Abstract
Over-expression of the c-MYC oncogene and Trp53 gene mutations are among the most common genetic alterations in human cancer and, when combined, result in highly aggressive malignant disease. Trp53 gene mutations produce over-expressed mutant TRP53 proteins that drive cancer growth through both loss of wild-type Trp53 tumor suppressor function and gain-of-function oncogenic properties. The Eμ-Myc mouse model provides a setting to study the functional interplay between c-Myc over-expression and mutant TRP53 proteins. Eμ-Myc transgenic mice carry a c-Myc transgene under the control of the immunoglobulin heavy chain gene enhancer (Eμ), recapitulating the chromosomal translocation underlying human Burkitt Lymphoma, and develop aggressive pre-B/B cell lymphoma with a high (~20%) spontaneous rate of Trp53 mutation.
The effect of five mouse mutant TRP53 proteins (V170M, I192S, G280, R246Q, R270H) was initially examined in three settings (Trp53-/-, Trp53+/- and Trp53+/+;Eμ-Myc) using a hematopoietic stem and progenitor cell (HSPC) reconstitution model. Each mutant TRP53 protein studied corresponds to a commonly re-occurring Tp53 mutation in human cancer. Retroviral over-expression enabled the comparison of mutant-specific and genotype-specific features for each mutant TRP53 protein. Mutant TRP53 expression did not accelerate lymphoma development in mice receiving Trp53-/- or Trp53+/- HSPCs. However, mice reconstituted with Trp53+/- HSPCs expressing the TRP53 mutants displayed an altered tumor spectrum compared to mice reconstituted with control Trp53+/- HSPCs. In contrast, mutant TRP53 markedly accelerated lymphoma development in mice receiving Trp53+/+;Eμ-Myc HSPCs, highlighting a synergy between c-Myc over-expression and Trp53 mutations in neoplastic transformation. Furthermore, inducible mutant TRP53 expression demonstrated a dependency on sustained expression of mutant TRP53 in established MYC-driven lymphomas. Notably, none of the c-MYC plus mutant TRP53 driven lymphomas exhibited spontaneous endogenous Trp53 mutations.
Despite the enhanced tumorigenesis, most established lymphomas from this model displayed sensitivity to TRP53-activating drugs consistent with a weak dominant negative effect over wild-type Trp53-induced apoptosis. Consistent with this finding, pre-malignant Trp53+/+;Eμ-Myc primary B-cells expressing mutant TRP53 were not protected against Trp53-induced apoptosis. Pre-malignant B-cells displayed a small increase in cell cycling and an expansion of the tumor-initiating pre/pro-B cell population. Most significantly, functional assessment of DNA damage in pre-malignant cells, using single cell gel electrophoresis (comet assay) and γ-H2AX staining, revealed increased DNA damage, suggesting an important role for defects in DNA repair during mutant TRP53-driven lymphoma development.
To investigate the nature of the dominant negative effect, mutant TRP53 protein was exogenously expressed in mouse Eµ-Myc Trp53+/+ lymphoma cell lines. The impact of mutant TRP53 on the transcriptional function of the endogenous wild-type TRP53 protein was then studied using the TRP53-activating compound, nutlin-3a. Surprisingly, in established lymphoma cell lines, mutant TRP53 impaired nutlin-3a-induced apoptosis despite substantial induction of the critical pro-apoptotic effector, PUMA. To explore this finding further, we globally characterized the dominant negative effect, and assessed for mutant TRP53-specific transcriptional targets, by performing whole transcriptome sequence (RNAseq) analysis after treatment with nutlin-3a. Analysis of known wild-type Trp53 target genes (n=283) demonstrated that the induction of these genes as a group was repressed in the presence of the mutant TRP53 protein (ROAST test, p=6.7e-04). Remarkably, however, mutant TRP53 significantly repressed only 57% of the nutlin-3a-induced Trp53 target genes. Analysis of these strongly repressed genes highlighted the importance of several pathways, including metabolism, DNA damage repair and negative feedback loops in TRP53 signaling. This suggests a previously unrecognized selectivity of the dominant-negative-effect for certain p53 pathways that may be important in cancer initiation. Additional mutant TRP53-specific transcriptional targets were also identified and are under further investigation.
Disclosures
No relevant conflicts of interest to declare.
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