FLT3 is overexpressed or coexpressed with FLT3 ligand in >90% of AML cases (3
), providing another mechanism of constitutive receptor activation. Therefore, specific targeting of activated FLT3 receptor by small molecule inhibitors is an attractive therapeutic approach for AML. Although several chemical compounds have been developed to inhibit FLT3 activity (46
), none of these inhibitors is directed solely to the FLT3 receptor; they can affect activities of other kinases, such as PKC, TrkA, VEGFR, KIT, or PDGFR, thus increasing the possibility of nonspecific toxicity (3
). The effectiveness of a given inhibitor may also vary depending on the actual mutation in the FLT3 receptor, raising a possibility of drug resistance. Therefore, elucidating the pathways downstream of FLT3 might lead to the development of better therapeutic approaches in AML.
The major known effect of all FLT3 inhibitors is the induction of apoptosis (47
). Ours is the first report demonstrating that treatment of FLT3 ITD cells with MLN518, in addition to apoptosis, can also trigger differentiation. We also showed that inhibition of the ERK1/2 pathway in FLT3 mutant AML line could achieve the same effect, whereas inhibition of other downstream pathways (Akt, Stat3/5) resulted only in apoptosis. Thus, activation of FLT3 affects signaling pathways controlling both differentiation and apoptosis.
Several clinical trials with various FLT3 inhibitors are currently in progress (21
). In one of them, oral administration of CEP-701 to 14 AML patients expressing FLT3-activating mutations demonstrated sustained FLT3 inhibition and clinical evidence of biologic activity in only 5 patients (22
). The same study also showed that two out of eight patients exhibited >90% inhibition in FLT3 receptor activation, but remained resistant to the clinical effect of this compound. The molecular effects of another FLT3 inhibitor, SU11248, were evaluated on patient samples and showed transient or sustained decrease in ERK1/2 activation in 80% of patients. Interestingly, activation of the upstream kinase, MEK1, was seen in only 39% patients. Thus, components of the FLT3–MEK1–ERK1/2 cascade can be dissociated in patients (21
Previously, we have identified C/EBPα as a transcription factor necessary and sufficient for neutrophilic differentiation (24
). It was logical to assume that such important molecule might be a target in pathogenesis of leukemia. In fact, the expression or function of C/EBPα are disturbed in various subtypes of leukemia (30
), providing an explanation for the block in differentiation.
In the course of this work, we show that, in mutant FLT3 AML, the differentiation-promoting function of C/EBPα is inhibited at yet another level: posttranslational modification by phosphorylation. We were able to show, using myeloid and 293T cells, that FLT3 activation induces phosphorylation of C/EBPα on S21, which inactivates C/EBPα function. Consistent with the known role of C/EBPα in promoting granulocytic differentiation, inhibition of FLT3 or introduction into the cells of S21A C/EBPα mutant rescued the differentiation block in AML cells. Furthermore, inhibition of FLT3 activity decreased the levels of S21 phosphorylation in FLT3 mutant cell lines and AML patients. Nonetheless, inhibition of FLT3 was less effective in decreasing the C/EBPα phosphorylation in patient samples when compared with cell lines. One possible explanation for this difference is that the activities of serine/threonine phosphatases, such as protein phosphatase 1 (PP1), or MAP kinase phosphatase-1 (MKP-1) may be lower in patients (57
Several groups previously generated 32Dcl3 stable lines expressing mutants of FLT3 (59
). In those cells, C/EBPα, was shown to be down-regulated at the mRNA level (60
). Also, two out of three FLT3 ITD-positive patients had very low levels of C/EBPα mRNA, which increased approximately twofold following the FLT3 inhibition therapy (60
). In our own studies with murine 32Dcl3 and 503 (PU.1−/−
line expressing endogenous C/EBPα; unpublished data) stable lines, we found that FLT3 ITD mutation did not lead to a strong activation of ERK1/2 and we did not observe an increase in C/EBPα phosphorylation on S21, which is located in a protein region highly conserved among mammalian species. Furthermore, in a mouse transplantation model, FLT3 ITD mutations resulted in development of myeloproliferative disease rather than AML (61
). These findings suggest that the signaling pathways activated by FLT3 are different in mice and humans. In fact, intrinsic differences have been reported between mouse and human control of hematopoiesis mediated by C/EBPα (62
). We cannot rule out the possibility that both C/EBPα-inactivating mechanisms (transcriptional repression and functional inhibition) may be operational in human FLT3 mutant AML, possibly depending on the differentiation stage of the transformed cell.
Overexpression or constitutive activation of the ERK pathway has been shown to play an important role in the pathogenesis and progression of various cancers (63
). Currently, preclinical trials for pancreas, colon, breast cancers, and leukemia are ongoing with compounds specifically inhibiting MEK1/2 component of this pathway (64
). Abnormal activation of the ERK pathway also occurs in leukemia because of the activating mutations in FLT3, Ras, as well as genes in other pathways (PI3K, PTEN, Akt) (65
). Thus, targeting the Ras–Raf–MEK–ERK pathway in leukemia may offer a potential alternative to standard chemotherapy. It has been shown that the primary effect of down-modulation of MEK–ERK pathway activation in AML primary blasts by a selective inhibitor of MEK1 (PD98059) was a cell cycle arrest followed by apoptosis (66
). Our results demonstrate that PD98059 could decrease pS21-C/EBPα levels in the FLT3/ITD AML lines and induce granulocytic differentiation. Our data provide strong indication that inhibitors of MEK–ERK cascade could have significant clinical benefit in the treatment of FLT3 mutant AML, especially in cases of resistance to FLT3 inhibitors. Furthermore, development of protein therapies based on transduction of constitutively active C/EBPα (such as the S21A mutant) may prove effective in treatment of subtypes of leukemia with inadequate expression/function of C/EBPα.