**4. Effect of FLT3 mutation on the microenvironment**

Normal hematopoietic stem cells drive hematopoiesis, but this process requires appropriate factors secreted by adjacent cells, adhesion molecules, neighboring cells such as mesenchymal stromal cells, osteolineage cells, and endothelial cells that exist in the microenvironment [113]. In agreement with the microenvironment mediating the tight control necessary for normal hematopoiesis, earlier studies have demonstrated that malfunction of microenvironmental cells can lead to the development of myeloproliferation, which represents one of the outcomes of aberrant hematopoiesis. Walkley et al. demonstrated 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 microenvironment can induce myeloid malignancies.

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].

migration and homing, *PIM1* modulates the resistance of FLT3/ITD+

likely to be beneficial to interfere with the communication of FLT3/ITD<sup>+</sup>

*CXCR4* signaling exists that accentuates the resistance to FLT3 inhibitors.

**4. Effect of FLT3 mutation on the microenvironment**

expression and migration of FLT3/ITD+

of *FLT3* inhibitors in FLT3/ITD+

132 Myeloid Leukemia

tate the migration of FLT3/ITD+

tion between FLT3/ITD+

promotes the resistance of FLT3/ITD+

increasing the interaction between FLT3/ITD+

tion [106] but also the proliferation of FLT3/ITD+

*CXCL12*. Together with *CXCL12* protecting FLT3/ITD+

An additional molecular machinery that specifically mediates the migration of FLT3/ITD<sup>+</sup>

is *PIM1* kinase. The expression and kinase activity of *PIM1* are upregulated in FLT3/ITD+

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

tors [21, 22]. Targeting *PIM1* synergizes with *FLT3* inhibition [111] and restores the sensitivity

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

regard, antagonizing PIM1 represents an additional therapeutic strategy to abrogate the interac-

resistant to *FLT3/ITD* inhibitors. Similarly, *ROCK1* enhances not only *CXCL12*-induced migra-

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

tors, the findings strongly indicate that reciprocal interaction between FLT3/ITD and *CXCL12/*

Normal hematopoietic stem cells drive hematopoiesis, but this process requires appropriate factors secreted by adjacent cells, adhesion molecules, neighboring cells such as mesenchymal stromal cells, osteolineage cells, and endothelial cells that exist in the microenvironment [113]. In agreement with the microenvironment mediating the tight control necessary for normal hematopoiesis, earlier studies have demonstrated that malfunction of microenvironmental cells can lead to the development of myeloproliferation, which represents one of the outcomes of aberrant hematopoiesis. Walkley et al. demonstrated

AML cells toward *CXCL12*. In addition to regulating

AML cells [21]. A recent study in abstract form indicated that

AML. The upregulated *PIM1* kinase, in turn, would facili-

AML cells and microenvironment cells. In this

cells [13]. Therefore, antagonizing *ROCK1* is

AML cells from the insult of FLT3 inhibi-

AML toward *CXCL12* by activating *CXCR4* signaling, thereby

AML cells and marrow niches, particularly for those that have become

cells

AML

AML cells to *FLT3* inhibi-

AML cells between the

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 bone marrow stromal cells by generating an inflammatory environment [122]. The same study also demonstrated that the expression of FLT3 mRNA and protein is absent in HSCs, strongly suggesting that FLT3/ITD protein is not expressed in most primitive HSCs, even if FLT3/ITD mutation exists in the FLT3 gene in HSCs. Because these HSCs harboring the FLT3/ITD gene but lacking the expression of *FLT3/ITD* protein would not be targeted by the *FLT3* inhibitors, they may represent a reservoir for the development of resistant clones, in which additional mutations can be accumulated. The lack of mutant *FLT3/ITD* protein in HSCs harboring FLT3/ ITD mutation on the FLT3 gene implies that current strategies targeting FLT3/ITD protein or activity would be ineffective. In this regard, disrupting the FLT3 gene, for instance, by using a gene-editing strategy, would represent an additional approach to eliminate HSCs containing FLT3/ITD mutation. Moreover, because FLT3/ITD+ AML restructures the marrow environment in favor of AML cells over normal HSCs, factors provided by FLT3/ITD+ AML cells that influence the marrow environment would represent a novel therapeutic target.

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