Abstract

Inactivation of the TP53 pathway is a frequent event in human cancers promoting tumorigenesis and resistance to chemotherapy. Inactivating TP53 mutations are uncommon in non-complex karyotype leukemias suggesting that the TP53 pathway must be inactivated by other mechanisms. ASPP proteins are a family of TP53-binding proteins consisting of three members: pro-apoptotic ASPP1 and ASPP2 and apoptosis-inhibiting iASPP. ASPPs control induction of apoptosis via direct interactions with TP53 but also other key proteins such as BCL-2/BXL-xl.

We here reveal universal dysregulation of ASPPs in AML correlating with chemotherapy failure and provide a novel therapeutic target.

mRNA and protein expression levels of ASPP1, ASPP2 and iASPP were determined in freshly isolated native patient samples (n=120) as well as healthy bone marrow donors (n=34). To mimick direct implications of the specific ASPP protein in response to chemotherapy, ASPPs were stably silenced in leukemia cell lines (MOLM14, Jurkat, HL60, NB4 or OCI-3), native patient blasts and bone marrow donor samples using a retroviral shRNA approach. Sensitivity towards standard chemotherapeutics was assessed using XTT viability and annexin V-based apoptosis assays. Transfection with an empty vector served as a negative control.

Interestingly, all family members, ASPP1 (p=0.001), ASPP2 (p=0.02) and iASPP (p=0.001) were found to be significantly altered in AML compared to healthy donors. Subgroup analyses revealed association with high-risk features and failure towards induction chemotherapy. Importantly, dysregulation was not exclusive and may affect several family members - which was associated with dismal outcome.

Retroviral ASPP1/2 -interference lead to perturbed proliferation capacities (up to 3-fold increase) and attenuated apoptosis in response to chemotherapy in all tested models. Vice versa, inhibition of iASPP resulted in attenuated proliferation and an increase in apoptosis.

For ASPP1/2, we demonstrate promoter methylation and restoration of protein expression upon treatment with aza-nucleosides. Additional BCL-2 inhibition (using venetoclax) consequently resulted in superadditive, proapoptotic efficacy compared to the knockdown cell strains.

To summarize, dysfunctional regulation of ASPP proteins is a common feature in high-risk leukemias associated with treatment failure. Restoring pro-apoptotic ASPP1/2 function using hypomethylating agents and combination with proapoptotic agents such as venetoclax may overcome therapy resistance in these settings.

Disclosures

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.