Figure 7. pG1 sensitizes resistant lymphoma cells to inhibition of PI3K independent of C481S BTK mutation.

A, schema for overriding the BTK^C481S mutation by dual inhibition of CDK4 and PI3K. B, confirmation of the BTK sequence (WT or C481S) reconstituted in DT40 BTK^−/− cells by Sanger sequencing. C, immunoblotting of indicated proteins in DT40 BTK^−/− cells stably infected with a lentivirus expressing human BTK^WT-V5, BTK^C481S-6xHis or the GFP-V5 control vector as described in the Supplementary Methods. D, Cell cycle analysis by propidium iodide staining of DT40_BTK^WT and DT40_BTK^C481S cells cultured with PD 0332991 (PD) (0.3 μmol/L) for 16 hours. Number in the FACS profile indicates the percentage of cells in S phase as analyzed by FlowJo. E, DT40_ BTK^WT and DT40_ BTK^C481S cells were cultured in the absence (Cntl) or presence of PD 0332991 (pG1) as in D before addition of GS-1101 or BAY 80-6946 for 32 hours at concentrations indicated in the continuous presence of PD 0332991. Cell death was determined by ToPro-3 staining and FACS analysis. The total number of live cells was determined by Trypan Blue staining (x10⁶ cells/ml). Error bars represent SD. Data are representative of 3 independent experiments.