De Novo ABL Kinase Domain Mutations in Murine Bone Marrow Cells Retrovirally Transduced with Wild-Type p210 BCR-ABL and Amplified in Culture with Imatinib Mesylate.
Authors: Flamant Stephane , Guillier Martine , Bonnet Marie-Laure , Lecouedic Jean-Pierre , Vainchenker William , Turhan Ali G. 
Published: October 2019

Imatinib mesylate (IM) is a tyrosine kinase inhibitor which is highly efficient in chronic myelogenous leukemia (CML), especially in the firts chronic phase of the disease. Recent data showed, however, that resistance to IM can develop in patients in more aggressive phases of their disease, which mainly occurs through mutations within the ABL kinase domain that interfere with IM binding, leading to IM-resistant relapses. The mechanisms of the occurrence of ABL kinase domain mutations in patients on IM therapy are not well understood, and in some of them, a mutation pre-existing to the introduction of IM was described, suggesting the possibility of a clonal selection under IM therapy.

To determine if ABL kinase domain mutations could be induced de novo in primary marrow cells, we used an ecotropic BCR-ABL retrovirus (MIGR-p210 vector, 5.105 viral particles / ml) and infected 5-FU-treated bone marrow cells from C57BL/6 mice. Retrovirally transduced cells (30% GFP+) were transplanted in lethally irradiated animals in which they induced lethal leukemia in 3 weeks. Both BCR-ABL-transduced and control C57BL/6 bone marrow cells were seeded in liquid cultures (104 cells/well) in the presence of 0.25 μM IM with weekly half medium changes during which the concentration of IM was increased gradually from 0.25 to 2 μM over 2 months. In these conditions, no growth could be obtained from normal bone marrow cells (0/192 wells) whereas in 10/192 wells containing BCR-ABL-transduced cells, we observed significant growth on IM. These cells were then amplified in the presence of murine stromal MS-5 cells and 2 μM IM for over 6 months and 2 clones (C3 and C10) exhibiting persistent growth were further characterized. At cytological analysis both cell lines had a typical mast cell morphology. Flow cytomery analyses demonsrated the presence of CD41 marker on both cell lines, with absence of myeloid (Gr1), erythroid (Ter119) and B-cell (B220) markers. Cells were not polyploid and interestingly, they exhibited higher growth rates in the presence of IM, with reduced growth upon IM deprivation. Both cell lines had evidence of BCR-ABL vector integration by PCR analysis and were highly GFP+. To explore the mechanisms of IM-resistance in these cells, we extracted high molecular weight genomic DNA and amplified a BCR-ABL fragment of 1236 bp encompassing the ABL kinase domain of the integrated construct. We then sequenced the ABL kinase domain using internal primers in both 5′-3′ directions. Plasmid DNA from the original MIGR-p210 BCR-ABL vector served as control. In both clones (C3 and C10) ABL kinase point mutations were readily detectable which were not found in the BCR-ABL retroviral vector. C3 carried two mutations interesting the C helix (E300K) and the SH2 contact region (E371K) of ABL kinase domain, whereas C10 carried a single mutation in the C helix (D295N). These mutations were previously detected in a random in vitro mutagenesis assay of BCR-ABL in bacterial systems. Thus, our model is the first demonstration of the occurrence of ABL kinase domain mutations and the concomittant generation of an IM-resistant phenotype in primary marrow stem cells transduced with BCR-ABL vector DNA and selected in the presence of IM. The mechanisms of the occurrence of these mutations in vitro are currently under study but these results suggest that de novo ABL kinase mutations could also occur in vivo in CML patients treated with IM.