Self-renewal and growth factor-independent proliferation are two important traits for oncogenesis. The involvement of FLT3 in proliferation of highly undifferentiated hematopoietic cells suggests the oncogenic potential of this signaling pathway. Clinical and experimental evidence both indicate that FLT3 is a proto-oncogene with the capacity to enhance survival and proliferation of leukemia blast cells. Wild type FLT3 is expressed in a wide range of hematopoietic malignancies, including acute lymphoid leukemia and mixed lineage leukemia; most notably, it is expressed in 70%–100% of AML [18, 45, 46]. The aberrantly activated FLT3 pathway is observed in about 29.9% of patients with AML . Two types of mutations have been attributed to the deregulated FLT3 receptor.
FLT3 Internal Tandem Duplication Mutation
An in-frame internal tandem duplication (ITD) mutation in the JM domain of the FLT3 receptor correlates with the highest frequency of FLT3 related AML cases. Clinical studies identify the FLT3-ITD mutation in 17%–26% of AML cases [48, 49]. The ITD was first identified when screening primary leukemia specimens for FLT3 expression, through reverse transcriptase-polymerase chain reaction (PCR), produced amplification products that were longer than expected in multiple patients . DNA sequencing revealed that the insertional mutation occurs within exon 14 and varies in length from 3 to >400 base pairs . The cause of the duplication event is unknown, although it has been proposed that the mutation results from a general failure in a slippage repair or a mismatch repair mechanism during DNA replication [52, 53]. FLT3-ITD has been identified in all French American British subtypes, a categorization of malignant cells based on genotype and morphology. It appears at the highest frequency in the M3 (promyelocytic) subtype and at the lowest frequency in the M2 (myeloblast with differentiation) subtype [50, 54–59].
The ITD mutation causes constitutive activation of the FLT3 receptor. The JM region of many RTKs acts through various mechanisms to regulate the kinase domain. The crystal structure of the normal FLT3 receptor offers direct insight into the mechanism used by the JM domain to regulate catalytic activity of the kinase domain and suggests how the ITD disrupts this mechanism. FLT3 activity occurs when normal phosphorylation of specific tyrosine residues prevents the JM domain from folding properly to induce autoinhibition . The structural analysis of the EphR2 receptor, of a related RTK subfamily, also suggests that the mechanism of induced phosphorylation occurs by disrupting the normal steric hindrance of the JM domain . Moreover, this repressive sequence is conserved in the JM domains of c-KIT and PDFGβ [61–63]. The ITD insertion generally occurs near the JM hinge region and may offset the JM orientation thereby causing a “leaky” autoinhibition of catalytic activity.
There is no correlation between the size of the insertional mutation and the degree of autophosphorylation . Some experiments indicated that the ITD mutation caused maximal activity of the receptor because exogenous FL expression failed to enhance the level of signaling, whereas other experiments observed enhanced pathway signaling through FL expression [64–66]. Such conflicting results may be reconciled by further analysis into the ratio of wild type to mutant receptors on the plasma membrane. This ratio may affect the degree of autophosphorylation because the FLT3-ITD receptor can homodimerize with mutant receptors or heterodimerize with wild-type receptors independent of the ligand.
Single-Amino Acid Mutations in FLT3
Missense point mutations in the kinase domain can also confer constitutive activation of the FLT3 receptor. The most common activating point mutation is the substitution of tyrosine for aspartic acid at position 835 within the activation loop of the kinase domain. Point mutations at other positions, such as 836 or 841, have also been associated with FL-independent activation [67, 68]. The FLT3-D835 mutation is observed in 7% of AML cases and is easily identified because it results in the loss of an EcoRV restriction site [47, 54, 69]. The normal aspartate residue is integral for regulating the activation loop, as evidenced by its conservation across several RTK subfamilies. Furthermore, point mutations at this site in related receptors, such as c-KIT, MET, and RET, are associated with deregulated signaling [70–72]. The mechanism for activation likely parallels that of related proteins whereby the substitution stabilizes the “open” ATP-binding conformation of the activation loop .
