CD20-Modified Exosomes Delivered E260 Inhibits Progression of Diffuse Large B-Cell Lymphoma By Restoring Hippo Activity

Introduction: Fer tyrosine kinase (Fer) is ubiquitously expressed in various cancer types and has been proved to play a pivotal role in tumorigenesis and metastasis. E260, as an effective inhibitor of Fer, is selective lethal to cancer cells. However, the effect of Fer and E260 in diffuse large B-cell lymphoma (DLBCL) remain elusive. Thus, we set out to investigate the function and mechanisms of E260 targeting Fer in DLBCL for a treatment purpose.

Methods: The impact of Fer expression to predict clinical outcome was evaluated in 50 newly diagnosed DLBCL and 30 healthy donors between 2014 and 2019 by immunohistochemistry. This study was approved by the ethics board of Shandong Provincial Hospital, and informed consent was obtained. Next, we assessed the oncogenic effect of Fer by using gain-and loss-of-function assays. Fer inhibitor E260 was used in the treatment of DLBCL cells. CD20 targeted exosomes were isolated from CD20 transfected HEK293T cells and then incubated with E260 in order to generate CD20-EXO-E260. The anti-tumor efficacy of E260 and CD20-EXO-E260 were assessed by cell proliferation, apoptosis, migration and drug sensitivity assays. To elucidate the regulatory mechanism, we test the expression of Hippo-YAP pathway and YAP location after E260 treated by western blotting and immunofluorescent staining. Interaction of the Fer and AJUBA was further validated by coimmunoprecipitation (Co-IP) assay. To further explore the clinical value of Fer, we isolated and test Fer expression in exosomes from the plasma of DLBCL patients. ROC curve was performed to test the diagnostic value of exosomal Fer in DLBCL.

Results: To explore the association of Fer expression with DLBCL tumorigenesis, we examined Fer expression using immunohistochemical staining. Fer was upregulated in DLBCL patients (Fig.1A) and correlated with adverse clinical parameters (Fig.1B) and shorter overall survival time (Fig.1C).

Since Fer was also increased in the DLBCL cell lines, we then set out to explore the biological function of Fer in DLBCL cells (Fig.1D). Transfected efficacy was verified by western blot (Fig2.A). In vitro gain-and loss-of-function assays demonstrated the stimulatory role of Fer on cell viability, migration and chemoresistance (Fig2.B-D).

Recently, exosomes have been emerged as promising drug delivery carriers. Thus, we generated CD20-modified exosomes as an alternative to targeting deliver E260 to DLBCL cells. This event not only facilitated the targeting efficacy of E260 (Fig.3A) but also showed higher efficacy than E260 in eliminating DLBCL progression through inhibiting cell proliferation and chemotaxis, enhancing cell apoptosis and drug sensitivity both in vitro and in vivo (Fig.3B-F).

In the view of GO and KEGG analysis, Fer mainly enrichment in regulating cell proliferation, adhesion, response to drug and Hippo pathway in cancer (Fig.4A). Consistent with this, we demonstrated that E260 activated Hippo pathway and leading to YAP cytoplasmic sequestration (Fig.4B-C). Co-IP assay confirmed the physical interaction between Fer and AJUBA. Furthermore, upregulated AJUBA reversed E260 mediated cell proliferation inhibition (Fig.4D). Thus, we suggest that E260 inhibits DLBCL progression by downregulating AJUBA which restores Hippo tumor-suppressor activity.

In the mice model, the levels of plasma exosomal Fer could reflex the tumor burden (Fig.5A). We next detected that exosomal Fer and AJUBA expression increased in the plasma of DLBCL patients (Fig.5B-C) and released after R-CHOP therapy (Fig.5D). Besides, Fer expression was positively related with AJUBA (Fig.5E). As shown in the Figure 5F-G, exosomal Fer could act as a promising indicator in early diagnosis and disease progression for DLBCL patients.

Conclusion: We demonstrated for the first time the oncogenic role of Fer in DLBCL progression and therefore provided an appealing therapeutic target. CD20-targeted exosomes delivery E260 are more effective in eliminating DLBCL progression through diminishing AJUBA and activating Hippo pathway. Furthermore, we provided circulating exosomal Fer as a novel non-invasive biomarker for DLBCL diagnosis.