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The p53 (TP53) tumor suppressor is the most frequently mutated gene in human cancers. Restoring expression of wild-type p53 has led to tumor growth suppression in a variety of tumor models that are p53 deficient. Other mechanisms, for example, upregulation of Mdm2, exist in tumors to inactivate the p53 pathway. Mdm2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation, is present at high levels in many tumors with wild-type p53. In this study, the effects of restoring p53 activity were probed in Mdm2-overexpressing tumors genetically using animal models. Here, it was demonstrated that elevated levels of Mdm2 and decreased levels of p53 act additively to dampen p53 activity in DNA damage response and tumor development. Our data further indicate that restoration of wild-type p53 expression in Mdm2-overexpressing angiosarcomas results in tumor stasis and regression in some cases. Finally, it was determined that restored p53 suppressed cell proliferation but did not elicit apoptosis in the Mdm2-overexpressing angiosarcomas.Implications: Restoration of wild-type p53 expression in Mdm2-overexpressing tumors suppresses tumor growth, which represents a potential clinical strategy to treat tumors with high levels of Mdm2.Visual Overview: http://mcr.aacrjournals.org/content/12/6/901/F1.large.jpg.Mol Cancer Res; 12(6); 901–11. ©2014 AACR.

PDF file - 354K, Figure S1. Tumor-free survival curves of the Mdm2Tg p53Neo/Neo and Mdm2Tg p53Neo/Neo CreER mice. Figure S2. Analysis of p53Neo recombination in tamoxifen-treated Mdm2Tg p53Neo/Neo CreER tumors. Figure S3. Initial tumor volumes of the Mdm2Tg p53Neo/Neo and Mdm2Tg p53Neo/Neo CreER angiosarcomas. Figure S4. p53 target gene p21 expression in tamoxifen-treated spontaneous Mdm2Tg p53Neo/Neo and Mdm2Tg p53Neo/Neo CreER angiosarcomas. Figure S5. PCR analysis of p53Neo allele recombination upon tamoxifen treatment in transplanted Mdm2Tg p53Neo/Neo CreER angiosarcomas. Figure S6. Effects of tamoxifen on the Mdm2Tgp53Neo/Neo angiosarcomas. Table S1. List of Mdm2Tg p53Neo/Neo and Mdm2Tg p53Neo/Neo CreER angiosarcomas monitored in the MRI study.

POLE germline mutations from the IRRDC and literature

Supplementary Figure S1 (related to Figure 1): PoleP286R mouse model design, validation, and tumor findings. Supplementary Figure S2 (related to Figure 1): PoleS459F mouse model design, validation, and tumor findings. Supplementary Figure S3 (related to Figure 2): WES analysis reveals a stochastic accumulation of mutations in Pole mutant tumors. Supplementary Figure S4 (related to Figure 2): Tumors from Pole mutant mice exhibit mutational signatures found in POLE driven human cancers. Supplementary Figure S5 (related to Figure 2): Exome data from tumor fractions from a single mouse were compared for 3 additional mice. Supplementary Figure S6. Determination of cell of origin for PoleS459F/S459F and PoleS459F/+ mice lymphomas (Related to Figure 3). Supplementary Figure S7. Characterization of T cell malignancies in PoleS459F/S459F and PoleS459F/+ mice (Related to Figure 3). Supplemental Figure S8. Extrinsic effect of 'Group B' malignant T cells on B cell population; (Related to Figure 3).

POLE mutations are a major cause of hypermutant cancers, yet questions remain regarding mechanisms of tumorigenesis, genotype–phenotype correlation, and therapeutic considerations. In this study, we establish mouse models harboring cancer-associated POLE mutations P286R and S459F, which cause rapid albeit distinct time to cancer initiation in vivo, independent of their exonuclease activity. Mouse and human correlates enabled novel stratification of POLE mutations into three groups based on clinical phenotype and mutagenicity. Cancers driven by these mutations displayed striking resemblance to the human ultrahypermutation and specific signatures. Furthermore, Pole-driven cancers exhibited a continuous and stochastic mutagenesis mechanism, resulting in intertumoral and intratumoral heterogeneity. Checkpoint blockade did not prevent Pole lymphomas, but rather likely promoted lymphomagenesis as observed in humans. These observations provide insights into the carcinogenesis of POLE-driven tumors and valuable information for genetic counseling, surveillance, and immunotherapy for patients.Significance:Two mouse models of polymerase exonuclease deficiency shed light on mechanisms of mutation accumulation and considerations for immunotherapy.See related commentary by Wisdom and Kirsch p. 5459

