**3. Functional interaction between FLT3/ITD and CXCR4 in the migration and homing of AML cells that are associated with resistance**

Because *CXCL12/CXCR4* provides a survival signal to FLT3/ITD+ AML cells, it suggests that *CXCL12/CXCR4* signaling accentuates FLT3/ITD signaling activity. By contrast, FLT3/ ITD regulates cell migration to *CXCL12* [50], indicating that FLT3/ITD modulates *CXCR4* signaling. Therefore, FLT3/ITD and *CXCL12/CXCR4* signaling mutually interacts. While an earlier study demonstrated that patients with FLT3/ITD+ AML have higher *CXCR4* expression than those with FLT3 wild-type AML [45], subsequent studies have demonstrated controversial findings. We and other groups have demonstrated that overexpressing FLT3/ITD in mouse Ba/F3 cells or human CD34+ cells significantly downregulated *CXCR4* expression [50, 59]. Incubating human CD34+ cells with *FLT3* ligand also decreased the expression of *CXCR4* [50]. Moreover, the mRNA expression of *CXCR4* was significantly lower in patients with FLT3/ITD+ AML than in those with wild-type FLT3 [9, 106]. These data indicate that FLT3/ITD can reduce the expression of *CXCR4* in contrast to the data of the earlier report. The mechanism responsible for the modulation of CXCR4 expression by FLT3/ITD remains subject to investigation. PIM1, which is activated by FLT3/ITD, upregulates *CXCR4* [107]. Similarly, *RUNX1*, which is elevated in FLT3/ITD+ AML, upregulates CXCR4 transcription [78]. On the other hand, *CEBPα*, a transcriptional factor that increases CXCR4 expression [108], is inactivated by FLT3/ITD [11, 109]. Therefore, the inactivation of *CEBPα* by FLT3/ ITD can decrease CXCR4 expression. Because FLT3/ITD inhibits *CEBPα* but enhances *PIM1* and/or *RUNX1* expression, the balance between the inactivation of *CEBPα* and activation of *PIM1* and/or *RUNX1* may determine the expression of *CXCR4* in FLT3/ITD+ AML.

Although the *FLT3* ligand, as well as FLT3/ITD, increases the migration of mouse hematopoietic cells to *CXCL12* [19, 50, 106], *FLT3* signaling can decrease the migration of CD34+ cells and mouse Ba/F3 cells toward *CXCL12* [50, 59]. Enhancing migration and decreasing migration in response to *CXCL12* by FLT3/ITD appear to be controversial, but the reduction of migration toward *CXCL12* is most likely a consequence of a decrease in *CXCR4* expression, which, in turn, induces the quantitative reduction of *CXCR4* signaling. Jacobi et al. reported that the transient expression of FLT3/ITD decreases *CXCR4* expression in human CD34+ cells, coincident with their reduced migration toward *CXCL12* [59]. This is consistent with the reduction in *CXCR4* expression in CD34+ cells or Ba/F3 cells incubated with *FLT3* ligand that is accompanied by a decrease in CXCL12-mediated migration [50]. These data indicate that FLT3/ITD, as well as normal *FLT3* signaling, can inhibit *CXCL12/CXCR4* signaling by downregulating *CXCR4* expression. By contrast, the sustained expression of FLT3/ITD enhances migration in response to *CXCL12*, even with a significant downregulation of the *CXCR4* level [50]. Augmentation in cell migration toward *CXCL12* despite the reduction in *CXCR4* expression suggests that the increase in migration was not due to the qualitative increase in *CXCR4* signaling. A subsequent study by Onishi et al. confirmed that enhanced migration by FLT3/ITD was mediated through the qualitative change in *CXCR4* signaling [106]. The data indicated that molecules and/or pathways downstream of *CXCR4* that are regulated in the presence of FLT3/ITD were overlapped but distinct from those regulated in the absence of FLT3/ITD, suggesting that FLT3/ITD regulates *CXCR4* signaling pathways functionally distinct from those of normal cells [106]. This implies that FLT3/ITD functionally alters *CXCR4* signaling. These findings strongly suggest that FLT3/ITD can negatively regulate *CXCR4* signaling by qualitatively decreasing *CXCR4* signaling by downregulating *CXCR4* expression, whereas it also increases *CXCR4* signaling activity by changing the global gene expression downstream of CXCR4 (**Figure 2**). One of the molecules responsible for the activation of *CXCR4* signaling by FLT3/ITD is Rho-associated kinase-1 (*ROCK1*). *ROCK1* promotes the migration of *CXCR4*<sup>+</sup> cells to *CXCL12*, whereas antagonizing *ROCK1* displays the opposite effect. *CXCL12* transiently upregulates *ROCK1* expression but subsequently downregulates its expression in the absence of FLT3/ITD. This downregulation is associated with the attenuation in cell migration to CXCL12, suggesting the presence of negative

