A Role of Autotaxin in Non-Mutational Resistance in Imatinib-Resistant Acute Lymphoblastic Leukemia (ALL) Cell Line SupB15_RT.

The Bcr-Abl oncogene is present in 30-40% of adult patients with acute lymphoblastic leukemia (ALL). Therapy with imatinib has become standard for Ph+ ALL but resistance to the tyrosine kinase inhibitor occurs for the majority of patients. In about 80% of patients with acquired resistance mutations in the tyrosine kinase domain (TKD) have been found. In contrast, primary resistance to imatinib appears to be multifactorial and precise mechanisms have been incompletely elucidated. We have established an imatinib-resistant cell line (SupB15_RT) which was derived from the previously well characterized SupB15 cell line (SupB15_WT) by gradually increasing the exposure to imatinib. We found that several commonly implicated mechanisms of imatinib resistance, i.e. Bcr-Abl gene amplification, point mutations in the TKD, Bcr-Abl overexpression, up-regulation of multidrug resistance gene proteins or ineffective inhibition of Bcr-Abl tyrosine phosphorylation do not play a role in conferring the imatinib-resistant phenotype in SupB15_RT cells. Thus, the SupB15_RT cells represent a suitable model for the analysis of resistance mechanisms in Ph+ ALL with primary imatinib resistance. Interestingly, SupB15_RT cells show cross-resistance to the second generation Abl kinase inhibitors Nilotinib and Dasatinib. Analysis of signal transduction pathways downstream of Bcr-Abl revealed that imatinib exposure was not associated with down-regulation of pSTAT-5 and pErk in the imatinib-resistant SupB15_RT cells, in contrast to SupB15_WT. Phosphorylation of Akt was inhibited by 0.5μM imatinib in SupB15_WT cells, whereas imatinib in concentrations up to 5μM failed to suppress Akt phosphorylation in SupB15_RT cells, indicating constitutive activation of Akt kinase during imatinib treatment. By comparative gene expression analysis of SupB15_WT vs. SupB15_RT cells using Affimetrix-Microarrays, we identified 29 differentially regulated (at least 3-fold) genes. One of the most highly up-regulated genes in imatinib-resistant SupB15_RT cells was Autotaxin (ATX), a nucleotide pyrophosphatase/ phosphodiesterase 2. This exo-enzyme was originally identified as a tumor cell autocrine motility factor, which is involved in tumor progression and migration in various tumor cell types. ATX is a lysophospholipase D which is involved in the synthesis of lysophosphatidic acid (LPA), a signaling molecule that promotes survival, growth, differentiation, and motility. We investigated if LPA imparted imatinib resistance in SupB15_WT cells by modulation of growth, survival and migration. When SupB15_WT cells were treated with LPA, alone or in combination with imatinib, SupB15_WT cell proliferation was increased both in the absence as well as in the presence of imatinib. The dose-dependent increase of proliferation after LPA treatment was 1.9–2.6-fold (1–10μM LPA) in the presence of 1μM imatinib. In addition we performed migration experiments using Transwell assays. We detected a 3-fold increase in migration of SupB15_RT vs. SupB15_WT cells. We found no influence on apoptosis in imatinib treated SupB15_WT cells treated with LPA compared with cells not treated with LPA. Taken together, our results indicate a role of ATX in imatinib-resistant SupB15_RT cells, preferentially by stimulating proliferation and migration through LPA signaling via LPA receptors and activation of PI3K and Akt.