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. Author manuscript; available in PMC: 2018 Aug 1.
Published in final edited form as: Mol Cancer Ther. 2017 May 3;16(8):1693–1704. doi: 10.1158/1535-7163.MCT-16-0821

Figure 4. MLN4924 combined with mTOR/PI3K inhibition causes complete MPM tumor growth suppression and significantly reduces the growth of NF2-mutant schwannoma.

Figure 4

(A) Merlin deficient Meso-33 and VAMT cells were transfected with control or DCAF1 siRNA and harvested after 72 hours for immunoblotting as indicated. (B) FC-1801 cells were transfected with Flag-HA-tagged (FH) Merlin or empty vector and control or DCAF1 siRNAs as indicated. Cells were also treated with the indicated concentration of MLN4924 or DMSO for 4 hours prior to lysis and immunoblotted as indicated. (C) The indicated Merlin-deficient MPM lines were treated with DMSO (left) or 5 nM rapamycin (right) for 4 hours and immunoblotted as indicated. (D) VAMT cells were treated with the indicated concentrations of GDC-0980 for three hours and immunoblotted as indicated. (E) 8–10 × 106 VAMT cells were injected bilaterally in the rear flanks of NSG mice. After xenografts exceeded 100 mm3, mice were treated with vehicle or the indicated inhibitors. MLN4924 treatments were 120 mg/kg (subcutaneously, twice per day, three times per week M-W-F). GDC-0980 treatments were 5 mg/kg (oral, once per day, five times per week). Data are means ± SEM (n=10). ***P < 0.001, unpaired T-test. (F) Primary Schwann cells from normal individuals (NF2 WT, left) and schwannoma cells from NF2 patients (NF2−/−, right) were treated with DMSO or 100 nM MLN4924 for 72 hours. Graph indicates the percentage of proliferating cells based on immunostaining for Ki-67 positivity. Data are means ± SEM (NF2+/+ n=3; NF2−/− n=4). ***P < 0.001, unpaired T-test. (G) Primary NF2−/− human schwannoma cells were treated with DMSO or the indicated concentrations of MLN4924, GDC-0980, or a combination of the two inhibitors as indicated. Ki-67+ cells were counted as in (F) and the graph indicates Ki-67+ relative to DMSO controls. Data are means ± SEM (n=3). *P < 0.05, unpaired T-test. (H and I) On-target mTORC1 and NAE inhibition in MSK-LX19 PDX. NSG mice bearing 200 mm3 MSK-LX19 PDXs were treated once with vehicle or the same doses of the indicated inhibitors as in (E). Tumors were isolated 7 hours after drug administration. (H) Representative images of tumors immunostained for phospho-S6 (P-S6) and counterstained with hematoxylin (H). (I) Lysates from the same tumors as (H) were immunoblotted as indicated to detect neddylated cullins (n=2). (J) Serially transplanted MSK-LX19 tumors were injected unilaterally in the rear flanks of NSG mice. After tumors exceeded 100 mm3, mice were treated as in (E). Data are means ± SEM (n=7). *P < 0.05, unpaired T-test.