Apoptosis Stimulating Protein of p53 (ASPP2) Haploinsufficiency Promotes Irradiation-Induced High Grade Lymphomas in a Mouse Model.

Apoptosis Stimulating Protein of p53 (ASPP2) binds the tumor suppressor protein p53 and promotes damage-induced apoptosis in part through stimulation of p53 apoptotic function. We have previously demonstrated that low ASPP2 levels correlate with poor clinical outcome in patients with diffuse large B-cell lymphoma treated with anthracycline-based chemotherapy, which is consistent with its role as a tumor suppressor. We targeted the ASPP2 allele in a mouse model by homologous recombination using a knockout vector that replaced exons 10–17 with a neoR gene. Although ASPP2−/− is an early embryonic lethal event, ASPP2+/– mice appeared developmentally normal and reproduce. Importantly, ASPP2+/– mice developed tumors spontaneously at a higher frequency than sibling ASPP2+/+ controls. We now show that after ionizing irradiation, ASPP2+/– mice develop high grade lymphatic malignancies compared to ASPP2+/+ mice. ASPP2+/+ and +/– mice were irradiated, with either 6 Gy or 10,5 Gy, 8 weeks after birth. Following irradiation, mice were monitored weekly by blood smears and flow cytometry if suspicious cells were observed. Tumor formation was detected as early as 17 weeks post-irradiation. In the 10,5 Gy group, ASPP2+/– mice developed thymic lymphomas (7/31), while no tumors were detected in ASPP2+/+ mice (0/14). Tumor-free survival was statistically significant (p= 0,045, log-rank test). In the 6 Gy dose group 9 out of 28 of the ASPP2+/– mice developed high-grade thymic lymphomas compared to 0/20 ASPP2+/+ mice. Tumor-free survival was statistically significant (p=0,024, log-rank test). Immunophenotyping and flow cytometry on mice with circulating tumor cells from whole unfractionated gated viable bone marrow (Bm) cells revealed that the Bm was typically almost completely replaced by CD4neg, CD5+, CD8+ leukemic/lymphoma cells that are negative for B-cell (B220) and myeloid markers (Mac-1/Gr-1). Preliminary analysis demonstrated that MEFs (mouse embryonic fibroblasts) from ASPP2+/– mice treated with doxorubicin, serum starvation or irradiation had an attenuated G1/G0 checkpoint arrest. Ongoing experiments are being conducted in thymocytes prepared from mice of different ASPP2 and p53 genotypes. Our data demonstrates that ASPP2 plays an important role in suppressing the development of lymphomas after irradiation in a mouse model. Consequently, disruption of the p53-ASPP2 axis may be important in lymphomagenesis and response to therapy. Furthermore, our data provides a rationale for the future study of ASPP2 expression in patients during the formation of secondary hematologic malignancies after therapies with DNA damaging agents.