The Transcription Factor TAp63 Exerts Pro-Survival Effects in Chronic Lymphocytic Leukemia Acting through the BCL2 Pathway

Recent evidence indicates that TAp63, the prevalent isoform of TP63 in Chronic Lymphocytic Leukemia (CLL), is implicated in disease pathogenesis. In CLL, TAp63 expression, modulated by both immune signaling and epigenetic modifications, promotes leukemic cell survival and homing to the bone marrow. In activated normal B cells, the TAp63 transcription factor binds the BCL2 gene, participating in an anti-apoptotic pathway (axis NF-κB/TAp63/BCL2) augmenting cell survival. In this study, we investigated the expression of TAp63 in a large cohort of CLL cases and its potential fluctuation during disease progression. Additionally, in order to further understand the pro-survival role of TAp63 in CLL, we interrogated at the molecular level the interplay betweenΤAp63 and BCL2.

Initially, using RT-qPCR we quantified TAp63 mRNA expression in 166 CLL patients, consisting of 89 with unmutated IGHV genes (U-CLL) and 77 with mutated IGHV genes (M-CLL), prior to administration of treatment. Significantly higher TAp63 mRNA levels were observed in U-CLL vs M-CLL (FD=13.83, p<0.0001). However, outliers were identified in both subgroups, prompting us to re-classify all cases into TAp63^high and TAp63^low subgroups using ROC curve and Youden index statistical procedures. TAp63^high patients displayed significantly shorter time-to-first-treatment (TTFT) (TAp63^highmedian TTFT: 1.58 years; TAp63^lowmedian TTFT: 4.07 years; p=0.03) and shorter overall survival (OS) (TAp63^highmedian OS: 7.825 years; TAp63^lowmedian OS: not yet reached; p=0.046). Next, we analyzed TAp63 mRNA expression in longitudinal samples of 25 U-CLL cases treated with either FCR or rituximab-chlorambucil. In each case, samples were collected at three 'landmarks'; diagnosis, first progressionand first relapse. Expression analysis by RT-qPCR showed that TAp63 levels significantly increased at disease relapse compared to diagnosis (FD=3.47, p=0.02).

We subsequently investigated links between TAp63 and BCL2 by measuring BCL2 mRNA levels in 56 U-CLL cases from the present cohort and found statistically significant correlation with the corresponding TAp63 mRNA levels (spearman rho=0.31, p=0.01). To validate this observation, we undertook functional studies in the MEC1 CLL cell line. Considering that MEC1 cells express high TAp63 mRNA levels, we generated a stable MEC1 cell line to inducibly downregulate TAp63, using CRISPR/dCas9-KRAB upon treatment with doxycycline (Dox). We used 2 different guide RNAs (sgRNAs; sgRNA1, sgRNA2) targeting 2 distinct regions of the endogenous TAp63 promoter. After 5 days of induction, the expression levels of both TAp63 and BCL2 were quantified by one step RT-qPCR in Tet-on-dCas9-KRAB-sgRNA-TAp63 MEC1 cells. Inducible downregulation of TAp63 expression (gRNA1: FD=1.7, gRNA2: FD=1.53) resulted in downregulation of BCL2 expression (gRNA1: FD=1.34, gRNA2: FD=1.12) with strong correlation (rho=0.97, p<0.0001) between TP63 and BCL2 mRNA levels. Furthermore, we also observed correlation between TAp63 and BCL2 protein expression in primary cells of one representative TP63^high CLL case (rho=0.94, p=0.01), in which TAp63 was silenced by RNA interference (RNAi) with 3 different siRNAs. Prompted by these results, we additionally assessed ex vivo the effect of the BCL2 inhibitor Venetoclax in primary CLL cells of both TAp63^high (n=8) and TAp63^low (n=6) cases. Cell viability was measured by flow cytometry at 24 and 48 hours after treatment. TAp63^high cases were more resistant to treatment with Venetoclax as they showed no statistically significant reduction in cell viability compared to the respective (DMSO-treated) controls, in contrast to TAp63^low cases (24h: FD=3.63, p=0.004; 48h: FD=7.17, p=0.005).

In conclusion, we provide evidence suggesting that up-regulated TAp63 expression represents a novel resistance mechanism to chemoimmunotherapy in CLL. The pro-survival role of TAp63 is supported by its strong association with BCL2. Indeed, based on the present findings, TAp63 appears to act as a positive modulator of BCL2 in CLL cells, rendering them less responsive to apoptosis induction with the BCL2 inhibitor Venetoclax.