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30 Physicochemical properties
Soluble calcium ion
HSC lacking the calcium receptor have hypocellular BM with reduced number of HSC. They fail to lodge near endosteum upon transplantation Low blood
perfusion, hypoxia
Dormant serially transplantable HSC reside in poorly perfused hypoxic niches and the endosteal niches are hypoxic
Table 1.3. Bone marrow niche factors. From (Levesque et al., 2010)
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31 12-CXCR4 signaling axis for homing and migration (Tabe and Konopleva, 2014).
However, pathways related to survival and proliferation are abnormally activated in LSC (Tabe and Konopleva, 2014)
IM has been successfully used for treatment of a majority of CP-CML patients.
However, several patients develop resistance to IM even after few years of remission, especially after discontinuation of IM intake, suggesting that a subpopulation of leukemic cells evade chemotherapeutic effects of IM and persist in the BM. It is proposed that the interactions between leukemic cells and the BM stromal cells play a crucial role in the development chemoresistance. Therefore, role of BM stromal cells and the mechanisms involved in the persistence of leukemic cells against IM and other TKIs need to be studied in detail.
Cytokines secreted by MSC, osteoblasts and other stromal components have been implicated in CML cell chemoprotection against IM and other TKIs. BM stromal cell conditioned media (CM) as well as addition of individual cytokines IL-6, IL-8, IL-11, macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony- stimulating factor (GM-CSF) and SDF-1 increased proliferation of CML cells and partially protected CML cells from the inhibitory effect of IM. The combination of these cytokines was more effective than individual cytokines indicating a cumulative role (Weisberg et al., 2008). MSC co-cultured with CML cells in a transwell system without direct physical contact had higher gene expression of IL-17 signaling related inflammatory cytokines such as IL-8, IL-1B and CCL2. Similar changes were observed when MSC were co-cultured in physical contact with CML cells (Civini et al., 2013). CM media from stromal cell line HS-5, protected K562 cells from IM, Nilotinib (NI) and Dasatinib (DS) induced cell death. Stromal CM increased the phosphorylated levels STAT3 in CML cells which was independent of BCR-ABL activity. K562 cells had increased protein levels of anti-apoptotic regulators BCL-xl, MCL-1 and Survivin when cultured in stromal CM and treated with IM (Bewry et al., 2008). In a recent study, a stroma secreted cytokine FGF2 was reported to protect K562 cells from IM induced cell death and confer IM resistance to K562 cells in long term culture. The IM resistance in K562 cells in presence of FGF2 could be abrogated by inhibition of FGFR and ABL
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32 tyrosine kinase activity in K562 cells. FGFR signaling by FGF2 restored Erk1/2 activity in long term K562 cultures in presence of IM which could have played an important role in IM resistance (Traer et al., 2014). Inhibition of stroma secreted placental growth factor (PlGF) slowed the progression of CML without affecting the disease characteristics or homing of the leukemic cells. Leukemia-stromal interaction by VLA-4-VCAM-1 binding upregulated NFkB signaling in the stromal cells resulting in high PlGF expression by the stromal cells. PlGF did not affect STAT5 phosphorylation which is a downstream target of BR-ABL signaling but increased phosphorylation levels of ERK1/2 (Schmidt et al., 2011).CXCR4 is involved in the migration of HSC towards the stromal cell in the BM.
CXCL-12 induces migration of leukemic cells in vitro (Zeng et al., 2006)which can be effectively inhibited by CXCR4 inhibitor plerixafor (AMD3100). AMD3100 reduced the adhesion of CML cells to fibronectin, endothelial cells and MSC (Weisberg et al., 2012).
CD34+ cells from CML-BP patients had lower membrane bound expression of CXCR4 compared to CD34+ cells form CP-CML patients or healthy individuals. BCR-ABL expression was reported to downregulate membrane bound CXCR-4 expression in BCR- ABL transduced MO7e cell line in a dose dependent manner (Geay et al., 2005).
Inhibition of BCR-ABL by IM increased CXCR4 expression in presence of MSC which might facilitate the migration of CML cell towards BM stromal cells. IM treatment increased the migration of CML cell towards stromal layer in a transwell assay which was abrogated by CXCR4 antagonist (Jin et al., 2008). CXCR4 antagonist AMD3100 reverted the chemoprotective effect of stromal cells on CML cells treated with TKIs (Dillmann et al., 2009; Weisberg et al., 2012). It was hypothesized that post IM treatment CML cells migrate towards the chemoprotective environment of the stromal cells which express copious amounts CXCL-12. And hence a combinatorial therapy using CXCR4 antagonist along with IM was proposed (Beider et al., 2014; Jin et al., 2008).
Physical interaction of CML cells with stromal microenvironment depends on adhesion molecules such as VLA-5, CD44, N-Cad. CD44 adhesion molecule which binds to E- selectin expressed on endothelial cells in the BM, was reported to be highly expressed in CML stem and progenitor cells. BCR-ABL+ CML cells lacking CD44 expression had reduced homing and engraftment in mouse model (Krause et al., 2006). Similarly lack of E-selectin in the recipient BM led to reduced engraftment of BCR-ABL+ cells (Krause et
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33 al., 2014). TKI treatment along with CD44 monoclonal antibody targeting significantly reduced CML-LSC in vivo in mouse model (Hellqvist et al., 2013).
Adhesion of CML cells to fibronectin via VLA-5 was implicated in adhesion dependent chemoprotection of CML cells, suggesting the role of ECM in CML chemoresistance (Damiano et al., 2001). CML-stromal interaction via VLA-4/VCAM-1 binding resulted in NFkB activation in stromal cells which led to expression of inflammatory cytokines by stromal cells (Schmidt et al., 2011).
CML and stromal cells also interact with each other via N-Cad which is expressed by CML as well as stromal cells. N-Cad expression in primary CML cells increased post IM treatment in presence of MSC. MSC adherent CD34+ CML cells had higher levels cytoplasmic N-Cad/β-catenin complex as well as nuclear β-catenin levels. Inhibition of Wnt signaling by DKK1 increased apoptosis in CML cells in co-culture with MSC (Zhang et al., 2013). However it was reported later that Wnt signaling is important for intrinsic TKI chemoresistance in CML cells but not for stroma mediated extrinsic chemoresistance (Eiring et al., 2015).
Thus, understanding the signaling mechanisms involved in the development of chemoresistance in CML, especially, the role of closely interacting bone marrow microenvironment will help in developing better therapeutic strategies to complete eliminate the leukemic cells.