Everolimus

For infection, cells were seeded 24 h before. GLV-1 h68 was diluted in the respective amount of DMEM supplemented with 2% (v/v) FCS to prepare the infection medium. The dilution ratio was calculated to ensure infection with a specific multiplicity of infection (MOI, effector target ratio, i.e. viral particles per cell). Cells were rinsed with phosphate buffered saline (PBS, Sigma-Aldrich) prior to infection, shortly before the respective amount of infection medium was added. Virus infection was allowed to take place for 1 h with swaying every 15 min. Then, infection medium was replaced with normal cell culture medium. Mock treatment was conducted with DMEM supplemented with 2% (v/v) FCS. For sole treatment with everolimus (Sellekchem, Munich, Germany), cell culture medium was replaced with medium containing everolimus at the respective concentration at 24 h post cell seeding. For combinatorial treatment with GLV-1 h68 and everolimus, infection medium was replaced with cell culture medium containing everolimus in the respective concentration.

MCF7/NeuT cells were seeded onto a 6-well plate and allowed to attach overnight. On the next day, cells were treated with different concentrations of LY294002 (Selleckchem), PD98059 (Selleckchem), U73122 (Tocris), everolimus (Selleckchem) or CHIR-99021 (Selleckchem). For induction of the oncogenic rat HER2 variant, NeuT in the MCF7 cells, doxycycline (Sigma) was added with a final concentration of 1 µg/ml per well to one half of the experiment setup one hour after the inhibitor addition [16 (link)]. Medium was changed every three days, and cells were harvested after three and seven days, for RNA and protein expression analyses as described above.
For the EDI3 expression studies in endogenous HER2 expressing cell lines, SKBR3 and HCC1954 cells were seeded onto a 6-well plate and allowed to attach overnight. On the next day, cells were treated with different concentrations of lapatinib, everolimus, CHIR-99021, compound 3i (666–15), C188-9, KC7F2, SC75741, 10074-G5 or SR18662 (all from Selleckchem). After one, two and three days (as well as 4 days for lapatinib), cells were harvested at approximately 80% confluency for RNA and protein expression analyses as described above. Medium was changed after two days.

Mice (female SCID/beige) were purchased from Charles River Laboratories. N = 160 mice were used for the studies. Mice were implanted subcutaneously with 2.5x10⁶ Caki-1 RCC cells plus Matrigel per mouse. When tumors reached an average of ~400 mm³, mice in the everolimus combination studies were randomized into the following 4 treatment groups: (i) vehicle, (ii) telaglenastat at 200 mg/kg and dosed orally twice daily (BID), (iii) everolimus at 1 mg/kg dosed orally once daily (QD), and (iv) telaglenastat and everolimus (Selleck Chemicals, #S1120) at 200 mg/kg and 1 mg/kg, respectively. For the cabozantinib (and other TKI combination studies), mice were randomized into the following 4 treatment groups: (i) vehicle, (ii) telaglenastat at 200 mg/kg and dosed orally BID, (iii) cabozantinib at 1 mg/kg dosed orally QD, and (iv) telaglenastat and cabozantinib (Selleck Chemicals, #S1119). Sunitinib (Selleck Chemicals, #51042) was dosed orally at 20mg/kg QD and axitinib (MedChemExpress, #HY-10065) at 25mg/kg QD. Statistical analyses were conducted using Ordinary ANOVA with Tukey’s multiple comparison tests.