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The tumor protein p53 (TP53, best known as p53) transcription factor is a critical tumor suppressor, but those p53-inducible genes most important for tumor suppression have remained unclear. Using unbiased RNA interference and CRISPR (Clustered Regularly Interspersed Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) screens, genetically engineered mouse models, human cancer genome analysis, and integrative eCLIP-sequencing and RNA-sequencing analyses, we reveal a new branch of p53-mediated tumor suppression involving the RNA splicing regulator Zinc finger Matrin-type 3, Zmat3.

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, −3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies.

The p53 transcription factor is a critical barrier to pancreatic cancer progression. To unravel mechanisms of p53-mediated tumor suppression, which have remained elusive, we analyzed pancreatic cancer development in mice expressing p53 transcriptional activation domain (TAD) mutants. Surprisingly, the p5353,54 TAD2 mutant behaves as a "super-tumor suppressor," with an enhanced capacity to both suppress pancreatic cancer and transactivate select p53 target genes, including Ptpn14. Ptpn14 encodes a negative regulator of the Yap oncoprotein and is necessary and sufficient for pancreatic cancer suppression, like p53. We show that p53 deficiency promotes Yap signaling and that PTPN14 and TP53 mutations are mutually exclusive in human cancers. These studies uncover a p53-Ptpn14-Yap pathway that is integral to p53-mediated tumor suppression.

summary Background: Oral cavity and in particular oral tongue cancers occur with a rising incidence in younger patients often lacking the typical risk factors of tobacco use, alcohol use, and human papilloma virus (HPV) infection. Their prognosis when treated with chemoradiation has not been well studied and responsible risk factors remain elusive. A viral etiology (other than HPV) has been hypothesized. Methods: First we analyzed outcomes from 748 head and neck cancer patients with locoregionally advanced stage tumors treated with curative-intent chemoradiation by anatomic site. Second, we analyzed seven oral tongue (OT) tumors from young, non-smokers/non-drinkers for the presence of viral mRNA using short-read massively-parallel sequencing (RNA-Seq) in combination with a newly-developed digital subtraction method followed by viral screening and discovery algorithms. For positive controls we used an HPV16-positive HNC cell line, a cervical cancer, and an EBV-LMP2A transgene lymphoma. Results: Younger patients with oral cavity tumors had worse outcomes compared to non-oral cavity patients. Surprisingly none of the seven oral tongue cancers showed significant presence of viral transcripts. In positive controls the expected viral material was identified. Conclusion: Oral cavity tumors in younger patients have a poor prognosis and do not appear to be caused by a transcriptionally active oncovirus.

The p53 transcription factor is a critical tumor suppressor in humans and mice. Despite this essential function, the molecular pathways through which p53 acts in tumor suppression remain enigmatic. To define the transcriptional programs through which p53 suppresses carcinogenesis, we have taken combined mouse genetic and genomic approaches. We previously generated a panel of p53 knock-in mouse strains expressing mutants in the first (p5325,26), second (p5353,54), or both (p5325,26,53,54) of two amino-terminal transcriptional activation domains (TADs). In terms of transcriptional activity, we have discovered that p5325,26 is severely impaired for transactivation of the majority of canonical p53 target genes (e.g. p21, Puma, Noxa), but retains the ability to activate a set of primarily novel p53 target genes, while p5325,26,53,54 lacks transactivation activity altogether. The p5353,54 mutant, in contrast, is uncompromised for transactivation of p53 target genes. The p5325,26,53,54 mutant is completely defective in tumor suppression, underscoring the importance of transcriptional activation for p53-mediated tumor suppression. Intriguingly, the p5325,26 mutant retains full activity in suppressing a variety of cancers, indicating that efficient transactivation of most canonical p53 target genes is dispensable for tumor suppression. As p5325,26 activates only a subset of p53-inducible genes, yet retains tumor-suppressor activity, it has helped pinpoint a small set of novel, direct p53-inducible tumor suppression-associated target genes (TSAGs) whose functions we are currently interrogating through genetic screens. Additionally, we have made the surprising discovery that the p5353,54 mutant suppresses pancreatic cancer more efficiently than wild-type p53, and that this capacity correlates with the ability of p5353,54 to hyperactivate a subset of p53 target genes. Indeed, our analysis of these genes has uncovered Ptpn14, a direct p53 target gene critical for mediating p53 function in suppressing pancreatic cancer cell growth. Ptpn14 encodes a protein tyrosine phosphatase that negatively regulates the oncoprotein Yap in both human and mouse cells. These studies thus reveal a p53-Ptpn14-Yap axis in pancreatic cancer. Together, these strategies will help to elaborate the transcriptional networks fundamental for p53 function in tumor suppression. Citation Format: Stephano Spano Mello, Kathryn Bieging-Rolett, Alyssa Kaiser, Elizabeth Joy Valente, Nitin Raj, Jacob McClendon, Brittany Maria Flowers, David Warren Morgens, Michael Cory Bassik, Laura Donatella Attardi. Deconstructing p53 pathways in tumor suppression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr IA07.

