PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cell Stem Cell. Author manuscript; available in PMC 2013 September 7.
Published in final edited form as:
PMCID: PMC3725984
NIHMSID: NIHMS389045

FLT3-ITD Knock-in Impairs Hematopoietic Stem Cell Quiescence/Homeostasis, Leading to Myeloproliferative Neoplasm

SUMMARY

Internal tandem duplication (ITD) mutations within the FMS-Like Tyrosine Kinase gene (FLT3) render the receptor constitutively active, driving proliferation and survival in leukemic blasts. Expression of Flt3-ITD from the endogenous promoter in a murine knock-in model results in progenitor expansion and a myeloproliferative neoplasm. In this study, we show that this expansion begins with over-proliferation within a compartment of normally quiescent long-term hematopoietic stem cells (LT-HSCs), which become rapidly depleted. This depletion is reversible upon treatment with the small molecule inhibitor Sorafenib, which also ablates the disease. Although the normal LT-HSC has been defined as Flt3-negative by flow cytometric detection, we demonstrate that Flt3 is capable of playing a role within this compartment by examining the effects of constitutively activated Flt3-ITD. This indicates an important link between stem cell quiescence/homeostasis and myeloproliferative disease while also giving novel insight into the emergence of FLT3-ITD mutations in the evolution of leukemic transformation.

INTRODUCTION

Internal tandem duplications (ITD) in FMS-like tyrosine kinase-3 (FLT3) comprise a group of the most common molecular mutations in acute myelogenous leukemia (AML) (Gilliland DG, 2002; Levis M, 2003; Stirewalt DL, 2003). Found in approximately 23% of AML cases, an ITD mutation abrogates the negative regulatory function of the juxtamembrane domain, rendering the receptor constitutively active, independent of its natural ligand (Fenski R, 2000; Kiyoi H, 1998; Mizuki M, 2000). This oncogenic mutation activates canonical receptor tyrosine kinase signaling, most prominently via STAT5, RAS/MAPK and PI3K, stimulating proliferation and anti-apoptotic pathways (Hayakawa F, 2000; Kiyoi H, 1998; Mizuki M, 2000; Tse KF, 2001).

The presence of a FLT3-ITD mutation is a poor prognostic feature in AML, predicting increased relapse rates and reduced overall survival (Frohling S, 2002 Kottaridis PD, 2001; Levis M, 2003; Meshinchi S, 2001; Nakao M, 1996; Thiede C, 2002). Though there are many small molecule FLT3 tyrosine kinase inhibitors (TKI) in various stages of clinical trials, responses have been largely heterogeneous and transient (Grundler R, 2003; Knapper S, 2006; Levis M, 2011; Stone RM, 2005; Weisberg E, 2002). After an initial response, patients relapse, suggesting that leukemia-initiating stem cells may be escaping TKI-induced cytotoxicity.

There has been increasing evidence linking FLT3-ITD with leukemic stem cells (LSCs), including the presence of the mutation within the CD34+/CD38 leukemia initiating cell fraction in most cases (Lapidot T, 1994; Levis M, 2005). The FLT3-ITD mutation also remains present at relapse in most cases, suggesting its presence in the cells that escape therapy (Cloos J, 2006; Shih LY, 2002). Furthermore, AML cases that have the FLT3-ITD mutation were shown to have the highest engraftment capacity in NOD/SCID mice (Lumkul R, 2002; Rombouts WJ, 2000a,b). Thus, understanding the role of FLT3-ITD mutations in LSCs is of great therapeutic interest.

FLT3 is an important molecule in normal hematopoietic development as well as in leukemia (Gilliland DG, 2002). FLT3 is expressed on common lymphoid progenitors (CLPs) and a minor fraction of common myeloid progenitors (CMPs) and is implicated in dendritic cell development (D’Amico A, 2003). Additionally, FLT3 is expressed during the early stages of hematopoiesis, indicating a potential involvement in stem cell function or maintenance. In murine hematopoiesis, where cell surface marker expression during stages of differentiation has been extremely well-defined, hematopoietic stem cell (HSC) activity is restricted to a small subset of the KSL compartment (Lin c-Kit+ Sca-1+)(Ikuta K, 1992; Spangrude G, 1988; Okada S, 1992 ; Katayama N, 1993). Yet, within this compartment, Flt3 is thought to be expressed only on multipotent progenitor cells (MPPs) (Christensen JL, 2001; Adolfsson J, 2001). Of the KSLs, MPPs have the lowest capacity for self-renewal and higher rates of proliferation (Passegué E, 2005), while the compartment defined as Flt3 contains HSCs capable of short-term (CD34+) or long-term (CD34) reconstitution of all hematopoietic lineages (Morrison SM, 1994; Osawa M, 1996; Randall, 1996). Thus the most primitive stem cell in the murine hematopoietic hierarchy, the Long-Term HSC (LT-HSC), has been classically defined by its lack of Flt3 expression. Flt3 has been previously reported to be dispensable for HSC maintenance and myeloid development (Sitnicka E, 2002) and Flt3 and Flt3-ligand (FL) knockout mice have only minor defects in HSC function (Mackarehtschian K, 1995; McKenna HJ, 2000). However, knockout studies fail to take into account the effects of overlapping or compensatory pathways, and few studies to date have addressed the effects of constitutive Flt3 activity on normal hematopoiesis or stem cell function.

