In acute myeloid leukemia (AML), about 25-30% of patients harbor a constitutively active receptor tyrosine kinase (RTK) FLT3 encoded by a FLT3 allele harboring internal tandem duplication (FLT3-ITD) mutation. The presence of FLT3-ITD correlates with poor prognosis in AML and it makes FLT3 an attractive therapeutic target in AML. Unfortunately, to date small-molecule inhibitors of FLT3 have resulted in only partial and transient clinical responses with residual leukemic blasts resistant to FLT3 inhibitors detected in blood or bone marrow. In this study, we investigated whether the RTK Axl is responsible for resistance of FLT3-ITD(+) AML cells to PKC412 and AC220, FLT3 inhibitors currently under clinical trials for FLT3-ITD(+) AML patients. Upon treatment with PKC412 or AC220, phosphorylation of Axl was significantly enhanced in the FLT3-ITD(+) MV4-11 AML cell line and in primary blasts from a FLT3-ITD(+) AML patient. Consistently, a PKC412-resistant AML cell line and PKC412-resistant primary blasts from FLT3-ITD(+) AML patients had significantly higher levels of constitutively phosphorylated Axl and total Axl when compared with a PKC412-sensitive AML cell line and PKC412-sensitive primary blasts from FLT3-ITD(+) AML patients. We also found that resistance of AML cells against the FLT3 inhibitor PKC412 and AC220 was substantially diminished by the inhibition of Axl via a small-molecule inhibitor TP-0903, a soluble receptor Axl fusion protein Axl-Fc or knockdown of Axl gene expression by shRNA. Collectively, our study suggests that Axl is required for resistance of FLT3-ITD(+) AML cells against the FLT3 inhibitor PKC412 and AC220, and that inhibition of Axl activation may overcome resistance to FLT3-targeted therapy in FLT3-ITD(+) AML.
Previous clinical studies have shown that the presence of receptor tyrosine kinase (RTK) FLT3 with activating mutation, FLT3-ITD (internal tandem duplication), translates into poor prognosis in acute myeloid leukemia (AML). 1 Several tyrosine kinase inhibitors (TKIs) of constitutively active FLT3 have been developed and tested in clinical trials. 1 So far, these small molecule inhibitors have resulted in only modest improvement in AML patients because of, in part, the presence of leukemic blasts showing resistance against FLT3 inhibitors. 2 Thus, devising an improved therapeutic strategy to overcome AML resistance to FLT3 inhibitors could improve the likelihood of achieving long-term survival of AML patients.Tumor suppressor p53 is a critical gatekeeper for cell growth and division, and regulates a large number of downstream targets in response to various oncogenes and genotoxic stresses, ultimately suppressing tumorigenesis. 3 Although mutations in the tumor suppressor p53 gene (TP53) are found in only about 5-10% of AML patients, inactivation of wild-type p53 protein frequently occurs through overexpression of its negative regulatory molecule, murine double minute 2 (MDM2), which targets p53 for ubiquitin-mediated degradation. 3 On genotoxic stimuli such as irradiation, p53 is phosphorylated and the interaction between p53 and MDM2 is blocked, which leads to an increased level of p53 and eventually inhibition of the growth of tumor cells. Recent studies have shown that FLT3-ITD blocks p53 activation in AML, and restoration of p53 activities sensitizes AML blasts to FLT3 inhibitor treatment. 4,5 In addition, BA/F3 cells expressing FLT3-ITD (BA/F3-FLT3-ITD) were resistant to FLT3 inhibitor PKC412 on being transfected with lentivirus encoding shRNA targeting p53. 4 This suggests that FLT3-ITD + AML cells acquire resistance to FLT3 inhibitors, at least in part, by inactivation of p53, which provides a rationale to target p53 and its regulatory network for overcoming AML resistance to FLT3 inhibitors.First, we tested whether activation of p53 by Nutlin-3 could be exploited for overcoming resistance of FLT3-ITD AML cells to FLT3 inhibitors. Nutlin-3 is a highly specific, nonConflict of Interest: The authors declare no conflict of interest. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript genotoxic MDM2 antagonist and functions as a competitive inhibitor of the p53-MDM2 interaction. 6 As shown in Figure 1a, Nutlin-3 was able to significantly suppress the growth of the AML cell line MOLM13-R-PKC412 that is resistant to the FLT3 inhibitor PKC412. Another PKC412-resistant AML cell line MV4-11R-PKC412 that was generated from PKC412-sensitive MV4-11 harboring mutant form of p53 (ref.7) was resistant to Nutlin-3. Consistently, Nutlin-3 increased the level of p53 and regulated the expression of well-known p53 target genes, p21 and Axl, 3 in MOLM13-R-PKC412 (wild-type p53) but not in MV4-11R-PKC412 (mutant p53) ( Figure 1b). In primary AML blasts from patients that showed intrinsic re...
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