The ErbB signaling pathway in acute myeloid leukemia.

Scheepers, E. (Ellen) (2013) The ErbB signaling pathway in acute myeloid leukemia. thesis, Medicine.

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Abstract

Background: Acute myeloid leukemia (AML) is characterized by a defect of hematopoietic stem and progenitor cells, leading to the accumulation of myeloid blast cells and cytopenia of the remaining normal hematopoietic lineages. Among children and young adults up to 18 years, the 5-year event free survival of AML is 60-70%. Currently, cytarabine in combination with antracycline, is the backbone of the chemotherapy. After achievement of a complete remission with one or two chemotherapy courses, continuation of the chemotherapy is necessary to reduce the relapse rate. Still, the relapse rate of the newly diagnosed children with AML is 40-50%. To improve cure and relapse rates, quality of life and reduce additional side effects for both patients and survivors, new treatment approaches are warranted. After the success of tyrosine kinase inhibitors on the improved survival of cancer patients as for example erlotinib and gefitinib for the treatment of lung and breast cancers, tyrosine kinase inhibitors are the current focus of new therapeutic approaches in many forms of cancer including AML. Recently, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib has been shown to induce complete remission of AML in two patients with concurrent non-small cellular lung cancer, suggesting a possible role for EGFR in AML. However, EGFR expression analysis in AML is poorly defined and the role of EGFR and other ErbB family members in AML is still unclear. Aim: In this study, we studied the role of ErbB signaling pathway in AML by determining the efficacy of ErbB inhibitors (canertinib, gefitinib, erlotinib and cetuximab) on cell survival and cellular downstream targets, and analyzing the presence of ErbB family members in AML cells. Results: Using WST-1 assays, we have shown that especially the least selective ErbB inhibitor canertinib induces cell death with a median LC50 of 7.0 µM (range: 2.6 - 15.2 µM canertinib in n=8 AML cell lines). To determine whether this reduction of cell survival could be induced via an ErbB dependent manner, we started with determining the cell membrane protein expression levels of the ErbB family members using flowcytometry. ErbB1 (EGFR) was only detectable in OCI-AML3 (23.2%) and in a lesser extent in EOL1, HL60 and THP1 cells (approximately 10%), whereas ErbB2 and ErbB4 were expressed in various degrees among the different AML cell lines (with a range of respectively 8.8-63.9% for ErbB2 and 18.8-90.6% for ErbB4). ErbB3 was not detectable by flowcytometry. No correlation between the cell membrane ErbB protein expression levels and their sensitivity towards ErbB inhibitors in AML cell lines was observed. However, canertinib treatment and in a lesser extent erlotinib treatment resulted in a reduction of phosphorylated Akt and Erk protein levels using western blot analysis in THP-1 and EOL-1 cells. Moreover, total and phosphorylated Akt and Erk levels are slightly increased in THP-1 cells after EGF stimulation. This suggests, together with the ErbB1 cell membrane protein expression in THP-1 cells, that ErbB1 could be present and functionally active in THP-1 cells. Focusing on ErbB1 in THP-1 cells, one hour of EGF stimulation reduces the degree of total ErbB1 antibody expression in THP-1 cells, probably due to internalization of the activated ErbB1 receptor. Western blot analysis did not show ErbB1 in THP-1 cells. Phosphorylated ErbB1in THP-1 was only detectable using immunoprecipitation combined with western blot analysis. However, because of antibody cross reactivity, further studies are needed to identify the observed receptor tyrosine kinase. Conclusion: In summary, these data suggest that ErbB family tyrosine kinase inhibitors is suggested to be a therapeutic strategy in AML. However, further research is warranted to determine the exact role of the ErbB signaling pathway in AML and how the presence of the ErbB family members could contribute in relation to disease progression, relapse as well as interaction with possible escape mechanisms in AML.

Item Type:	Thesis (Thesis)
Supervisor name:	Bont, Prof. Dr. E.S.J.M. de
Faculty:	Medical Sciences
Date Deposited:	25 Jun 2020 11:07
Last Modified:	25 Jun 2020 11:07
URI:	https://umcg.studenttheses.ub.rug.nl/id/eprint/2689

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