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L. Mazzera et al.
To verify that the anti-tumor activity of pan-AKI and the synergizing effects of c-Abl inhibitors observed on cul- tured/isolated MM cells could be reproduced in vivo, we set up a multidrug-resistant xenograft mouse model of human MM. Consistent with our in vitro results, imatinib significantly potentiated the anti-tumor activity induced by pan-AKI in this in vivo setting, while having no effect as a single agent in vivo in a multidrug-resistant xenograft
mouse model of human MM (Figure 10A). Animal sur- vival was also significantly improved in mice treated with the combination imatinib/pan-AKI versus those that received monotherapies or vehicle alone (P<0.0015) (Figure 10A and Online Supplementary Table S4).
Immunobloting analyses on tumor masses harvested after five days post treatment confirmed decreases in the phosphorylation levels of Aurora kinases, enhancement of
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Figure 7. Accumulted NF-κB-inducing kinase (NIK) activates cytoplasmic c-Abl. (A) Stable clones of RPMI-8226 transfected with empty vector (EV) or with plasmid expressing NIK (NIK) were treated with MK-0457 (0.4 μM) or NIK siRNA, respectively. After 24 hours (h) cytoplasmic and nuclear extracts were prepared and equal amount of Cytoplasmic (cyto) and nuclear (nuc) cell lysates (10 μg) were immunoblotted against NIK, phospho-c-Abl (Thr735), phospho-c-Abl (Tyr245), c-Abl, β Tubulin and Histone H3 as loading control of cytoplasmic and nuclear fraction, respectively. Bands were subjected to densitometric scanning: cytoplasmic and nuclear blots were normalized to total β tubulin and Histone H3, respectively. In the graph below the relative fold change of cytoplasmic or nuclear c-Abl, phospho-c-Abl (Thr735) and phospho-c-Abl (Tyr245) levels was normalized with respect to empty vector (EV) control condition, which was taken as 1. The ratio of cytoplasmic to nuclear c- Abl, phospho-c-Abl (Thr735) and phospho-c-Abl (Tyr245) protein expression (Cyto/Nuc) is shown. The Cyto/Nuc ratio of phosphorylated and non-phosphorylated c- Abl in empty vector (EV) control condition (CONT) was set as 1. In histogram are shown average quantification results±Standard Deviation (SD) of three independent blots [#P<0.01, °P<0.005, *P<0.001 vs. (EV) control condition, Dunnett test]. (B) Stable clones of RPMI-8226 transfected with empty vector (EV) untreated or treat- ed with MK-0457 (0.4 μM) and of RPMI-8226 expressing NIK (NIK) electroporated with non-specific control siRNA (CONT) or with NIK siRNA were harvested after an incubation of 24 h for cytospins and stained for c-Abl or were formalin fixed and paraffin embedded in cytoblocks for NIK staining. The microphotographs shown are representative of similar observation in three independent experiments (20x, 40x and 100x original magnifications). (C) OPM-2 and JJN3 cells were transfected with non-specific control siRNA (Cont) or NIK siRNA and after 3 h multiple myeloma (MM) cell lines were treated with MK-0457 (0.4 μM). After 48 h whole cell lysates were prepared and immunoblotted against NIK, phospho-c-Abl (Thr735), c-Abl and Actin as loading control. Bands were subjected to densitometric scanning. Levels of Thr735-phosphorylated c-Abl were normalized to overall c-Abl levels and c-Abl phosphorylation under non-specific siRNA control condition was set to 1. Histogram below represents the mean±SD of three independent experiments. (°P<0.02, *P<0.005, **P<0.0005 vs. control siRNA condition, Dunnett test).
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haematologica | 2019; 104(12)