Cellular Response to Cytarabine Is Modulated by the DNA Mismatch Repair Pathway: Implications for Treatment of Acute Myeloid Leukemia
Sarah E Fordham*,1,
Elizabeth C Matheson*,1,
Julie Irving1 and
James M Allan1
1 Northern Institute for Cancer Research, Newcastle University, Newcastle Upon Tyne, United Kingdom, 2 Department of Biology, University of York, York, United Kingdom
The DNA mismatch repair (MMR) pathway is responsible for repairof spontaneous errors arising during DNA replication, thus maintainingthe integrity of the genome. DNA MMR is frequently dysregulatedin some forms of leukemia. We and others have shown that microsatelliteinstability, the hallmark of dysfunctional DNA MMR, is presentin up to 90% of therapy-related myeloid leukemia, 50% of relapsedmyeloid leukemia, but is rarely seen in de novo leukemia. Paradoxically,functional MMR mediates the cytotoxicity of certain chemotherapeuticagents, particularly methylating agents and the nucleoside analogue6-thioguanine (6-TG), and dysregulation of the MMR pathway conferstolerance to these agents. In the present study, using celllines harboring defects in MMR components, we show that MMRstatus also modulates response to the nucleoside analogue cytarabine(Ara-C) and to other therapeutic nucleoside analogues commonlyused in the treatment of leukemia. We initially determined geneand protein expression levels of the major MMR components (MSH2,MSH3, MSH6, MLH1 and PMS2) in two psuedo-isogenic cell linepairs, and investigated the ability of cell extracts to bindto defined mismatches in electrophoretic mobility shift assays.In cytotoxicity assays, the cell lines HL-60R (which demonstrates200-fold overexpression of MSH3) and MT-1 (which lacks functionalMSH6 due to bi-allelic gene mutation) were tolerant to the cytotoxiceffects of a methylating agent, methylnitrosourea (MNU), and6-TG, relative to their respective parental cell lines. We alsogenerated a panel of fully isogenic MMR-defective cell linesin which either MSH2, MSH3 or MSH6 protein was reduced to almostnegligible levels using short hairpin RNA-mediated gene knockdown.Knockdown of either MSH2 or MSH6 conferred tolerance to thekilling effects of MNU and 6-TG by virtue of loss of MutS activity(a heterodimer of MSH2 and MSH6 responsible for recognitionof base:base mispairs), whereas knockdown of MSH3 did not affectcellular response to these agents. Consistent with a role forMMR in affecting cellular response to other nucleoside analoguesused to treat leukemia, the cell lines also displayed differentialtoxicity to the killing effects of Ara-C, clofarabine, cladribineand fludarabine compared to their MMR-proficient parental counterparts,however the exact response was dependent on the specific natureof the MMR defect. Cell lines with a reduction in MSH2 proteindemonstrated hypersensitivity to cytotoxicity induced by thesenucleoside analogues. Conversely, MSH3 knockdown conferred resistanceto the cytotoxic effects of these agents. These data suggestthat DNA MMR can affect response to nucleoside analogues viamultiple mechanisms, and may also involve interaction of DNAMMR components with other DNA repair pathways. One possibilityis that these agents induce base lesions in DNA recognized byDNA MMR components. Consistent with this model, we have shownthat Ara-C induces DNA polymerase slippage in vitro, generatinga substrate potentially recognized by MutSβ (a heterodimerof MSH2 and MSH3 responsible for recognition of small insertionsand extrahelical loops). Furthermore, we have also shown thatAra-C is mutagenic at the thymidine kinase and hypoxanthine-guaninephosphoribosyltransferase loci in the TK6 cell line. Taken together,these data suggest that cellular MMR status affects responseto nucleoside analogues and furthermore, the specific natureof the defect is important in determining the exact response.These findings have implications for the use of nucleoside analoguesin the treatment of cancers where MMR dysfunction has been identifiedto occur with high frequency, such as therapy-related and relapsedacute myeloid leukemia.
Disclosures: No relevant conflicts of interest to declare.
* Asterisk with author names denotes non-ASH members.