In order to determine whether the Flt3-ITD mutation affects HSC function, our lab has generated a Flt3-ITD knock-in mouse model that has an 18bp insertion in the juxtamembrane domain of murine Flt3 (Li L, 2008). These mice express the germline ITD mutation under control of the endogenous murine Flt3 promoter and develop a myeloproliferative neoplasm (MPN) characterized by splenomegaly, leukocytosis and myeloid hypercellularity with a median survival of 10 months. Here, we describe isolation of an MPN-initiating stem cell within the signaling lymphocytic activation molecule (SLAM) compartment (LinCD48CD150+) (Kiel M 2005) of Flt3-ITD knock-in marrow that is capable of full engraftment and complete recapitulation of the MPN phenotype. Furthermore, we demonstrate that Flt3 transcripts and Flt3 protein are detectable in the SLAM-defined LT-HSC compartment and constitutive activation significantly perturbs HSC quiescence/homeostasis. We find that Flt3-ITD expression under the endogenous promoter results in changes in gene expression within the LT-HSC compartment including molecules involved in increased proliferation and activation of canonical FLT3 signaling pathways. Moreover, we demonstrate in vivo restoration of normal HSC function and simultaneous ablation of the MPN by treatment of mice with the FLT3 small molecule inhibitor Sorafenib. These data suggest a previously unrecognized role of Flt3 in LT-HSC homeostasis and demonstrates an intrinsic link between normal stem cell quiescence/homeostasis and MPN development as well as providing novel insights into the occurrence of Flt3 mutations in leukemogenesis.

RESULTS

Physiological expression of Flt3-ITD results in changes in composition of the hematopoietic stem-progenitor cell (HSPC) compartment

Bone marrow was harvested from mice expressing the Flt3-ITD mutation under the control of its endogenous promoter. When stained for cell surface marker expression, Flt3-ITD bone marrow consistently displayed a 2-10-fold expansion in the total number of lineage-negative progenitor cells as compared to wildtype (WT) littermate controls (Figure 1A,B,and F). Within this expanded compartment, Flt3-ITD marrow showed increased numbers of cells expressing both c-Kit and Sca-1, resulting in an approximate 4-8-fold increase in total number of KSLs - a population enriched for multipotent HSCs (Figure 1C,D) (Spangrude G, 1992; Okada S, 1992; Katayama N, 1993). Upon further breakdown of the KSL compartment by Flt3 and CD34 expression, Flt3-ITD mice appeared to have an increase in the frequency of multipotent progenitors (MPPs) as well as an uncharacterized population of cells expressing Lin Kit+Sca+ Flt3+CD34 not observed at a significant frequency in WT littermates (Figure 1E).

Figure 1
Expression of FLT3-ITD from the endogenous promoter disrupts HSPC composition in knock-in mice

An alternative method for phenotypic identification of HSCs eliminates Flt3 as a necessary surface marker by utilizing SLAM marker expression to identify these rare cells. SLAM-defined HSCs (Lin CD48CD150+) have previously been described as highly enriched for long-term engraftment capacity (Kiel M, 2005). Strikingly, Flt3-ITD mice consistently displayed a 2-5-fold decrease in this SLAM population despite increases observed in total bone marrow cellularity (Figure 1 F-H). This decrease was even greater for the KSL-SLAM compartment (Figure 1I,J) – believed to be even further enriched for LT-HSCs (Kiel M, 2005). This observed progenitor expansion with concomitant decrease in phenotypic LT-HSCs suggests that the expression of Flt3-ITD might perturb normal HSC homeostasis.

Another method for HSC identification is the frequency of side population (SP) cells as determined by MDR1-mediated Hoechst dye efflux, commonly associated with functional HSC activity (Goodell MA, 2004). Again Flt3-ITD bone marrow displayed a consistent 5-fold reduction in number of SP-defined HSCs (Figure 2A,B). When examined for overlap with cell surface marker phenotype, we observed that a significantly smaller proportion of Flt3-ITD KSL cells were SP cells, as compared to results in WT littermate controls (Figure 2C), suggesting that fewer Flt3-ITD KSLs were functional stem cells.

Figure 2
FLT3-ITD bone marrow demonstrates reduced Side Population and HSC function

Flt3-ITD expression results in an observed reduction in HSC activity

To investigate the functional effects of the Flt3-ITD mutation on HSCs, we transplanted several cell surface marker-defined hematopoietic populations. Unfractionated, lineage-depleted and KSL bone marrow populations from Flt3-ITD mice demonstrated decreased engraftment capacity in comparison to WT marrow (Figure 2D). The presence of the ITD mutation did not confer any abnormal long-term engraftment capacity to any progenitor population examined, including CMP, GMP, MPP, ST-HSC, or the uncharacterized KSL Flt3+CD34 compartment (Table S1).

To explore the possibility that Flt3-ITD expression was affecting engraftment primarily by altering the homing ability of HSCs, intrafemoral injections were performed to bypass the need for HSCs to traffic to the bone marrow. As seen with intravenous injection, intrafemorally injected Flt3-ITD Lin cells resulted in significantly reduced engraftment levels when compared to WT controls (Figure S1). This data suggests that homing to the bone marrow is not the primary mechanism behind FLT3-ITD engraftment defects.

