Figure 8: RAS-PI3K crosstalk mediates AKT reactivation in the absence of p-ErbB3.

(a) 6L1G but not TZB treatment blocks both RAS-GTP formation and RAS–p110α interaction. RAS-GTP pull-down assay using GST-RBD beads (top) and co-immunoprecipitation of p110α and RAS with pan-RAS immunoblot detection (bottom) after 72 h of treatment of BT474 cells with 5 μM of RAS farnesyltransferase inhibitor lonafarnib alone or in combinations with 100 nM TZB and 6L1G. (b–e) Combination treatment of TZB with RAS farnesyltransferase inhibitor blocks p-AKT rebound and triggered apoptosis. Inhibition of XTT cell proliferation (b), cell cycle (c), and downstream signalling (d) after 72 h of treatment with 5 μM tipifarnib in combination with 100 nM TZB or 6L1G (Supplementary Fig. 5G). The p-AKT rebound was quantified over 48 h in e. In b–e, tipifarnib was used at 5 μM concentration in BT474, AU565, HCC1419 and at 1 μM in SKBR3 cells. Note that ERK phosphorylation was inhibited by both 5 μM tipifarnib and lonafarnib (two-sided, unpaired Welch’s t-test). (f) Selective RAS inhibitors RAS104 and RAS107, blocking the RAS-RBD binding interface, induce apoptosis in combination with TZB after 3 days treatment in BT474 cells. The non-interfering RAS binder RAS109 or the non-targeted DARPin E3_5 were used as controls with minute or no pro-apoptotic activity, respectively. BT474 cells were transfected with plasmids expressing RAS104, RAS107, RAS109 or E3_5 GFP-fusions and treated with 100 nM TZB 24 h later (Supplementary Fig. 5B). The PFA-fixed cells were stained with Vybrant Ruby stain, and the transfected GFP-positive population was analysed by FACS for cell cycle distribution.