Figure 7.
Concurrent loss of activity of MCL-1 inhibitors is mediated via MDR1-driven drug efflux in CARF-resistant myeloma cells. (A) Surface CD243 (MDR1) expression on proteasome inhibitor sensitive, IXA-resistant as well as CARF-resistant RPMI8226 cells. (B) Drug combination studies of S63845 and established inhibitors of MDR1 demonstrated a reversal of the observed cross-resistance. Viability was assessed 72 hours after treatment initiation. Graphs show the mean ± standard deviation (SD) of 3 independent experiments performed in triplicate. Cells treated with 0.1% DMSO served as the control. Asterisks indicate statistically significant differences between CARF-resistant cells treated with single-agent DMSO or S63845 at the indicated concentrations. *P < .05; **P < .01; ***P < .001. (C) Intracellular S63845 concentrations were determined 6 hours after treatment via a mass spectrometry–based assay in RPMI8226-sensitive (DMSO) and CARF-resistant cells in the presence or absence of established MDR1 inhibitors (tariquidar and nelfinavir; n = 3 biological replicates). **P < .01; ***P < .001. (D) BAK activity status in CARF-sensitive and -resistant cells 4 hours after treatment with MCL-1 inhibitors in the presence or absence of MDR1 inhibitors (tariquidar and nelfinavir; n = 3 biological replicates. *P < .05; ***P < .001. (E) Dose-response curves of A-1210477 and S63845 in CARF-sensitive, CARF-resistant, and CARF-resistant-MDR1-knockout cells. Viability was assessed 72 hours after treatment. Graphs represent the mean ± SD of 3 independent experiments performed in triplicate. (F) Proposed mechanisms of MCL-1 inhibitor resistance in myeloma. In sensitive cells, MCL-1 inhibitors disrupt the binding between MCL-1 and proapoptotic molecules (BIM and/or BAK), which leads to the activation and oligomerization of BAK, release of cytochrome c and induction of apoptosis. Multiple modes of resistance exist in MCL-1 inhibitor insensitive cells. Downregulation of proapoptotic molecules (BAK, BAX, and BIM) or upregulation of antiapoptotic molecules (MCL-1, Bcl-2, and Bcl-xL) raise the threshold for apoptosis induction. The same is true of drug efflux–driven resistance, which limits the availability of intracellular MCL-1 inhibitors. In addition, altered binding patterns of pro- and antiapoptotic molecules (eg, Bcl-2:BIM > MCL-1:BIM complexes) affect MCL-1 inhibitor sensitivity levels, which are also mediated via dynamic responses such as treatment-induced sequestration of BAK and BIM via Bcl-xL.

Concurrent loss of activity of MCL-1 inhibitors is mediated via MDR1-driven drug efflux in CARF-resistant myeloma cells. (A) Surface CD243 (MDR1) expression on proteasome inhibitor sensitive, IXA-resistant as well as CARF-resistant RPMI8226 cells. (B) Drug combination studies of S63845 and established inhibitors of MDR1 demonstrated a reversal of the observed cross-resistance. Viability was assessed 72 hours after treatment initiation. Graphs show the mean ± standard deviation (SD) of 3 independent experiments performed in triplicate. Cells treated with 0.1% DMSO served as the control. Asterisks indicate statistically significant differences between CARF-resistant cells treated with single-agent DMSO or S63845 at the indicated concentrations. *P < .05; **P < .01; ***P < .001. (C) Intracellular S63845 concentrations were determined 6 hours after treatment via a mass spectrometry–based assay in RPMI8226-sensitive (DMSO) and CARF-resistant cells in the presence or absence of established MDR1 inhibitors (tariquidar and nelfinavir; n = 3 biological replicates). **P < .01; ***P < .001. (D) BAK activity status in CARF-sensitive and -resistant cells 4 hours after treatment with MCL-1 inhibitors in the presence or absence of MDR1 inhibitors (tariquidar and nelfinavir; n = 3 biological replicates. *P < .05; ***P < .001. (E) Dose-response curves of A-1210477 and S63845 in CARF-sensitive, CARF-resistant, and CARF-resistant-MDR1-knockout cells. Viability was assessed 72 hours after treatment. Graphs represent the mean ± SD of 3 independent experiments performed in triplicate. (F) Proposed mechanisms of MCL-1 inhibitor resistance in myeloma. In sensitive cells, MCL-1 inhibitors disrupt the binding between MCL-1 and proapoptotic molecules (BIM and/or BAK), which leads to the activation and oligomerization of BAK, release of cytochrome c and induction of apoptosis. Multiple modes of resistance exist in MCL-1 inhibitor insensitive cells. Downregulation of proapoptotic molecules (BAK, BAX, and BIM) or upregulation of antiapoptotic molecules (MCL-1, Bcl-2, and Bcl-xL) raise the threshold for apoptosis induction. The same is true of drug efflux–driven resistance, which limits the availability of intracellular MCL-1 inhibitors. In addition, altered binding patterns of pro- and antiapoptotic molecules (eg, Bcl-2:BIM > MCL-1:BIM complexes) affect MCL-1 inhibitor sensitivity levels, which are also mediated via dynamic responses such as treatment-induced sequestration of BAK and BIM via Bcl-xL.

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