Figure 6. BH3-mimetics therapeutic inhibition of the BCL-2/BCL-XL programmed cell death pathway “Achilles' heel” to eradicate “early” TKI-resistant lung tumor survivor cells.
A, siRNA-mediated knockdown of BCL-2 and BCL-XL in HCC827 cells. WCLs at day 2 and day 6 post-siRNA transfection were then extracted for Western blotting to verify efficient gene knockdown of the target protein(s) expression.
B, BCL-2/BCL-XL RNAi knockdown or BH3-mimetic ABT-737 (2μM) in conjunction with erlotinib (1μM), remarkably suppressed the emergence of “early” EGFR-TKI resistant tumor-evader cells in HCC827. Representative photomicrographs from the triplicate experiments are shown here. Mag: 50×.
C, Proapoptotic BH3-mimetic ABT-737 eradicated the H1975 early tumor prosurvival resistance against CL-387,785. H1975 cells that were pretreated with 6 days of CL-387,785(1μM) were replated at full confluence, followed by further treatments as indicated for 3 additional days in triplicate, either with CL-387,785(1μM), ABT-787(2μM), or ABT-737+CL-387,785, followed by crystal violet cell staining (Top). U, Untreated control. (Bottom), Induction of proapoptotic marker cleaved-PARP by BH3-mimetic in the CL-387,785-resistant early tumor survivor H1975 cells.
D, ABT-737, Obatoclax and HA14-1 eradicated the H1975 early tumor prosurvival resistance against dual-TKIs inhibition by erlotinib/SU11274 (ERL/SU). The experiment was performed with H1975 cells similar to (C) above, except that cells were pre-treated with dual EGFR-MET inhibitors here, i.e. Erlotinib(1μM)/SU11274(1μM). B H 3-mimetic used in treatment Days 7–9 were all 2μM in concentration. (Top), Crystal violet cell survival staining assay. (Bottom), BH3-mimetic treatment of the dual ERL/SU-resistant tumor cells induced a proapoptotic response.
E, In vivo EGFR inhibition with erlotinib in conjunction with BH3-mimetic ABT-737 led to significantly more durable tumor response and prolonged remission in HCC827-luc lung adenocarcinoma xenograft. Left: Schematic outline of treatment conditions of the in vivo HCC827-luc xenografts. Middle (Upper): In vitro HCC827_ERL-D9.R early resistant TKI-evader cells emerged after 6 days of erlotinib(1μM) treatment, were eradicated by co-targeting BH3-mimetic inhibition using ABT-737(2μM), Obatoclax(2μM) or HA14-1(2μM)+/-ongoing erlotinib from days 7–9. Middle (Lower): In vivo HCC827-luc tumor xenograft growth, under treatment conditions as in Groups I-IV, was monitored by BLI as described in the Materials and Methods. Error bar, ± SEM.
Erlotinib-alone (III) vs ABT-737+Erlotinib (IV): *, p=0.0009. Erlotinib-alone (III) vs Diluent Control (I), p<0.0001 (†); ABT-737+Erlotinib (IV) vs Diluent Control (I), p<0.0001 (†). Group II (ABT-737-alone) vs Group IV (ABT-737+Erlotinib), p=0.0004 (††).
Right, BH3-mimetic ABT-737 in vivo treatment in conjunction with EGFR-TKI (Group IV) significantly abolished lung tumor recurrence.