PF239 COMBINED TARGETING OF BCL-2 AND SET-PP2A INTERACTION INDUCES POTENT AND SYNERGISTIC ANTI-LEUKEMIC EFFECTS ON ACUTE MYELOID LEUKEMIA

Abstract

Background: Acute myeloid leukemia (AML) is an aggressive malignancy that arises from the transformation of hematopoietic stem cells. Most AML patients are older than 60 years, and in this group only 5–15% of cases are cured (Döhner et al., 2015). Therefore, it is necessary to explore new therapeutic options. Overexpression of the anti-apoptotic BCL-2 protein is a common finding in AML. ABT-199 (Venetoclax), a potent and selective inhibitor of BCL-2, has been recently developed. However, clinical activity of ABT-199 monotherapy is modest in AML (Konopleva et al., 2016), which could be explained by the genetic and epigenetic heterogeneity of AML. Our group has shown that inactivation of PP2A is present in 78% of AML patients, and that FTY720 treatment restores PP2A activity by disrupting the SET-PP2A interaction, thereby inhibiting proliferation of AML cells (Pippa et al., 2014). Since PP2A promotes apoptosis by affecting the phosphorylation state of a variety of pro- and anti-apoptotic factors, we hypothesized that BCL-2 targeting could synergize with PP2A re-activation and induce synergistic anti-leukemic effects. Aims: To investigate whether the combination of ABT-199 with FTY720 could induce synergistic anti-leukemic effects in vitro and in vivo. Methods: A panel of six human AML cell lines was used to perform cell culture experiments. To generate zebrafish AML xenograft models, AML cell lines were treated for 24 h with ABT-199, FTY720 or their combination. Subsequently, cells were stained with a fluorescent cell tracker and injected into the yolk sac of 48 hours-post-fecundation wild type zebrafish embryos. After injection, cell proliferation and colonization index were measured. Colony-Forming-Unit (CFU) assays in semi-solid medium were performed on patient-derived AML cells isolated from three patients with AML at diagnosis. Results: GI50 values for ABT-199 and FTY720 showed that HL-60, MV4–11 and MOLM-13 were the most sensitive cell lines to ABT-199, while OCIAML-3 and HEL cells were resistant to BCL-2 specific inhibition (GI50 value greater than 10 μM). Regarding FTY720 treatment, we observed that OCIAML-3 and MV4–11 were the most sensitive cell lines, while HEL cells were FTY720-resistant. By western-blot analyses, we ascertained that sensitivity to ABT-199 was dependent on BCL-2, MCL-1 and BCL-XL expression levels, and sensitivity to FTY720 was dependent on PP2A and SET expression. Next, survival of AML cells was analyzed after exposure to ABT-199, FTY720, or their combination at different concentrations (accordingly on the above GI50 calculated values). Analyses of the combination index values showed that combination of ABT-199 and FTY720 had a potent and synergistic anti-leukemic effect, even in OCI-AML3 cells, which were ABT-199-resistant cells. Mechanistically, combination of ABT-199 and FTY720 induced a synergistic pro-apoptotic activity by increasing significantly the number of Annexin/IP positive cells, the activity of Caspases 3/7, and PARP cleavage. Compared to single treatments, treatment with ABT-199 plus FTY720 also reduced significantly cell proliferation and/or cell invasive potential of HL-60, MOLM-13, and MV4–11 cells in zebrafish xenograft models. Furthermore, we demonstrated in primary-AML cells that the combined treatment significantly decreased the number as well as the size of CFUs. Summary/Conclusion: Combined targeting of BCL-2 and the SET-PP2A interaction has potent and synergistic anti-leukemic effects in vitro and in vivo. Our findings provide a rational basis for further testing of novel combined therapies with ABT-199 and FTY720 in AML patients.