1010 - The interplay between BcR signaling and the p53 pathway upon DNA damage in primary CLL cells

Background: In the clinical management of chronic lymphocytic leukemia (CLL), inhibitors targeting the B-cell receptor (BcR) are considered to act independently of the p53 pathway. However, different frequencies of TP53 gene defects in CLL subgroups harboring distinct antigen receptors suggested a potential interplay between BcR signaling and p53. We aimed to study whether the therapeutical inhibition of BcR signaling may affect this interplay and whether potential off-target effects of BTK kinase inhibitors affect p53 phosphorylation and function.

Methods: To explore the potential BcR-p53 crosstalk, we performed in-silico differential expression analysis after BcR activation using publicly available transcriptomic data (PMID: 33833385). Subsequently, we examined the effect of BcR signaling inhibitors (ibrutinib, idelalisib) in combination with DNA damage agents (doxorubicin, fludarabine) on p53 protein level by western blot analysis in 37 primary CLL cell samples with wild-type TP53 locus. In addition to monitoring p53 level, we analyzed the p53 phosphorylation pattern by Zn(II)Phos-Tag method followed by western blots. The transcriptional activity of p53 was assessed using qPCR focused on the expression of four p53 targets: BAX, BBC3, CDKN1A, and GADD45A.

Results: In-silico analysis of publicly available data showed that activated BcR signaling led to significant changes (FDR < 0.05; logFC≥|2|) in the expression of 13 out of 116 typical p53 targets. Next, we investigated the effect of BcR signaling inhibition on the p53 protein stabilized with DNA-damaging drugs in wt-TP53 primary CLL cells. While idelalisib showed a heterogeneous effect on the p53 level with either of DNA damaging drugs, ibrutinib combined with doxorubicin led to a significant and homogeneous decrease in the p53 level compared to a DNA-damaging drug alone. As the stability of p53 is dependent on its phosphorylation, we further explored whether ibrutinib affects the p53 phosphorylation pattern, and we found that the p53 phosphorylation pattern remained unchanged. Next, we used qPCR to evaluate whether decreased p53 level affected the expression of p53 target genes (BAX, BBC3, CDKN1A, and GADD45A). As expected, p53 activation by doxorubicin led to a significant increase in the expression of four target genes. However, samples treated with the combination of doxorubicin and ibrutinib showed either no significant difference in the expression level (CDKN1A, BBC3) or exhibited a higher expression of p53 target genes (BAX, GADD45A) compared to doxorubicin alone.

Conclusion: We documented the interplay between BcR signaling and the p53 pathway in primary CLL cells. While BTK inhibition in DNA-damaging conditions led to the reduction of p53 level, the expression of p53 target genes remained stable. It suggests that p53 target genes were either activated via compensatory mechanisms involving alternative p53-independent pathways or that strong activation by upstream signaling cannot be overcome solely by reducing p53 levels in the presence of persistent DNA damage.