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chemoresistant murine leukemia cells (Trendowski et al., 2015). At high concentration of 1µg/ml, CYD alone was sufficient to induce apoptosis of K562 cells whereas at lower concentration of 0.3µg/ml, there was no effect on cell death. Interestingly, when combined with IM, CYD at non-toxic concentration of 0.3µg/ml resulted in increased apoptosis of adherent K562 cells. Actin is regulated in cells by Rho GTPase through ROCK (Amano et al., 2010; Sit and Manser, 2011) and addition of ROCK inhibitor Y27632, similar to CYD reduced adhesion of K562 to stromal cells. Dominant negative RHOA expression also significantly reduced K562 adhesion to stromal cells confirming the role of actin cytoskeleton in cell-cell interaction of CML cells with the stromal cells.
As proposed by others, that mobilization of persistent LSC using alternative methods might render them more susceptible to chemotherapeutic agents (Buss et al., 2011;
Trumpp et al., 2010), actin cytoskeleton inhibitors at low concentration might be combined with the treatment regime to obtain better outcomes.
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VLA-4/ VCAM-1 interaction played an important role in chemoresistance against standard chemotherapeutic agents (Jacamo et al., 2014). Similarly, inhibition of Etoposide induced NFκB activation by treatment with NFκB inhibitor BAY 11-7082, rendered K562 cells more sensitive to Etoposide induced apoptosis (Morotti et al., 2006).
On the other hand, Schmidt et al reported that CML cells activated NFκB pathway in stromal cells via VLA-4/VCAM-1 interaction, which resulted in higher secretion of proliferative cytokines by the stromal cells (Schmidt et al., 2011). Based on these reports we expected that NFκB inhibition in our experimental set up might be effective in abrogating chemoprotective effect of the stromal cells. However, addition of BAY 11- 7082 did not have any additive effect on IM induced apoptosis of control or stroma adherent K562 cells. Furthermore, BAY 11-7082 treatment did not inhibit the adhesion of K562 cells to the stromal layer. This seeming contradiction might be because, NFκB signaling might chemoprotect against standard chemotherapeutic drugs in ALL cells but not TKI induced apoptosis in CML cells which is independent of NFκB signaling.
Moreover, unlike Etoposide, IM treatment did not result in NFκB activation in CML cells.
A potential role of ERK1/2 MAPK pathway in chemoresistance of CML was highlighted by other studies as well. Increased pERK1/2 levels were reported in CD34+ CML cells upon IM treatment (Chu et al., 2004). Similarly, induction of FGF2 mediated chemoresistance in K562 cells against IM was also associated with high levels of pERK1/2 (Traer et al., 2014). Furthermore, PlGF secreted by stromal cells activated ERK1/2 signaling in CML cells, which lead to CML disease progression (Schmidt et al., 2011). Besides, BCR-ABL fusion protein is also responsible for activation of ERK1/2 MAPK (Sattler et al., 2002). As mentioned earlier, in our study, IM treatment downregulated phospho ERK1/2 levels in control cells whereas in stroma adherent K562 cells, there was a significant increase in pERK1/2 levels. ERK inhibition by treating the cells with U0126 significantly increased the sensitivity of AD-K562 cells to IM treatment. This coincides with the assertion by other studies that ERK1/2 signaling pathway might be a key mediator in CML chemoresistance to IM and inhibition of ERK signaling might have an important effect in preventing disease progression in CML.
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Another signaling pathway that was active in CML cells and was found to be of importance in chemoresistance was BMP/SMAD signaling. Goldman et al reported that BMP signaling was important in embryonic hematopoiesis and for HSC maintenance in adult BM (Goldman et al., 2009). Higher levels of BMP2 and BMP4 in CML BM microenvironment were reported to be responsible for maintenance and expansion of LSC and myeloid progenitors (Laperrousaz et al., 2013). BMP-SMAD signaling are BCR-ABL independent as pSMAD1/8 levels were unaffected by IM treatment in CML cells. BMP signaling inhibitor LDN193189 was used to inhibit BMP-SMAD signaling in the CML cells. LDN193189 treatment reduced adhesion of K562 cells to the stromal layer in a dose dependent manner. To further confirm the role of BMP signaling in CML cell adhesion to the stromal cells, K562 cells were transduced with BMP4 and BMP2 genes. BMP2 and BMP4 transduced K562 cells showed significantly higher adhesion to the stromal layer. BMP inhibitor LDN193189 treatment increased the susceptibility of control K562 cells to IM induced cell death. Interestingly, addition of LDN193189 to AD-K562 cells made them significantly more susceptible to IM treatment confirming the important role of BMP-SMAD signaling in stroma mediated chemoresistance.
Thus, our study has shown that actin cytoskeleton, RhoA, ERK1/2 MAPK and BMP- SMAD signaling were involved in chemoprotection of CML cells through interaction with stromal cells. We propose that a combinatorial approach might be a more effective strategy in eradicating chemoresistant CML cells that persist long after IM treatment. To test this in our study, we used different combinations of inhibitors in addition to IM to induce cell death in AD-K562 cells. We found that several combinations of inhibitors such as actin inhibition with BMP inhibition (CYD+LDN193189), ERK inhibition with BMP inhibition (U0126+LDN193189) and ROCK inhibition with BMP inhibition (Y27632/LDN193189) in the presence of IM had maximum effect in inducing cell death in the stroma adherent K562 cells. Although single pathway inhibitors had an additive effect on apoptosis induction by IM treatment, we found that combinatorial approach was more effective.
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