Fig. 4

Rb phosphorylation and cell cycle suppression with the triple FGFR1, ER, and CDK4/6 blockade. a Western blot showing the phosphorylation (Ser780) of the Rb protein 48 h after treatments. For each cell line (MCF7 or T47D plus their LTED-R and FGFR1-overexpressing variants, and the FGFR1-amplified HCC1428 and its LTED-R variant), 7 conditions were tested: vehicle (VEH), fulvestrant (F), rogaratinib (R), palbociclib (P), fulvestrant plus rogaratinib (F + R), fulvestrant plus palbociclib (F + P), and fulvestrant plus palbociclib plus rogaratinib (F + P + R). Drug concentrations: MCF7 cell lines: F, 0.5 nM; P, 50 nM; R, 1 μM; T-47D cell lines: F, 1.5 nM; P, 100 nM; R, 1 μM; HCC1428 cell lines: F, 5 nM; P, 100 nM; R, 1 μM. Total Rb and beta-actin are shown for control purposes. It can be appreciated that in the FGFR1-high models (either because of engineered overexpression, LTED-R phenotypes, or primary amplification), the maximum suppression is achieved in the rogaratinib-containing combinations. Whereas the doublet (F + R) is highly active in most models, the triplet (F + P + R) is the only one that achieves suppression in all the models. Conversely, wild-type MCF7 and T-47D do not require rogaratinib for suppression, and actually, the greater effect is achieved by palbociclib. CCND1 protein levels showing that the effects on pRb are mediated by activation/inhibition of Cyclin D/CDK4/6 axis. b Charts showing BrdU incorporation (S-phase) of the same models and concentrations as in a, evidencing the translation of the effects over Rb phosphorylation in the cell cycle. The maximum effects in the FGFR1-high models are achieved by the triplet in all cases. Error bars: standard error. ***P < 0.001; **P < 0.01; *P < 0.05