A dual PI3K/mTOR inhibitor leads to decreased tumor burden and improved survival in GEM and primary orthotopic xenograft models of MYCN-driven neuroblastoma. (A) Viability and proliferation screens: MYCN-amplified cells [human primary culture (SFNB-06) and three cell lines (IMR32, Lan-5, and SK-N-BE2)] were sensitive to NVP-BEZ235 compared with nonamplified cells [two human primary cultures (SFNB-05 and SFNB-07) and cell lines (SK-N-SH, SY5Y, and SHEP)]. *P < 0.05, Student’s t test. (B) Tumor-bearing TH-MYCN mice were treated with NVP-BEZ235 (35 mg/kg) or PEG300 vehicle once daily by oral gavage for 28 days. Kaplan-Meier analysis demonstrates that NVP-BEZ235 enhanced mice survival (n = 5 for each group; P < 0.003, log-rank test). Arrow indicates cessation of therapy at 28 days. (C) A heavily pretreated primary MYCN-amplified human tumor (SFNB-06) was established orthotopically, and mice were treated with NVP-BEZ235 or vehicle, as above, for 28 days. NVP-BEZ235 prolonged survival (n = 10 for each group; P < 0.049, log-rank test). Arrow again indicates cessation of therapy at 28 days. (D and E) Tumor burden: tumor-bearing TH-MYCN mice (D) and orthotopic xenografts of SFNB-06 (E) were treated with NVP-BEZ235 (35 mg/kg) or PEG300 vehicle once daily by oral gavage for 14 days. NVP-BEZ235 treatment led to decreased tumor volume and weight in both models (*P < 0.05, Student’s t test).

A dual PI3K/mTOR inhibitor suppresses both proliferation of tumor cells and angiogenesis in neuroblastoma. (A to H) Tumors from mice transgenic for TH-MYCN were treated with NVP-BEZ235 (35 mg/kg) or PEG300 vehicle once daily by oral gavage, and mice were killed at 14 days of treatment. Endo-thelial cell density (CD31), pericyte density (α-SMA), proliferation of tumor cells (Ki67), and H&E staining were assessed. Scale bars, 50 μm. n = 3 for each arm. (I) Quantification of (A) to (C) and (E) to (H) using ImageJ (*P < 0.0008, Student’s t test). (J to P) Mice carrying MYCN-amplified human orthotopic xenografts (SFNB-06) were treated with NVP-BEZ235 or vehicle, collected, and analyzed as in (A) to (I). (R) Quantification of (J) to (L) and (N) to (P) with ImageJ (*P < 0.0008, Student’s t test). n = 3 for each arm. In all quantitations, vehicle arms were normalized to 100%; NVP-BEZ235 arms were graphed as percent of vehicle.

NVP-BEZ235 blocks PI3K/mTOR and destabilizes MYCN in vivo. (A) Tumor-bearing mice transgenic for TH-MYCN were treated daily with NVP-BEZ235 or vehicle for 7 or 14 days (same dosage as in Fig. 2). Western analysis demonstrates decreased levels of MYCN protein in tumor lysates from animals treated with NVP-BEZ235 in comparison with control. (B) Western analysis of lysates from human orthotopic tumors in Fig. 1, I to O, confirms expression of MYCN in primary tumor and in vehicle-treated orthotopic xenografts, with decreased levels of MYCN protein in lysates from two different xenografts, treated with NVP-BEZ235 for 14 days. (C) Real-time qRT-PCR: NVP-BEZ235 (7 and 14 days) had no significant effect on levels of MYCN mRNA in the TH-MYCN [from (A)]. P = 0.56 for 7 days, P = 0.75 for 14 days. (D) Real-time qRT-PCR: primary human SFNB-06 tumors treated with vehicle or NVP-BEZ235 for 14 days [from (B)]. P = 0.62, Student’s t test. Vehicle arms were normalized to 100%. NVP-BEZ235 arms were graphed as percent of vehicle.

