BCL-2 Inhibitor Venetoclax Enhances Temozolomide Sensitivity in AML

Introduction:

Many older patients with acute myeloid leukemia (AML) are ineligible for intensive chemotherapy due to frailty and co-morbidities; for such patients, existing treatments are often ineffective and new treatments are needed. Temozolomide (TMZ) is an alkylating agent that causes DNA methylation at O⁶ guanine, generating single strand break leading to apoptosis. However, the efficacy of TMZ depends on the DNA repair protein O⁶-methylguanine methyltransferase (MGMT), that maintains the genomic integrity by removing the O⁶-methyl group and restoring guanine nucleobase, thereby enhancing resistance to TMZ. Previous clinical trials in AML found that responses to TMZ correlated with low MGMT expression; however, even in those with low MGMT expression complete response rates were only in the 25% range. BCL-2, an anti-apoptotic protein, is overexpressed in AML cells. Direct inhibition by the selective BCL-2 inhibitor venetoclax (Venet) promotes apoptosis. This study evaluated the ability of Venet to enhance TMZ sensitivity in AML cells, including those with MGMT overexpression.

Methods:

KG1, MV4-11 and MOLM13 AML cell lines were studied, as well as bone marrow blast cells collected from AML patients. Western blot was used to measure MGMT and BCL-2 expression. The cells were incubated with TMZ at varying concentrations in combination with a fixed concentration of Venet. After 48 hours, cell viability and apoptosis assays were performed using spectrophotometry and flow cytometry, respectively. Synergy was evaluated by the Chou-Talalay method. Cleaved-PARP was measured by Western blot in selected combination doses after 3 hours in MV4-11 and MOLM13 and after 6 hours in KG1.

Results:

KG1 cells expressing high MGMT demonstrated strong resistance to TMZ; however, co-incubation with 1 uM Venet resulted in a marked enhancement of sensitivity to TMZ. Similarly, in MV4-11 and MOLM13 cell lines, which demonstrated very low or absent MGMT expression. Venet 2.5 nM in combination with TMZ markedly increased the cytotoxicity to TMZ. A synergistic effect was demonstrated in all cell lines with combination index (CI) < 1. Cells overexpressed annexin V and propidium iodide (PI) apoptotic marker after drug combination in all cell lines. Apoptotic effect with the drug combination was verified by cleaved-PARP expression. Most (6/8) AML patient samples which were resistant to TMZ in vitro became sensitized to TMZ in combination with 1 uM Venet, including those with moderate to high MGMT expression.

Conclusion:

Venetoclax synergizes with TMZ and induces cytotoxicity in all AML cell lines and in most AML patient samples, including those in whom MGMT was highly expressed, by activating apoptotic pathways to trigger cell death. This combination represents a potentially promising new treatment. Further studies evaluating this combination in animal models are in progress.