The Flt3-ITD MPN-initiating cell is contained within the SLAM compartment

As SLAM-defined HSCs have been previously shown to be highly enriched for cells with high long-term engraftment capacity (Kiel M, 2005), we hypothesized that the observed engraftment defect may be due to the depletion of this SLAM population (Figure 1G,H). Once sorted by lineage, CD150 and CD48 expression, WT and Flt3-ITD SLAM cells demonstrated equivalent engraftment capacity, suggesting that in this model, SLAM cells best represent the functional HSC (Figure 3A).

Figure 3
Transplantation of SLAM cells recapitulates MPN phenotypes

We next sought to determine whether Flt3-ITD SLAM cells were capable of recapitulating the MPN observed and previously reported in mice harboring the knocked-in mutation (Li L, 2008). Transplant recipients of either Flt3-ITD or WT sorted SLAM cells were sacrificed 8 weeks post-transplant and examined for signs of disease. Flt3-ITD SLAM recipients exhibited both splenomegaly and myeloid expansion (Figure 3B,D). In addition, transplant recipients recapitulated the stem cell immunophenotypic abnormalities observed in primary Flt3-ITD knock-in animals. Flt3-ITD SLAM recipients displayed a significant expansion in lineage-negative and myeloid progenitor cells, as observed in the donor mice (Figure 3C, D). Interestingly, despite receiving an equivalent number of donor SLAM cells, Flt3-ITD recipients demonstrated a 2-4 fold depletion of the SLAM compartment by 8 weeks post-transplant (Figure 3E). These data reveal that Flt3-ITD SLAM cells have the ability not only to engraft, but also to autonomously lead to the development of a MPN.

Flt3 mRNA and Flt3 protein are present at detectable levels in long-term engrafting SLAM cells

In mice, the long-term hematopoietic stem cell has been classically defined as having a LinKit+Sca+ CD34Flt3 immunophenotype (Christensen JL, 2001; Osawa M, 1996). Due to the observed defect in engraftment in the Flt3-ITD knock-in mice and the correlated depletion of the SLAM compartment, we hypothesized that although Flt3 surface protein may not be abundant enough for detection by flow cytometry in the LT-HSC, low levels of expression may still play a role in stem cell homeostasis. To determine whether Flt3 was transcribed in the long-term engrafting and MPN-initiating stem cell, SLAM cells from both WT and Flt3-ITD mice were sorted and Flt3 transcript levels were quantified by qRT-PCR. Flt3 transcripts were detectable in both WT and Flt3-ITD SLAM cells, with an approximate 4-fold increase in Flt3 mRNA expression in the Flt3-ITD SLAM compartment (Figure 4A).

Figure 4
Flt3 is detected in SLAM cells and capable of exerting functional effects in LT-HSCs

As Flt3 protein is thought to be absent or undetectable on the cell surface of classically defined primitive LT-HSCs we were interested in determining if the observed levels of Flt3 mRNA resulted in detectable Flt3 protein. When we measured the levels of total Flt3 protein (including intracellular) in the SLAM-defined HSC compartments of WT and Flt3-ITD mice, we were able to detect Flt3 protein in a fraction of both SLAM compartments (Figure 4B).

The effect of Flt3-ITD on LT-HSC homeostasis is cell autonomous

We next sought to determine whether our observed Flt3-ITD phenotype in the LT-HSC was due to its direct expression within these primitive stem cells or if the effect was an indirect microenvironmental one (i.e. through cytokine/growth factor release from Flt3-ITD expressing cells in the bulk bone marrow or stromal environment). We transplanted recipient mice with a mixture of WT and Flt3-ITD Lin bone marrow cells that resulted in approximately equal numbers of WT and Flt3-ITD SLAM cells. We could then examine the effects on both stem cell populations in a single environment (in the presence of both WT and Flt3-ITD marrow). At 10 weeks post-transplant, bone marrow from these mice was examined for the presence of WT (CD45.1) and Flt3-ITD (CD45.2) SLAM cells. Strikingly, only the Flt3-ITD SLAM cells were depleted, with the frequency of WT SLAM cells remaining at a normal level (Figure 4C). Within a single recipient, WT SLAM cells were present at frequencies ranging from 3 to 20-fold higher than Flt3-ITD SLAM cells (Table S2), suggesting that the observed SLAM depletion was a direct cell autonomous effect.

Constitutive Flt3 activation leads to increased proliferation within the normally quiescent LT-HSC compartment

In an effort to determine the mechanism of Flt3-ITD-driven SLAM cell depletion, we hypothesized that the oncogenic mutation may drive these normally quiescent cells to proliferate. To further investigate, we measured HSC proliferation rates in WT and Flt3-ITD bone marrow through in vivo BrdU incorporation assays. Bone marrow was harvested 4 hours post BrdU injection and co-stained with cell surface markers to examine proliferation in multiple HSPC subsets. Despite observed expansion of the Lin subset, Flt3-ITD mice unexpectedly displayed decreased BrdU incorporation in this fraction (Figure 5A-C). In striking contrast however, subsets more highly enriched for LT-HSCs from Flt3-ITD mice demonstrated an unusually high level of BrdU incorporation. Flt3-ITD KSLs and KSL-SLAM cells had a greater than 2-fold increase in BrdU incorporation rate over WT controls. To further establish whether the Flt3-ITD mutation was driving normally quiescent HSCs to proliferate, we examined cell cycle status by measuring PI staining in stem cell subsets. While WT and FLT3-ITD Lin progenitors demonstrated equivalent numbers of cells in S+G2/M phase, Flt3-ITD SLAM cells showed significant increases in cycling cells as compared to WT littermates (Figure 5D).

