BI D1870 has previously been demonstrated to inhibit Aurora T and the cellcycle specialists PLK1, although at greater concentrations than RSK inhibition. MCF7 cells expressing GFP, AKT1, RSK3, or RSK4 were handled with BEZ235 or BI D1870 for 24 hours. V5 labeled proteins pifithrin were run using the same blot, but bands were noncontiguous because of variations in protein size. AU565 and mcf7 cells were treated with BEZ235 and/or BI D1870 for 24-hours. Asterisks suggest non-specific band. MCF7 cells expressing GFP, RSK3, or RSK4 were handled with BEZ235 or BI D1870 for twenty four hours and subjected to cell cycle analysis to determine induction of apoptosis. Growth assay of breast cancer cells AU565 and HCC1143 transfected with siRNAs targeting RSK4 or get a grip on handled with BEZ235 and GDC 0941 for 24 hours, examined by CellTiter Glo. HCC1143 and au565 cells transfected with siRNA targeting RSK4 or get a handle on treated with BEZ235 or GDC 0941 for 24-hours and subjected to cell cycle analysis to examine induction of apoptosis. phenotype and using ERK path inhibitors to over come resistance. Mouse xenograft try out MCF7 Cellular differentiation cells overexpressing RSK4 or GFP control. Rats were treated 6 times weekly with BEZ235 or car for 24 days. Box plots represent cancer quantities, with whiskers showing minimum and maximum. The 2 treated populations are compared by a 2 tailed Students t test. Tumors were harvested at 24 days and examined by IHC for phosphorylation of rpS6235/236 and RSK4 expression. Representative images are shown in top section. H Score quantification of IHC examination of rpS6235/236, bottom panel. A 2 tailed Students t test compares the 2 treated populations. R 0. 01. Original magnification, 40, 400. Mouse xenograft assay with MCF7 cells overexpressing RSK4 or GFP get a handle on. Mice were treated 6 times per week with one agent BEZ235 or MEK162 or in combination. Containers represent tumor volume difference, lines represent mean tumor volume, bars represent SEM. A 2 tailed Students ALK inhibitor t test compares the treated versus untreated tumors. To try this hypothesis, we mixed PI3K inhibitors with the MEK inhibitor NVP MEK162 or the container RSK certain inhibitor dihydropteridinone. In MCF7 cells, RSK3 or RSK4 expression decreased reaction to therapy with some of the PI3K inhibitors alone. However, the mix of PI3K inhibition with MEK162 or BI D1870 completely reversed the resistance of RSK expressing cells. We addressed AKT overexpressing cells with combined PI3K inhibitors and RSK or MEK inhibitors, to confirm the specific efficacy of BI D1870. MCF7 cells overexpressing AKT1 were refractory to combined PI3K and MEK/RSK inhibition, confirming the particular efficacy of this mixture for cells with activation of the MEK/ERK/RSK pathway, as expected.