Drug resistance in acute myeloid leukemia : Clinical and experimental studies

Abstract: The long-term result of chemotherapy in acute myeloid leukemia (AML) is poor, even though a majority of AML patients initially respond to chemotherapy and achieve complete remission (CR). Initially resistant disease as well as relapse is a result of resistance to chemotherapy drug resistance. The aim of this thesis was to further explore the relevance of three putative mechanisms of drug resistance in AML -regarding drug transport, drug target and apoptosis - as well as the immediate effect of drug exposure on the expression of three markers of drug resistance in leukemic cells in vitro. Topoisomerase IIalpha (topo IIalpha) is the target of topoisomerase poisons, which are widely used in AML treatment. We investigated topo IIalpha at the protein level, in relation to cell cycle phases, as well as the mRNA level, in samples from patients with acute leukemia. We found that, in contrast to the situation with normal cells, topo IIalpha was expressed not only in the S/G2/M cell cycle phases (mean 76% topo IIalpha positive cells) but also in G0/G1 (mean 39% positive cells). Despite an association between low topo IIalpha protein expression and drug resistance in vitro, we found no association between topo IIalpha mRNA or protein expression and clinical outcome. The recently described efflux pump breast cancer resistance protein (BCRP) was determined in a material of 40 AML patients. The majority of samples showed lower BCRP mRNA expression than that seen in a drug sensitive reference cell line, MCF-7. Overall we found no significant association between BCRP mRNA and clinical outcome; however we did find an association between BCRP mRNA levels and survival in a subgroup of patients responding to intial chemotherapy (n=28, median survival 18 months in the high-BCRP group vs 52 months in the low- BCRP group, p=0.047), suggesting a predictive value for BCRP mRNA in AML. The initial effect of drug exposure on the expression of the efflux pumps Pglycoprotein (Pgp) and BCRP as well as on the cytosolic enzyme glutathion-Stransferase pi(GSTpi) was investigated in vitro using HL-60 leukemic cell lines with different levels of drug resistance. After 10 min exposure to cytarabine, Pgp mRNA had increased (1.7-3.1-fold); and Pgp protein was detectable in the initially Pgpnegative HL-60 S cell line after 8 hours exposure. Since cytarabine is administered simultaneously with the Pgp-substrate daunorubicin in standard treatment of AML, this finding may be of clinical importance but needs to be further studied in vivo. P53 is a key protein regarding apoptotic response to cytostatic treatment. The protein p14ARF stabilizes p53 via the ARF-MDM2-p53 pathway and, in theory, high levels of p14ARF would make the cell more prone to apoptosis. High levels of p14ARF mRNA have been associated with longer survival in AML. We investigated p14ARF mRNA expression in 57 AML patients with normal karyotype and found an association with survival. In a multivariate Cox regression analysis, the hazard ratio for death for patients with low p14ARF mRNA expression was 3.83 (95% CI 1.13-9.44). In vitro, patient samples with low expression of p14ARF mRNA tended to be more sensitive to the recently described compound PRIMA-1, suggesting a role for this compound for patients with altered regulation of the ARF-MDM2-p53 pathway.