Fig. 6: Targeting of KRAS–SHANK3 interaction by anti-SHANK3 nanobodies induces apoptosis and inhibits KRAS-driven tumour growth.
From: SHANK3 depletion leads to ERK signalling overdose and cell death in KRAS-mutant cancers
a–c The inhibitory function of anti-SHANK3 SPN nanobodies on KRAS–SHANK3 interaction. a The effect of two independent SHANK3 SPN nanobodies (A01 and E01) at the indicated concentration on the binding of His-tagged SHANK3 SPN to GST-tagged KRAS-GTP as measured by ELISA (data are mean ± s.d. of the Eu-signal; three independent experiments; Unpaired two-tailed Student’s t-test with Welch’s correction). b Top: The experimental outline showing His-SHANK3 SPN pulldown of GST-KRAS-GTP in the presence of anti-SHANK3 SPN nanobodies. Bottom: A representative blot showing loss of SHANK3–KRAS interaction in the presence of the nanobodies (n = 2 independent experiments). c Specificity analysis of anti-SHANK3 SPN nanobodies. A549 cells expressing mCherry alone (control; Ctrl) or mCherry-tagged anti-SHANK3 SPN nanobodies (A01, E01) were subjected to IP. A representative western blot, probed with the indicated antibodies is shown (n = 2 independent experiments). d, e, Analysis of cell viability in the presence of anti-SHANK3 nanobodies. Representative flow cytometry assay histograms (d) and quantification (e) of apoptosis (Annexin V-FITC-positive (+) cells) in PANC-1, A549 and H441 cells, expressing mCherry (Ctrl) or mCherry-tagged anti-SHANK3 SPN-nanobodies, 4 days after transfections [mean ± s.d.; n = 3 (PANC-1) and 4 (A549) independent experiments; two-way ANOVA and Sidak’s post hoc test; example scatter plots and gating strategy are shown in Supplementary Fig. 12]. f Analysis of tumour growth with HCT-116 cells transiently expressing mCherry-tagged anti-SHANK3 SPN nanobodies (A01 and E01) or mCherry (Ctrl) and inoculated on CAM membranes (data represent individual tumours and the mean ± s.d.; n = 18 (Ctrl, E01) or 21 (A01) tumours/treatment group; Kruskal-Wallis one-way ANOVA and Dunn’s post hoc test). g Schematic model of SHANK3-controlled cell fate in KRAS-mutant cancers. SHANK3 directly interacts with active KRAS and competes with RAF for KRAS binding to sustain oncogenic RAS-MAPK/ERK signalling at an optimal level (i.e. below toxic oncogenic signalling) in KRAS-mutant cancers. SHANK3 loss (1) or inhibition of SHANK3–KRAS interaction (2) drive KRAS-mutant cells into cell death. Source data are provided as a Source Data file.
