Axl blockade inhibits growth and induces apoptosis of AML cells via Bcl-2 and Puma. (A) Lentiviral overexpression of Axl compared with control-infected cells indicated a growth advantage of MV4-11 leukemia cells (n = 3; *P < .05, #P < .05). (B) In contrast, silencing of Axl by shRNA reduced growth of AML cells (n = 3; *P < .05; #P < .05). (C) Immunoblot showing increased Axl phosphorylation Gas6+ OCI-AML5 and MV4-11 cells induced by serum starvation (SF). Data are also provided as densitometric quantification of (pAxl/β-actin)/(tAxl/β-actin) normalized to cells cultured in normal medium (NM; n = 3; *P < .05). (D) In Gas6− HL60 cells phosphorylation of Axl was not upregulated in SF conditions. Densitometric quantification of (pAxl/β-actin)/(tAxl/β-actin) was normalized to NM cells (n = 3; *P < .05). (E) Treatment with BGB324 induced upregulation of pro-apoptotic Puma mRNA levels in AML cells compared with control (n = 3; *P < .05). (F) Immunoblot of anti-apoptotic Bcl-2 that was decreased by BGB324 treatment. Densitometric quantification of (cleaved caspase 3)/(β-actin) is normalized to untreated cells (n = 3; *P < .05). (G) Downmodulation of Puma reduced numbers of Annexin V+ cells after treatment with BGB324. Data are presented as percentage of mean Annexin V+ cells ± SEM (n = 3; *P < .05). (H) Overexpression of Bcl-2 reduced the fraction of Annexin V+ apoptotic cells after treatment with BGB324. Data are presented as percentage of mean Annexin V+ cells ± SEM (n = 3; *P < .05). (I) BGB324 was more efficient in primary AML cells expressing high levels of Axl (n = 10; r2 = 0.84; P < .05).