Figure 5
Flt3-ITD HSCs demonstrate increased proliferation and cell cycle entry

Downstream targets of Flt3 are abnormally activated in Flt3-ITD SLAM cells

To further validate that the observed expression of Flt3-ITD in the hematopoietic stem cell resulted in a functional signal, we measured the activation of known Flt3 downstream signaling molecules in these cells. As FLT3-ITD signaling is known to activate the STAT5 transcription factor (Hayakawa F, 2000), we measured the expression of Stat5 targets by qRT-PCR in Flt3-ITD SLAM cells (Basham B, 2008). When compared to WT SLAM cells, several Stat5 targets were significantly overexpressed in the Flt3-ITD SLAM compartment - most prominently Fos, JunB, Mkp-1, and Socs3 (Figure 5E). Additionally, we examined several genes that have been found to be upregulated by FLT3-ITD signaling in CD34+ cells transduced with Flt3-ITD (Kim KT, 2007; Li L, 2007). We found that Pim1 was overexpressed on average 5-fold within the Flt3-ITD SLAM compartment compared to WT SLAM cells. Finally, a positive regulator of cell cycle progression Cyclin F was found to be overexpressed in the Flt3-ITD SLAM compartment, while negative cell cycle regulator p57 was downregulated. This data indicates that constitutively activated Flt3 alters gene expression patterns and proliferation status within the most primitive compartment of HSCs.

Inhibition of Flt3-ITD signaling using the small molecule inhibitor Sorafenib leads to restoration of normal stem cell activity and ablation of the MPN

As the depletion of LT-HSCs in the ITD mice is driven by Flt3-ITD signaling, we hypothesized that attenuating Flt3-ITD signaling may restore normal stem cell activity in the knock-in mice. Sorafenib has been shown to have significant activity against FLT3 (Auclair D, 2007; Zhang W, 2008), and is approved for use in targeting Raf-1 in renal cell carcinoma (Escudier B, 2007). Several clinical trials involving Sorafenib have shown promising activity in AML patients with FLT3 mutations (Safaian NN, 2009; Tong FK, 2006; Ravandi F, 2010; Crump M, 2010). Sorafenib also appears to have selective inhibitory effects on mutant FLT3-ITD over WT FLT3 (Zhang W, 2008). Treating mice with Sorafenib has been shown to induce regression of MV4;11 (a human AML cell line expressing FLT3-ITD) tumors in nude mice (Auclair D, 2007).

To determine whether the loss of LT-HSCs observed in Flt3-ITD mice could be prevented, we sought to attenuate Flt3-ITD signaling early in development. WT females were bred to males heterozygous for the mutation, and pregnant mothers were treated with Sorafenib (20mg/kg) beginning at day 15 of pregnancy. Mothers were continually treated during a 2-week feeding period after giving birth, enabling pups to receive Sorafenib doses through breast milk. At 2 weeks of age, Flt3-ITD pups from untreated mothers displayed splenomegaly (Figure 6A), myeloid expansion (Figure 6B, C), a 5-fold reduction in KSL-SLAM cells and a 2-fold reduction in SLAM cells. In contrast, Flt3-ITD pups from treated mothers displayed no clinical signs of disease and their bone marrow contained equivalent numbers of SLAM and KSL-SLAM cells to those of WT littermates (Figure 6D, E).

Figure 6
Early treatment with Sorafenib prevents MPN and preserves stem cell numbers

Having demonstrated that stem cell numbers could be preserved and MPN prevented if mice were treated during development of the hematopoietic system, we next examined whether HSC numbers and function could be restored after the MPN was already established in adult mice. Adult heterozygous mice were treated with 10mg/kg Sorafenib for two weeks. Post-treatment, these mice demonstrated near complete resolution of the MPN phenotype, including a reduction in spleen size (Figure 7A) and a decrease in progenitor numbers within the Lin and KSL compartments to levels comparable to WT controls (Figure 7B, C). Strikingly, the numbers of SLAM and KSL-SLAM cells were also largely restored to the levels of WT controls (Figure 7D, E). Histopathology of the BM and spleen also demonstrated the restoration of cellularity and architecture in these tissues in the Flt3-ITD treated mice (Figure 7G). In spleens from Flt3-ITD mice treated with Sorafenib, we observed reductions in overall cellularity, nucleated cells and white pulp areas as well as a restoration of red pulp areas. The marrow of Flt3-ITD treated mice displayed decreased cellularity, collapsing venous sinuses and restoration of the number of mature red blood cells in the marrow (Figure 7G). Thus, small molecule inhibition of Flt3-ITD simultaneously led to reversal of disease phenotype and restoration of normal stem cell homeostasis in adult mice.