The p53 tumor suppressor is a stress sensor, driving cell cycle arrest or apoptosis in response to DNA damage or oncogenic signals. p53 activation by oncogenic signals relies on the p19(Arf) tumor suppressor, while p53 activation downstream of acute DNA damage is reported to be p19(Arf)-independent. Accordingly, p19(Arf)-deficient mouse embryo fibroblasts (MEFs) arrest in response to acute DNA damage. However, p19(Arf) is required for replicative senescence, a condition associated with an activated DNA damage response, as p19(Arf)-/- MEFs do not senesce after serial passage. A possible explanation for these seemingly disparate roles for p19(Arf) is that acute and chronic DNA damage responses are mechanistically distinct. Replicative senescence may result from chronic, low-dose DNA damage responses in which p19(Arf) has a specific role. We therefore examined the role of p19(Arf) in cellular responses to chronic, low-dose DNA-damaging agent treatment by maintaining MEFs in low oxygen and administering 0.5 G y γ-irradiation daily or 150 μM hydroxyurea, a replication stress inducer. In contrast to their response to acute DNA damage, p19(Arf)-/- MEFs exposed to chronic DNA damage do not senesce, revealing a selective role for p19(Arf) in senescence upon low-level, chronic DNA damage. We show further that p53 pathway activation in p19(Arf)-/- MEFs exposed to chronic DNA damage is attenuated relative to wild-type MEFs, suggesting a role for p19(Arf) in fine-tuning p53 activity. However, combined Nutlin3a and chronic DNA-damaging agent treatment is insufficient to promote senescence in p19(Arf)-/- MEFs, suggesting that the role of p19(Arf) in the chronic DNA damage response may be partially p53-independent. These data suggest the importance of p19(Arf) for the cellular response to the low-level DNA damage incurred in culture or upon oncogene expression, providing new insight into how p19(Arf) serves as a tumor suppressor. Moreover, our study helps reconcile reports suggesting crucial roles for both p19(Arf) and DNA damage-signaling pathways in tumor suppression.

An iron-dependent form of cell death called ferroptosis has been implicated as a component of the tumour-suppressor activity of p53, providing fresh insight into how this protein prevents cancer development. See Article p.57

The p53 transcription factor is mutated in the majority of human pancreatic cancers, underscoring its critical role in suppressing pancreatic cancer development. Although p53 can restrain neoplastic cell expansion by inducing cell-cycle arrest or apoptosis in response to diverse stress signals, the molecular pathways through which p53 acts in tumor suppression remain largely elusive. To define the transcriptional networks underlying p53 tumor suppressor function, we have used mouse genetic and genomic approaches. We generated a set of p53 knock-in mouse strains expressing mutants in the first (p53 25,26), second (p5353,54), or both (p5325,26,53,54) of two transcriptional activation domains (TADs). Using a combination of ChIP-sequencing and expression profiling on cells expressing these mutants, we have been able to define a limited list of direct p53 target genes whose expression is tightly correlated with tumor suppression. Specifically, we found that p5325,26 is severely compromised for transactivation of most classical p53 target genes (e.g. p21, Puma), but retains the ability to activate a subset of p53 target genes, while p5325,26,53,54 lacks transactivation activity completely. The p5325,26,53,54 mutant is completely inactive in tumor suppression, highlighting the importance of transactivation for p53-mediated tumor suppression. Interestingly, however, p5325,26 retains full activity in suppressing various cancers, including pancreatic cancer, indicating that efficient transactivation of most canonical p53 targets is dispensable for tumor suppression. As p5325,26 activates only a subset of p53-dependent genes, yet retains tumor suppressor activity, it has helped pinpoint a small set of novel, direct p53-inducible tumor suppression-associated genes (TSAGs) whose functions we are currently interrogating through genetic screens. In addition, we have made the surprising discovery that the p5353,54 mutant suppresses pancreatic cancer more effectively than wild-type p53, and that this capacity correlates with the ability of p5353,54 to hyperactivate a subset of p53 target genes. Indeed, our analysis of these genes has unveiled p53 targets critical for mediating p53 function in suppressing mouse and human pancreatic cancer cell growth, including a protein tyrosine phosphatase that negatively regulates Yap signaling. Collectively, these approaches will delineate the transcriptional networks fundamental for p53 function in tumor suppression, an approach that will ultimately facilitate the design of novel therapies for pancreatic cancers with p53 mutation. Citation Format: Laura D. Attardi, Stephano Spano Mello, Elizabeth J. Valente, Pawel K. Mazur, Nitin Raj, Kathryn Bieging-Rolett, Hannes Vogel.{Authors}. Deconstructing p53 transcriptional networks in pancreatic cancer suppression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr PR07.

