Caspase-8 Determines Chemosensitivity of ALL Cells and Mediates Favorable Interactions of Cytotoxic Drugs,

Abstract 3511

Sensitivity of tumor cells towards chemotherapy mainly determines the prognosis of patients suffering from acute lymphoblastic leukemia (ALL); nevertheless, underlying mechanisms regulating chemo-sensitivity remain poorly understood. Here, we aimed at characterizing the role of Caspase-8 for chemo-sensitivity of B- and T-ALL cells.

Several different drugs of routine anti-leukemia therapy were tested in vitro. All drugs that induced cell death also activated and cleaved Caspase-8. Caspase-8 was activated independently from extrinsic apoptosis signaling suggesting a downstream amplifier role of Caspase-8 upon drug-induced apoptosis in ALL cells. Most importantly, Asparaginase, Cyclophosphamide, Dexamethasone and Doxorubicin induced apoptosis in a Caspase-8 dependent manner as knockdown of Caspase-8 inhibited drug-induced apoptosis. Accordingly in primary ALL cells, the protein expression levels of Caspase-8 correlated with cell death sensitivity towards these cytotoxic drugs in vitro. In contrast, Cytarabin, Etoposid and others induced apoptosis via Caspase-8 independent signaling. Thus protein expression of Caspase-8 should be evaluated as a potential biomarker for risk stratification in ALL.

The expression of Caspase-8 is frequently downregulated in tumor cells mostly due to epigenetic silencing by promoter hypermethylation. In previous work, we had shown that Methotrexate is able to upregulate the expression of epigenetically downregulated Caspase-8 which is mediated by the transcription factor p53 (Ehrhardt et al, Oncogene 2008). Here we found that Methotrexate (MTX) was able sensitize B- and T-cell leukemia cell lines for apoptosis induction by the Caspase-8 dependent drugs Asparaginase, Cyclophosphamide, Dexamethasone and Doxorubicin. Sensitization by MTX for drug-induced apoptosis was mediated by p53 and Caspase-8 as shown by stable expression of respective small hairpin RNAs introduced by lentiviral transduction. Accoordingly to the data obtained in cell lines, in patient-derived ALL cells with low expression of Caspase-8, MTX sensitized for induction of apoptosis by Asparaginase, Cyclophosphamide, Dexamethasone and Doxorubicin. Transient transfection of siRNA into patient-derived ALL cells revealed that synergistic apoptosis induction by MTX and these drugs was dependent on p53 and Caspase-8.

Our results indicate that Caspase-8 is crucial for the high anti-leukemic efficiency of numerous routine cytotoxic drugs and drug combinations. Re-expression of epigenetically downregulated Caspase-8 represents a promising approach to increase efficiency of anti-leukemic therapy. Retrospectively, our data might explain on a molecular level, why clinical empirical studies already revealed a high anti-leukemic efficiency for some of these drug combinations over decades. Routine, decades-known cytotoxic drugs activate signaling mechanisms recognized rather recently such as reversing epigenetic silencing.