Figure 4.

Therapeutic Targeting of the MYCN/Aurora-A Interaction Inhibits Tumor Growth and Prolongs Survival in GTML/Trp53^KI/KI Mice

(A) Proximity ligation assay (PLA) analyzing MYCN/Aurora-A complexes in GTML/Trp53^KI/KI neurospheres following MLN8237 treatment (48 hr). Left panel shows close proximity (<40 nm) of antibody conjugated PLA probes that have been ligated, amplified, and detected with complementary fluorescent probes. Red dots represent the presence of MYCN or Aurora-A protein, or MYCN/Aurora-A interactions as indicated. Antibodies used are indicated by white text (2° Ab, secondary antibody control). Scale bar, 20 μm. Right panel shows mean values of signals (red dots) per cell representing MYCN expression or MYCN/Aurora-A interactions. Values are derived from triplicate biological replicates, and error bars represent SDs. p, unpaired t test.

(B) Kaplan-Meier survival for GTML/Trp53^KI/KI mice treated with MLN8237 (n = 10), GDC-0449 SHH antagonist (n = 4), or vehicle (n = 11) as indicated. (p, log rank test.)

(C) Longitudinal MRI analysis of tumor volume (n = 4) on the axial plane (top). Representative MRIs of the axial plane of MLN8237-treated animals compared to vehicle as indicated at day 0 and last day of treatment (bottom).

(D) H&E and immunohistochemical staining indicating levels of MYCN protein, cell proliferation (Ki-67), apoptosis (cleaved caspase 3), or mitotic activity as measured by phosphorylated Ser10 on histone H3 (H3 p-S10) after treatment with GDC-0449 or MLN8237. Scale bar, 50 μm.

(E) Immunoblotting of MYCN protein levels, and total and phosphorylated Thr288 on Aurora-A (p-T288 Aurora-A) in MLN8237-treated tumor tissues. For (D) and (E), animals were treated with vehicle, GDC-0449, or MLN8237 for 48 hr, and samples were taken 2 hr after final administration of agent.

Error bars represent mean ± SD. See also Figure S4.