In the present study we questioned whether the sustained activation of NF-κB often observed in Acute Myeloid Leukemia (AML) cells can efficiently be modulated by the proteasome inhibitor bortezomib. Cells from AML patients (n=20) were exposed to 20 nM bortezomib for 24 hrs. Cytotoxic effects were observed in 75% of the patients, but surprisingly we observed that not the AML CD34+ but the CD34- cells were most sensitive (survival CD34- vs. CD34+, 45% ± 18% vs. 75% ± 18%, p = 0.03). Limited cytotoxicity of AML CD34+ to bortezomib was also observed in long-term Ms5 culture assays. Since the AML CD34+ fraction is typically enriched for leukemic stem cells, we questioned whether the difference might be linked to a difference in cell cycle activity. However, by using the CFSE assay it was observed that CFSE bright (non-dividing) and dim (dividing) AML cells were equally sensitive to bortezomib. Since NF-κB is an important target of the proteasome, NF-κB activation (p65) was determined in nuclear extracts of AML CD34+ and CD34- cells. Bortezomib treatment reduced the NF-κB activity, whereby the reduction was much more pronounced in AML CD34-cells as compared to AML CD34+ cells (fold decrease respectively 1.77 vs. 1.18, p = 0.06). Combination of bortezomib with the NF-κB inhibitor BMS-345541 reduced the survival of AML CD34+ cells to similar levels as in AML CD34- cells, demonstrating that sensitivity to bortezomib was at least related to inhibition of NF-κB activation. Micro-array studies of AML CD34+ and CD34- cells (Leukemia 2011;12:1815) revealed that NF-κB activation in AML CD34- cells is much more dependent on cytokine and chemokine signaling compared to AML CD34+ cells, suggesting alternative activation pathways in both fractions. This might clarify the difference in response of AML CD34+ and CD34- cells to bortezomib. Finally it was shown that MCL-1 is also an important target of bortezomib in AML cells. Western blots demonstrated that MCL-1 accumulated in AML cells upon bortezomib treatment. Targeting MCL-1 by RNAi or the inhibitor obatoclax sensitized AML CD34+ cells (n=4) to the effects of bortezomib (fold decrease = 2.2, p = 0.05). In conclusion, we demonstrated that AML CD34- cells are most sensitive to bortezomib treatment. Resistance of AML CD34+ cells was related to insufficient inhibition of NF-kB activation and accumulation of MCL-1. The limited effectiveness in AML CD34+ cells can be overcome by co-targeting the NF-κB or MCL-1 pathway.
|Nummer van het tijdschrift||8|
|Status||Published - 1-aug-2012|