secreted from the microenvironment modulate the function of FLT3/ITD+

Because *CXCL12/CXCR4* provides a survival signal to FLT3/ITD+

earlier study demonstrated that patients with FLT3/ITD+

Similarly, *RUNX1*, which is elevated in FLT3/ITD+

**3. Functional interaction between FLT3/ITD and CXCR4 in the** 

**migration and homing of AML cells that are associated with resistance**

that *CXCL12/CXCR4* signaling accentuates FLT3/ITD signaling activity. By contrast, FLT3/ ITD regulates cell migration to *CXCL12* [50], indicating that FLT3/ITD modulates *CXCR4* signaling. Therefore, FLT3/ITD and *CXCL12/CXCR4* signaling mutually interacts. While an

sion than those with FLT3 wild-type AML [45], subsequent studies have demonstrated controversial findings. We and other groups have demonstrated that overexpressing FLT3/ITD

*CXCR4* [50]. Moreover, the mRNA expression of *CXCR4* was significantly lower in patients

FLT3/ITD can reduce the expression of *CXCR4* in contrast to the data of the earlier report. The mechanism responsible for the modulation of CXCR4 expression by FLT3/ITD remains subject to investigation. PIM1, which is activated by FLT3/ITD, upregulates *CXCR4* [107].

[78]. On the other hand, *CEBPα*, a transcriptional factor that increases CXCR4 expression [108], is inactivated by FLT3/ITD [11, 109]. Therefore, the inactivation of *CEBPα* by FLT3/ ITD can decrease CXCR4 expression. Because FLT3/ITD inhibits *CEBPα* but enhances *PIM1* and/or *RUNX1* expression, the balance between the inactivation of *CEBPα* and activation of

Although the *FLT3* ligand, as well as FLT3/ITD, increases the migration of mouse hematopoi-

mouse Ba/F3 cells toward *CXCL12* [50, 59]. Enhancing migration and decreasing migration in response to *CXCL12* by FLT3/ITD appear to be controversial, but the reduction of migration toward *CXCL12* is most likely a consequence of a decrease in *CXCR4* expression, which, in turn, induces the quantitative reduction of *CXCR4* signaling. Jacobi et al. reported that the transient

their reduced migration toward *CXCL12* [59]. This is consistent with the reduction in *CXCR4*

a decrease in CXCL12-mediated migration [50]. These data indicate that FLT3/ITD, as well as normal *FLT3* signaling, can inhibit *CXCL12/CXCR4* signaling by downregulating *CXCR4* expression. By contrast, the sustained expression of FLT3/ITD enhances migration in response to *CXCL12*, even with a significant downregulation of the *CXCR4* level [50]. Augmentation in cell migration toward *CXCL12* despite the reduction in *CXCR4* expression suggests that the increase in migration was not due to the qualitative increase in *CXCR4* signaling. A subsequent study by Onishi et al. confirmed that enhanced migration by FLT3/ITD was mediated through

cells or Ba/F3 cells incubated with *FLT3* ligand that is accompanied by

*PIM1* and/or *RUNX1* may determine the expression of *CXCR4* in FLT3/ITD+

expression of FLT3/ITD decreases *CXCR4* expression in human CD34+

etic cells to *CXCL12* [19, 50, 106], *FLT3* signaling can decrease the migration of CD34+

AML than in those with wild-type FLT3 [9, 106]. These data indicate that

this hypothesis remains yet to be proven.

in mouse Ba/F3 cells or human CD34+

[50, 59]. Incubating human CD34+

with FLT3/ITD+

130 Myeloid Leukemia

expression in CD34+

AML cells, although

AML cells, it suggests

AML have higher *CXCR4* expres-

AML, upregulates CXCR4 transcription

AML.

