Analysis of Dasatinib-Induced Molecular Mechanisms of Apoptosis in Hypoxia-Adopted CML Cells Utilizing Quantitative Proteomics Technology.
Authors: Tabe Yoko , Jin Linhua , Iwanami Hiroko , Kazuno Saiko , Fujimura Tsutomu , Matsushita Hiromichi , Ueno Takashi , Miida Takashi , Andreeff Michael , Konopleva Marina , Kimura Shinya 
Published: October 2019

Abstract
Abstract 2773
Development of the second-generation ABL tyrosine kinase inhibitor (TKI) dasatinib in CML aimed at overcoming resistance to imatinib, to eliminate persistent residual disease and thus prevent recurrence of active leukemia after TKI discontinuation in chronic myeloid leukemia (CML). Hypoxia has recently been reported as an essential component of the leukemia BM microenvironment that promotes leukemia cell homing, survival and chemoresistance (Benito et al, PlosOne 2011). In this study, we investigated the anti-CML efficacy and molecular mechanisms of action of dasatinib under hypoxic conditions.
We developed a hypoxia-adopted subclone of the KBM5 CML cell line (KMB5-HA), which was selected under 1.0% oxygen conditions, and an imatinib-resistant KBM5 subline bearing the T315I mutation (KBM5-T315I). KBM5-HA cells cultured under hypoxia accumulated in G0/G1 and exhibited moderate spontaneous apoptosis compared to KBM5 parental cells in normoxia (sub G1 %: KBM5 3.8±0.8, KBM5-HA 8.7±0.9; p=0.01; proportion of cells in G0/G1: KBM5 36.5±1.1%, KBM5-HA 52.4±6.6%; p=0.02, PI analysis). These cells displayed higher sensitivity to dasatinib than parental KBM cells (IC50; 1.3 nM for KBM5, 0.3 nM for KBM5-HA, at 48hrs by MTT). Low-dose dasatinib (0.5nM) which failed to cause inhibition of proliferation in parental KBM5 cells, caused significant apoptosis induction with cell cycle arrest in KBM-HA cells (sub G1 %: control 8.7±0.9, datatinib 49.1±16.9; p=0.02, G0/G1 %: control 52.4±6.4%, dasatinib 74.1±3.5%; p=0.01). In KBM5-T315I cells, dasatinib induced more prominent cell growth inhibition under hypoxia compared to normoxia (IC50 at 48hrs: normoxia 16.2 nM, hypoxia 3.7 nM). Treatment with 5nM dasatinib, which did not affect KBM5-T315I cell growth under normoxia, induced significant apoptotic effects under hypoxia (sub G1 %: control 6.9±0.9, dasatinib 20.3±3.3; p=0.05, G0/G1 %: control 57.3±5.8, dasatinib 67.4±8.9; p=0.41).
We next investigated dasatinib-induced changes of Stat-5 and ERK activation in CML cells by immunoblotting Treatment with 0.5 nM dasatinib resulted in marked down-regulation of phosphorylated (p-) Stat-5 and p-ERK in both, KBM5 and KBM5-T315I cells. In KBM5-HA cells, however, no baseline expression of p-Stat-5 or p-ERK was detected. These results suggest that in KBM5-HA cells dasatinib induces apoptosis and cell cycle arrest via Stat-5 or ERK-independent pathways. To investigate the candidate signaling factors responsible for dasatinib effects on KBM-HA, we performed the proteomic analysis utilizing proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ, Applied Biosystems) coupled with two-dimensional-liquid chromatography-tandem mass spectrometry. A total of 1,234 proteins were detected, and 296 proteins were found to be significantly up-regulated in response to dasatinib treatment in KBM5-HA cells. Among the up-regulated proteins, we found 39 proteins involved in apoptosis induction including Cytochrome C and Cytochrome C oxydase subunits (p<0.001). Mass spectrometry further revealed the up-regulation of apoptosis related mitochondrial chaperones HSP10 and HSP60 (p<0.001), suggesting a role of HSP60/HSP10 in mitochondrial membrane permeabilization.
In summary, these findings unravel novel mechanisms of action of dasatinib under conditions mimicking the hypoxic BM microenvironment via induction of Cytochrome C oxydase and mitochondrial chaperones HSP10 and HSP60, which lead to loss of mitochondrial transmembrane potential and release of apoptogenic Cytochrome C in CML cells.

Disclosures:
Tabe: Bristol-Myers Squibb: Research Funding.