Supplemental Titles and Figure Legends

Replicative senescence is the terminal growth arrest that most normal human cells enter into after a fixed number of divisions in vitro, limiting the proliferative potential of a cell and preventing genomic instability caused by critically short telomeres. Thus, senescence presents a tumor-suppressive mechanism and a barrier to tumor formation. However, senescent cells are inherently resistant to apoptosis and, as they accumulate in aging tissues, may contribute to organ dysfunction and promote tumor progression as part of the stromal environment. Replicative life span in normal human cells can be extended by inactivation of the tumor suppressor gene p53 or its direct target, the cyclin-dependent kinase inhibitor p21, suggesting a direct role for this pathway in senescence. However, p53 recruitment to promoters of target genes during replicative senescence has not been shown in live cells. In this study, we used chromatin immunoprecipitation to determine that p53 preferentially occupied the promoters of growth arrest genes p21 and GADD45 in senescent normal human diploid fibroblasts but not the promoters of other target genes that recruited p53 following doxorubicin-induced DNA damage, such as apoptosis regulators TNFRSF10b, TNFRSF6, and PUMA. This differential recruitment of p53 in senescent versus doxorubicin-treated fibroblasts was accompanied by differences in post-translational modification of p53. These data provide mechanisms for both the growth arrest mediated by p53 and the resistant nature of senescent cells to apoptosis despite p53 activity. (Cancer Res 2006; 66(17): 8356-60)

Supplementary Tables 1-2 from p53 Is Preferentially Recruited to the Promoters of Growth Arrest Genes p21 and GADD45 during Replicative Senescence of Normal Human Fibroblasts

Mutations in the exonuclease domain of POLE are associated with tumors harboring very high mutation burdens. The mechanisms linking this significant mutation accumulation and tumor development remain poorly understood. Pole+/P286R;Trp53+/– mice showed accelerated cancer mortality compared to Pole+/P286R;Trp53+/+ mice. Cells from Pole+/P286R mice showed increased p53 activation, and subsequent loss of p53 permitted rapid growth, implicating canonical p53 loss of heterozygosity in POLE mutant tumor growth. However, p53 status had no effect on tumor mutation burden or single base substitution signatures in POLE mutant tumors from mice or humans. Pten has important roles in maintaining genome stability. We find that PTEN mutations are highly enriched in human POLE mutant tumors, including many in POLE signature contexts. One such signature mutation, PTEN-F341V, was previously shown in a mouse model to specifically decrease nuclear Pten and lead to increased DNA damage. We found tumors in Pole+/P286R mice that spontaneously acquired PtenF341V mutations and were associated with significantly reduced nuclear Pten and elevated DNA damage. Re-analysis of human TCGA (The Cancer Genome Atlas) data showed that all PTEN-F341V mutations occurred in tumors with mutations in POLE. Taken together with recent published work, our results support the idea that development of POLE mutant tumors may involve disabling surveillance of nuclear DNA damage in addition to POLE-mediated hypermutagenesis.

Cancer cells survive chemotherapy and cause lethal relapse by entering a senescent state that facilitates expression of many phagocytosis/macrophage-related genes that engender a novel cannibalism phenotype. We used biosensors and live-cell imaging to reveal the basic steps and mechanisms of engulfment by senescent human and mouse tumor cells. We show filamentous actin in predator cells was localized to the prey cell throughout the process of engulfment. Biosensors to various phosphoinositide (PI) species revealed increased concentration and distinct localization of predator PI(4) P and PI(4,5)P2 at the prey cell during early stages of engulfment, followed by a transient burst of PI(3) P before and following internalization. PIK3C2B, the kinase responsible for generating PI(3)P, was required for complete engulfment. Inhibition or knockdown of Clathrin, known to associate with PIK3C2B and PI(4,5)P2, severely impaired engulfment. In sum, our data reveal the most fundamental cellular processes of senescent cell engulfment, including the precise localizations and dynamics of actin and PI species throughout the entire process.