Figure 7
Treatment of adult mice with Sorafenib restores stem cell activity and reverses signs of disease

To ensure that the observed increase in phenotypic SLAM cells was also indicative of a restoration of stem cell function, Lin progenitors from treated and untreated WT and Flt3-ITD mice were transplanted into lethally irradiated recipients. Recipient mice were treated with 10 mg/kg Sorafenib for an additional 2 weeks after irradiation to continue inhibition of Flt3-ITD signaling during the engraftment period. At 4 weeks post-transplant, in contrast to untreated Flt3-ITD marrow, treated Flt3-ITD Lin bone marrow demonstrated greatly increased engraftment capacity, equivalent to that of WT controls (Figure 7F).

DISCUSSION

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.

EXPERIMENTAL PROCEDURES

Mice

Generation of mice harboring a Flt3-ITD mutation knocked-in to the endogenous Flt3 locus has been previously described (Li L, 2008). The mice used in these experiments were bred with a CMV-Cre line to flox out the neo selection cassette. Except for Sorafenib treated pups that were 2 weeks of age, all mice were evaluated at 6-10 weeks of age with age-matched, littermate controls. All experiments were performed in accordance with IACUC-approved protocols.

Bone Marrow Isolation

Bone marrow was harvested by crushing tibias, femurs, hips and spine of each mouse with a mortar and pestle (Lo Celso, 2007). Red blood cell lysis was performed according to the manufacturer’s protocol using RBC lysis buffer (eBioscience).

Transplantation

Congenic C57BL/6 CD45.1+ recipient mice were irradiated with a single dose at a lethal (10 cGy) level, delivered >4 hours before injections. For in vivo intrafemoral injections, cells were injected into the femoral cavity of anesthetized mice with a Hamilton needle (Sigma Aldrich). Sca1 depleted helper cells were obtained from double positive CD45.1+CD45.2+ bone marrow flushed from femurs stained with Sca1-APC and depleted using anti-APC microbeads (Miltenyi Biotech) bound to LD columns (Miltenyi Biotech). Similarly, lineage depletions were conducted using biotinylated monoclonal antibodies (Ter119, Gr-1, B220, and CD3) (BD Biosciences) and depleted with anti-biotin microbeads (Miltenyi Biotech).

To create chimeric mice harboring both Flt3-ITD and WT hematopoiesis, recipients were injected with CD45.2 Flt3-ITD and CD45.1 WT Lin cells in a 4:1 (ITD:WT) ratio. Donor contributions were assessed by FACS post-transplant (Table S4).

Sorafenib treatment

Sorafenib was formulated and administered as previously described (Li L, 2011). Adult mice were orally gavaged at a dose of 10mg/kg daily for 2 weeks with a schedule of 5 days on and 2 days off and sacrificed immediately for analysis after the 2 week period. For functional studies after treatment, Lin BM was isolated by magnetic cell separation as described above and 100,000 Lin cells were injected with 1×106 red blood cell lysed WT CD45.1 WBM helper cells. For the in utero experiment, pregnant mothers were gavaged with 20mg/kg of Sorafenib starting at E.15 and continuing for 2 weeks after birth of pups.

Flow cytometry and cell sorting

Cell preparation, staining and analysis were performed as previously described (Li L, 2008). . Sorting of different hematopoietic subpopulations was also conducted on a FACS Aria (BD Biosciences) and analyzed using FlowJo software (Flow Jo). Side population staining was conducted as previously described with Hoechst 33342 (Invitrogen) (Goodell MA, 2004) and analyzed on a MoFlow Cytometer (Dako Cytomation).

BrdU and Flt3 intracellular staining

BrdU incorporation experiments were carried out using a BrdU Assay kit (BD Biosciences) with cell surface staining prior to fixation, permeabilization and DNAse treatment. Each of the three independent experiments was conducted as per manufacturer’s protocol.

Flt3 intracellular stains were conducted with the FoxP3/Transcription Factor Staining Buffer set (eBioscicences). Flt3-PE antibody (BD Biosciences) and PE Rat IgG2aκ (BD Biosciences) isotype control was used for these experiments.

RNA preparation and Quantitative Polymerase Chain Reaction

RNA was isolated by using a miRNeasy Mini RNA kit (Qiagen). cDNA synthesis and qPCR was performed as described previously using a CFX-96 RealTime PCR system (BioRad) (Li L, 2008). Primers for genes are listed in the supplemental data (Table S4). RPS16 was used as an internal loading control. Each PCR was run in triplicate and gene expression values are expressed as a fold change calculated by 2−ΔCt. A total of three independent PCR reactions were run for each gene target.

Statistical Analysis

All p values were calculated using a student’s t-test, two tailed, unpaired analysis.

HIGHLIGHTS

  • Flt3-ITD perturbs normal mouse hematopoietic stem cell homeostasis
  • Flt3 is expressed and capable of exerting functional effects in murine LT-HSCs
  • Flt3-ITD leads to depletion of murine LT-HSCs through loss of quiescence
  • MPN and HSC defects are simultaneously reversed by Sorafenib treatment

Supplementary Material

01

ACKNOWLEDGEMENTS

We would like to thank members of the Small Lab and Brown Lab for helpful discussions, the flow cytometry core in the Department of Oncology at Johns Hopkins University, most notably Lee Blosser and Ada Tam. Moflo studies were completed with the help of Hao Zhang in the Dept of MMI. This work was supported by grants from the National Cancer Institute (CA90668, CA70970), Leukemia & Lymphoma Society and the Giant Food Pediatric Cancer Research Fund. Dr. Small is also supported by the Kyle Haydock Professorship.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REFERENCES

