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Reactivation of mutant p53 by SLMP53-2: a promising therapeutic option against hepatocellular carcinoma

ABSTRACT: In order to pursue the search for new reactivators of mutant p53, a small library of molecules was synthesized starting from a previously established lead (SLMP53-1). The molecules were tested in various cell-based assays and one, herein referred to as SLMP53-2, displayed a marked reduction of the IC50 values in NCI-H1299 cells expressing the p53 mutants (mutp53) R175H, Y220C, G245S, R248Q, R273C, and R273H. The activity of SLMP53-2 was then investigated using cancer cells lines expressing endogenous mutant p53 and focusing on hepatocellular carcinoma HuH-7 and breast ductal carcinoma HCC1419 cells endogenously expressing mutp53-Y220C. In HuH-7 cells, SLMP53-2 displayed a concentration-dependent growth inhibitory effect on colony formation, restored wt-like p53 protein conformation and transcriptional activity and displayed in vivo antitumour activity in a xenograft mouse model, with no apparent toxicity for normal tissues. To investigate more deeply the impact of SLMP53-2 treatments on gene expression in HuH-7 cells we performed microarray-based analysis using two different concentrations of the compound, corresponding to 2x and 3x IC50. We also employed the MDM2 inhibitor Nutlin alone or in combination with 2x IC50 SLMP53-2.

ORGANISM(S): Homo sapiens

PROVIDER: GSE124021 | GEO | 2020/04/13

REPOSITORIES: GEO

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Project description:Missense mutations in the TP53 gene are frequent genetic alterations in human tumor tissue and cell lines. In contrast to wild-type p53, the mutant p53 (mutp53) protein has lost the transcriptional activity towards pro-apoptotic and growth arrest genes, but retained the property to interact with DNA in a structure-specific fashion. Expression of mutp53 is advantageous for tumor cells, however the molecular mechanism of mutp53 action is still not known. We used the glioblastoma-derived U-251 MG human cell line to analyze DNA binding of mutant p53 (R273H mutation) on a Nimblegen custom 135k tiling array and to correlate mutp53 binding regions with the epigenetic state and occupation by other transcription factors (ETS1 and SP1). We found that mutp53-binding regions are G/C-rich and are located around transcriptional start sites (TSS) of many protein-coding genes, which in most cases are active, but are not always regulated upon transient mutp53 depletion. We propose a model which does not only rely on interactions of mutp53 with diverse transcriptional regulators at active promoters, but primarily is based on a DNA binding activity of mutp53. We designed a Nimblegen custom 135k tiling array that covers a large set of putative and known p53 (wild-type and mutant) target genes in the human genome. For analysis of the epigenetic state of genes covered by the tiling array in control and mutant p53-depleted U251 cells we focused on changes in active histone marks, H3K4me3 and H3K9Ac, and RNA polymerase II recruitment and processivity. H3K4me3 and H3K9Ac marks are enriched in active promoter regions and the phosphorylation of serine 5 (S5-P) and serine 2 (S2-P) in the CTD of RNA polymerase II have been described to define initiated and elongating complexes, respectively. We performed the ChIP-chip experiments for H3K4me3, H3K9Ac, RNA polymerase II (S5-P) and RNA polymerase II (S2-P) from U251 cells transfected with p53-specific siRNA or control siRNA (2 biological replicates each). To analyze binding of mutant p53 to the genes covered by the tiling array we performed mutant p53 ChIP-chip experiments in 4 biological replicates of. In addition, we analyzed the distribution of SP1 and ETS1 binding sites in 3 biological replicates.

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Reactivation of mutant p53 by SLMP53-2: a promising therapeutic option against hepatocellular carcinoma

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