Mutations in the exonuclease domain of POLE are associated with tumors harboring very high mutation burdens. The mechanisms linking this significant mutation accumulation and tumor development remain poorly understood. Pole+/P286R;Trp53+/- mice showed accelerated cancer mortality compared to Pole+/P286R;Trp53+/+ mice. Cells from Pole+/P286R mice showed increased p53 activation, and subsequent loss of p53 permitted rapid growth, implicating canonical p53 loss of heterozygosity in POLE mutant tumor growth. Somewhat surprisingly, however, p53 status had no effect on tumor mutation burden or single base substitution signatures in POLE mutant tumors from mice or humans. Pten has important roles in maintaining genome stability. We find that PTEN mutations are highly enriched in human POLE mutant tumors, including many in POLE signature contexts. One such signature mutation, PTEN-F341V, was previously shown in a mouse model to specifically decrease nuclear Pten and lead to increased DNA damage. We found tumors in Pole+/P286R mice that spontaneously acquired PtenF341V mutations and were associated with significantly reduced nuclear Pten and elevated DNA damage. Further analysis in cells suggests the presence of the mutant polymerase triggers DNA damage in specific contexts. Taken together, our analysis supports the idea that POLE-mediated hypermutagenesis is necessary, but not entirely sufficient, for tumorigenesis. Disabling surveillance of nuclear DNA damage is a likely sufficient factor. Citation Format: Meijuan J. Sun, Vivian S. Park, Wesley D. Frey, Leonard G. Williams, Karl P. Hodel, Juliet D. Strauss, Sydney J. Wellens, James G. Jackson, Zachary F. Pursell. Mouse model and human patient data suggest critical roles for Pten and p53 in suppressing POLE mutant tumor development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1504.

We and others have previously shown that the breast cancers most difficult to eradicate with chemotherapy are TP53 wild-type, and these are among the most lethal breast cancers. For instance, chemotherapy-treated TNBC patients with TP53 wild-type tumors have a median overall survival of 45 months, vs 263 months for TP53 mutant tumors. Treatments fail to eradicate these cancers for two reasons: the tumor cells 1) avoid intrinsic cell death, and 2) escape immune clearance. We have shown that p53 drives a program of senescence that arrests the cell cycle to prevent intrinsic modes of cell death such as mitotic catastrophe, apoptosis and nutrient deprivation. However, it is not currently understood how breast cancer cells that enter senescence to survive chemotherapy escape immune clearance. Here, we show tumor cells from mice and human patients that survive chemotherapy activate complex programs of immune modulation. Surprisingly, the surviving, senescent tumor cells are highly enriched for many antigen presentation genes and positive regulators of T cell activation, suggesting they have switched from immunologically “cold” to “hot”. Unfortunately, these senescent tumors concurrently upregulate redundant expression of many T cell inhibitory checkpoint genes, including CD80 and PD-L1. scRNA-seq and cell imaging revealed that CD80 and PD-L1 each mark unique populations of cells in the treated tumor, typified by p53 signaling or interferon signaling, respectively. In p53 wild-type, syngeneic, orthotopic mouse mammary tumor models that recapitulate human breast cancer response to chemotherapy, treatment of tumors with chemotherapy followed by targeting of the PD-L1 and/or CD80 axes improved response, including complete eradication in some instances. Unfortunately, however, even combination strategies failed to elicit a cure in the majority of cases. Our findings reveal the formidable challenge of eliminating residual disease populated by senescent cells that express multiple redundant immune inhibitory pathways and suggest rational strategies are needed based on the specific checkpoint pathways expressed in residual disease. Citation Format: Ashkan Shahbandi, Fang-Yen Chiu, Nathan A. Ungerleider, Ashlyn Y. Anderson, Heather L. Machado, Zachary F. Pursell, Raegan Kvadas, Sonia G. Rao, James G. Jackson. Chemotherapy-induced senescence activates robust, parallel programs of immune checkpoint expression that can be targeted with immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1297.