  • Adolfsson J, Borge OK, Bryder D, Theilgaard-Monch K, Astrand-Grundstrom I, et al. Upregulation of FLT3 expression within the bone marrow Lin(−)Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of self-renewal capacity. Immunity. 2001;15:659–69. [PubMed]
  • Akala OO, Clarke MF. Hematopoietic stem cell self-renewal. Curr Opin Genet Dev. 2008;16:496–501. [PubMed]
  • Auclair D, M. D, Yatsula V, Pickett W, Carter C, Chang Y, et al. Antitumor activity of sorafenib in FLT3-driven leukemic cells. Leukemia. 2007;21:439–445. [PubMed]
  • Basham B, S. M, Grein J, McClanahan T, D’Andrea A, Lees E, Rascle A. In vivo identification of novel STAT5 target genes. Nucleic Acids Res. 2008;36:3802–3818. [PMC free article] [PubMed]
  • Boyer SW, Schroeder AV, Smith-Berdan S, Forsberg EC. All Hematopoietic Cells Develop from Hematopoietic Stem Cells through Flk2/FLT3-Positive Progenitor Cells. Cell Stem Cell. 2011;9:65–73. [PMC free article] [PubMed]
  • Buza-Vidas N, Cheng M, Duarte S, Charoudeh HN, Jacobsen SE, Sitnicka E. FLT3 receptor and ligand are dispensible for maintenance and post-transplantation expansion of mouse hematopoietic stem cells. Blood. 2009;102:881–886. [PubMed]
  • Buza-Vidas N, Woll P, Hultquist A, Duarte S, Lutteropp M, et al. FLT3 expression initiates in fully multipotent mouse hematopoietic progenitor cells. Blood. 118:1544–8. [PubMed]
  • Chao MP, Seita J, Weissman IL. Establishment of a normal hematopoietic and leukemia stem cell hierarchy. Cold Spring Harb Sym Quant Biol. 2008;73:439–49. [PubMed]
  • Choudhary C, O. J, Brandts C, Cox J, Reddy PN, et al. Mislocalized activation of oncogenic RTKs switches downstream signaling outcomes. Mol Cell. 2009;36:326–339. [PubMed]
  • Christensen JL, Weismsman I. Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells. Proc Natl Acad Sci USA. 2001;25:14541–14546. [PubMed]
  • Cloos J, Goemans BF, Hess CJ, et al. Stability and prognostic influence of FLT3 mutations in paired initial and relapsed AML samples. Leukemia. 2006;20:1217–1220. [PubMed]
  • Crump M, Hedley D, Kamel-Reid S, Leber B, et al. A randomized phase I clinical and biologic study of two schedules of sorafenib in patients with myelodysplastic syndrome or acute myeloid leukemia: a NCIC (National Cancer Institute of Canada) Clinical Trials Group Study. Leuk Lymphoma. 2010;51:252–60. [PubMed]
  • D’Amico A, Wu L. The early progenitors of mouse dendritic cells and plasmacytoid predendritic cells are within the bone marrow hemopoietic precursors expressing FLT3. J Exp Med. 2003;198:293–303. [PMC free article] [PubMed]
  • Escudier B, E. T, Stadler WM, Szczylik C, Oudard S, Siebels M, et al. Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med. 2007;356:125–134. [PubMed]
  • Fenski R, F. K, Serve S, et al. Constitutive activation of FLT3 in acute myeloid leukaemia and its consequences for growth of 32D cells. Br J Haematol. 2000;108:322–330. [PubMed]
  • Frohling S, S. R, Breitruck J, et al. Prognostic significance of activating FLT3 mutations in younger adults (16 to 60 years) with acute myeloid leukemia and normal cytogenetics: a study of the AML Study Group Ulm. Blood. 2002;100:4372–4380. [PubMed]
  • Gilliland DG, G. J. The roles of FLT3 in hematopoiesis and leukemia. Blood. 2002;100:1532–1542. [PubMed]
  • Goodell MA, M.-F. S, Camargo FD. Isolation and characterization of side population cells. Methods Mol Biol. 2004;290:343–352. [PubMed]
  • Greenblatt S, Li L, Slape C, Nguyen B, et al. Knock-in of a FLT3/ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model. Blood. 2012 Epub Feb 8. [PubMed]
  • Grundler R, Thiede C, Miething C, Steudel C, et al. Sensitivity toward tyrosine kinase inhibitors varies between different activating mutations of the FLT3 receptor. Blood. 2003;102:646–651. [PubMed]
  • Hayakawa F, T. M, Kiyoi H, et al. Tandem-duplicated FLT3 constitutively activates STAT5 and MAP kinase and introduces autonomous cell growth in IL-3-dependent cell lines. Oncogene. 2000;19:624–631. [PubMed]
  • Hoelbl A, Kovacic B, Kerenyi MA, Simma O, Warsch W, et al. Clarifying the role of Stat5 in lymphoid development and Abelson induced transformation. Blood. 2006;107:4898–4906. [PMC free article] [PubMed]
  • Ikuta K, Weissman IL. Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation. Proc Natl Acad Sci USA. 1992;89:1502–1506. [PubMed]
  • Jan M, Snyder TM, Corces-Zimmerman, et al. Clonal Evolution of Pre-Leukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia. Blood. 2011;118 Abstract 4. [PMC free article] [PubMed]
