Bone Marrow Stromal Cells Modulate Sensitivity of CXCR4-Deficient CML Cells to Pharmacological Inhibitors of PI3K/AKT Signaling.
Authors: Jin Linhua , Tabe Yoko , Tanaka Nobuko , Xu Yuanyuan , Kato Atsushi , Kantarjian Hagop , Andreeff Michael , Konopleva Marina 
Published: April 2020

Abstract
Chronic myelogenous leukemia (CML) is driven by constitutively activated BCR-ABL tyrosine kinase that reportedly activates downstream PI3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) signaling. The inhibitory role of p210BCR-ABL on stromal adhesion is speculated to cause abnormal circulation and proliferation of CML progenitors. Recently, it has been reported that p210BCR-ABL expression downregulates membrane expression of the CXC chemokine receptor 4 (CXCR4) which may be associated with migration defects in CML, and that tyrosine kinase inhibitor Imatinib restores CXCR4 expression in CML blast crisis cells (Cancer Res2005;65:2676). The goal of this study was to elucidate the functional roles of CXCR4, PI3K/Akt and MAPK/ERK signaling on proliferation and apoptosis of Imatinib-sensitive (IS) and Imatinib-resistant (IR) CML cell lines in the co-culture system with human mesenchymal stem cells (MSC) mimicking the bone marrow microenvironment. Imatinib(0.2μM) inhibited Akt phosphorylation and significantly increased CXCR4 membrane expression in IS KBM-5 cells co-cultured with MSC (p=0.02), which resulted in a more pronounced G1 cell cycle arrest in co-culture system. Similarly, PI3K inhibition (LY294002, 20μM) was significantly more effective in its ability to induce apoptosis and G1 cell cycle arrest in KBM-5 cells co-cultivated with MSC in direct cell-to-cell contact. MSC co-culture by itself diminished AKT phosphorylation but did not affect ERK phosphorylation, and did not modulate sensitivity to the specific MEK inhibitor PD98059(40μM). Additive apoptosis induction was observed by combination therapy of LY294002/Imatinib and PD98059/Imatinib in stromal co-culture system. In contrast, Imatinib failed to suppress pAKT in IR KBM5-R cells, and did not affect the constitutively low CXCR4 levels. MSC co-culture of KBM5-R cells enhanced constitutively activated Akt and ERK signaling in these cells. This translated into increased apoptosis induction by both, LY294002 (p=0.008) and PD98059 (p=0.01) in stromal co-cultures, with no further growth inhibition when combined with Imatinib. In summary, these results suggest that Imatinib modulates CXCR4 expression in sensitive CML cells which may enhance their homing to stroma and contribute to the inhibition of CML proliferation. Of importance, we observed that Imatinib treatment of CML patients in vivo caused transient abrupt upregulation of CXCR4 expression on CD34+CD38- cells from 4 CML blast crisis patients, which correlated with a dramatic drop in their WBC and % of peripheral blasts. In contrast, IR cells are deficient in their interactions with stroma, however they may respond to stroma-secreted cytokines via activation of PI3K/AKT and MEK/ERK signaling. Notably, both IS and IR CML cells are highly sensitive to pharmacological inhibitors of the PI3K/AKT pathway when co-cultured with stromal cells indicating that CML cells within bone marrow microenvironment are critically dependent on PI3K/Akt activation for their survival. These results provide rationale for the use of pharmacological inhibitors of PI3K/AKT signaling in IR CML cells, and combination strategies in IS CML.