| Chronic myeloid leukemia (CML) arises from the neoplastic transformation of a hematopoietic stem cell, which is characterized by the Philadelphia chromosome. A P210BCR-ABL protein is generated by the BCR-ABL fusion gene, which is a constitutively active tyrosine kinase that drives cell proliferation and apoptosis through multiple downstream signal pathways. Since the BCR-ABL is considered essential for the disease progression, targeting the tyrosine kinase activity of BCR-ABL shows an attractive therapeutic strategy. The BCR-ABL kinase inhibitor imatinib, because of the safety and effectivity, has become the first line therapy of CML. But still some patients show primary or acquired resistant to imatinib therapy, the most frequently described mechanism of acquired resistance to imatinb is the occurrence of point mutations in the kinase domain of BCR-ABL. Patients who are intolerant or resistant to imatinib have the effective salvage therapy of the second line ABL kinase inhibitors nilotinib and dasatinib. Both of them target most mutants except the cross resistant BCR-ABLY3151mutation.Ponatinib, from the Ariad pharmacy company, is a multitargeted kinase inhibitor that can even inhibit BCR-ABLT3151, which is the third line therapy for patients with CML. BCR-ABL kinase domain mutation-mediated ponatinib resistance has been investigated in vitro. A pivotal phase II trial is underway, almost94%CML patients have previously failed at least2tyrosine kinase inhibitors, after ponatinib treatment half of the patients achieved major cytogenetic response, but there are still some patients resistant to ponatinib therapy.Here, we developed ponatinib resistant, BCR-ABL positive cell lines lacking a kinase domain mutation and investigated mechanisms of resistance, trying to possibly in advance of full mechanistic understanding in the clinic, to search and investigate new tyrosine kinase inhibitor, and to restore ponatinib resistant paitent’s response.Two BCR-ABL+cell lines,32D(BCR-ABL) and K562, were exposed to escalating concentrations of ponatinib to generate ponatinib-resistant cells. BCR-ABL kinase domain sequencing of resistant cells confirmed BCR-ABL to be unmutated. MTS cell proliferation assay of sensitive and resistant cells showed that ponatinib resistant cell also exhibited resistance to imatinib, nilotinib and dasatinib. In each of the ponatinib-resistant cell lines, BCR-ABL tyrosine kinase activity was pharmacologically silenced by low concentrations of ponatinib and yet the cells survived. All data indicated a BCR-ABL kinase independent mechanism of resistance.Based on western blot, tyrosine phosphorylation of the BCR-ABL downstream target STAT5was substantially maintained despite ponatinib-mediated inhibition of BCR-ABL1catalytic activity in32D (BCR-ABL)ponR cells. A small molecule inhibitors screen was applied to the32D(BCR-ABL)ponR resistant cell in the presence or absence of ponatinib to assess synthetic lethality. Only dual combinations of ponatinib and certain JAK inhibitors are lethal to resistant cell. Knocking down the BCR-ABL by shRNA mimicked the ponatinib effect, resistant cell exhibited single agent sensitivity to some JAK inhibitors, which suggested that JAK/STAT co-critical signaling pathway may be involved in this BCR-ABL independent mechanism of resistance. Sensitive and resistant cells were used for gene microarray analysis, and validated by the real time PCR. IL-3expression is up-regulated in resistant cell (p<0.01), and the IL-3neutralizing antibody could reduce the tolerance of resistant cell to ponatinib. Synthetic lethality screening identified MEK/ERK co-critical signaling pathway in K562ponR cells, only ponatinib synergizes with MEK/ERK inhibition to induce synthetic lethality in ponatinib resistant K562ponR cells.We analyzed one ponatinib treatment failure patient sample recently, only BCR-ABLT3151mutation was found in this patient and BCR-ABLT3151activity was inhibited ex vivo by low concentration of ponatinib. Synthetic lethality siRNA screening showed that combination of ponatinib and knockdown CSFlR drastically reduces CML cell viability, which suggested that CSFlR co-critical signaling pathway was involved in this BCR-ABL independent mechanism of resistance.Our findings indicate a BCR-ABL independent mechanism of ponatinib resistance, some relapsed CML patients may benefit from targeting both BCR-ABL and co-critical signaling pathways. |
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