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The inactivation of p53 functions enhances the efficiency and decreases the latency of producing induced pluripotent stem cells (iPSC) in culture. The formation of iPSCs in culture starts with a rapid set of cell divisions followed by an epigenetic reprogramming of the DNA and chromatin. The mechanisms by which the p53 protein inhibits the formation of iPSCs are largely unknown. Using a temperature sensitive mutant of the p53 (Trp53) gene, we examined the impact of the temporal expression of wild type p53 in preventing stem cell induction from somatic cells. We also explored how different p53 mutant alleles affect the reprogramming process. We found that little or no p53 activity favors the entire process of somatic cell reprogramming. Reactivation of p53 at any time point during the reprogramming process not only interrupted the formation of iPSCs, but also induced newly formed stem cells to differentiate. Among p53-regulated genes, p21 (Cdkn1a), but not Puma (Bbc3) played a partial role in iPSCs formation probably by slowing cell division. Activation of p53 functions in iPSCs induced senescence and differentiation in stem cell populations. High rate of birth defects and increases in DNA methylation at the IGF2-H19 loci in female offspring of p53 knockout mice suggested that the absence of p53 may give rise to epigenetic instability in a stochastic fashion. Consistently, selected p53 missense mutations showed differential effects on the stem cell reprogramming efficiency in a c-Myc dependent manner. The absence of p53 activity and functions also contributed to an enhanced efficiency of iPSC production from cancer cells. The production of iPSCs in culture from normal and cancer cells, although different from each other in several ways, both responded to the inhibition of reprogramming by the p53 protein. Cancer Res; 72(21); 5635–45. ©2012 AACR.

Among patients with neuroendocrine tumors, males are more likely than females to develop liver metastatic disease. We show that this male bias toward liver metastasis is also observed in a mouse model for pancreatic neuroendocrine tumorigenesis (PanNETs). Interestingly, primary tumors from mice with metastatic disease display hepatomimicry, expressing genes normally expressed in liver. Both male and female primary tumors from mice with metastatic disease show hepatomimicry, although one of the liver genes, complement C5, is only induced in males. In mice knocked out for the complement C5 gene, the frequency of liver metastasis dropped significantly; moreover, males and females lacking a gene for complement C5 developed liver metastasis at the same frequency. Liver metastasis was also prevented by PMX53, a small molecule antagonist of C5aR1/CD88, which is the receptor for complement C5a. Thus complement C5 is epistatic to metastasis and promotes gender bias of liver metastatic disease. Citation Format: Tanupriya Contractor, Chang Chan, Shinta Kobayashi, Richard Clausen, Yvonne Sun, Edaise da Silva, Evan Vosburgh, Arnold J. Levine, Laura Tang, Chris R. Harris. Complement C5 promotes male bias of pancreatic neuroendocrine tumor metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3185. doi:10.1158/1538-7445.AM2015-3185

Loss of the tumor suppressor, p53, occurs in over 50% of cancers. The ability for p53 to protect the cell from tumorigenesis is based on its ability to induce cell cycle arrest, apoptosis, and senescence following genotoxic stress. Furthermore, genetic deletion of p53 in mice gives rise to spontaneous thymic (T-cell) lymphomas and sarcomas. In this study, we used the p53-knockout (KO) mouse as a model for progression of tumorigenesis. We first analyzed genomic DNA of p53-KO and WT normal thymus at various ages and p53-KO thymic lymphomas using TCRβ sequencing, allowing us to determine the clonality of the T cell repertoire. These data suggest that p53-KO thymic lymphomas were mostly oligoclonal, with some tumors comprising of a dominant clone. We further analyzed tumors with dominant clones for genomic instability by exome sequencing. Sequencing analysis revealed that there were many non-synonymous mutations occurring in each tumor, but with no similarities between tumors. On the other hand, all of the murine tumors had deletion of Pten and amplification of Cdk6. This is in stark contrast to what is seen in most human T cell lymphoblastic leukemias, which have activating mutations in Notch1 and loss of Arf. Interestingly, loss of Pten not only occurred in all tumors, but also in most, if not all, clones in the tumor. This suggests that Pten loss is an early event driving T cell transformation in a p53-KO background. Using this data, we found the somatic mutation rate in the tumors to be 1000-fold greater than the germline mutation rate. Loss of p53 also caused chromosomal abnormalities as seen with increased copy number variations. Taken together, our work suggests that, with initial loss of p53, the path to T cell tumorigenesis is vastly different from that of human tumors. Without p53, the somatic genome increases in mutation rate and copy number variations, thus solidifying p53's role as guardian of the genome. Citation Format: Crissy Dudgeon, Chang Chan, Yvonne Sun, Arnold J. Levine. Exome sequencing of murine p53-knockout T-cell lymphomas reveals Pten loss and Cdk6 amplification as key steps toward cellular transformation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-80. doi:10.1158/1538-7445.AM2014-LB-80

The tumor suppressor p53 is nonfunctional in over 50% of cancers. The ability for p53 to protect the cell from tumorigenesis is rooted in its ability to induce cell cycle arrest, apoptosis, and senescence following genotoxic stress. Furthermore, loss of p53 in mice gives rise to spontaneous thymic lymphomas and sarcomas. In this study, we used the p53-KO mouse as a model for progression of tumorigenesis. We first analyzed genomic DNA of p53-KO and WT normal thymus at various ages using TCRβ sequencing. This procedure allowed us to determine the clonality of the T cell repertoire. We then analyzed the clonality of p53-KO thymic lymphomas. Our results indicated that while the highest T cell clone was 0.08% for normal thymus, the highest T cell clone for the p53-KO tumor was 70%, indicating that the p53-KO thymic lymphomas were monoclonal. Building on this data, we analyzed the mutations occurring in the thymic lymphomas by mouse exome sequencing. We believe that combining TCRβ sequencing with mouse exome sequencing will allow for a better understanding of the clonality and genetic mutations required for cellular transformation. Citation Format: Crissy Dudgeon, Chang Chan, Yvonne Sun, Arnold J. Levine. Utilizing TCRβ and whole exome sequencing to determine the clonality and mutational spectrum of p53-null thymic lymphomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 796. doi:10.1158/1538-7445.AM2013-796