A Novel Mechanism for Imatinib Mesylate (STI571) Resistance in CML Cells: Contribution of TC-PTP to Modulating Signals Down-Stream from the BCR-ABL Fusion Protein.
Authors: Shimizu Takatsune , Miyakawa Yoshitaka , Iwata Satoshi , Kuribara Akiko , Tiganis Tony , Morimoto Chikao , Ikeda Yasuo , Kizaki Masahiro 
Published: October 2019

Acquired resistance to imatinib mesylate (STI571) in chronic myelogenous leukemia (CML) patients has become a serious problem. To adress the novel molecular mechanism for imatinib-resistance in CML, we previously established imatinib-resistant sublines (designated KTR cells) from the CML cell line KT-1. We have analyzed p-glycoprotein expression, the number of bcr-abl fusion gene and the sequence of ATP binding site of ABL kinase domain. However, these were not responsible for imatinib-resistance in KTR cells. Interestingly, T-cell protein tyrosine phosphatase (TC-PTP) protein levels were markedly down-regulated in all KTR cells as compared to parental KT-1 cells. Therefore, we examined whether the suppression of TC-PTP expression might contribute to imatinib-resistance in KTR cells. We transduced the nuclear isoform of TC-PTP (TC45) and catalytically inactive TC45-D182A cDNA into KTR cells by retroviral gene transfer. Subsequently, we analyzed the sensitivity to imatinib by MTT proliferation assays. We also studied the signaling pathways in all transduced cells by Western blottings. KTR cells successfully expressed TC45 and TC45-D182A protein (designated KTR-TC45 and KTR-D182A cells, respectively). In MTT proliferation assays, the proliferation of KTR-TC45 cells restored their sensitivity to imatinib, but not in KTR-mock or KTR-D182A cells, indicating that transduced catalytically active TC45 restored the sensitivity to imatinib in KTR cells. In KTR2-mock cells, the percentage of annexin V positive apoptotic cells was 8% in the control and was increased to 25% upon imatinib treatment. In KTR-TC45 cells, the percentage of apoptotic cells was increased from 12% to 56% by the treatment with imatinib, suggesting that TC45 expression in KTR cells restored the susceptibility to apoptosis by imatinib mesylate. Taken together, these results indicate that the sensitivity to imatinib in KTR cells can be modulated by TC-PTP expression. In parental KT-1 cells, phosphorylation of STAT5 was abolished with the treatment of 0.5 μM imatinib for 1 hour. In contrast, STAT5 phosphorylation in KTR cells was stronger than that of KT-1 cells and only slightly suppressed upon exposure to 0.5 μM imatinib. In KTR-mock and KTR-D182A transduced cells, STAT5 phosphorylation was augmented compared to KTR-TC45 transduced cells. Upon treatment with 0.5 μM imatinib for 1 hour, phosphorylation of STAT5 was abolished in KTR2-TC45 cells whereas it remained elevated in KTR-mock and KTR-D182A cells. The expression of TC-PTP had no effect on the phosphorylation of the JAK2 or BCR-ABL in KTR cells. Besides, expression of TC-PTP did not alter protein kinase PKB/AKT or mitogen-activated protein kinase signaling in KTR cells. These results indicate that the loss of TC-PTP could enhance tyrosine phosphorylation of STAT5 and was involved in the acquired resistance to imatinib in KTR cells. In conclusion, we demonstrated that reconstitution of TC-PTP in imatinib-resistant KTR cells restored the sensitivity to imatinib. Although it will be necessary to ascertain the relevance of our studies in primary samples, we would like to propose that the loss of TC-PTP may represent a novel mechanism by which CML cells can acquire imatinib-resistance.