P610: SYNERGISTIC ACTIVITY OF FTO INHIBITOR FB23-2 WITH IBRUTINIB IN XENOGRAFT MURINE MODEL OF CHRONIC LYMPHOCYTIC LEUKEMIA

Abstract

Background: Several studies have documented that FB23-2, as the selective inhibitor of RNA N6-methyladenosine (m6A) demethylase FTO, exerts crucial role in dampening tumorigenesis. Indeed, FTO deactivation was proved to render resistant leukemia cells sensitive to targeted agents, which suggests a potential combination therapy strategy of FB23-2. Aims: The present study was aimed to identify the synergistic utility of FB23-2 combination with ibrutinib in CLL. Methods: A disseminated leukemia xenograft murine model was established to explore the efficacy of FB23-2 in CLL. Leukemia burden of CLL mice was quantified by luciferase intensity using bioluminescence imaging. Bone marrow and spleen infiltration of leukemic cells was assessed by flow cytometry. And cell survival was determined by cell counting kit-8. Results: To investigate the combinatory effect of FB23-2 and ibrutinib, cell viability assay was performed firstly. FB23-2 displayed potent synergistic activity with ibrutinib in promoting cytotoxicity in CLL cells (Figure 1A). Consistently, FTO silencing mediated by lentivirus exhibited exquisite sensitivity to ibrutinib in CLL cells (Figure 1B). Besides, CLL primary cells and MEC-1 cells with treatment of FB23-2 and ibrutinib simultaneously triggered increased cell apoptosis compared to single agent (Figure 1C). Further underlying cooperative mechanism investigation demonstrated an aberrant activation of DNA damage pathway after serial dilution of FB23-2 treatment. Hence, enhanced expression of p-ATM, p-chk2 and p-H2AX was detected in FTO knockdown and ibrutinib co-treatment group, indicating FTO inhibition inducing ibrutinib-sensitivity via regulating DNA damage (Figure 1D). In addition, we investigated the efficacy of FB23-2 alone and corporation in CLL xenograft murine model. Compared to ibrutinib alone, combination treatment with FB23-2 significantly prolonged the lifespan of CLL mice (median survival time 20 versus 22.5 days, respectively, p=0.02, Figure 1E). FB23-2+ibrutinib group showed an obviously declined proportion of CLL cells compared to ibrutinib-treated alone both in bone marrow(20.93%±2.95% versus 37.17%±4.22%, p=0.02) and spleen (23.58%±6.03% and 54.58%±9.01%, p=0.03, Figure 1F-G). Similar results were obtained with three treated group relative to control group (all p<0.05). Moreover, FB23-2 combined with ibrutinib diminished leukemia burden and splenomegaly in CLL mice (Figure 1H-I). As shown in Figure 1J, leukemia involvement and malignant proliferation were more evident in control group, confirming the potent anti-leukemic activity of FB23-2. Image:Summary/Conclusion: Taken together, our investigation provides pre-clinical evidence for the utility of FB23-2 in CLL, especially combinatory benefit with ibrutinib. FB23-2 represents a promising strategy to novel treatment optimum in CLL.