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Introduction: p53 is an important tumor suppressor, and loss of p53 pathway function is a common event in human cancer. ASPP2 is a damage-inducible p53 binding protein that enhances apoptosis, at least in part, by selective stimulation of p53 pro-apoptotic target genes. Low ASPP2 expression occurs in many human tumors and as we have previously demonstrated, correlates with poor clinical outcome in patients with B-cell lymphomas. However, the mechanisms by which ASPP2 suppresses tumor formation remain unknown. Methods: To rigorously study ASPP2 in vivo function, we targeted the ASPP2 allele in a mouse model by homologous recombination by replacing exons 10–17 with a neoR gene. Aging mice were followed for spontaneous tumor formation. Additionally, six week old mice were irradiated at different doses and followed for tumor development. To explore the functional consequences of low ASPP2 expression, ASPP2+/− and ASPP2+/+ thymocytes were subjected ex vivo to 5Gy ionizing irradiation and apoptosis was assessed by Annexin-V/PI staining and flow cytometry. We also irradiated ASPP2+/+ and ASPP2+/−MEFs (mouse embryonic fibroblasts) with 5Gy and performed cell cycle analysis. Furthermore, we conducted global gene expression profiling between the unirradiated and irradiated ASPP2+/+ and ASPP2+/−MEFs using an Affymetrix GeneChip® Mouse Gene 1.0 ST Array. Moreover, phosphoproteomic analysis was performed on unirradiated and irradiated ASPP2+/+ and ASPP2+/−MEFs using 2-dimensional gel electrophoresis, fluorescent phosphoprotein dye Pro-Q Diamond staining, and liquid chromatography tandem mass spectroscopy. Results: We were unable to generate ASPP2−/− mice due to an early embryonic lethal defect. However, ASPP2+/− mice appear developmentally normal and reproduce. We observed an increased formation of spontaneous tumors in aging ASPP2+/− mice compared to ASPP2 +/+ mice (43% vs. 22%, at 115 weeks, p=0.011). Additionally we were able to show that after ionizing radiation, ASPP2+/− mice develop high-grade lymphomas in a dose-dependent manner at a significantly higher incidence at 50 weeks compared to ASPP2+/+ mice (p = 0.024 and p = 0.045, 6 Gy and 10.5 Gy respectively). Immunophenotyping demonstrated that these were high-grade T-cell lymphomas of thymic origin. Since ASPP2 is damage-inducible and promotes apoptosis, we wished to determine the extent to which reduction in ASPP2 expression attenuated the cellular damage response. We therefore gamma-irradiated ex vivo ASPP2+/+ and ASPP2+/−thymocytes in short-term culture and found a two-fold reduction in apoptosis in ASPP2+/− thymocytes compared to ASPP2+/+ thymocytes. Additionally, after 5 Gy gamma-irradiation, ASPP2+/− MEFs exhibited an attenuated G0/G1 checkpoint compared to ASPP2+/+ MEFs. Preliminary analysis of global gene expression in ASPP2+/+ and ASPP2+/− MEFs shows specific differences in gene expression patterns after damage. Likewise, preliminary analysis of phosphoproteomics between ASPP2+/+ and ASPP2+/− MEFs after damage, demonstrate differences in the phosphophorylation status of 170 proteins. Conclusions: ASPP2 is a haploinsufficient tumor suppressor, and reduction in ASPP2 expression attenuates both cell cycle checkpoints and apoptosis-induction after damage. These results suggest that reduction of ASPP2 levels modulate the cellular damage response, possibly at transcriptional as well as post-transcriptional levels, and provide a functional explanation for the increased tumor incidence in ASPP2+/− mice---since attenuated damage-response thresholds would lead to an impaired ability to eliminate cells that have accumulated tumorigenic mutations. Our study provides important insights into the p53-ASPP2 axis, and opens new avenues for investigation into its role in tumorigenesis and response to therapy.

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.

ASPP2 interacts with the tumor suppressor protein p53 and promotes damage-induced apoptosis in part through stimulation of p53-mediated apoptosis. 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. Moreover, reduced ASPP2 expression has been demonstrated in other tumor types. These findings led us to hypothesize that ASPP2 may function as a tumor suppressor. To further explore this, we targeted the ASPP2 allele in a mouse by homologous recombination using a knockout vector that replaced exons 10–17 with a neoR gene. Two separate ES clones were used for blastocyst injections to generate several chimeras that were used to generate ASPP2 heterozygous mice. ASPP2+/− mice appear developmentally normal and reproduce. However ASPP2−/− mice could not be generated. Genotype analysis as early as Ed 6.5 did not detect ASPP2−/− embryos---which implies an early embryonic lethal defect in the homozygote. ASPP2+/− (n=135) and ASPP2+/+ (n=63) sibling mice were observed for spontaneous tumor formation. Overall median tumor-free survival was 117 weeks in the ASPP2+/− mice verses 125 weeks in the ASPP2+/+mice (p = 0.035 log-rank test). Overall tumor incidence (at 115 weeks) for ASPP2+/− and ASPP2+/+ mice was 43% and 22%, respectively. The incidence of tumor types, from all tumors detected, was similar between ASPP2+/− and ASPP2+/+ mice: 34% versus 33% (lymphoma), 18% versus 14% (sarcoma), and 47% versus 52% (carcinoma), respectively. Compound p53+/−;ASPP2+/− mice did not exhibit accelerated tumor formation relative to p53+/−;ASPP2+/+ mice. Additionally, a tet-Myc:ASPP2+/− lymphoma mouse model did not exhibit accelerated lymphomagenesis. However, preliminary data suggests that ASPP2+/− mice may have an increased incidence of irradiation-induced leukemia/lymphoma when compared to ASPP2+/+ mice, and confirmatory studies are ongoing. In response to ionizing radiation, doxorubicin, or serum-starvation, preliminary analysis reveals a G0/G1 checkpoint defect in ASPP2+/− MEFs compared to ASPP2+/+ MEFs. Our results provide in vivo evidence that ASPP2 can function as a tumor suppressor. Further studies are underway to determine the mechanism of this observation.