Dysregulation of NF-kb and p73 signal axes in chronic lymphocytic leukemia with gain-of-function mutated p53

Abstract Background: Genetic alterations in TP53 are common in CLL (10% at diagnosis, 40% at relapse) and are thought to be the foremost marker for adverse prognoses, typically presenting as genomic deletions encompassing the gene locus of TP53 on chromosome 17 (del17p). Biallelic alteration of TP53 occurs in up to 80% of CLL patients harboring del17p and is often observed as hot-spot missense mutations (85% of cases) in the DNA binding domain (DBD) of TP53 on the remaining allele. This commonly results in gain-of-function (GOF) p53 mutants such as R175H, R273H, and R248Q (7%, 6%, and 5% of all TP53 point mutations in CLL, respectively). Yet, there is little knowledge surrounding the functional changes of GOF p53 mutants in CLL, and a need to better understand the associated biological consequences given their prevalence. Methods: Mutant p53 cell lines were generated using CRISPR/Cas9 knock-in to induce either R175H or R248Q point mutation in the OSUCLL cell line. Whole cell lysates were collected for proteomic analysis. Findings from this dataset, as well as exploration of key pathways from ingenuity pathway analysis (IPA) were assessed via immunoblotting. Changes in mRNA levels were evaluated via quantitative real-time PCR (qPCR). Additionally, flow cytometry was used to analyze differences in surface protein expression. Results: Immunoblot assessment of classical NF-kB signaling revealed a modest increase of nuclear p65 in mutant p53 cell lines (60% and 40%, R175H and R248Q, respectively, p<0.05). mRNA and surface expression levels of downstream targets PD-L1, CD86, and VCAM-1 were also increased, relative to WT (100%, 50%, and 250%, respectively, p<0.05). In addition to classical NF-kB, we observed an increase in alternative NF-kB signaling via expression of total p100 protein (90% for both mutants, p<0.05) in GOF mutant p53 cells lines with concomitantly elevated levels of p52 in R175H and R248Q mutants (70% and 60% increase, respectively, p<0.05). Further downstream, we detected an increase in mRNA levels and surface expression of CD122 (IL2rb), a gene target of p52. Proteomic analysis revealed heightened expression of p73 in GOF p53 cell lines, compared to WT (1.6-fold increase, p<0.0001). This finding was validated via immunoblotting for total p73 (500% increase, both mutants, p<0.05). By nature, p73's effects are pleiotropic, so we sought to determine whether differential expression was due to a specific isoform. Immunoblotting for N-terminally truncated (DN) isoforms of p73 suggests augmented expression in our models (600% increase, p<0.05). Lastly, preliminary coimmunoprecipitation experiments support this notion and suggest p53 harboring the R175H mutation interacts more favorably with p73 and has diminished downstream p21 protein expression. Conclusion: Work in solid tumors has demonstrated a diminished regulatory capacity of GOF p53 over NF-kB, resulting in sustained NF-kB signature in the presence of GOF p53 mutants. We believe our data reflect a similar abrogation in mutant GOF p53 oversight of NF-kB activity in CLL, as we observed heightened NF-kB signaling in CLL harboring GOF p53 mutations. These trends were associated with concomitantly increased p73 expression, namely the DN isoforms, which can be driven through NF-kB signaling. Interestingly, it has previously been reported that overexpression of p73 is correlated with del17p in CLL. In the context of GOF mutants in CLL, we believe increased DNp73 expression drives disease aggression by repressing transcription of classical p53 target genes in p53 GOF mutated CLL. This may be potentiated by GOF mutants mitigating p53's transactivation ability through enhanced cytosolic sequestration of p53 through R175H mutant's more favorable interaction with p73. It is also possible that DNp73 disrupts p53 through alternative mechanisms, such as promoter sequestration, though further studies are needed to support this notion. Altogether, our data demonstrate potential avenues of increased disease aggression in CLL harboring GOF mutations in p53 and support the continued investigation of NF-kB and p73 signaling in CLL with GOF p53 mutations.