  • Katayama N, Shih JP, Nishikawa S, Kina T, Clark SC, Ogawa M. Stage-specific expression of c-kit protein by murine hematopoietic progenitors. Blood. 1993;82:2353–60. [PubMed]
  • Kiel M, Yilmaz OH, Iwashita T, Terhost C, Morrison SJ. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell. 2005;7:1109–1121. [PubMed]
  • Kikushige Y, Yoshimoto G, Miyamoto T, et al. Human FLT3 is expressed at the hematopoietic stem cell and the granulocyte/macrophage progenitor stages to maintain cell survival. J Immunol. 2008;180:7358–7367. [PubMed]
  • Kim KT, B. K, Davis S, Piloto O, Levis M, Li L, et al. Constitutive Fms-like tyrosine kinase 3 activation results in specific changes in gene expression in myeloid leukaemic cells. Br J Haematol. 2007;138:603–615. [PubMed]
  • Kiyoi H, O. R, Ueda R, Saito H, Naoe T. Mechanism of constitutive activation of FLT3 with internal tandem duplication in the juxtamembrane domain. Oncogene. 2002;21:2555–2563. [PubMed]
  • Kiyoi H, T. M, Yokota S, et al. Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product. Leukemia. 1998;12:1333–1337. [PubMed]
  • Knapper S, Burnett AK, Littlewood T, et al. A phase 2 trial of the FLT3 inhibitor lestaurtinib (CEP701) as first-line treatment for older patients with acute myeloid leukemia not considered fit for intensive chemotherapy. Blood. 2006;108:3262–3270. [PubMed]
  • Kottaridis PD, G. R, Frew ME, et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood. 2001;98:1752–1759. [PubMed]
  • Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994;367:645–8. [PubMed]
  • Levis M, Murphy KM, Pham R, Kim KT, Stine A, et al. Internal tandem duplications of the FLT3 gene are present in leukemia stem cells. Blood. 2005;106:673–680. [PubMed]
  • Levis M, R. F, Wang ES, Baer MR, Perl A, Coutre S, Erba H, Stuart RK, Baccarani M, Cripe LD, et al. Results from a randomized trial of salvage chemotherapy followed by lestaurtinib for patients with FLT3 mutant AML in first relapse. Blood. 2011;117:3294–301. [PubMed]
  • Levis M, S. D. FLT3: ITDoes matter in leukemia. Leukemia. 2003;17:1738–1752. [PubMed]
  • Li L, P. O, Kim KT, Ye Z, Nguyen HB, Yu X, et al. FLT3/ITD expression increases expansion, survival and entry into cell cycle of human haematopoietic stem/progenitor cells. Br J Haematol. 2007;137:64–75. [PubMed]
  • Li L, P. O, Nguyen HB, et al. Knock-in of an internal tandem duplication mutation into murine FLT3 confers myeloproliferative disease in a mouse model. Blood. 2008;111:3849–3858. [PubMed]
  • Li L, Zhang L, Fan J, Greenberg K, Desiderio S, Rassool FV, Small D. Defective nonhomologous end joining blocks B-cell development in FLT3/ITD mice. Blood. 2011;117:3131–9. [PubMed]
  • Lo Celso C, Scadden D. Isolation and transplantation of hematopoietic stem cells (HSCs) J Vis Exp. 2007 [PubMed]
  • Lumkul R, G. N, Malehorn MT, Hoehn GT, Zheng R, Baldwin B, et al. Human AML cells in NOD/SCID mice: engraftment potential and gene expression. Leukemia. 2002;16:1818–1826. [PubMed]
  • Mackarehtschian K, H. J, Moore KA, Boast S, Goff SP, Lemischka IR. Targeted disruption of the flk2/FLT3 gene leads to deficiencies in primitive hematopoietic progenitors. Immunity. 1995;3:147–161. [PubMed]
  • McKenna HJ, S. K, Miller RE, et al. Mice lacking FLT3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells. Blood. 2000;95:3489–3497. [PubMed]
  • Meshinchi S, W. W, Stirewalt DL, et al. Prevalence and prognostic significance of FLT3 internal tandem duplication in pediatric acute myeloid leukemia. Blood. 2001;97:89–94. [PubMed]
  • Mizuki M, F. R, Halfter H, et al. FLT3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways. Blood. 2000;96:3907–3914. [PubMed]
  • Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity. 1994;1:661–673. [PubMed]
  • Nakao M, Y. S, Iwai T, et al. Internal tandem duplication of the FLT3 gene found in acute myeloid leukemia. Leukemia. 1996;10:1911–1918. [PubMed]
  • Okada S, Nakauchi H, Nagayoshi K, Nishikawa S, Miura Y, Suda T. In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells. Blood. 1992;80:3044–50. [PubMed]
  • Orford KW, Scadden DT. Deconstructing stem cell self-renewal: genetic insights into cell cycle regulation. Nat Rev Genet. 2008;9:115–28. [PubMed]
  • Osawa M, H. K, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34low/negative hematopoietic stem cell. Science. 1996;5272:242–245. [PubMed]
  • Passegue E, Wagers AJ, Guiriato S, Anderson W, Weissman IL. Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates. J Exp. Med. 2005;202:1599–1611. [PMC free article] [PubMed]
