Abstract
Introduction: Although ABL tyrosine kinase inhibitor (TKI), imatinib, nilotinib and dasatinib have demonstrated the potency against chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph) positive acute lymphoblastic leukemia (Ph+ALL) patients, resistance to ABL TKI can develop in the many patients. It has already reported that ABL kinase domain mutations have been implicated in the pathogenesis of ABL TKI resistance, however, it is fully not known the molecular mechanism of drug resistance ABL TKIs. Therefore, new approach against ABL TKI resistant cells may improve the outcome of Ph-positive leukemia patients. Interleukin-1 (IL-1) is a proinflammatory cytokine and central mediator of innate immunity. IL-1β also controls essential cell responses. Because enhanced IL-1β signaling is reported in patients of hematological malignancies, IL-1β may be the promising therapeutic value in ABL TKI resistant CML patients.
Materials and methods: In this study, we established ABL TKI-resistant in vitro cell line models (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R). We also investigated whether IL-1β was involved in ABL TKI resistant Ph-positive leukemia cells and cytokines were induced by IL-1β in human umbilical vein endothelial cells (HUVEC).
Results: We analyzed the relationship of IL-1β signaling pathways and ABL TKI sensitivity by microarray gene expression data from the online Gene Expression Omnibus (GEO). IL-1β is related to imatinib sensitivity and resistant in CML patients from the public microarray datasets of GSE14671. We next examined ABL TKI resistant cell lines (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R) in this study. BCR-ABL point mutation was not found in ABL TKI resistant cells. BCR-ABL expression levels were not increased in ABL TKI resistant K562 cells. These cells were highly resistant to ABL TKIs compare to K562 cells (K562 imatinib-R: imatinib 2μM, nilotinib-R: nilotinib 2μM, dasatinib-R: dasatinib 100nM, ponatinib-R: ponatinib 50nM). We investigated gene expression profiles in cultured ABL TKI resistant K562 cells by DNA microarray. We found gene expression of IL-1β and IL-1β mediated signaling pathway was increased ABL TKI resistant K562 cells. IL-1β gene amplification was confirmed by RT-PCR analysis. Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB) acts as a central mediator of inflammatory responses. Because bortezomib is a proteasome inhibitor whose anti-cancer action is partly mediated through inhibition of NF-κB, we examined the bortezomib in ABL TKI resistant cells. Combined treatment of ABL TKI resistant cells with ponatinib or imatinib and bortezomib caused more cytotoxicity than each drug alone. Caspase 3/7 activity and cellular cytotoxicity was also increased. ABL TKIs are also associated with vascular adverse events (VAEs) in CML, we next investigated the in vitro effects of ABL TKIs on cultured HUVEC. We found gene expression of IL-1β was increased after ABL TKI especially dasatinib and ponatinib treatment. IL-1β was increased in the cell culture supernatant after ABL TKIs treatment. In the immunoblot analysis, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation was increased by IL-1β in the time dependent manner. We also found that gene expression of IL-6, IL-8, intercellular adhesion molecule 1 (ICAM1) and monocyte chemotactic protein-1 (MCP-1) was enhanced by IL-1β stimulation.
Conclusion: The IL-1β signaling pathway is involved in ABL TKI sensitivity and drug resistant in CML cells and plays a key role in cytokine production of the HUVEC. We also provide the promising clinical relevance as a candidate drug for treatment of ABL TKI resistant leukemia patients.
Disclosures
Ohyashiki: Asahikase: Research Funding; Taiho Pharmaceutical KK: Honoraria, Research Funding; Dainippon Sumitomo KK,: Honoraria, Research Funding; MSD,: Honoraria, Research Funding; Bristol Meyer Squibb KK,: Honoraria, Research Funding; Ono Pharmaceutical KK,: Honoraria, Research Funding; Celegene KK,: Honoraria, Research Funding; Pfizer KK,: Honoraria, Research Funding; Kyowakko Kirin KK,: Research Funding; Nihon-Seiyaku,: Research Funding; Eizai,: Research Funding; Chugai KK,: Honoraria, Research Funding; Takeda Pharmaceutical KK,: Honoraria, Research Funding; Asteras KK,: Research Funding; Jansen Pharma KK,: Research Funding; Nippon-shinyaku,: Honoraria, Research Funding; Novartis KK,: Honoraria, Research Funding.
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