FLT3 Mutant Signaling
Parallels between the FLT3 receptor and related RTKs suggest that activating mutations disrupt an autoinhibitory mechanism that is critical for maintaining the inactive conformation of the kinase domain. The mutant receptor activates effector proteins to mediate proliferation and survival and blocks differentiation through ligand-independent autophosphorylation. Identifying molecules of the mutant signaling pathway offers insight into its role in the pathogenesis of leukemia (Fig. 2).
Aberrant stimulation of proteins that confer a proliferative advantage was first identified in mutant-FLT3 myeloid leukemia cells. In vitro, the 32D mouse myeloid cell line harboring the ITD-mutation showed factor-independent proliferation and resistance to radiation-induced apoptosis [73, 74]. Both STAT5 and Ras pathways were shown to be activated. Constitutive phosphorylation of mitogen-activated protein (MAP) kinase has been identified in FLT3-ITD transfected Ba/F3 lymphoid cell line and likely participates in the Ras pathway, causing factor-independent growth and survival . Evidence that inhibition of MAP kinase suppressed colony formation further supports the necessity of this pathway, although other studies indicate that its upregulation alone is insufficient to induce proliferation. Increased expression of the protein kinase B (AKT) has likewise been attributed to promoting survival and proliferation. Although expression levels of MAP kinase and AKT increased weakly in mutant FLT3, their distinction is supported by evidence of their transient phosphorylation following normal FLT3 activation. Brandts et al. recently confirmed prior evidence by Scheijen et al.  that AKT inhibits apoptotic signaling by phosphorylating Forkhead family member Foxo3α in FLT3-ITD Ba/F3 cells . The inactivation of Foxo3α prevents transcription of its proapoptotic target genes in the Bcl2 family. A similar pathway involving the Foxo family members has been observed in Bcr-abl-mediated transformation .
Constitutively phosphorylated STAT5 has been observed in primary AML blasts and in immortalized cell lines Ba/F3 and 32D that express the mutated FLT3 receptor [64, 73, 74, 78, 79]. The phosphorylation of STAT5 contrasts most clearly with the wild-type signaling pathway because normal FL-activated of FLT3 does not cause significant STAT5 activation. This evidence suggests that mutant FLT3 receptors recruit unique downstream phosphorylation targets to induce the abnormal phenotype . It remains unclear whether mutant FLT3 receptors promote leukemogenesis by increasing quantitative signaling of normal effector proteins or by causing qualitative differences in downstream targets.
STAT5 is known to activate PIM-1, and RNA microarray data identified a consistent increase in PIM-1 expression in FLT3-ITD cells . Quantitative PCR further implicated PIM-1 as a target of aberrant STAT5 expression by correlating a 10-fold decrease in its expression with FLT3 inhibition. PIM-1 is normally induced by a number of FL-related cytokines, including G-CSF and IL-3, to increase cell mitogenesis and survival. The effect of PIM-1 activity occurs through phosphorylation of a diversity of substrates including Cdc25A, which is key to cell cycle progression, and Bad, which blocks proapoptotic signaling. PIM-1 has also been reported to cooperate within antiapoptotic pathways in chronic myeloid leukemia (CML) cell lines.
FLT3 and Differentiation
FLT3 activity may also regulate differentiation pathways. Differentiation is tightly associated with the loss of proliferative capacity and increased propensity toward apoptosis. Normal myeloid progenitor cells proceed along a differentiation gradient to arrive at a terminally differentiated cell type with a limited lifespan. Blocking differentiation has been identified as an important mechanism for cancer progression. The preferential expression of FLT3 in primitive hematopoietic cells suggests its role in regulating differentiation. FL activation of 32D cells transfected with wild-type FLT3 mitigated their progression toward differentiated neutrophils but could not induce a complete block . However, 32D cells transfected with FLT3-ITD showed a complete block to morphologic differentiation, demonstrated at the molecular level by inhibition of prodifferentiation and myeloid proteins. Further investigation identified the inhibition of CCAAT/enhancer-binding protein α (C/EBPα) as a critical pathway to escape differentiation . Yet the study also found primary cells from one AML patient to reveal high levels of C/EBPα, which indicates that other contributing mutations may be more dominant inhibitors of differentiation. The capacity of FLT3 to block differentiation is additionally questioned by evidence that mice transplanted with FLT3 mutant cells fail to develop AML blasts phenotypes due in part to differentiation .
