Attenuation of Apoptosis-Stimulating Protein of p53-2 (ASPP2) Promotes IL-3-Independent Growth and Enhances Cytogenetic Abnormalities after Gamma-Irradiation in Mouse Hematopoietic Cells - Mimicking Early Leukemogenesis of Therapy-Associated Human AML

Inactivation of tp53 by chromosome/gene loss or loss-of function mutation is frequently observed in therapy-associated acute myeloid leukemia. Tp53 mutations associate with complex and monosomal karyotypes and provide the strongest predictive risk factor in this subgroup. ASPP2 is an independent tumor suppressor. We have previously shown that ASPP2 expression is attenuated in acute leukemia via methylation by unknown mechanisms. We now provide evidence that attenuation of ASPP2 expression may act as an early leukemia-initiating event - facilitating acquisition of structural chromosomal aberrations and loss of its binding partner p53.

We found low ASPP2 mRNA and protein expression levels in complex karyotype AML, arguing for a role of ASPP2 loss in leukemogenesis. To evaluate for malignant transformation we engineered IL3-dependant murine Ba/F3 cells and stably silenced ASPP2 expression using retroviral-induced shRNA, Interestingly, only ASPP2(ko) cell strains could be successfully weaned from IL3 dependence - suggesting that loss of ASPP2 expression promoted cellular transformation. In contrast, empty vector control cells did not survive the weaning process. As expected, cellular proliferation was enhanced in the ASPP2(ko) cell lines as measured by standard XTT and CSFE protocols. To study how loss of ASPP2 could promote transformation, we performed cytogenetic analysis of the chromosome set of parental (+IL3) vs. gamma-irradiated empty vector controls (+IL3) and ASPP2(ko) cell strains (-IL3) with and without irradiation (+/- stress enforcement using gamma irradiation, 2 x 5Gy). Gamma irradiation did not significantly alter cytogenic analysis between the empty vector controls compared to the parental cells. Tantalizingly however, we found a consistent chromosomal loss of chr8, der8 and der19 - and, most intriguingly, loss of chromosome 11 (which locates tp53 in mice), and a monosomal karyotype in the ASPP2-attenuated cells. In addition, several marker chromosomes (6-8 in ASPP2i vs. 0-2 in empty vector controls were identified. We are now in the process of verifying our findings in knockout native human bone marrow blasts.

Conclusions: Our findings provide novel evidence that loss of ASPP2 contributes to malignant transformation by promoting genomic instability as measured by increased cytogenetic abnormalities after gamma-irradiation. These findings may have important relevance for the pathogenesis of therapy-associated AML - but also of hypodiploid acute lymphoblastic leukemia, which is as well associated with tp53 loss-of-function and predicted for a dismal outcome. Studies are ongoing to elucidate the mechanisms of how loss of ASPP2 expression promotes human AML, as well as restore ASPP2 function as a novel therapeutic pathway to exploit in treating AML.