APG115 Enhances the Sensitivity of Ibrutinib Via Promoting p53 to Target MCL-1 in Chronic Lymphocytic Leukemia

Introduction: APG115 is an orally active, selective, potent small molecule inhibitor of the MDM2-p53 interaction, which destabilizes MDM2-p53 complex and promotes p53 activation. Previous studies reported that APG115 promoted cell apoptosis in several types of cancer. However, its role and mechanism in the development of CLL are still unclear.

Methods: Peripheral blood samples from de novo CLL patients were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong University. The effects of APG115 on cell proliferation, apoptosis and cycle were detected by CCK8, Annexin V-PE/7AAD staining and PI/RNase staining respectively. Related protein expression of p53 pathway in CLL cell lines and primary cells were detected by Western blot. Protein interactions were verified by COIP. Immunofluorescence was applied to detect protein localization.

Results: To study the role of APG115, we added APG115 into CLL cell lines and primary cells to select appropriate concentration according to IC₅₀. The cells viability of gradually decreased along with the extension of time or concentration augment, which suggested that CLL cells were time-dose-dependent on APG115. In addition, the protein levels of MDM2, p53, PUMA and p21 increased under the treatment of APG115, indicating that APG115 relieved the inhibition of MDM2 on p53 and activated downstream apoptotic pathways. Meanwhile, the APG115 induced cell apoptotic elevation and cell cycle arrest in G0/G1 phase.

Subsequently, aimed to explore whether APG115 affects the ibrutinib sensitivity in CLL cells, the cell viability of combination group under both APG115 and ibrutinib was obviously suppressed than groups with single drug intervention. Immunofluorescence images revealed that p53 promoted MCL-1 into cytoplasm under the action of APG115. However, on the basis of ibrutinib intervention, APG115 facilitated the degradation of MCL-1 by p53.

Furthermore, the invasion range of CLL cells in the combination group was smaller than that in the single agent group, and the invasion range of CLL cells in the single agent group was lower than that in the control group in vivo(Figure A). After injection of medication, both the monotherapy group and the combination group were lower than the control group, but there was no significant difference between the monotherapy group and the combination group. The survival rate of combination group was higher than that of single agent group (Figure B). Immunohistochemistry of the dissected spleens of mice showed that the malignancy of the cells in the single agent group was lower than that in the control group, while that in the combination group was lower than that in the monotherapy group.

Finally, ibrutinib-resistant cell line was established to study the effect of APG115 on chemoresistance. After adding APG115, it was found that the survival rate of drug-resistant strain was lower than that of sensitive strain. It was proved that p53 induced the ubiquitination process when combined with MCL-1 to reduce the inhibitory effect of apoptosis via CO-IP. After APG115 was added, the apoptosis rate of drug-resistant strains was higher than that of sensitive strains. When APG115 was added on the basis of ibrutinib, the difference in apoptosis rate of drug-resistant strains was greater than that of sensitive strains.

Conclusions: In summary, this study first revealed anticancer effects of the novel MDM2-p53 inhibitor APG115 in CLL. Promisingly, APG115 is expected to be a potential agent to improve the effects of BTK inhibitor treatment in CLL.

No relevant conflicts of interest to declare.