However, RGS2 mRNA, a regulator of G-protein signaling, was observed to be significantly repressed in the majority of primary AML bone marrow samples that harbor the FLT3-ITD mutation compared with AML samples lacking the mutation . Several myeloid cell lines have been shown to induce RGS2 during granulocytic differentiation, and RGS2 overexpression in 32D cells expressing FLT3-ITD overcomes the block to differentiation. The study suggests that FLT3-ITD signaling represses differentiation to some degree in AML cases, depending on the cellular context.
FLT3 and In Vivo Models of Leukemia
At the molecular level, FLT3 appears to recruit many proteins involved in proliferation, self-renewal, and anti-apoptosis. The expression profiles of mutant FLT3 cell lines support its association with leukemogenesis by identifying a number of genes involved with growth and survival. However, the diversity of protein function and the redundancies in cellular processes prevent clear correlations between proteins and their function.
FLT3 mutations are insufficient to induce leukemia blasts in vivo  despite the diversity of avenues that the FLT3 receptor mutations promotes deregulation. FLT3-ITD transduction into primary murine hematopoietic progenitors induced a myeloproliferative phenotype but could not confer cells with the necessary arrested differentiation that is associated with AML. The Knudson Two Hit Model is therefore supported by evidence that FLT3-ITD can only promote AML when expressed concurrently with other selected mutations. Kelly et al. transduced FLT-ITD into bone marrow cells of PML/RARα transgenic mice and observed complete penetrance and rapid progression of acute promyelocytic leukemia (APL) compared with the long latency period for leukemogenesis in transgenic mice without FLT3 mutation . The association of the PML/ RARα mutation with block to differentiation suggests that although mutant FLT3 may effect differentiation, transformation depends on a more potent mechanism. The block to differentiation may be a critical limitation for leukemogenesis through aberrant FLT3 signaling.
Nevertheless, these studies are critical to implicating the mutated FLT3 receptor in leukemogenesis. As a single mutation, FLT3 induces a myeloproliferative phenotype, and primary AML cells expressing the FLT3-ITD receptor engraft into NOD/SCID mice with greater efficiency that those lacking the mutation .
Clinical Implications of FLT3 Mutations in AML
Substitution mutations in the tyrosine kinase domain (TKD) and insertional mutations in the JM domain of FLT3 represent the most frequent genetic abnormality associated with AML. Both mutations lead to constitutively active FLT3, although studies indicate that each mutation presents unique clinical features.
The FLT3-ITD mutation is recognized at diagnosis by leukocytosis, with the mean white blood cell (WBC) count typically elevated to levels significantly higher than that of patients without the mutation . The incidence of FLT3-ITD mutations correlates with the age of AML patients. Its frequency in pediatric studies ranges from 5%–16% [55, 58, 88–90], whereas FLT3-ITD occurs in 23%–35% of adult AML cases [48, 49, 51, 68, 69, 91–94]. Three studies on the pediatric subgroup indicate that FLT3-ITD appears at a higher frequency in children over 10 years, which corresponds to the adult frequency, whereas studies on the adult AML population show no increase in FLT3 mutation frequency with increased patient age. The incidence of FLT3-ITD mutations is also observed at various frequencies that correlate with other genetic factors. The mutation appears less frequently in patients with poor-risk cytogenetics and with core-binding factor translocations [t(8;21) and (INV16)] .
