Fig. 2: Imparied Puma activation contributes to TKI resitance SCLL.

Western blot analysis of PUMA protein levels in the murine SCLL BBC1 cell line (A, above) or human KG1 cell line, after treatment at the indicated concentrations of either the ponatinib or BGJ398 FGFR1 inhibitors, show a dose dependent increase in the parental cells, while no changes are seen in the resistant cells that carry FGFR1 mutations in the inhibitor binding site. The same effect is seen in in cell lines CEP2A and ZNF112, which are resistant due to Pten deletions (B, above). RT-PCR analysis in all four cell lines shows that transcription levels of PUMA increases significantly in the cells treated (A, B, below) with the FGFR1 inhibitors at the maximum concentrations shown in A and B (above). Flow cytometric analysis of BBC1 and CEP2A cells shows an increase in 7AAD/Annexin V+ cells in the parental cells treated with BGJ398 at the IC50, but no changes are observed in the resistant cells (C). Western blot analysis (D) of signaling intermediates demonstrates consistent Akt activation and Puma suppression in the resistant cells (see text) and phosphorylation of Foxo3a is suppressed in parental, but not in the resistant cells treated with BGJ398. Student’s t-test was used for statistical comparison between groups. **p < 0.01; ***p < 0.001; ns not significant.