Figure 5.
Targeting PAK4 by KPT-9274 induces significant cell death in FGFR3-expressing, t(4:14)-positive HMMCLs. (A) Public data from Jonathan Keats’ laboratory (keatslab.org) were used to evaluate the presence of cytogenetic abnormalities, FGFR3 mutations, and FGFR3 expression level in the 23 MM cell lines tested for KPT-9274 sensitivity. Two groups were established based on the presence versus absence of t(4;14), FGFR3 mutation, and high level of FGFR3 expression (superior to the median calculated in the 23 MMCL). Fourteen MMCL were at least positive for t(4;14), high FGFR3 expression, or presence of FGFR3 mutation, whereas 9 MMCL were negative for all. We next compared the KPT-9274 IC50 average between the 2 groups using an unpaired Student t test and observed that MMCL with high FGFR3 and/or FGFR3 mutation and/or t(4;14) were significantly more sensitive (P = .0324). (B) CD138-positive and -negative cells from a t(4;14) myeloma patient with FGFR3/IGH fusion were treated with different concentrations of KPT-9274 for 48 hours. Cell viability was assessed by CTG and presented as percent of viable cells compared with control. IC50 analysis was performed using GraphPad software. (C) Nude mice were subcutaneously inoculated with MM1S (left panel) or OPM2 (right panel) MM cell lines. Treatment started following detection of tumor (∼2 weeks from cell injection). Mice were treated with either 100 mg/kg of KPT-9274 or vehicle orally once per day, 5 d/wk. Tumors were measured in 2 perpendicular dimensions by caliper. (D) Comparison of tumor volume in control and treated mice at day 13 after initial assessment of tumor appearance and start of treatment in MM1S and OPM2 injected mice respectively. (E) A nitrocellulose filter arrayed with antibodies against 400 signal transduction proteins was blotted with lysates of RPMI 8226 cells transfected with GFP-tagged PAK4. The multiprotein complexes were detected by immunoblotting with a horseradish peroxidase–conjugated anti-GFP antibody and visualized by chemiluminescence. (F) Nitrocellulose filters immobilized with 24 antibodies were incubated with whole cell lysates from cells untreated or treated with KPT-9274. Data are presented as signal intensity. TR, treated.

Targeting PAK4 by KPT-9274 induces significant cell death in FGFR3-expressing, t(4:14)-positive HMMCLs. (A) Public data from Jonathan Keats’ laboratory (keatslab.org) were used to evaluate the presence of cytogenetic abnormalities, FGFR3 mutations, and FGFR3 expression level in the 23 MM cell lines tested for KPT-9274 sensitivity. Two groups were established based on the presence versus absence of t(4;14), FGFR3 mutation, and high level of FGFR3 expression (superior to the median calculated in the 23 MMCL). Fourteen MMCL were at least positive for t(4;14), high FGFR3 expression, or presence of FGFR3 mutation, whereas 9 MMCL were negative for all. We next compared the KPT-9274 IC50 average between the 2 groups using an unpaired Student t test and observed that MMCL with high FGFR3 and/or FGFR3 mutation and/or t(4;14) were significantly more sensitive (P = .0324). (B) CD138-positive and -negative cells from a t(4;14) myeloma patient with FGFR3/IGH fusion were treated with different concentrations of KPT-9274 for 48 hours. Cell viability was assessed by CTG and presented as percent of viable cells compared with control. IC50 analysis was performed using GraphPad software. (C) Nude mice were subcutaneously inoculated with MM1S (left panel) or OPM2 (right panel) MM cell lines. Treatment started following detection of tumor (∼2 weeks from cell injection). Mice were treated with either 100 mg/kg of KPT-9274 or vehicle orally once per day, 5 d/wk. Tumors were measured in 2 perpendicular dimensions by caliper. (D) Comparison of tumor volume in control and treated mice at day 13 after initial assessment of tumor appearance and start of treatment in MM1S and OPM2 injected mice respectively. (E) A nitrocellulose filter arrayed with antibodies against 400 signal transduction proteins was blotted with lysates of RPMI 8226 cells transfected with GFP-tagged PAK4. The multiprotein complexes were detected by immunoblotting with a horseradish peroxidase–conjugated anti-GFP antibody and visualized by chemiluminescence. (F) Nitrocellulose filters immobilized with 24 antibodies were incubated with whole cell lysates from cells untreated or treated with KPT-9274. Data are presented as signal intensity. TR, treated.

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