The effects of the therapeutic agents ponatinib and VS-5584 on chronic myeloid leukemia leukemogenesis

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by cells with the BCR-ABL1 fusion protein, which exhibits continuous tyrosine kinase activity. Therefore, tyrosine kinase inhibitors are used in the treatment of CML. Ponatinib is an ATP-competitive tyrosine kinase inhibitor capable of inhibiting BCR-ABL1 autophosphorylation, including T315I-mutant. Leukemia stem cells are malignant cells with similar properties to normal hematopoietic stem cells. Since tyrosine kinase inhibitors are not effective on primitive leukemia stem cells, the efficacy of treatment may be low. In addition, low tyrosine kinase inhibitor doses that use to prevent severe side effects, do not suppress alternative survival pathways that are active in CML cells. It is clear that there is a need to develop multi-targeted treatment approaches instead of single drug ponatinib therapies for patients with CML who exhibit sub-optimal response to tyrosine kinase inhibitor treatment. VS-5584, an inhibitor of the PI3K/Akt/mTOR pathway known to be active in CML, is a potent, selective dual inhibitor that preferably kills cancer stem cells. In this thesis study, it was aimed to determine an in vitro combination therapy which could overcome the main causes of failure of CML therapy. For this purpose, the effects and roles of simultaneous inhibition of BCR-ABL1 and PI3K/Akt/mTOR pathways by combination of ponatinib and VS-5584 on elimination of CML and leukemic stem cells has been investigated at the molecular level. 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 IC50, IC75, ED50, ED75 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 and by cyclinD1, p27 antibodies with western blot. Changes in the activities of the PI3K/Akt/mTOR pathway proteins were measured by quantitation of the phosphoprotein levels with the antibody array. 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 of the altered transcription factors. Ponatinib and VS-5584 were found cytotoxic for K562 and LSC cells in time and dose dependent manner. Unlike ponatinib, VS-5584 was shown to be significantly less cytotoxic in the healthy HSC cell line. ED50 doses were additive, ED75 and ED90 showed synergistic effect. In addition to ponatinib-induced apoptosis, VS-5584 was shown to cause significant G0/G1 arrest. Especially in LSC cell line it was shown that while ponatinib was insufficient to suppress Akt pathway, combination of ponatinib and VS-5584 inhibited PI3K/Akt/mTOR pathway through inhibiting the phosphorylation of several proteins, particularly S6, S6K, BAD. It was found that the Elk-1/SRF, AP-1 and NFkB transcription factors displayed markedly elevated activity in leukemia and leukemia stem cell models. In addition to these transcription factors which may be a selective intervention point, suppression of Myc/Max, CREB, STAT3 and E2F/DP1 transcription factors was also observed in K562 and LSC cells after the combination treatment. It was determined that the activities of C/EBP and FOXO1 transcription factors in K562 cells and of C/EBP and p53 transcription factors in LSC cells increase following the application of the combination. 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 can be a promising treatment strategy that can selectively inhibit the uncontrolled proliferation of CML blasts and stem cells and reduce the known serious side effects of ponatinib. Keywords: Chronic Myeloid Leukemia; PI3K/Akt/mTOR pathway; Ponatinib