cells, coincident with

cells and

cells significantly downregulated *CXCR4* expression

cells with *FLT3* ligand also decreased the expression of

**Figure 2.** Quantitative and/or qualitative regulation of CXCR4 signaling by FLT3/ITD. *CXCL12/CXCR4* signaling augments FLT3/ITD activity, but in contrast, FLT3/ITD modulates *CXCL12/CXCR4* signaling, indicating that *CXCL12/ CXCR4* and FLT3/ITD signaling mutually interacts. Regulation of CXCR4 signaling by FLT3/ITD is classified into two categories: one is quantitative regulation and the other is qualitative mechanism. FLT3/ITD regulates expression of *CXCR4*, depending on the transcriptional mediators or kinases. For instance, inactivation of CEBPα by FLT3/ITD can decrease *CXCR4* expression, whereas activation of *PIM1* and/or *RUNX1* can increase *CXCR4* expression. Downregulation of CXCR4 diminishes cell migration to CXCL12, whereas upregulation of *CXCR4* expression leads to enhancement in cell migration to *CXCL12*. On the other hand, FLT3/ITD modulates global gene expression downstream of *CXCR4*, which leads to the enhancement of cell migration to *CXCL12*. Classification of genes that are regulated by *CXCL12* in FLT3/ ITD<sup>−</sup> cells and those in FLT3/ITD+ cells based on the molecular pathways or biological process demonstrated that they are functionally overlapped but distinct. The data suggest that FLT3/ITD functionally alters *CXCL12/CXCR4* signaling. For instance, downregulation of *ROCK1* expression by *CXCL12* that is normally observed in control cells is abrogated by FLT3/ITD, which is responsible for the enhancement in cell migration to *CXCL12* by FLT3/ITD.

feedback in *CXCL12/CXCR4* signaling mediated by modulating *ROCK1* expression to prevent excessive migration in normal cells. By contrast, FLT3/ITD or *FLT3* ligand enhances the expression and prevents the subsequent downregulation of the *ROCK1* level that is normally induced by CXCL12, thereby abrogating the negative feedback generated by *CXCL12* and *ROCK1*. The loss of negative feedback on *ROCK1* expression induced by *FLT3* signaling resulted in the sustained activation of *CXCL12/CXCR4* signaling, thereby enhancing the migration of FLT3/ITD+ cells toward *CXCL12*. Enhanced chemotaxis is also mediated through RAS [58].

that the loss of retinoic acid receptor gamma (PARγ) resulted in myeloproliferation in mice; however, the transplantation of the marrow cells into PARγ-deficient cells did not cause myeloproliferation in wild-type recipients, whereas the transplantation of wildtype marrow cells caused myeloproliferation in PARγ-deficient recipients, indicating that myeloproliferation caused by the loss of PARγ was microenvironmental [114]. The microenvironmental effect on aberrant myeloproliferation is also supported by experiments using Rb-deficient cells. Knocking out Rb resulted in myeloproliferation in mice; however, the genetic defect in both hematopoietic cells and the microenvironment was necessary for the development of myeloproliferation [115]. Furthermore, deletion of DICER1 in primitive osteolineage cells led to myelodysplastic syndrome and AML [116], indicating that malfunction of *DICER1* in the niche component was sufficient to cause myeloid malignancy. These findings indicate that the genetic alteration and/or malfunction of the micro-

Molecular Interaction Between the Microenvironment and FLT3/ITD+ AML Cells Leading to the…

