In Vitro Derivation of ARA-C Resistant BXH-2 AML Cell Lines: Association of RAS/MAPK and P53-Regulated Pathways with Chemoresistance.

Abstract

Chemotherapy resistance in acute myeloid leukemia (AML) is proposed to be due to insufficient intracellular concentration of nucleoside analog triphosphates, inability to achieve sufficient alterations in DNA strands or dNTP pools, or defective induction of apoptosis. Mouse AML models could be used to study mechanisms of AML chemoresistance and feature isogenic backgrounds, the availability of genetic manipulation, controlled conditions of disease induction and treatment regimens, and the possibility of using forward genetic screens for treatment resistance. The BXH-2 mouse strain has a high incidence of AML caused via chronic infection by a B-ecotropic murine leukemia virus, which has been used a molecular tag to identify AML-associated genes. Nine BXH-2 derived AML cell lines were assayed for responses to Ara-C. Expression levels of multi-drug resistance gene family members showed no correlation to Ara-C response. However, in general, cell lines with Nf1 tumor suppressor gene loss, and concomitant ERK hyperactivation, were more Ara-C resistant. Furthermore, U0126 treatment, which was shown to inhibit ERK activity in these cells, partially sensitized Nf1-defective AML cells to Ara-C, but did not have this effect in Nf1 wild-type cells. These data suggest that upregulated ERK activity represents one mechanisms of de novo Ara-C resistance and that inhibition of ERK in leukemic cells may partially overcome chemoresistance. To study mechanisms of acquired chemoresistance, we have derived eight Ara-C resistant subpopulations from two of the BXH-2 AML cell lines via in vitro consecutive selection with increasing doses of drug per selection cycle. This resulted in derivative lines with an IC50 about 800-fold higher than the parental lines. Although we could not detect a difference in activation of ERK, p21Cip was dramatically downregulated in one of the selected Ara-C resistant lines. Interestingly p21Cip is not expressed in one of the de novo Ara-C resistant cell lines. Also, Ara-C induction of p53, a well-known upstream activator of p21Cip, was decreased in all resistant lines tested. Examination of one resistant line by RT-PCR and DNA sequencing showed that repeated Ara-C treatment selects for a subpopulation of cells that harbor a 66-bp genomic deletion of the splice acceptor of exon 4 of the p53 gene, resulting in production of an mRNA encoding a deletion mutant lacking parts of both the transactivation and DNA-binding domains. Retroviral transduction of a dominant-negative p53 cDNA rendered the parental cells more resistant to Ara-C treatment. These data indicate that p53 loss-of-function could explain at least partly the acquired resistance to Ara-C. Recent work shows that primary BXH-2 AML growing in histocompatible mice can be suppressed for several weeks by high dose Ara-C treatment of mice. This model will be used to test candidate genetic pathways for Ara-C responsiveness in vivo. This work was sponsored by the Leukemia and Lymphoma Society of America (LLS 7019-04, Specialized Center of Research).