Fig. 3. Genetic and therapeutic suppression of TORC1 overcomes resistance to neratinib.

(A) Immunoblot analysis of parental and neratinib-resistant 5637 cells treated with the indicated drugs combinations for 24 hr; alpelisib (PI3Ka inhibitor), everolimus (TORC1 inhibitor), selumetinib (MEK1/2 inhibitor), buparlisib (pan-PI3K inhibitor), MK2206 (AKT inhibitor). (B) Heatmaps representing 12-point dose response assays of 5637^NR and OVCAR8^NR cells treated with the indicated single agents or combinations. For single agents, cells were treated with increasing concentrations (3-fold) of the drug, up to 1 μM. For combination assays, all cells were treated with 1 μM neratinib and increasing concentrations of the second drug. (C) Representative images of cells seeded in 12-well plates, treated with the indicated drug combination; drugs and media were replenished every 72 hr. When control cell monolayers reached ~90% confluency, they were stained with crystal violet and imaged. (D) Immunoblot analysis of parental and neratinib-resistant MCF7^V777L cells treated with the indicated drugs for 24 hr. (E) Growth of organoids established from neratinib-resistant ST1616B (HER2^D769Y) breast cancer PDX. Organoids were treated with vehicle, neratinib (100 nM), everolimus (30 nM) or the combination. Viability was assessed 6 days later by measuring ATP levels and normalized to vehicle treated control. Values are mean ± SEM from three independent experiments, Student’s t-test. Representative images of organoids from each treatment group are shown on the right, scale bar = 1 mm. (F) Immunoblot analysis of neratinib treated 5637^NR and OVCAR8^nr cells transfected with the indicated siRNAs. (G) Viability of neratinib-treated cells transfected with the indicated siRNAs. When control cells reached ~90% confluency, cells were trypsinized and counted using a Coulter counter. Values are mean ± SEM from three independent experiments, one-way ANOVA. (H) Immunoblot analysis of lysates from 5637, OVCAR8, MCF7^L755S and MCF7^V777L cells stably transfected with TSC2 shRNA or empty-vector control. Cells were treated with increasing concentrations of neratinib; 24 hr later, cell lysates were prepared and tested by immunoblot with the indicated antibodies. MCF7^L755S and MCF7^V777L cells were tested under estrogen-free conditions. (I) Viability of 5637, OVCAR8, MCF7^L755S and MCF7^V777L cells transfected with TSC2 shRNA or empty vector and treated with neratinib. Drug and media were replenished every 72 hr. MCF7 and MCF7^V777L cells were treated with neratinib under estrogen-free conditions. When control cells reached ~90% confluency, monolayers were trypsinized and cell number measured using a Coulter counter. Each bar represents mean cell viability ± SEM from three independent experiments, Student’s t-test. See also Figure S3.