MYCN drives paracrine signaling between tumor vascular cells. (A to C) SHEP cells were stably transduced with MYCN^WT or MYCN^T58A. (A) Cycloheximide (CHX) pulse-chase assay: cycloheximide (50 μg/ml) with NVP-BEZ235. MYCN^T58A protein showed a prolonged half-life. (B) Western blot: MYCN^T58A protein was resistant to NVP-BEZ235. (C) Vehicle-treated MYCN^WT and MYCN^T58A were normalized to 100%. NVP-BEZ235–treated arms were graphed as percent change of vehicle (raw data in fig. S3, F to H). Proliferation and viability assays: MYCN^T58A conferred resistance to NVP-BEZ235 versus MYCN^WT (*P < 0.026; **P < 0.05). VEGF ELISA: NVP-BEZ235 suppressed levels of VEGF in MYCN^WT cells, with blunting response in MYCN^T58A cells (***P < 0.002). HUVEC migration: HUVECs (upper chamber) with MYCN^WT or MYCN^T58A cells (bottom chamber). NVP-BEZ235 reduced migration was blunted in MYCN^T58A cocultures (****P < 0.008). (D to F) MYCN-amplified Kelly cells were transduced with shRNA MYCN (Sh-MYCN) and shRNA control vector (Sh-Control). (D and E) Western blot: Sh-MYCN (#3) was more efficient than NVP-BEZ235, without altering p-Akt/p-rpS6. (F) Proliferation and viability: Sh-MYCN recapitulated the effects of NVP-BEZ235 (*P < 0.001; **P < 0.007, Sh-MYCN versus shRNA vehicle; ^#P < 0.04, Sh-MYCN versus NVP-BEZ235). VEGF ELISA: Sh-MYCN and NVP-BEZ235 similarly reduced VEGF (***P < 0.005, Sh-MYCN versus shRNA control; ^##P < 0.04, Sh-MYCN versus NVP-BEZ235). HUVEC migration: Sh-MYCN and NVP-BEZ235 blocked migration (****P < 0.002, Sh-MYCN versus shRNA control; ⁺P < 0.0001, all assays with Sh-MYCN + NVP-BEZ235 versus Sh-Control + vehicle).

Stabilization of MYCN protein blocks the efficacy of NVP-BEZ235. (A) Kelly MYCN-amplified neuroblastoma cells were stably transduced with three different HUWE1 shRNAs or shRNA control. Western analysis demonstrates that shRNA in lane 2 (Sh-1) knocked down HUWE1 without affecting the PI3K target p-Akt or mTOR target p-rpS6. Sh-1 was used for subsequent experiments. (B) Western blot: HUWE1 knockdown promoted partial resistance to NVP-BEZ235 (lane 4 versus lane 2). (C) Cycloheximide pulse-chase assay: cycloheximide was added over a 2-hour time course. HUWE1 knockdown promoted longer half-life of MYCN versus control. (D) Vehicle-treated arms from both Sh-Control and HUWE1 knockdown lines were normalized to 100%, and NVP-BEZ235–treated arms were graphed as percent change compared to vehicle-treated (original graphs in fig. S9, C to E). Proliferation and viability: with NVP-BEZ235, HUWE1 knockdown cells were resistant versus Sh-Control cells (*P < 0.003; **P < 0.003). VEGF ELISA: NVP-BEZ235 blocks VEGF in Sh-Control, with significant resistance mediated by HUWE1 knockdown (***P < 0.03). HUVEC migration: HUWE1 or Sh-Control cells (lower chamber) with HUVECs (upper chamber). With NVP-BEZ235 treatment, HUWE1 knockdown cells were significantly resistant versus Sh-Control cells (****P < 0.003).

Tumors derived from HUWE1 knockdown cells were resistant to treatment with NVP-BEZ235 in vivo. (A to S) HUWE1 knockdown (Sh-1) or shRNA control Kelly lines were transplanted orthotopically into renal capsules of nude mice. Daily treatment with NVP-BEZ235 (35 mg/kg) or vehicle was initiated on day 14. Tumors were collected for analysis after 2 weeks of treatment. n = 5 for each arm. Graphs were normalized as in Fig. 4C. (A) Tumor burden (weight and volume): NVP-BEZ235 blocked shRNA control tumors, whereas HUWE1 knockdown tumors showed significant growth despite treatment with NVP-BEZ235 (*P < 0.003, Student’s t test, for difference). (B) Representative images of whole tumors under each condition. White ovals indicate kidney in each sample. Scale bar, 5 mm. (C to R) Histological analysis: CD31 (endothelial cells), Ki67 (proliferating cells), DAPI (nuclei), and H&E. Scale bars, 50 μm. (S) Quantification of data from (C) to (N). Vascular density (CD31) and proliferation (Ki67) in HUWE1 knockdown tumors were resistant to treatment with NVP-BEZ235 relative to shRNA control tumors (**P < 0.003, Student’s t test). n = 3 mice in each arm. Three representative images per mouse were quantified using ImageJ.