  • Pinheiro RF, de Sa Moreira E, Silva MR, Alberto FL, Chauffaille Mde L. FLT3 internal tandem duplication during myelodysplastic syndrome follow-up: a marker of transformation to acute myeloid leukemia. Cancer Genet Cytogenet. 2008;183:89–93. [PubMed]
  • Randall TD, F. E. Lund, Howard MC, Weissman IL. Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells. Blood. 1996;87:4057–4067. [PubMed]
  • Ravandi F, Kantarjian H, Faderl S, et al. Outcome of patients with FLT3-mutated acute myeloid leukemia in first relapse. Leuk Res. 2010;34:752–756. [PMC free article] [PubMed]
  • Ravandi F, Cortes JE, Jones D, Fader S, Garcia-Manero G, et al. Phase I/II study of combination therapy with sorafenib, idarubicin, and cytarabine in younger patients with acute myeloid leukemia. J Clin Oncol. 2010;28:1856–62. [PMC free article] [PubMed]
  • Rombouts WJ, B. I, Lowenberg B, Ploemacher RE. Biological characteristics and prognosis of adult acute myeloid leukemia with internal tandem duplications in the FLT3 gene. Leukemia. 2000a;14:675–683. [PubMed]
  • Rombouts WJ, M. A, Ploemacher RE. Identification of variables determining the engraftment potential of human acute myeloid leukemia in the immunodeficient NOD/SCID human chimera model. Leukemia. 2000b;14:889–897. [PubMed]
  • Safaian NN, C. A, Bruns I, Fenk R, Reinecke P, Dienst A, et al. Sorafenib (Nexavar((R))) induces molecular remission and regression of extramedullary disease in a patient with FLT3-ITD(+) acute myeloid leukemia. Leuk Res. 2009;33 [PubMed]
  • Santaguida M, S. K, King B, Sabnis AJ, Forsberg EC, Attema JL, et al. JunB protects against myeloid malignancies by limiting hematopoietic stem cell proliferation and differentiation without affecting self-renewal. Cancer Cell. 2009;15:341–352. [PMC free article] [PubMed]
  • Shih LY, Huang CF, Wu JH, et al. Internal tandem duplication of FLT3 in relapsed acute myeloid leukemia: a comparative analysis of bone marrow samples from 108 adult patients at diagnosis and relapse. Blood. 2002;100:2387–2392. [PubMed]
  • Sitnicka E, Bryder D, Theilgaard-Monch K, Buza-Vidas N, Adolfsson J, Jacobsen SE. Key role of FLT3 ligand in regulation of the common lymphoid progenitor but not in maintenance of the hematopoietic stem cell pool. Immunity. 2002;17:463–472. [PubMed]
  • Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science. 1988;241:58–62. [PubMed]
  • Stirewalt DL, Radich JP. The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer. 2003;3:650–665. [PubMed]
  • Stone R, Paquette RL, et al. Phase IB study of PKC412, an oral FLT3 kinase inhibitor, in sequential and simultaneous combinations with daunorubicin and cytarabine induction and high-dose cytarabine consolidation in newly diagnosed patients with AML. Blood. 2005:121a.
  • Stubbs MC, Kim YM, Krivtsov AV, Wright RD, Feng Z, Agarwal J, et al. MLL-AF9 and FLT3 cooperation in acute myelogenous leukemia: development of a model for rapid therapeutic assessment. Leukemia. 2008;22:66–77. [PMC free article] [PubMed]
  • Thiede C, S. C, Mohr B, et al. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood. 2002;99:4326–4335. [PubMed]
  • Tong FK, C. S, Hedley D. Pharmacodynamic monitoring of BAY 43-9006 (Sorafenib) in phase I clinical trials involving solid tumor and AML/MDS patients, using flow cytometry to monitor activation of the ERK pathway in peripheral blood cells. Cytometry B Clin Cytom. 2006;70:107–114. [PubMed]
  • Tse KF, Novelli E, Civin CI, Bohmer FD, Small D. Inhibition of FLT3-mediated transformation by use of a tyrosine kinase inhibitor. Leukemia. 2001;15:1001–1010. [PubMed]
  • Weisberg E, Boulton C, Kelly LM, et al. Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412. Cancer Cell. 2002;1:433–443. [PubMed]
  • Wilson A, L. E, Oser G, van der Wath RC, Blanco-Bose W, et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell. 2008;135:1118–1129. [PubMed]
  • Yoshimoto G, Miyamoto T, Jabbarzadeh-Tabrizi S, Iino T, Rocnik JL, Kikushige Y, et al. FLT3-ITD up-regulates MCL-1 to promote survival of stem cells in acute myeloid leukemia via FLT3-ITD specific STAT5 activation. Blood. 2009;114:5034–43. [PubMed]
  • Zhang W, K. M, Shi YX, McQueen T, Harris D, Ling X, et al. Mutant FLT3: a direct target of sorafenib in acute myelogenous leukemia. J Natl Cancer Inst. 2008;100:184–198. [PubMed]
  • Zola H. High-sensitivity immunofluorescence/flow cytometry: detection of cytokine receptors and other low-abundance membrane molecules. Curr Protoc Cytom. 2004 Chapter 6: Unit 6.3. [PubMed]