Ibrutinib (PCI-32765) is an irreversible BTK (Bruton's tyrosine kinase) kinase inhibitor that has been extensively used as a tool compound to validate the role of BTK kinase in B cell related malignances.1, 2.Ibrutinib has been shown in preclin. studies to inhibit the proliferation of diffuse large B-cell lymphoma cells, mantle cell lymphoma cells, chronic lymphocytic leukemia cells and multiple myeloma cells by blocking BTK kinase activity; ibrutinib was recently approved for the clin. application on mantle cell lymphoma and chronic lymphocytic leukemia cells.3, 4, 5, 6, 7.Ibrutinib has also exhibited anti-inflammatory effects in preclin. models.8, 9.Recently, it has been reported that ibrutinib is also effective against epidermal growth factor receptor mutant-pos. non-small cell lung cancers through inhibition of epidermal growth factor receptor kinase activities.10.In addition, there is evidence showing that BTK is also an important target for Acute Myeloid Leukemia (AML).11.Despite the evidence that BTK knockdown impaired AML cancer cell growth, which suggested that BTK was important for AML cell proliferation, BTK kinase inhibition through use of a small mol. inhibitor like ibrutinib led only to moderate inhibition of proliferation of U937 cells with no apparent activity against other AML cell lines such as HL-60, TF-1 and THP-1.11.To further investigate the potency and Interestingly, we found that only FLT3-internal tandem duplication (ITD) mutant AML cell lines (MOLM13, MOLM14 and MV4-11) were sensitive to ibrutinib (Figure 1a and Supplementary Table 1).This is similar to what has been observed with the highly potent and selective FLT3 inhibitor, AC220, but differs from that observed for the targeted BTK kinase inhibitor, CGI-1746.12, 13 The selective antiproliferative activity was further confirmed in the clonogenic assay (GI50: 170-478 nM) (Supplementary Figure 1).This suggests that ibrutinib might target FLT3 kinase in addition to BTK kinase.We then investigated the effect of ibrutinib on FLT3-ITD mediated signaling in drug-sensitive cell lines.(Figure 1b) Ibrutinib potently inhibited FLT3-ITD auto-phosphorylation (EC50: 318, 39 and 356 nM resp. for MOLM13, MOLM14 and MV4-11) (Supplementary Figure 2A).Phosphorylation of STAT5, a well-established downstream target, also was significantly inhibited with an EC50 between 100 and 200 nM (Supplementary Figure 2B).In addition, c-Myc expression was inhibited with an EC50 between 185 and 315 nM.(Supplementary Figure 2C) However, in FLT3 wt-expressing cell lines such as NOMO-1, HL-60, OCI-AML-3 and U937 cells, FLT3 signaling was not affected by ibrutinib upon FLT3 ligand stimulation (Supplementary Figure 2D).Interestingly, despite the expression of the FLT3 wt kinase in all four cell lines, the induction of FLT3 auto-phosphorylation upon FLT3 ligand treatment was not observed in NOMO-1 and HL-60 cells like what was observed in the OCI-AML-3 and U937 cells, which might be due to the different genetic background and require further detailed mechanistic study.The FLT3 inhibitor AC220 demonstrated similar inhibitory activity to ibrutinib in FLT3-mediated signaling pathways in the FLT3-ITD pos. cell lines.However, the BTK inhibitors, CGI-1746 and AVL-292, did not exhibit any effect on the signaling14 (Figure 2b and Supplementary Figure 2D).As all of these cell lines express BTK kinase, we then studied the effect of ibrutinib on BTK signaling.Upon anti-IgM (IgM) stimulation of B-cell receptor to active the BTK kinase signaling pathway, we found that only in the FLT3 wild-type (wt)-expressing cell lines (U937 and OCI-AML3), downstream mediators of BTK kinase were affected despite BTK phosphorylation having been inhibited in both FLT3 wt and FLT3-ITD cell lines.9 This suggests that FLT3-ITD mutant cell lines might not rely on BTK signaling for growth (Supplementary Figure 3A).Interestingly, except NOMO-1 and U937 cells, the phosphorylation of BTKY223 of MOLM14, MOLM13, MV4-11, OCI-AML-3 and HL-60 cells did not respond to anti-IgM stimulation apparently, which might be due to the basal-level expression of the IgM in these AML cells (Supplementary Figure 3B).AC220 did not affect BTK kinase activity and its direct downstream target PLCγ but instead inhibited phosphorylation of protein kinase B and extracellular-signal-regulated kinase.The BTK inhibitors, CGI-1746 and AVL-292, were effective against BTK kinase and PLCγ kinase phosphorylation, but had no effect on the phosphorylation of protein kinase B and extracellular-signal-regulated kinase.These results suggest that ibrutinib may mainly exert its inhibitory activity through FLT3 kinase but not BTK kinase in the FLT3-ITD pos. cells.To further confirm this, we knocked down BTK kinase in MOLM14 (FLT3-ITD), MOLM13 (FLT3-ITD), human erythroleukemia cell (HEL) (FLT3 wt) and NB4 (FLT3 wt) cells and found that the growth of MOLM14 and MOLM13 was only minimally affected; however NB4 cells were significantly inhibited and HEL cells were moderately inhibited (Figures 2a and b).The fact that BTK knockdown in HEL cells did not lead to suppressed growth may be due to the fact that mutant JAK2 is the oncogenic driver in this cell line.15 In addition, the finding that ibrutinib still significantly blocked the growth of BTK-knockdown MOLM14 and MOLM13 cells further validated that the growth inhibitory effect of ibrutinib is likely dependent mainly on FLT3-ITD inhibition.However, BTK kinase inhibition may also exert a combinatorial effect (Figure 2c).It has recently been reported that BTK kinase is activated by FLT3-ITD in FLT3-ITD pos. cell lines and dual inhibition of BTK and FLT3 kinase demonstrates combinatorial effects; these findings further confirm this observation.16 Previous kinase selectivity profiling revealed that FLT3 wt might be an addnl. target of ibrutinib.9 The biochem. ADP-Glo assay confirmed that ibrutinib has an IC50 of 205.8 nM/65.65 nM against FLT3 wt and FLT3-ITD, resp. (Supplementary Figure 4A).In the FLT3-ITD-dependent isogenic BaF3 cell line, ibrutinib demonstrated a GI50 of 120 nM; this inhibitory effect could be completely rescued by interleukin-3 treatment (Supplementary Figure 4B).Interestingly, it also potently inhibited the FLT3-D835H-BaF3 isogenic cell (GI50: 0.063 μM) and moderately inhibited FLT3-D835Y-BaF3 as well as TEL-FLT3-wt-BaF3 isogenic cells (GI50: 0.5 μ/0.46 μ), however was less active against ITD-D835Y, ITD-F691L and FLT3-K663Q mutations.(Supplementary Figure 5A) In addition, ibrutinib did not affect the growth of other oncogene-expressing Ba/F3 cells, such as c-Kit, PDGFRα, BCR-ABL and NRAS (Supplementary Figure 5B).Although there are cysteine residues in the ATP-binding pocket, binding mode examination with mol. modeling demonstrates that unlike the mode of inhibition against BTK kinase, ibrutinib inhibits FLT3 kinase through reversible binding and this is further confirmed by the washing-out experiment (Supplementary Figure 4C and D).Ibrutinib induced apoptotic cell death in a concentration- and time-dependent manner in FLT3-ITD cell lines but not FLT3 wt-expressing cell lines (Supplementary Figure 6).Cell cycle anal. revealed that ibrutinib, but not the highly selective BTK kinase inhibitor, CGI-1746, could induce a concentration-dependent manner-arrest cells in G0/G1 phase (Supplementary Figure 7).In addition, ibrutinib exhibited dose-dependent anti-proliferation activity against FLT3-ITD mutant-expressing primary patient cells but not FLT3 wt-expressing and multiple mutants such as FLT3-ITD/FLT3-D835Y and FLT3-D835Y/E mutants expressing samples, which further confirms that ibrutinib is FLT3-ITD selective (Figure 2d).AML still remains a serious unmet medical need and the FLT3-ITD mutant has been found in ∼30% of AML patients.Besides standard chemotherapy, there is no targeted therapy approved in the clinic to date despite several inhibitors that are presently under clin. investigation.17 Currently, ibrutinib is under extensive clin. investigation against a variety of different B-cell malignancies.Given the fact that the safety profile of ibrutinib is tolerated in the patients, our results might help to expand the application of this drug to AML patients harboring the FLT3-ITD mutant.Interestingly, despite the fact that the effective concentration of ibrutinib in the in vitro studies in the FLT3-ITD pos. cell lines falls within the range of the peak plasma concentration used in human clin. trials (around 150 ng/mL at 560 mg/day dosage), the concentration required for the induction of apoptosis and cell cycle arrest for ibrutinib is higher, which suggests that an alternative formulation might be needed such as nano technol., to allow the increase of the plasma concentrations in the human patients while simultaneously retaining the safety profile for the potential clin. application.18.
