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c-Abl/NIK limits the efficacy of Aurora inhibitors
was significantly inhibited by pan-AKI, thereby indicating that Src kinase, of which c-Abl and STAT3 are direct downstream substrates and effectors,16-18 may compensate for or obscure the pan-AKI-induced NIK-dependent c-Abl activation.
Based on its off-target activity against wild-type and mutatedBCR-ABLincludingtheimatinib-/nilotinib-/dasa- tinib-resistant T315I-BCR-ABL, MK-0457 has shown clin- ical efficacy in chronic myelogenous leukemia patients bearing T315I mutated BCR-ABL.48-50 However, it has been
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Figure 12. Functional inhibition of PIM kinases enhances the anti-myeloma effects of Aurora inhibitors. (A) Multiple myeloma (MM) cell lines were incubated with the pan-PIM kinase inhibitor (SMI-4a) at 10 μM for 3 hours (h), and then were treated with MK-0457 (0.4 μM) or PHA-680632 (1 μM) in absence or presence of HS- 5 cells (+HS-5). After 48 h cell death was measured by flow cytometry analysis of Annexin V-FITC/PI or Annexin V-PE/7-AAD staining. Values represent means±Standard Deviation (SD) of three independent experiments. (#P<0.05, *P<0.01, **P<0.005 vs. pan-AKI-treated MM cell lines; Dunnett test). (B) MM cell lines cells were transfected with non-specific control siRNA (Cont) or cotransfected with siRNA targeting PIM1 and PIM2 and after 3 h MM cell lines were treated with MK-0457 (0.4 μM) or PHA-680632 (1 μM). After 48 h whole cell lysates of transfected MM cell lines were prepared and immunoblotted against PIM2, PIM1 and Actin. At the same time point, cell death was measured by flow cytometry analysis of Annexin V-FITC/PI or Annexin V-PE/7-AAD staining. Values in the graph represent means±SD of 3 independent experiments. (*P<0.05 vs. non-specific control siRNA; Dunnett and Tukey-Kramer tests). (C) PIM2, PIM1 and Actin western blot analysis in CD138-purified plasma cells from three patients with MM (samples MM#5, MM#6, MM#13) and peripheral blood mononuclear cells (PBMC) from two healthy vol- unteers (samples V#1, V#2) treated with pan-AKI. Bands were subjected to densitometric scanning and normalized to Actin; changes (folds increase) in the levels of each protein relative to untreated control, which was taken as 1 and values are shown below each lane. (D) CD138-purified plasma cells from ten patients with MM seeded in in presence of HS-5 cells and PBMC from five healthy volunteers were incubated with the pan-PIM kinase inhibitor (SMI-4a) at 10 μM for 3 h, and then with MK-0457 (0.4 μM) or PHA-680632 (1 μM). After 24 h cell death was measured by annexin-V staining and/or sub-G1 DNA content. Because of heterogeneous levels of basal cell death, the data of all ten primary samples and PBMC tested are expressed as % of specific cell death with the formula % Specific cell death = 100 x (induced cell death−basal cell death)/(100−basal cell death) and are shown in box plot format (median line in box delimited by 25th and 75th) (*P<0.005 vs. either treatment alone; Dunnett test).
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