Abstract

Acute myeloid leukemia (AML) cannot be cured by chemotherapy in approximately 60% of cases. Several prognostic factors have been evaluated, such as cytogenetic changes or molecular mutations. Length mutations of the FLT3-gene (internal tandem duplications, FLT3-ITD) confer a significantly worse prognosis with an increased rate of relapsed and refractory disease upon chemotherapy. The high rate of induction failure and of relapse upon chemotherapy in FLT3-ITD positive patients raises the question whether dysregulation at the level of the apoptotic machinery promotes resistance of AML blasts. Myeloid cell leukemia-1 (Mcl-1) protein is an anti-apoptotic member of the Bcl-2 family and blocks cytochrome c-release from mitochondria by interacting with proapoptotic members of the BCL-2 protein family, e.g. BAX and BAK, thereby preventing their activation and mitochondrial outer membrane permeabilization (MOMP). By Western blotting, high levels of Mcl-1 protein expression could be demonstrated in 6/6 FLT3-ITD positive patient samples versus 2/6 in FLT3-wildtype patient samples. Upregulation of Mcl-1 at a RNA and protein level could also be demonstrated in FLT3-ITD positive cell lines using transfected murine 32D cells (32D-FLT3-ITD vs 32D-FLT3- wt) and human FLT3-ITD positive cell lines (MV4;11 (ITD positive) vs RS4;11 (ITD negative)). To functionally investigate the role of Mcl-1 overexpression in resistance to chemotherapy, 32D-FLT3-ITD cells were transfected with a murine Mcl-1-wildtype construct. 32D-FLT3-ITD positive cells stably expressing Mcl-1 and controls were tested for induction of apoptosis upon cytotoxic treatment using various apoptosis assays (TMRE, AnnexinV-Staining, DNA content analysis by FACS). Overexpression of Mcl- 1 in 32D-FLT3-ITD cells conferred a striking decrease in induction of apoptosis upon chemotherapy (daunorubicine/cytarabine) and tyrosine kinase inhibitor treatment in comparison to the empty vector control. To analyze the influence of Mcl-1 expression on drug resistance in primary blasts, we perfomed siRNA knockdown experiments on primary AML blasts; siRNA silencing of Mcl-1 expression in primary AML-blasts was shown to result in increased apoptosis rates of up to 25% upon growth factor starvation or treatment with cytotoxic drugs. Constitutively activated FLT3-receptor phosphorylates and activates downstream signaling nodes as AKT and ERK, which are known upstream modifiers of Mcl-1. Thus, we hypothesized that phosphorylation of Mcl-1 by these pathways may be involved in differential Mcl-1-expression in FLT3-ITD positive AML. To investigate the role of Mcl-1 phosphorylation on drug resistance, a wildtype MCL-1 construct was mutagenized at different phosphorylation sites (serine/threonine to alanine). Experiments analyzing the functional role of mutated Mcl-1 when stably expressed in the hematopoietic cell line 32D-FLT3-ITD are in progress and will be presented. In conclusion, we here present evidence that Mcl-1 is critically involved in mediating resistance in FLT3-ITD positive AML. Our findings provide a rationale to clinically investigate agents that inactivate Mcl-1 in FLT-ITD positive AML.

Disclosures: No relevant conflicts of interest to declare.

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