Multiple studies indicate that FLT3-ITD mutations confer a poor prognosis in AML patients less than 60 years of age [48, 49, 51, 68, 69, 91–94]. The prognostic impact of FLT3-ITD likely fluctuates among patients because the mutation is often identified in conjunction with other contributing genetic abnormalities associated with the highly heterogeneous disease. For example, the incidence of FLT3-ITD mutations in patients with moderate and good-risk cytogenetics correlates with a significantly reduced clinical outcome, whereas the prognosis of APL patients harboring FLT3-ITD appears minimally affected . However, a recent multivariate analysis of 250 adult AML patients indicated that FLT3-ITD is an independent marker of poor prognosis , a correlation that is also attributed to the MLL gene  and the ETS-related gene  in AML. The data suggest that despite the substantial diversity of molecular abnormalities documented in AML, certain mutations, such as FLT3-ITD, exert substantial influence on tumor progression. Moreover, all but 1 of 16 studies on adult AML found a significant decrease in overall survival, disease-free survival, and event-free survival . Two studies that examined 1,000 patients 4 years after receiving comparable therapies demonstrated that FLT3-ITD expression correlated with decreased overall survival [48, 69]. Another study, examining 224 AML patients with normal cytogenetics and a median follow-up of 34 months, found that patients expressing the FLT3-ITD mutation had a significantly lower overall survival . Two other studies observed no effect of the FLT3-ITD mutation on overall survival, although these results may be explained by the relatively early follow-ups of 11.1 and 12.2 months [51, 68].
Multiple studies used PCR to correlate high ratios of mutant to wild-type FLT3 with decreased overall survival [48, 68]. The mutant to wild-type ratio often increased at relapse and a retrospective analysis of minimal residual disease (MRD) status for 11 AML patients indicated an increased tendency for relapse in those with FLT3-ITD positive MRD status . This provides evidence that FLT3 expression levels significantly affect prognosis and relapse. This suggests that the pathway exerts a strong influence on leukemia progression. However, the heterogeneity in AML blast gene expression and the notable fraction of AML patients that lose the FLT3-ITD mutation in relapse indicate that multiple factors determine disease progression and that FLT3-ITD should not be exclusively used as a marker for MRD.
In pediatric cases, the ITD mutation provides a more negative and independent prediction of prognosis. Data from six combined pediatric studies, which examined 461 pediatric AML patients, identified a 19% survival rate for cases associated with FLT-ITD compared with a 58% survival rate for patients with wild-type FLT3 receptors [50, 100]. One of the studies reported a remission induction rate of 40% and an event-free survival of only 7%, compared with 74% and 44%, respectively, in patients without the mutation . Unlike adult AML findings, pediatric studies also found a significant decrease in complete remission of AML harboring the FLT3-ITD mutation [58, 88].
The clinical pathogenesis of FLT3-TKD expressing AML is less understood than the FLT3-ITD due to its lower frequency in patients, which limits the number of statistically significant conclusions [55, 68, 88, 101–103]. Conflicting clinical results have also prevented substantial inferences on the effects of FLT3-TKD. For example, one clinical study of 201 AML patients found no significant correlation of increased mean WBC counts or age-dependent frequency of FLT3-TKDs but associated FLT3-TKD with a shorter disease-free survival . However, a larger clinical study of 979 AML patients less than 60 years of age found that FLT3-TKD was associated with elevated mean WBC counts without observing correlation with disease-free survival . A recent meta-analysis of four major studies of FLT3 in AML patients indicated that TKD mutations confer a significant decrease in overall survival and are comparable to ITD mutations in lowering disease-free survival . Although this analysis provides interesting insights, the heterogeneity and the abstracted nature of the patient data requires cautious interpretation. Genetic analysis of FLT3-TKD samples indicated that levels of FLT3 expression are greater than in either FLT3-ITD or wild-type FLT3 . Studies have also identified a higher frequency of FLT3-TKD mutations in patients with MLL duplications or a double-stranded break in the MLL gene. Future clinical studies must examine larger AML populations to achieve significant insight into potential effects of FLT3-TKD mutations.