Evaluation of Targeted Selective Inhibitors to Enhance Temozolomide Treatment Sensitivity in Acute Myeloid Leukemia

Abstract

Temozolomide (TMZ) is an alkylating agent with limited activity in acute myeloid leukemia (AML). The DNA repair enzyme O6-methylguanine methyltransferase (MGMT) enhances tumor cell resistance to TMZ. B-cell lymphoma-2 (BCL-2) and mouse double minute 2 homology (MDM2) are proteins involved in AML cell survival and chemotherapeutic resistance. BCL-2 is an antiapoptotic protein that prevents cell apoptosis, and MDM2 is a ubiquitin E3 ligase that mediates p53 degradation. Inhibiting BCL-2 and restoring p53 functions may affect AML treatment. Venetoclax (VEN) is a small molecule that promotes cell apoptosis through inhibition of the BCL-2 protein. Idasanutlin (IDA) is an MDM2-specific inhibitor that disrupts p53:MDM2 interactions and restores p53 functions. This study established the need to enhance TMZ sensitivity in AML cells with high and low MGMT expression levels by incorporating VEN and IDA into TMZ treatment. AML human cell lines and blast-containing mononuclear cells were used to evaluate MGMT and target proteins for each inhibitor. Cell viability, cell apoptosis, and DNA damage were assessed with TMZ alone and in combination with a pre-selected inhibitory concentration of 50% (IC50) of a VEN or IDA inhibitor. Further, VEN inhibitor was applied in vivo using mouse model. Leukemic engrafted mice treated with selected TMZ and VEN concentrations, alone and in combination, were evaluated for survival and CD45+ and CD33+ expression in the bone marrow. In high and low MGMT-expressing AML cell lines, co-incubation of TMZ with VEN at IC50 resulted in a marked enhancement of TMZ sensitivity. In VEN+TMZ treated cell lines, apoptosis was induced and severe DNA damage was observed compared to TMZ alone. AML patient samples were resistant to TMZ alone but became sensitized to TMZ+VEN in combination, including those with high MGMT expression levels. The combination-treated mice had the longest survival among the group; however, by the time of death, no difference was seen in CD45+ cells compared to the single drug or untreated groups. In high and low MGMT-expressing cells, co-incubation of TMZ with IDA at IC50 did not improve TMZ sensitivity, regardless of p53 status. In both high and low MGMT-expressing cells, there was no evidence of DNA damage in the TMZ+IDA combination groups compared to TMZ alone. AML patient samples were resistant to TMZ alone and highly sensitive to IC50 IDA. IDA did not enhance the TMZ sensitivity, as insignificant cell death was observed in IDA alone and TMZ combination. VEN enhances TMZ cytotoxicity in high and low MGMT cells by decreasing cell viability, increasing cell apoptosis, and inducing DNA damage. The opposite was observed in IDA and TMZ combination-treated cells. Overall, we found VEN and TMZ combination to be promising to pursue further for clinical trails.