Hypoxia is one of the essential components of the leukemia bone marrow (BM) microenvironment that promotes leukemia cell homing, survival and chemoresistance (Benito, PlosOne 2011). Tyrosine kinase inhibitors (TKIs) do not eradicate the total mass of chronic myeloid leukemia (CML) cells including primitive and quiescent cells. Persistence of CML cells in the hypoxic BM niche after cessation of TKI therapy may result in disease relapse. We have reported that hypoxia adapted CML cells acquire TKI resistance associated with higher glyoxalase-1 (Glo-1) enzyme activity which detoxifies methylglyoxal, a cytotoxic by-product of glycolysis (Takeuchi, Cell Death Differ 2010).
In this study, we employed a proteomic approach based on isobaric tags for relative and absolute quantification (iTRAQ, Applied Biosystems) to investigate additional molecular mechanisms of CML adaptation to hypoxia and acquired resistance to dasatinib under hypoxic conditions. The specific pathway alterations were identified by KEGG(Kyoto University) and MetaCore (GeneGo). We utilized two hypoxia-adapted (HA) subclones of CML cell lines, KCL22-HA and KBM5-HA cells, which were selected over a month under 1.0 % oxygen culture conditions. The growth rate of both HA-CML cell lines was slower than that of the corresponding parental cells (ratio of incremental increase in cell numbers, 0.39 for KCL22-HA/KCL22, 0.54 for KBM5-HA/KBM5 at 48hrs). Although parental KCL22 cells were sensitive to dasatinib, KCL22-HA cultured under hypoxia acquired resistance to dasatinib (IC50: KCL22 0.1 nM, KCL22-HA >20nM, at 48hrs by MTT). In contrast, dasatinib induced more prominent cell growth inhibition in KBM5-HA cells cultured under hypoxia compared to KBM5 parental cells (IC50: KBM5 1.3 nM, KBM-5/HA 0.3 nM). Dasatinib effectively downregulated the phosphorylation levels of Stat5 and ERK in parental KCL22 and KBM5 cells. Notably, the baseline levels of p-Stat5 and p-ERK were markedly diminished in both KCL22-HA and KBM5-HA cells. We next performed iTRAQ proteomic analysis and detected more than 1,300 proteins in each cell type. Comparison of the basal proteome of KCL22 vs KCL22-HA and KBM5 vs KBM5-HA cells showed differential expression of 54 proteins in KCL22 isogenic cells (37 upregulated, 17 downregulated) and of 159 proteins in KBM5 cells (56 upregulated, 103 downregulated). These alterations included consistent activation of glycolysis and gluconeogenesis pathway (p<0.0001) along with increased ATP synthase (p=0.02) and hypoxia induced HIF1 activation (p<0.0001) in both cells adapted to grow under hypoxia. In dasatinib-resistant KCL-HA cells, these changes were partially reversed by dasatinib which affected expression of 19 proteins (5 up-regulated / 14 down-regulated) with significant decrease in oxidative phosphorylation (p=0.01), ATP synthase (p=0.02), spliceosome (p=0.02) and Cytochrome C (p=0.04). On the contrary, in dasatinib-sensitive KBM5-HA cells dasatinib upregulated 296 proteins including 39 apoptogenic proteins such as Cytochrome C (p<0.001) and apoptosis related mitochondrial chaperones HSP10 and HSP60 (p<0.001), along with further upregulation of the enzymes involved in the glycolytic energy production (p<0.001) and stimulation of nuclear mRNA splicing via spliceosome (p<0.001), known to play a crucial role in the control of gene expression of the apoptosis-regulating genes. We observed the consistent upregulation of glycolysis and increases of ribosome and spliceosome after dasatinib treatment in dasatinib-sensitive parental KCL22 and KBM5 cells. Interestingly, dasatinib-resistant KBM5-STI cells harboring T315 Bcr-Abl mutation exhibited significantly lower levels of baseline gluconeogenesis (p<0.0001) and translation regulation of translation initiation pathways (p<0.0001) compared to the parental KBM5 cells.
In summary, these findings demonstrate the dramatic metabolic changes in hypoxia adjusted CML cells and indicate that hypoxia adapted / dasatinib resistant CML cells repress their stimulated glycolic metabolic state and oxidative phosphorylation along with spliceosomal mRNA splicing after dasatinib exposure, which allow them to enter quiescent stage and escape from apoptosis. Altogether these data indicate novel mechanisms of acquired bone marrow microenvironment-mediated resistance to TKI in CML.
Tabe:Bristol-Myers Squibb: Research Funding.
Asterisk with author names denotes non-ASH members.