Abstract

ASPP1 belongs to a family of p53-binding proteins and enhances apoptosis by stimulation of p53-transactivation of selected proapoptotic target genes. It is preferentially expressed in hematopoietic stem cells (HSC) and together with p53 preserves the genomic integrity of the HSC pool. Consequently, attenuated expression of ASPP1 which is linked to methylation of the promoter region has been associated with malignant transformation and development of acute lymphoblastic leukemia and lymphomas.

We now provide evidence that ASPP1 is highly altered in AML suggesting a role in leukemogenesis as well as therapy response. ASPP1 mRNA and protein expression levels of freshly isolated native patient samples (68) and healthy bone marrow donors (29) were determined by qRT-PCR and western immunoblotting. Statistical analyseswere performed. To explore implications of attenuated ASPP1 levels with regard to apoptosis induction and proliferation, ASPP1-expressing leukemia cell lines (MOLM14, Jurkat), native patient blasts or native bone marrow donor samples were stably silenced using a retroviral shRNA approach. Vice versa, ASPP1 was stably overexpressed in AML cell lines expressing per se low ASPP1 levels. Expression was thereby confirmed by qRT-PCR and western blotting. XTT viability and annexin V-based apoptosis assays were performed using standard chemotherapeutics in comparison to empty vector controls. Decitabine was used as an epigenetic sensitizer via hypomethylation of the promoter region.

ASPP1 mRNA expression was found to be frequently and highly statistically significantly (p=0.001) attenuated in AML. Low ASPP1 mRNA levels thereby translated into attenuated protein expression. Retroviral ASPP1-interference lead to perturbed proliferation capacities (up to 3-fold increase) and attenuated apoptosis upon standard chemotherapeutics in leukemia cell lines as well as native leukemia blasts. As expected, overexpression of ASPP1 resulted in significantly attenuated proliferation and higher induction of apoptosis in all tested cell lines and patient blasts.

Intriguingly, epigenetic therapy using the hypomethylating agent decitabine resulted in upregulation of ASPP1 expression in leukemia cells with originally low basal ASPP1 levels as confirmed by qRT-PCR and western blotting. Consequently, decitabine pretreatment sensitized these patient samples towards chemotherapy with a favorable proapoptotic overall efficacy compared to chemotherapy alone.

Our results demonstrate that dysfunctional regulation of ASPP1 expression likely contributes to the biology of leukemogenesis and to primary therapy resistance in a subgroup of patients with acute leukemia and seems to be linked to hypermethylation. Prospective clinical studies are warranted to evaluate the roleas a biomarker for risk stratification in leukemia patients and for monitoring therapy responses.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.