Imatinib Treatment Induces Growth and Survival Promoting MAPK1/2 in Hematopoietic Cell Lines Expressing BCR-ABLT315I.
Authors: Haertel Nicolai , Klag Thomas , Hanfstein Benjamin , Mueller Martin C. , Hehlmann Ruediger , Hochhaus Andreas , La Rosee Paul 
Published: October 2019

The Abl-kinase-inhibitor imatinib is current standard treatment in chronic myelogenous leukemia (CML). Resistance to imatinib in most cases is governed by BCR-ABL mutations. Mutation T315I prevents binding of imatinib through steric hindrance. Patients with advanced Ph+ leukemia carrying T315I-mutant BCR-ABL rapidly progress on continuous imatinib therapy. The growth advantage of T315I-expressing cells may be solely explained by selection. However, the rapid onset of progression led us to ask, whether an additional imatinib-dependent mechanism may be accounted for this phenomenon. In imatinib responding cells, BCR-ABL-inhibition leads to effective blockade of BCR-ABL-depending singalling cascades such as the Ras/MAPK-pathway. However, paradoxical MAPK-activation in response to imatinib has been reported as a possible cytokine dependent resistance regulating factor. Using lymphoid hematopoietic cell lines (Baf3), we studied the effect of imatinib and the second generation inhibitor dasatinib on MAPK1/2 in interleukin 3 (IL3) dependent Baf3 cells. Baf3 cells transformed to IL3-independence by unmutated (p210) or mutant BCR-ABL (p210T315I) were treated with Abl-inhibitors in the presence or absence of additional IL3. Cellular BCR-ABL- and MAPK-activity were studied by Western blot analysis. Proliferation assays were used to study the functional significance of imatinib-induced signalling alterations in p210T315I -expressing cells. Imatinib at 5μM for 24hrs leads to significant MAPK1/2 inhibition in Bafp210wt cells in the absence of additional IL3 ([fold change vs untreated control] MAPK1/2: 0.07±0.06, p<0.05). In contrast, IL3 prevents imatinib from interfering with MAPK-signalling as observed in the presence of IL3 (MAPK1/2: 0.88±0.37, ns). Prevention of MAPK-inhibition correlates with rescue of Bafp210wt cells treated with imatinib in the presence of IL3 in viability count proliferation assays. Surprisingly, imatinib treatment in Bafp210T315I cells induced significant (p<0.05) and dose dependent activation of MAPK1/2 (1μM: 1.2±0.3; 5μM: 1.4±0.3) in the absence of IL3. This was not seen in cultures where IL3 was added. We further tested equipotent doses of the combined Src/Abl-inhibitor dasatinib in Bafp210T315I cells. Contrary to imatinib, dasatinib does not lead to MAPK-activation in Bafp210T315I cells (2.5nM: 0.9±0.8 n.s.; 12nM: 0.9±0.6 n.s.). Asking whether imatinib-induced MAPK-activation in p210T315I expressing cells would translate into a growth or survival advantage of these cells, proliferation studies with imatinib alone or combined chemotherapy were performed. Whereas imatinib alone did not consistently modulate cell growth, combined treatment studies with imatinib (5μM) and increasing concentrations of the chemotherapeutic agent decitabine demonstrated a slight but consistent growth advantage of combined vs decitabine only treated cells.

Conclusions: MAPK-activation in imatinib-treated p210T315I -expressing cells indicates off-target activity of imatinib leading to activation of growth and survival promoting signalling intermediates. The combined Src/Abl-inhibitor dasatinib differs from imatinib in that it does not alter cellular MAPK-activity in Baf p210T315I cells. Different target profiles may explain this differential inhibitor effect. Antagonistic antiproliferative activity of imatinib/decitabine points to a possible functional link of imatinib-induced MAPK-activation leading to increased survival of imatinib-resistant cells treated with imatinib.