This model, in which the Flt3-ITD mutation is knocked-in to the murine gene under the control of its endogenous promoter, provides a powerful tool to study the function of constitutive FLT3 activity in the murine hematopoietic system. Our results demonstrate that the Flt3-ITD mutation results in major perturbations in HSPC composition as well as functional defects in stem cell homeostasis and reconstitution capacity. The observed functional alterations in the LT-HSCs point to a previously unrecognized role for Flt3 in governing the dynamics of this population.
These results are somewhat surprising in light of the previous paradigm that Flt3 protein is undetectable by flow cytometry on the cell surface of LT-HSCs. Flt3 knock-out mice display minor defects in stem cell fitness, revealed only through serial competitive transplant experiments (Mackarehtschian K, 1995
). Additionally, previous studies seem to rule out Flt3 and its ligand as critical regulators of HSC maintenance and post-transplantation expansion in Flt3 and FL knockout mice (Buza-Vidas N, 2009
). However, Flt3 signaling is known to partially overlap with other receptors (i.e. c-Kit) and compensatory regulation by these other pathways may explain why the role of Flt3 in stem cell function has been masked in previous knock-out studies.
Multipotent progenitors with detectable surface Flt3 are thought to have less self-renewal capacity, yet give rise to all hematopoietic lineages including myeloid progenitors as well as lymphoid-primed multipotent progenitors (Christensen JL, 2001
; Adolfsson J, 2001
; Boyer SW, 2011
). However, recent data from Flt3-cre driven fate mapping has suggested that Flt3
transcriptional activity initiates in the phenotypic KSL-SLAM HSC compartment (Buza-Vidas N, 2011). This raises the possibility that Flt3 can be expressed and potentially functional without abundant cell surface protein present, as the limit of sensitivity for conventional FACS detection is typically 2000 molecules/cell (Zola H 2004
). These and our data do not preclude the possibility that these Flt3+
cells within the SLAM and KSL-SLAM compartments are non-self-renewing progenitors. Rather, our results demonstrate that Flt3
mRNA, total Flt3 protein (including intracellular), and activation of Flt3 signaling are all detectable within SLAM-defined LT-HSCs. These results are congruent with the previous paradigm defining LT-HSCs as Flt3-negative, in that Flt3 cell surface protein may not be present at a level detectable by flow cytometry in these cells. Our mouse model demonstrates a comparison of Flt3 constitutive activity in the HSC to that of genetically identical siblings and that Flt3 is capable of playing a functional role in this compartment, despite its low expression levels.
WT Flt3 must homodimerize in the presence of ligand to propagate downstream signaling. Flt3-ITD molecules in contrast, are constitutively active and can homodimerize or heterodimerize with WT molecules, resulting in activation of downstream effectors in the absence of ligand (Kiyoi H, 2002
, Koch S, 2008; Choudhary C, 2009
). This allows even small amounts of the activated mutant receptor to exert strong effects on signaling. This property of Flt3-ITD may explain the strong phenotype seen in our mouse model despite low levels of cell surface protein, as well as why the role of Flt3 in LT-HSCs would be difficult to observe in the absence of a constitutively-active mutant.
Our model demonstrates that Flt3-ITD is acting within the LT-HSC itself as opposed to exerting its effects on this population through the microenvironment. After a mixed bone marrow transplant we produced recipients harboring both WT and Flt3-ITD hematopoiesis within a single bone marrow environment. We observed depletion of only the ITD SLAM cells and not the WT SLAM cells from this mixed environment. This data suggests that the depletion of the SLAM compartment in ITD mice is not the result of secondary environmental effects but rather a direct effect of the mutation in this compartment.
As further evidence of Flt3 activity within this extremely rare LT-HSC population, we noted a significant increase in known downstream signaling, including increased expression of Stat5 targets and cell cycle promoters, along with a decrease in cell cycle inhibitor p57
. The result of Flt3-ITD signaling in the LT-HSC is a depletion of the pool of primitive stem cells. This oncogenic mutation disrupts HSC homeostasis, driving increased proliferation within this normally quiescent compartment and causing a greater proportion of HSCs to enter the cell cycle. This result is even more striking within the context of an overall decreased proliferation rate in Flt3-ITD Lin−
bulk progenitors. The general decreased proliferation may be due to negative feedback signals resulting from an abundance of myeloid progenitors present in the blood and marrow. Aberrant over-proliferation of the normally quiescent stem cell compartment leads to HSC exhaustion (Orford KW, 2008
; Akala OO, 2008
). In our model, it also results in the overproduction of various lineages (particularly of myeloid origin) that give rise to our MPN phenotype, highlighting once again the essential link between quiescence, HSC homeostasis and disease progression.
