Proliferating CLL Cells Express Abundant But Transcriptionally Compromised TP53 Protein
Authors: Samuel Jesvin J , Wignall Alice H , Najeeb Hishyar , Majid Aneela , Jayne Sandrine , Jones George D , Macip Salvador , Dyer Martin JS 
Published: October 2019

Abstract
Current models of CLL pathogenesis invoke specialized microenvironments within the lymph nodes and bone marrow that harbor proliferating cells. Such proliferating CLL cells are more resistant to current immuno-chemotherapeutic regimens than cells in the peripheral blood and are thought to be the cause of resistant disease. Various models have been used to recapitulate these CLL proliferation centers in vitro, including stimulating cells with CD154 and IL4. We studied 40 patients (6 with p53 mutations/deletions) using this system and observed that >50% of CLL cells undergo proliferation after 72 hours of stimulation, as assessed by Ki67 staining. Unexpectedly, under these conditions we also observed a 30-40 fold induction of TP53 protein in all cases of CLL analyzed, irrespective of TP53 mutational status. Nearly all cells showed increased TP53 protein expression (Figure 1), suggesting that such high-level TP53 protein expression did not hinder cell proliferation. Given that induction of wild-type TP53 protein usually induces cell cycle arrest if not apoptosis, we examined for transcriptional up-regulation of TP53 target genes using a combination of qRT-PCR, RNA arrays and RNA-Seq approaches. 4 out of 12 cases showed induction of some TP53 target genes, but overall there was no consistent pattern of transcriptional up-regulation of target genes, suggesting that the induced TP53 is transcriptionally compromised in CLL cells following CD154/IL4 stimulation. In contrast, DNA damage induced by doxorubicin in CD154/IL4 stimulated cells induced wild type TP53 protein to even higher levels, resulting in TP53 target gene up-regulation and apoptosis, as expected. CD154/IL4 stimulation also induced a 10-fold elevation in ROS levels in all cases. This resulted in significant oxidative DNA damage, as measured by a modified comet assay, which could explain the induction of TP53 in proliferating cells. qRT-PCR and RNA-Seq experiments failed to show a significant increase in TP53 mRNA levels, indicating that elevation of TP53 protein levels was occurring post-transcriptionally. Increased phosphorylation of TP53 at S15 was seen in all cases, which may account for the observed increased protein stability through dissociation from MDM2. All TP53 mRNA isoforms expressed retained transcriptional activation and DNA binding domains. In view of these results, we propose a model whereby oxidative stress induced by proliferation in CLL triggers TP53 protein expression. TP53 becomes phosphorylated but, for reasons that remain unclear, is unable to transactivate its target genes normally and induce cell-cycle arrest. Apoptosis could be suppressed by high-level expression of anti-apoptotic BCL2 proteins. However, TP53 remains able to trigger a full apoptotic response after further DNA damage and a higher threshold of protein levels is reached. Reactivation of the full transcriptional activities of wild-type TP53 in proliferating CLL cells may provide a new therapeutic approach.Figure 1CD154/IL4 stimulation increases CLL proliferation and induces TP53 expression. Top panel: Time course of Ki67 and TP53 expression in CD19+ CLL cells stimulated with rhCD154 and rhIL4. Bottom panel: Representative immunoblot of TP53 expression in CLL cells after 1, 3 and 7 days of co-culture with mouse L cells (NTL) or rhCD154 transfected-L Cells and rhIL4.Figure 1. CD154/IL4 stimulation increases CLL proliferation and induces TP53 expression. Top panel: Time course of Ki67 and TP53 expression in CD19+ CLL cells stimulated with rhCD154 and rhIL4. Bottom panel: Representative immunoblot of TP53 expression in CLL cells after 1, 3 and 7 days of co-culture with mouse L cells (NTL) or rhCD154 transfected-L Cells and rhIL4.

Disclosures:
No relevant conflicts of interest to declare.