (C) or primary clinical samples from patients with the indicated lymphoid neoplasms (D-E) were treated for 5 days with OSI-027 or rapamycin as indicated (C-D) or with 5μM OSI-027 versus 10nM rapamycin (E), MTS reduction was assayed. MTS reduction of cells treated with diluent (0.1% DSMO) for 5 days was set at 100% in each assay. Error bars in panels A through C, mean ± SEM of 3 independent assays.
Figure 5
Figure 5
OSI-027 induces apoptosis in malignant lymphoid cell lines and clinical lymphoma samples. (A-C) Jeko and Mino cells were treated for 48 hours with the indicated concentrations of OSI-027 (B) or rapamycin (C) and assayed using annexin V and PI as illustrated in panel A. Values in panels B and C represent the percentage of total cells that were negative for staining with annexin V and PI. (D) Jurkat were treated for 48 hours with the indicated concentrations of OSI-027, lysed in citric acid under conditions where fragmented DNA is extracted, stained with propidium iodide (PI) and subjected to flow microfluorimetry. Results of this and additional assays are summarized in Figure 6A. (E-G) Jeko, SeAx and Jurkat cells were treated for 24-96 hours with the indicated concentrations of OSI-027 (E-F) or 10nM rapamycin (G) and assayed as illustrated in panel D. (H) After treatment with diluent or OSI-027 for 48 hours, lysates from Jeko cells, Mino cells, or MCL samples from 3 separate patients were subjected to SDS-PAGE and probed with antibody to PARP1. Arrow, previously described caspase-induced PARP cleavage fragment. Error bars in panels B, C, E, and F, mean ± SEM of 3 independent assays.
Figure 6
Figure 6
OSI-027 induces Puma- and Bim-dependent apoptosis. (A) Parental Jurkat cells and Jurkat variants lacking critical apoptotic pathway components (inset) were treated for 48 hours with OSI-027, stained with PI and analyzed as illustrated in Figure 5D. Error bars, mean ± SEM of 3 independent assays. (B,D) Twenty-four hours after transfection with control siRNA or siRNA targeting Bax or Bak (B) or Bim or Puma (D) along with plasmid encoding EGFP-histone H2B (to mark successfully transfected cells), Jurkat cells were harvested to assess target down-regulation or treated for 48 hours with the indicated concentration of OSI-027, stained with APC–annexin V, and analyzed by 2-color flow cytometry to assess annexin V binding in EGFP-histone H2B+ cells. Error bars, mean ± SEM of 3 independent assays. Insets, immunoblots (B) or qRT-PCR (D) demonstrating down-regulation of the targeted protein or message, respectively. (C,E) After Jurkat (C) and Jeko cells (E) were treated for 48 hours with diluent (lanes 1); OSI-127 at 1.25, 2.5, 5, 10 or 20μM (lanes 2-6); or 10nM rapamycin (lane 7) in the presence of the caspase inhibitor Q-VD-OPh (5 μM) to inhibit apoptosis, whole cell lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies that recognize the indicated antigen. PARP1 served as a loading control. (F-G) After Jurkat cells were treated for 48 hours with diluent, OSI-027 at the indicated concentration or 10nM rapamycin in the presence of 5μM Q-VD-OPh, RNA was isolated and subjected to conventional RT-PCR or qRT-PCR. Noxa and GAPDH served as controls. Results in panel F, geometric mean of 3 independent experiments. (H) Beginning 24 hours after transfection with the indicated reporter construct, Jurkat cells were treated for 48 hours with diluent or OSI-027 at the indicated concentration in the presence of 5μM Q-VD-OPh and cell lysates were assayed for firefly luciferase. Error bars, mean ± SD of 4-6 independent experiments.
Figure 7
Figure 7
Activity of OSI-027 against Jeko xenografts in vivo. (A,C) Whole cell lysates prepared from separate xenografts of untreated mice (lanes 1-3) or xenografts harvested 4 hours after treatment with OSI-027 on day 3 (lanes 4,5), day 5 (lanes 6-8) or day 6 (lanes 9,10) were subjected to SDS-PAGE and probed with antibodies to the indicated antigen. (B) qRT-PCR for Bim and Puma mRNA in xenografts harvested before treatment (Pre) or on days 3, 5 and 6 at 4 hours after OSI-027. Each symbol represents the mean of 3 values from 1 xenograft. Horizontal bars, geometric mean of samples from that day. (D) Mice bearing established xenografts were treated with 3 cycles of OSI-027 at 58 mg/kg orally on a 6 days on/2 days off schedule. Results shown are the geometric mean of 5 masses from control mice and 6 masses in 5 treated mice, with each value normalized to its pretreatment value. The mass that became undetectable was arbitrarily assigned a value of 10% of its pretreatment value. (E) Appearance of Jeko xenografts in diluent- and OSI-027–treated mice on day 12 when control animals needed to be killed because masses had reached institutional limits for size. (F) Relative volumes of each Jeko xenograft on day 12 when control animals were killed and on day 23 at the end of treatment. (G) Relative volumes of Jeko xenografts on day 13 (when control animals needed to be killed, closed circles) and day 23 (end of treatment, open circles) in a separate xenograft experiment in which animals were randomized to diluent, OSI-027 50 mg/kg or rapamycin 2 mg/kg on days 1-6, 9-14, and 17-22. Horizontal bars, geometric mean of 6 (OSI-027) or 5 (other groups) mice in treatment group. (H) Proposed mechanism of cytotoxicity of OSI-027 in lymphoid cells. Steps inhibited by rapamycin, OSI-027 and Bcl-2 are shown. Dashed lines indicate that up-regulation of Bim is more variable than Puma among different lymphoid cell lines.
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