Murine models of activating tyrosine kinase mutations and fusion proteins implicated in leukemia provide instructive platforms to examine their effects in vivo. While informative, the use of retroviral transduction or transgenic systems employing exogenous promoters in many of these models precludes accurate physiologic expression levels of these oncogenes and may ultimately affect the resultant biological and phenotypic outcome (Ren, 2004
). This concept is supported in results from prior bone marrow transplant studies (Kelly et al., 2002b
) and transgenic animals modeling activated FLT3 mutations including our vav
FLT3-ITD mice (Lee et al., 2005
) and a recently reported Tel-FLT3 model (Baldwin et al., 2007
) wherein the expression of a constitutively activated FLT3 in the form of a Tel-FLT3 fusion protein is driven by an exogenous cytomegalovirus (CMV) promoter. While all of these models have provided valuable insights into FLT3-mediated leukemogenesis, the variable phenotypes observed among these aforementioned systems support the notion that promoter choice (e.g., FLT3-ITD: vav
; Tel-FLT3: CMV
) plays an important role in both the nature and severity of disease phenotype.
To more precisely assess the effects of activated FLT3 expression at more appropriate physiologic levels, we have generated a murine model in which expression of a constitutively activated form of the FLT3 tyrosine kinase is under the control of the endogenous murine Flt3 promoter, representing to our knowledge, one of the few ‘knock-in’ leukemogenic tyrosine kinase models.
Examination of these animals has revealed insights into the in vivo
effects on immature hematopoietic stem and progenitor cell populations of the FLT3-ITD, one of the most common activating tyrosine kinase mutations implicated in leukemia. Our murine data demonstrate that the leukemogenic properties conferred by FLT3-ITD are due, in part, to increased multipotent stem and progenitor cell cycling and enhanced survival properties within these compartments. These findings are supported by recent lentiviral-based FLT3-ITD expression studies in human hematopoietic stem and progenitor cells that reported increased survival and proliferation in FLT3-ITD transduced human cell populations (Li et al., 2007
While enhanced cell survival was observed in both FLT3-ITD positive myeloid progenitor (Lin-
) and more primitive LSK (Lin-
) populations containing long-term and short-term HSC, as well as multipotent progenitors cells, increased cell cycling appeared specific to the LSK population (Supplemental Figure 5
). Interestingly, this LSK-specific cell cycle aberrancy is nearly identical to that observed in triply null FoxO1, FoxO3, FoxO4
animals when conditionally deleted in the hematopoietic system (Tothova et al., 2007
). Prior reports have demonstrated that activated FLT3 receptor signaling induces phosphorylation of FoxO family members (FoxO3a) in Ba/F3 cells, with resultant nuclear exclusion of FoxO proteins and transcriptional repression of FoxO-target genes critical to normal cell survival and proliferation (Brandts et al., 2005
; Scheijen et al., 2004
). Taken together, our data suggests that enhanced cell proliferation within the LSK compartment of our mutant FLT3 mice is mediated, at least in part, through inactivation of the FoxO transcription factor family, although we have not formally examined if there are alterations among FoxO family members within these mice.
Our studies also illustrate that constitutive signaling by FLT3-ITD at physiologic levels directs hematopoietic differentiation towards the myeloid/monocytic lineage with concomitant suppression of megakaryocyte-erythroid development (Supplemental Figure 5
). Moreover, while the levels of common lymphoid progenitors (CLP) appear largely indiscernible between mutant FLT3 animals and their wild-type littermates, constitutive FLT3 signaling appears to induce a block in normal B cell development, which is consistent with the prevalence of FLT3-ITD mutations associated with acute myeloid versus acute lymphoid leukemia. This finding is also intriguing given the fact that absence of Flt3
also leads to deficiencies in normal B cell development, indicating that constitutive FLT3-ITD signaling likely interferes with normal FLT3 gene function (Mackarehtschian et al., 1995
The phenotypic consequences of the aforementioned alterations in hematopoietic stem and progenitor cell populations results in a chronic MPD in Flt3+/ITD
animals resembling human CMML which prompted us to look for the presence of activating FLT3 mutations in patient samples. Our findings along with previous reports (Lin et al., 2006
; Pardanani et al., 2003
), indicate that although the incidence of FLT3
mutations is relatively uncommon in chronic MPD, in the instances where isolated FLT3 lesions in chronic MPD are detected, these patients may frequently present with CMML. Although it would be premature to conclude that CMML with FLT3-ITD is a “unique” subtype of this disease that is associated with particular clinical and laboratory features at the current time, our data suggest that this molecular feature may represent an independent negative prognostic factor in CMML. Clearly, analysis of larger cohorts will need to be examined to see whether this notion bears out in multivariate analysis models. Importantly, however our findings support that molecular assessment for the presence of FLT3 mutations should be performed in individuals with CMML as their survival may be improved by inhibition of FLT3-ITD with available small molecule compounds, which have shown efficacy in AML (Smith et al., 2004
; Stone et al., 2005
; Wadleigh et al., 2005
). As imatinib mesylate has demonstrated considerable clinical efficacy in the small subset of CMML associated with PDGFRβ rearrangements involving chromosome 5q31-q33 (Apperley et al., 2002
; David et al., 2007
), similar effects might be predicted with the use of available FLT3 small molecule inhibitors in patients with FLT3-ITD associated CMML.
The disease phenotype findings in prior FLT3 murine models (Baldwin et al., 2007
; Kelly et al., 2002a
; Kelly et al., 2002b
; Lee et al., 2005
) and our current ‘knock-in’ model suggest that in patients with isolated FLT3 mutations, progression to AML is unlikely in the absence of pre-existing or the subsequent development of additional genetic lesions. These data also suggest that subsequent mutations must complement constitutive FLT3 activation to ensure full transformation of hematopoietic cells and that certain combinations of mutations are not only cooperative but also interdependent in the pathogenesis of AML (Supplemental Figure 5
). Our model will serve as a valuable biological tool to explore the individual contributions of these different classes of leukemogenic mutations in the development of AML and molecular therapeutic regimens that target them.