When Flt3-ITD signaling is attenuated through treatment with the small molecule inhibitor Sorafenib, the intricate link between stem cell homeostasis and disease becomes even more apparent. The restoration of stem cell numbers after only 2 weeks of treatment demonstrates the surprising resiliency and plasticity of hematopoietic stem cells. Although LT-HSCs normally divide only once every five months, there is growing evidence that this dynamic population is capable of rapidly responding to injury and then quickly returning to a largely quiescent steady state (Wilson A, 2008
). The rapid response of Flt3-ITD-harboring LT-HSCs to Sorafenib treatment reveals a surprising level of sensitivity within the hematopoietic system. While Flt3 inhibition appears non-toxic to normal HSCs, we demonstrate that Sorafenib is capable of targeting signaling within a rare population of MPN-initiating SLAM cells in Flt3-ITD mice, indicating the drug may be capable of targeting FLT3-ITD-containing leukemic stem cells in AML patients as well.
While the role of FLT3 is less completely understood in human hematopoiesis than in murine hematopoiesis, there is growing evidence that FLT3 is present in both normal human HSCs and AML LSCs (Kikushige Y, 2008
; Sitnicka E, 2003; Small D, 1994; Levis M, 2005
). While FLT3-ITD mutations are present in only about 5% of MDS cases, the mutation is more frequently observed at the time of progression to AML, suggesting a role in transformation from MDS to AML (Cloos J, 2006
; Shih LY, 2002
; Pinheiro RF, 2008
). In the majority of AML cases, ITD mutations are present in de novo
disease with normal cytogenetics. These patients achieve similar remission rates to patients lacking the mutation, yet FLT3-ITD patients are more likely to relapse, with a median survival of less than 5 months post-relapse (Kottaridis PD, 2001
; Frohling S, 2002
; Schnittger S, 2002; Thiede C, 2002
; Ravandi, 2010
; Levis, 2011
). While FLT3-ITD alone does not fully transform a cell to leukemia, growing evidence implicates this mutation in the process of disease progression and LSC survival and maintenance (Pinheiro RF, 2008
; Yoshimoto G, 2009
). We and others have shown that the combination of Flt3-ITD with other oncogenic mutations can lead to the development of a full-blown leukemia in mice (Stubbs MC, 2008
; Greenblatt S, 2012
) lending credence to the “two-hit” model of leukemogenesis (Gilliland G, 2002
). Our knock-in model studies suggest that one reason Flt3-ITD mutations are not sufficient to induce a leukemia may be due to ITD-driven depletion of the LT-HSC compartment. Thus, ITD mutations in an otherwise normal LT-HSC are unlikely to result in leukemia as they would instead lead to rapid elimination of the clone, with strong selective pressure against stem cells bearing this mutation. In other stages of development or in the presence of additional genetic lesions however, FLT3-ITD may instead cooperate to confer proliferative advantages (Stubbs MC, 2008
). This may explain why Flt3 mutations do not seem to be early hits in primitive HSC compartments but rather acquired in later stages of leukemogenesis in human AML. This may also explain recent findings suggesting Flt3-ITD mutations are not present in human pre-leukemic clones, further implicating this mutation as a secondary hit (Jan M, 2011
Novel insight into the role of FLT3 activating mutations in the overall etiology of transformation to leukemia may reveal new potential therapeutic strategies. In order to effectively target this molecule in AML patients it becomes necessary to consider the function of FLT3 in both disease development and stem cell maintenance, as well as the oncogenic dependence of leukemic blasts on FLT3 signaling for continued survival and proliferation. In LSCs strongly dependent on FLT3 signaling, inhibition may allow opportunities for blast clearance. In other cases FLT3 inhibition with sorafenib may simply return LSCs to a more normal proliferative state. As such, in the case of human disease, FLT3 may be considered for long-term combination therapy or even chronic inhibition, increasing the likelihood of durable remissions.