Apr‐246 triggers ferritinophagy and ferroptosis of diffuse large b‐cell lymphoma cells with distinct tp53 mutations

Purpose: Recently, seven diffuse large B-cell lymphoma (DLBCL) genotypes were defined, among which the A53 type showed adverse outcomes. Thus, it is necessary to explore promising anticancer drugs and therapies for TP53 dysfunction patients. APR-246 is a promising compound to combat mutant p53 in solid and hematological tumors, but the effect of APR-246 on DLBCL remains unclear. Methods: A set of 2464 DLBCL cases from multiple cohorts including our center, was integrated to identify the type and localization of TP53 mutations and clinical impacts. APR-246 was applied in TP53-mutated DLBCL cells and xenograft mouse models to explore the anti-tumor effect and the underlying mechanism. Results: Of all patients, 16% contained TP53 mutations. TP53 mutations correlated with poor overall survival (OS) and progression-free survival (PFS) in all cases. Notably, TP53single mutations in the DBD (exons 5-8) resulted in poor OS and PFS, but those on the exons (3-4, 9-11) outside the DBD. DLBCL cells carrying exons 5 and 7 or intact TP53 mutations showed more sensitivity to APR-246 than those containing spliced mutations or null TP53. DLBCL cells with knocked-out TP53 showed less sensitivity to APR-246. APR-246 also significantly inhibited the DLBCL tumor growth in xenograft animal model. The decreased cell viability induced by APR-246 was almost completely rescued by either the iron chelater deferoxamine (DFO) or ferrostatin-1 (Fer-1), indicating that ferroptosis mediates cell death. APR-246-induced cell death is an action involving ROS production, which was not altered by knockout of TP53 in OCI-LY7. However, only ferroptosis was induced by APR-246 in OCI-LY3, TMD8 and TP53-knockout OCI-LY7 cells, indicating that APR-246-induced autophagy in OCI-LY7 was TP53-dependent. Further, the cells re-expressing wild-type p53 showed the similar response as OCI-LY3 harboring wild-type p53 to APR-246, while the cells with re-expressed p53 mutant showed the similar response as OCI-LY7 to APR-246. The expression of ALOX5, ALOX12, and ALOX15 were increased in DLBCL cells after APR-246 treatment for 24 h. The LC3-II level was enhanced while the autophagy substrate SQSTM1 was reduced by APR-246 treatment, confirming APR-246 induces autophagy in OCI-LY7 cells. Additionally, FTH1, NCOA4 and HERC2 were also examined in TP53 knockout DLBCL cells, and the data demonstrated that APR-246 had no effect on the level of these proteins, indicating that APR-246 restored p53 was involved in ferritinophagy in OCI-LY7 cells. These findings were further confirmed in OCI-LY7 TP53-KO cells with re-expressing mutant or wild-type p53. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Diagnostic and Prognostic Biomarkers, Genomics, Epigenomics, and Other -Omics No conflicts of interests pertinent to the abstract.