Identification of Novel Transcriptional Consequences of Activation and Inactivation of TP53 in Multiple Myeloma.

The genetic lesions important in the pathogenesis and prognosis of multiple myeloma (MM) continue to be elucidated. TP53 is deleted in approximately 10% of cases and is associated with a poor prognosis in MM. As a transcription factor, TP53 regulates the expression of genes involved in a variety of cellular functions, including cell-cycle arrest, DNA repair, and apoptosis. In the current study, we identified novel TP53 target genes and demonstrated the clinical relevance of alterations in this signaling pathway in disease progression. First, we examined the expression of 122 genes that were identified as direct TP53 target genes by paired-end diTags (PET) analysis and whose p53-dependent expression had been validated. The target genes were profiled in a collection of 351 newly diagnosed MM samples using the Affymetrix U133Plus2.0 microarrays. In this set of tumors, TP53 expression in the lowest 10^th percentile was associated with a significantly shorter event-free (P = 0.0004) and overall survival (P = 0.0001). However, expression of only a few of the previously identified TP53 target genes correlated with TP53 expression in MM cells. This suggested that TP53 may regulate a distinct set of genes in MM. To elucidate the TP53 regulatory networks in MM, we used lentiviral transduction to over express TP53 in four MM cell lines (OCI-MY5, JJN3, ARP-1 and Delta 47). An increase in steady-state TP53 level was confirmed by Western blot analysis 24 hours post lentiviral transduction and massive cell death occurred within 36 hours. Gene expression profiling revealed that a total of 85 genes were affected by TP53 expression: 50 were up-regulated and 35 were down-regulated 1.5-fold or greater in at least 3 of 4 MM cell lines. Expression analysis of primary MM revealed that the 85 genes also exhibited differential expression in comparison of those with the lowest relative to the highest TP53 expression, suggesting that TP53 may directly or indirectly regulate the expression of these genes. Of the 85 genes, 4 genes (ZMAT3, TNFRSF10B, TRIM22, and NOTCH1) were in the group of 122 P53 target genes identified by PET analysis. Consistent with known TP53 cellular functions, we found 69 of the 85 TP53 regulated genes in MM to be involved in apoptosis, cell cycle regulation, cell growth and differentiation, DNA repair and chromatin modification, and transcription regulation. Unsupervised hierarchical clustering of 351 newly diagnosed MM and 90 relapsed MM with the 85 TP53 associated genes identified 2 subgroups in each analysis. The subtype associated with lower TP53 expression had a significantly shorter EFS (P = 0.0006) and OS (P = 0.0010) in newly diagnosed MM and shorter post-relapse survival in relapsed disease (P < 0.0001). Thus we have identified MM-specific TP53 target genes by expression profiling in both cultured myeloma cells and primary tumors that correlate with clinical outcome. The identification and characterization of these pathways may lead to a better understanding of the critical role of TP53 loss in cancer.