Selinexor Induces DLBCL Cell Senescence Mediated By USP1/MDMX/p53 Axis

Background:

Despite the increasing number of treatment options available for Diffuse Large B-Cell Lymphoma (DLBCL), only about half of patients achieve long-term clinical benefits. Selinexor, the selective XPO1 inhibitor, received approval as single agent for relapsed or refractory (R/R) DLBCL. Abundant evidence indicates that the nuclear accumulation of wild-type p53 plays a pivotal role in Selinexor's anti-tumor effect. A comprehensive understanding of Selinexor's involvement in inhibiting tumor cell growth by TP53 will further facilitate the advancement of precision therapy.

Methods:

XPO1 is a critical receptor in nuclear-cytoplasmic transport, responsible for exporting proteins from the nucleus to the cytoplasm. Hence, we employed data-independent acquisition (DIA) proteomics to gain insight in Selinexor-induced changes of the nuclear and cytoplasmic protein. To investigate the mechanistic impacts of the nuclear and cytoplasmic accumulation of wild-type p53, we used senescence-associated β-galactosidase (SA-β-gal) assays followed by cell cycle, apoptosis, co-IP, and ubiquitination assay.

Results:

By analyzing the results of the proteomics data, we identified that differentially expressed proteins of both nuclear and cytoplasmic were significantly enriched for ‘p53 signaling pathway’ and ‘cell cycle’ compared with control group. Gene Set Enrichment Analysis (GSEA) of proteomics data revealed that gene sets related to p53 pathway were up-regulated, while E2F targets were down-regulated in the Selinexor treated cells compared to control cells. This finding indicated that Selinexor induces a senescence-like state in DLBCL cells. For further validation, SA-β-gal staining showed positive upon Selinexor treatment in vitro.

Similarly to previous study reported, our proteomics analysis and sequential validation indicated that p53 protein was blocked in the nucleus after Selinexor treatment. But likewise, we found that p53 protein was accumulated in the cytoplasm of the DLBCL cells. We further investigated the molecular basis with how the treatment of Selinexor regulates p53 expression and cellular senescence. Through a series of in vivo and in vitro experiments, we discovered that Selinexor could upregulate p53 protein stability by reducing MDMX-p53 interaction. Based on this evidence, we consider it likely that reactivation of p53 protein induced by Selinexor could regulate tumor cell growth inhibition and cellular senescence.

In addition, the treatment of Selinexor in DLBCL cells could impact ubiquitination of MDMX protein. To analysis the Pearson correlation with the expression of XPO1, we focused on a deubiquitinating enzyme, USP1, that could be an effective therapeutic strategy for the treatment of DLBCL. To validate this hypothesis, we knocked down USP1, which resulted in a substantial decline in MDMX protein level. Consistently, knockdown of USP1 could also induce cellular senescence of DLBCL cell lines.

Conclusions:

Our study investigated the function and pathogenic mechanism of p53 protein in the treatment of Selinexor. We demonstrated that the treatment of Selinexor could promote p53 protein stability to further induce a senescence-like state in DLBCL cells by regulating the USP1/MDMX/p53 axis. Overall, this study highlights that reactivating p53 play important roles in the treatment of Selinexor for DLBCL patients.