Figure 5. In vivo targeting of HER3 in the context of different PIK3CA mutants.

(A) Bar chart representing the ratio of helical domain (E545 and E542) mutations as compared to kinase domain mutations (H1047) across TCGA PanCancer Altas studies represented in cBioPortal (68). Line graph showing the mRNA expression (RSEM) for NRG1 across the same studies. (B) Correlation of Log2 HER3 interaction levels from AP-MS experiments and Log2 HER3 Y1197 phosphorylation levels from immunoblot analysis. All values are normalized by FLAG-PIK3CA levels in their respective experiments. Mutations marked in red were selected for in vivo experiments. (C-D) CAL-27 cells expressing inducible PIK3CA variants were transplanted into athymic nude mice. Mice were fed with doxycycline to induce PIK3CA expression. When tumor volumes reached approximately 100 mm³, mice were treated with vehicle (PBS) or CDX3379 (10mg/kg, twice a week) for approximately 15 days, as indicated. Shown are (C) tumor growth curves, (D) representative tumor images, and (C) last day tumor volume (****P < 0.0001 when compared with the control-treated group). (E) Quantification of immunoblot analysis of signaling events in the same CAL-27 cells in vitro. PIK3CA variant expression was induced by doxycycline (1μg/ml in culture medium), cells were treated with CDX3379 (1μg/ml, 1hr), and lysates were analyzed by immunoblotting as indicated. Densitometry analysis of western blots was performed using ImageJ. Data are represented as mean ± SEM, n= 3 in each group. (*P < 0.05 when compared with the control-treated group).