Different Roles of Two Autotaxin Isoforms in Proliferation, Migration and Adhesion in the Non-Mutational Tyrosine Kinase Inhibitor Resistant Acute Lymphoblastic Leukemia Cell Line SupB15.

The Bcr-Abl oncogene is present in 30–40% of adult patients with acute lymphoblastic leukemia (ALL). The Abl kinase inhibitor imatinib-based therapy has become standard for this subset ALL. Acquired resistance to imatinib occurs frequently and is associated with mutations in the tyrosine kinase domain (TKD) approximately in about 80% of patients. In contrast, TKD mutations are uncommon in primary imatinib resistance which appears to be multifactorial, although the underlying mechanisms have been incompletely elucidated.

We have established a Ph+ cell line for the analysis of non-mutational resistance mechanisms of imatinib resistance: SupB15_RT, a Bcr-Abl expressing lymphoblastic cell line derived from SupB15_WT cell line by gradually increasing the exposure to imatinib. SupB15_RT shows cross-resistance to the second generation Abl kinase inhibitors Nilotinib and Dasatinib. We have shown that several commonly implicated mechanisms of imatinib resistance do not play a role in conferring the imatinib resistance in SupB15_RT cells. By comparative gene expression analysis of SupB15_WT vs. SupB15_RT cells using Affymetrix- Microarrays, we identified 29 differentially regulated genes. Autotaxin (ATX) is one of the most highly up-regulated genes in imatinib resistant SupB15_RT cells, and suggested a contribution to imatinib resistance.

ATX is an exo-enzyme (pyrophosphophatase/phosphodiesterase). It plays a role in tumor progression and migration as a tumor cell autocrine motilty factor in various solid tumor cell types. ATX is involved in the synthesis of the signaling molecule, lysophosphatidic acid (LPA) which promotes survival and motility. It was the aim of this study to determine whereas ATX plays a functional role for imatinib resistance in Ph+ ALL.

Using RT-PCR we demonstrated that 2 isoforms of ATX are expressed in SupB15_RT cells: ATX_short and ATX_long. ATX_long (863 aa) contains highly basic insertion in the catalytic domain (52 residues). We retroviraly transfected BaF3 cells with p185 and/or ATX_short or ATX_long to analyze its influence on growth, adhesion and migration in mouse cell model. In comparison to wild type BaF3 cells the proliferation of BaF3 cells expressing ATX_short is enhanced (1,5-fold), whereas ATX_long expressing BaF3 cells showed no difference in proliferation in comparison to Mock infected cells. The proliferation of p185 expressing BaF3 cells co-expressing ATX_short or ATX_long is not inhibited by the treatment with 1μM imatinib after 3 days in contrast to p185 expressing BaF3 cells. In adhesion experiments, BaF3 cells expressing ATX_short showed a higher attachment independent of p185 expression. We also performed migration experiments using transwell assays. These assays showed more migration with cells co-expressing p185 and ATX_long compared to p185 alone. This is in agreement with our results for SupB15_RT vs. SupB15_WT with a 3-fold migration increase of SupB15_RT. Application of 10% fetal calf serum (FCS) in migration experiments resulted in a 1,5-fold higher migration of the ATX_long expressing BaF3 cells compared to culture without FCS. One explanation for this finding may be that FCS contains lysophosphatidic choline (LPC) which is converted to LPA by ATX.

Although expression of both 2 isoforms of ATX is important for the increased proliferation, it seems that the 2 isoforms have different cellular functions in Ph+ lymphoblastic cells. ATX_short seems to enhance adhesion whereas ATX_long plays an important role in motility. Taken together our results indicate a role for ATX in TK- inhibitor resistant SupB15_RT cells through LPA signaling via LPA receptors. The ratio between ATX_short and ATX_long probably is important for the intracellular signaling and has to be explored.