Burkitt’s lymphoma (BL) is characterized by translocation of the MYC gene to an immunoglobulin locus. Transgenic mouse models have been used to study the molecular changes that are necessary to bypass tumor suppression in the presence of translocated MYC. Inactivation of the p53 pathway is a major step to tumor formation in mouse models that is also seen in human disease. Human BL is often highly associated with Epstein-Barr virus (EBV). The EBV latency protein latent membrane protein 2A (LMP2A) is known to promote B cell survival by affecting levels of pro-survival factors. Using LMP2A transgenic mouse models, we have identified a novel mechanism that permits lymphomagenesis in the presence of an intact p53 pathway. This work uncovers a contribution of EBV to molecular events that have documented importance in BL pathogenesis, and may underlie the poorly understood link between EBV and BL.

The activation of the p53 protein by cellular stress signals is fundamental for tumor suppression but also promotes pathological states, such as provoking the side effects of genotoxic cancer therapies. To better understand the mechanisms of p53 action in different contexts, we have leveraged both mouse genetic and genomic approaches. First, we have used mouse genetics to define transcriptional programs involved in p53 function in different in vivo settings, specifically by generating a panel of p53 transcriptional activation domain mutant knock-in mouse strains. These include strains expressing p53 mutants in the first (p5325,26), second (p5353,54), or both transactivation domains (p5325,26,53,54). We have observed that p5325,26 is severely compromised for transactivation of most classical p53 target genes, but retains the ability to activate a subset of p53 targets, while p5325,26,53,54 lacks transactivation activity completely. Interestingly, although unable to induce apoptosis or cell cycle arrest in response to acute DNA damage signals, p5325,26 retains full activity in suppressing cancers of a wide range of types, indicating that robust transactivation of most canonical p53 targets is dispensable for tumor suppression. Importantly, as p5325,26 activates only a subset of p53-dependent genes, yet retains tumor suppressor activity, it has helped to define a small set of novel p53-inducible tumor suppression-associated genes, which we are currently analyzing in detail. Second, we have utilized genomic approaches to better understand p53 function. Using ChIP-sequencing and RNA-sequencing to analyze transcriptional programs in acute DNA damage-treated mouse embryo fibroblasts, our studies have revealed an extensive p53-regulated autophagy program that contributes to p53 responses. Together, these approaches will help better define the transcriptional networks important for p53 action in different settings. Citation Format: Colleen A. Brady, Daniela Kenzelmann Broz, Dadi Jiang, Stephano Spano Mello, Kathryn Bieging, Thomas M. Johnson, Leslie A. Jarvis, Margaret M. Kozak, Shashwati Basak, Laura D. Attardi. Deconstructing p53 pathways in vivo. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA4.

Although Epstein-Barr virus (EBV) is linked to Burkitt's lymphoma (BL), the role of the virus in lymphomagenesis is unclear. LMP2A, encoded by EBV, is detected in BL biopsies and has pro-survival functions. We generated mice expressing MYC and LMP2A in B cells. Pretumor LMP2A/λ-MYC mice have significantly enlarged spleen size. In addition, LMP2A/λ-MYC mice show greatly accelerated tumor onset compared to λ-MYC mice. Inactivation of the p53 pathway and failure to induce the pro-apoptotic BH3-only protein, BIM are two major paths to tumor formation in MYC tumor models. Similar to previous work, we found p53 mutations in λ-MYC tumors, however we detected no p53 mutations in the rapidly arising LMP2A/λ-MYC tumors. We further demonstrate that the p53 pathway is functionally intact in LMP2A/λ-MYC tumors, which show increased levels of PUMA and sensitivity to p53 activation by Nutlin. We also show that BIM is induced in LMP2A/λ-MYC tumor cells. Despite the presence of p53 mutations in EBV+ human BL, our mouse model can be used to understand BL development in humans. We believe that LMP2A is important early in lymphomagenesis. The pro-survival function of LMP2A leads to an initial expansion of cells that have acquired a MYC translocation. This expansion is shown in our mouse model by enlarged spleens in pre-tumor mice. In humans, early expansion of cells increases the probability that a p53 mutation will occur in a cell with translocated MYC. Once the p53 mutation occurs, weak immune surveillance may select against high levels of LMP2A, consistent with low levels of LMP2A transcripts found in tumor biopsies. We have identified a novel mechanism that permits lymphomagenesis in the presence of an intact p53 pathway. This work also uncovers a contribution of Epstein-Barr virus (EBV) to molecular events that have documented importance in BL pathogenesis, and may underlie the poorly understood link between EBV and BL. Citation Information: Cancer Res 2009;69(23 Suppl):B30.