Activating mutations in FLT3 receptor tyrosine kinase are among the most common mutations in AML and indicate poor prognoses (1
). Thus, development of small molecule inhibitors specifically targeting FLT3 seemed to be a promising approach for treating a large number of AML cases (15
). Although different activating mutations in FLT3 exhibit divergent sensitivities toward different FLT3 inhibitors (30
), the preclinical and clinical trials involving the first generation of drugs (PKC-412, MLN518, CEP-701, SU11248, and AC220) showed biologic activity and favorable toxicity (15
). However, in several cases, patients demonstrated primary resistance to the drugs (3
). Alternatively, an initial response was soon followed by emergence of secondary mutations, for example, mutations N676K and D835Y in the kinase domain of FLT3 (38
). To bypass this problem, a search for a new generation of FLT3 inhibitors is under way. In the meantime, combination therapies with selective FLT3 inhibitors and conventional cytotoxic chemotherapy are being investigated, but those have been characterized by unacceptable toxicity and poor tolerance (40
). Alternative treatments of FLT3 mutant AML may involve inhibitors of the downstream pathways activated by FLT3ITD mutations. For example, we demonstrated before that inhibition of the ERK1/2 pathway in FLT3ITD-expressing AML cells leads to their differentiation (21
). The multitude of downstream signaling pathways activated by mutant FLT3 receptors (5
) may increase the repertoire of possible drug combinations.
A majority of the FLT3ITD downstream pathways control survival and apoptosis, while ERK1/2 signaling plays a role in the differentiation block by phosphorylating the C/EBPα transcription factor on serine 21 and inhibiting its function (17
). Interestingly, only a fraction of FLT3ITD patients exhibited activation of the ERK1/2 pathway (22
). Of note, due to technical difficulties in examining the ERK activity in FLT3ITD leukemias, additional studies are needed to determine whether activation of ERK can serve as a specific biomarker of FLT3 signaling in primary leukemias (22
). Moreover, we observed serine 21 phosphorylation on C/EBPα in cells with an FLT3ITD mutant receptor, which is disabled in ERK1/2 activation (mutant N51; refs. 23
). We hypothesized that a kinase other than ERK1/2 may be responsible for C/EBPα phosphorylation and differentiation block. In this report, we identified CDK1 (also known as CDC2) as the kinase specifically modifying C/EBPα on serine 21 in AML with FLT3ITD mutations. Thus, our data provide a potential molecular mechanism explaining the maturation block in FLT3ITD cases without activation of ERK1/2. However, in addition to ERK1/2, CDK1 is another modulator of C/EBPα differentiation function, and we cannot discard the contribution of other mediators to the differentiation block seen in FLT3ITD AML. Further, our observations do not rule out a potential interplay between ERK1/2 and CDK1 activity on C/EBPα function in certain FLT3ITD AML cases. The FLT3ITD and CDK1 connection was previously reported by Odgerel et al. (41
). While they reported that CDK1 is partially inactivated in FLT3ITD AML cell lines, our work concludes that CDK1 can be activated by FLT3ITD mutations. This apparent contradiction could be explained by the use of different FLT3 inhibitors (PKC412 and MLN518, respectively) and the concentrations used, resulting in either effects in apoptosis (41
) or differentiation (this study).
Several studies described modulation of C/EBPα activity by phosphorylation on various residues (17
). However, phosphorylation of a single amino acid, serine 21, seems to have the most remarkable effect by shifting the activity of C/EBPα from a granulocytic differentiation-promoting factor (unphosphorylated) to a dominant negative form (phosphorylated) (17
). Serine 21 can be phosphorylated by ERK1/2 (17
), p38 MAPK (20
), and CDK1 (this report). It is the only phosphorylation site on C/EBPα with clinical significance identified so far. Hyperphosphorylation of serine 21 in leukemic blasts blocks their maturation (21
). P38MAPK-mediated phosphorylation of serine 21-C/EBPα acts as a switch inhibiting neutrophilic differentiation of CD34+
progenitors while permitting their eosinophilic maturation and may be responsible for disturbed neutrophilic development in severe congenital neutropenia (45
). In liver cells, however, phosphorylation of serine 21 by p38 MAPK increases the activity of C/EBPα on promoters of genes involved in gluconeogenesis, thus possibly contributing to diabetes (20
CDK1 belongs to a family of cyclin-dependent kinases, which are critical regulators of cell division. While individual CDK proteins may substitute for each other’s function, gene-targeting experiments demonstrated that CDK1 is the only family member whose role in promoting mitosis cannot be substituted by any other CDK (47
). Misregulation of CDK1 expression/activity in solid tumors is well documented, and several clinical trials with CDK1 inhibitors are currently under way. In contrast, there is very little known about the role of CDK1 during leukemogenesis. Higher expression levels of CDK1 were detected in leukemic cells with del(5q) (48
). Also, a recent report described upregulation of CDK1 in leukemia with translocation liposarcoma/ETS-related gene (TLS-ERG) and attributed increased expression of CDK1 to the differentiation block (49
). Similarly, we found that in AML with constitutively active FLT3 receptor, CDK1 can contribute to the maturation block by inhibiting function of the transcription factor C/EBPα, which is required for granulopoietic development. Our findings open the door to the possibilities of using pharmacological inhibitors of CDK1 in leukemia as well.
Studies described herein demonstrated that various CDK1 inhibitors affect C/EBPα phosphorylation and promote the differentiation process to variable degrees. This might be due to their broad spectrum of action; by inhibiting multiple pathways at the same time, they can rapidly induce apoptosis. While we believe that much of the activity of these inhibitors is through their effects on C/EBPα phosphorylation, our experiments do not rule out effects on other pathways as well. We also found that activation of the CDK1 pathway by FLT3ITD involves upregulation of cyclin B1 rather than upregulation of CDK1 itself (49
). While these data suggest that cyclin B1 is involved in the activation of CDK1, we cannot rule out that other CDK1 regulators, such as Myt1 and cdc25c, could also be involved (41
). NU6102 demonstrated the best differentiation-promoting activity, perhaps because of the higher specificity against CDK1 versus other CDKs. The knockdown experiments are in accord with this hypothesis. Cells expressing lower levels of EGFP and presumably lower levels of shRNA showed more pronounced maturation, while the cells with higher levels of EGFP (and presumably the highest levels of shRNA) exhibited mainly apoptosis (this study).
In summary, we demonstrate that constitutive activation of the FLT3 receptor can lead to abnormal activation of multiple downstream signaling pathways (Figure ), which are capable of inhibiting the function of C/EBPα, contributing to the differentiation block. Inhibiting either FLT3 receptor, MEK1 kinase, or CDK1 can restore the activity of C/EBPα and induce myeloid maturation of leukemic blasts.
Effect of constitutive activation of the FLT3 receptor in leukemogenesis.