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.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.