http://dx.doi.org/10.5772/intechopen.71676

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Reports have demonstrated that HSCs regulate their own niches by instructing neighboring stromal cells to produce supportive factors or alter the overall microenvironment [117–119]. While the marrow niche supports leukemia cell proliferation or protects cells from chemotherapeutic insult by providing various survival signals, recent evidence has demonstrated that leukemia cells modulate the marrow environment to create a supportive niche favoring survival for AML cells, just as healthy HSCs regulate their niche. Zhang et al. demonstrated that chronic myeloid leukemia (CML) cells modulate the microenvironment in favor of CML cells over healthy HCS by modulating *CXCL12* expression and alter the localization of HSCs. CML cells modulate cytokine expression in the microenvironment, such that they support CML cells [120]. A study by Schepers et al. identified that myeloproliferative neoplasia (MPN) remodels endosteal bone marrow niches by stimulating mesenchymal stem cells to produce functionally altered osteoblastic lineage cells. This results in the creation of a self-reinforcing leukemic niche that impairs normal hematopoiesis and favors leukemic stem cell function [121]. Several cytokines, such as thrombopoietin and CCL3, that direct cell-cell interaction, alteration of TGF-β, and Notch and inflammatory signaling were involved in the expansion and/ or remodeling in osteoblastic lineage cells. The osteoblastic lineage cells remodeled by myeloproliferation compromised normal HSCs but effectively support leukemia stem cells [121]. Similarly, the latest study by Mead et al. demonstrated that FLT3/ITD modulates the marrow microenvironment and impaired the number of HSCs. In the marrow of FLT3ITD/ITD mice, FLT3/ ITD-induced myeloproliferation was associated with a progressive decline in the HSC compartment. Notably, when FLT3ITD/ITD marrow cells were transplanted with marrow competitor cells from wild-type mice into healthy recipients, the HSCs derived from the competitor cells were significantly reduced, demonstrating the presence of a cell extrinsic mechanism that diminishes the competitor HSC. Loss of competitor cells in the recipient mice that developed FLT3/ITD-induced myeloproliferation was coincided with the disruption of stromal cells in the recipient marrow, an activity that was associated with reduced numbers of endothelial and mesenchymal stromal cells showing increased inflammation-associated gene expression. The study finally discovered that tumor necrosis factor (TNF), a cell-extrinsic negative regulator of HSCs, was overexpressed in the marrow niche cells in FLT3ITD/ITD mice, and anti-TNF treatment partially rescued the loss of HSCs. These data clearly demonstrate that FLT3/ITD compromises HSCs through an extrinsically mediated mechanism of disrupting HSCs that support

environment can induce myeloid malignancies.

An additional molecular machinery that specifically mediates the migration of FLT3/ITD<sup>+</sup> cells is *PIM1* kinase. The expression and kinase activity of *PIM1* are upregulated in FLT3/ITD+ AML cells [110]. Enhanced *PIM1* activity induced by FLT3/ITD is essential for the migration and homing of AML cells [107]. The effect of *PIM1* on the migration and homing of FLT3/ITD cells is mediated by the increase in *CXCR4* owing to its recycling by the phosphorylation of serine 339 on *CXCR4*. These data indicate that *PIM1* activity is essential for the proper *CXCR4* surface expression and migration of FLT3/ITD+ AML cells toward *CXCL12*. In addition to regulating migration and homing, *PIM1* modulates the resistance of FLT3/ITD+ AML cells to *FLT3* inhibitors [21, 22]. Targeting *PIM1* synergizes with *FLT3* inhibition [111] and restores the sensitivity of *FLT3* inhibitors in FLT3/ITD+ AML cells [21]. A recent study in abstract form indicated that the microenvironment-induced expression of *PIM* kinase supports chronic leukemia (CLL) survival and promotes *CXCR4*-dependent migration [112]. Although this was investigated in CLL, the data suggest that microenvironmental factors increase the expression of *PIM1* kinase, which promotes the resistance of FLT3/ITD+ AML. The upregulated *PIM1* kinase, in turn, would facilitate the migration of FLT3/ITD+ AML toward *CXCL12* by activating *CXCR4* signaling, thereby increasing the interaction between FLT3/ITD+ AML cells and microenvironment cells. In this regard, antagonizing PIM1 represents an additional therapeutic strategy to abrogate the interaction between FLT3/ITD+ AML cells and marrow niches, particularly for those that have become resistant to *FLT3/ITD* inhibitors. Similarly, *ROCK1* enhances not only *CXCL12*-induced migration [106] but also the proliferation of FLT3/ITD+ cells [13]. Therefore, antagonizing *ROCK1* is likely to be beneficial to interfere with the communication of FLT3/ITD<sup>+</sup> AML cells between the marrow niches and inhibit their proliferation. These data suggest that FLT3/ITD increases the communication with the bone marrow microenvironment by enhancing the chemotaxis toward *CXCL12*. Together with *CXCL12* protecting FLT3/ITD+ AML cells from the insult of FLT3 inhibitors, the findings strongly indicate that reciprocal interaction between FLT3/ITD and *CXCL12/ CXCR4* signaling exists that accentuates the resistance to FLT3 inhibitors.
