ELIMINATION OF MUTATED P53 PROTEIN AND INDUCTION OF P53 DOWNSTREAM GENES IN CHRONIC LYMPHOCYTIC LEUKEMIA CELLS USING PRIMA-1MET

Background

In chronic lymphocytic leukemia (CLL), TP53 mutations represent the most adverse predictive and prognostic factor. As we have reported, particularly poor prognosis is associated with missense mutations located in DNA-binding motifs of the p53 protein. These alterations frequently lead to accumulation of mutated p53 protein with presumably oncogenic properties. Thus, the affected patients could potentially benefit from innovative therapy targeted directly to the mutated p53 protein. PRIMA-1^MET (Tocris Bioscience) is a small molecule currently tested in clinical trials, which has been declared to be able to change mutated p53 to wild-type (wt) conformation and rescue its activity.

Aims
To test a possibility of subtle molecular manipulation with accumulated mutated p53 in CLL cells using small molecule PRIMA-1^MET.

Methods
CLL patients’ peripheral blood mononuclear cells (leukemic cells’ proportion over 85%) used for reactivation study were chosen from the cohort characterized for TP53 status by FASAY and direct sequencing. The samples were analyzed for p53 protein level by western blot (WB). A metabolic activity (viability) after PRIMA-1^MET treatment was determined by WST-1 assay. Impact of 48 h PRIMA-1^MET exposure on p53 protein level was analyzed by WB. The p53 downstream genes’ induction was assessed by qRT-PCR.

Results
Firstly, the impact of PRIMA-1^MET on CLL cells’ viability was assessed to determine appropriate concentration range for the reactivation. We observed clear concentration-dependent curve of viability in all studied samples (n=5) between 0.5 and 4µM. Therefore, 2µM and 4µM were further applied for the testing of reactivation at molecular level. Five out of eleven mutated samples with high baseline p53 level showed clearly diminished or even absent p53 protein after 4µM PRIMA-1^MET treatment and four other samples manifested partially reduced level. The effects at 2µM were also apparent, but less pronounced. The best response was observed for mutations p.Y205H, p.R248W, and p.C275R, in which the p53 protein was completely eliminated even at 2µM concentration. The p53 downstream genes’ expression was heterogeneous among the samples, with the induction of CDKN1A (p21) and GADD45 being much more prominent compared to PUMA. The expression induction reached up to 11800% (p21) and 4500% (GADD45) in comparison with untreated control set at 100%. The BAX and MDM2 genes’ expression was not increased after treatment. This indicates that observed elimination of mutated p53 is not probably caused through the induction of the p53 basic negative regulator MDM2. Concerning the specificity of observed effects with respect to TP53 status, we noted the following: (i) in line with other studies, we noted similar impact of PRIMA-1^MET on both p53-wt and p53-mut samples’ viability, and (ii) certain induction of p53 target genes was also evident in p53-wt samples. In addition, we also analyzed 10 samples with TP53 mutation, but without p53 protein stabilization, for induction of the downstream genes. Again, the effects of PRIMA-1^MET were apparent, especially in case ofp21 and GADD45.

Summary
We have been able to demonstrate that PRIMA-1^MET can significantly diminish or even completely eliminate p53 protein accumulated due to the high-risk mutations in TP53 gene. It still remains to be elucidated, what is the specificity of the p53 pathway activation with respect to TP53 status. Supported by grants NT/13519-4/2012 and MUNI/A/1180/2014.

Keyword(s): Chronic lymphocytic leukemia, P53

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