Effects of Tyrosine Kinase and mTOR/PI3K Dual Inhibitors on Transcription Regulation in Chronic Myeloid Leukemia
Project no. 6 · Status: Kapanmış · 2015
Summary
Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by cells with the BCR-ABL1 fusion protein. The tyrosine kinase inhibitor (TKI) ponatinib can inhibit many BCR-ABL1 autophosphorylation, including T315I. Since the TKIs cannot suppress alternative active survival pathways and are not effective on leukemia stem cells, the efficacy of the treatment may be low. There is a need to develop multi-targeted treatment approaches for patients with CML who exhibit sub-optimal response to TKI treatment. VS-5584, an inhibitor of the PI3K/Akt/mTOR pathway known to be active in CML, is a potent dual inhibitor that preferably kills cancer stem cells. In this study, it was aimed to clarify the molecular processes in the treatment of CML by the simultaneous inhibition of BCR-ABL1 and PI3K/ mTOR with combinations of VS-5584 and ponatinib. Cytotoxic effects of ponatinib, VS-5584, and their combinations on CML cell model K562, leukemia stem cell model LSC, and normal hematopoietic stem cell model HSC were measured with WST-8 test. Combination indices were evaluated by CalcuSyn isobologram analysis. Apoptotic effects of determined doses were evaluated with Annexin V and active caspase-3 assays by using flow cytometry, effects on cell cycle were evaluated by using PI assay with flow cytometry. Changes in the activity of transcription factors were quantified by dual luciferase activity after transfection of appropriate vectors. qRT-PCR was performed to detect changes in mRNA levels of the target genes. In K562 and LSC cells ED50 doses were additive, ED75 showed synergistic effect. In addition to ponatinib-induced apoptosis, VS-5584 was shown to cause significant G0/G1 arrest. Suppression of Elk- 1/SRF, AP-1, NFkB, Myc/Max, CREB, STAT3 and E2F/DP1 and increment in C/EBP activity was also observed in leukemia cells after the combination treatment. Many genes whose expressions are regulated by these transcription factors have also been shown altered mRNA levels. The results showed that regulation of transcription factors involved in survival pathway with ponatinib and VS-5584 contributed to anti-proliferative and apoptotic processes. We believe that the combination of ponatinib and VS-5584 may be a promising treatment strategy that can selectively inhibit alternative survival pathways in CML cells.