Histone Deacetylase Inhibitor Combined with Flumatinib Promotes Anti-Leukemia Sensitivity in Philadelphia-Positive ALL By Targeting PI3K/AKT Signaling

Introduction Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) is the most common subtype of B-ALL in adults with an increased incidence over age and has a poorer prognosis compared with Ph-negative ALL. Flumatinib (FLU), a small molecule protein tyrosine kinase inhibitor (TKI), induces cell proliferation arrest and apoptosis in Ph+ALL and chronic myeloid leukemia (CML). Histone deacetylase inhibitors (HDACi) have been reported to display promising anti-leukemic properties alone or in combination with other agents. Chidamide (CHI), a novel HDACi, is reported to be effective in hematological malignancies. However, it is still undetermined about the effect of CHI in ALL, particularly the combination of CHI with TKI (FLU) in Ph+ALL. Here, we explored the effect of CHI with FLU on cell proliferation arrest and apoptosis in Ph+ALL and the underlying mechanisms.

Methods Cell Counting Kit-8 assay and Annexin V + propidium iodide (PI) staining following flow cytometry analysis were performed to examine cell proliferation arrest and apoptosis in Sup-b15 Ph+ALL cells treated with indicated drugs for 48 hrs. Synergy effects were determined by combination indices using CompuSyn software. Ph+ALL patient cohorts from our institute and the GEO database were used to explore the expression of PIK3CA and AKT2. Student T-test analysis was performed for differences between groups.

Results Sup-b15 cells were treated with various doses of CHI or FLU, respectively, and a dose-dependent and time-dependent cell proliferation arrest were observed for the two drugs in the cells (Fig. 1A); and the 50% inhibitory concentrations (IC50) of CHI and FLU were 1.5±0.2 μmol/L and 83.2±19.7 nmol/L, respectively. Different doses of FLU combined with IC50 or 1/2 IC50 of CHI significantly increased the cell proliferation arrest of the cells compared with the single drug control (P<0.05) (Fig. 1B). CompuSyn analysis showed the synergistic effect of CHI+FLU (Fig. 1C). Moreover, a combination of CHI (1μM) with FLU (100nM) significantly increased the apoptosis in the cells compared with the single drug controls (Fig. 1D). Consistently, the expression of pro-apoptotic genes (BAX and Caspase-3) were also significantly down-regulated upon the combination treatment compared to the single drug controls in both mRNA and protein levels (Fig. 1E, 1F). We also observed that oncogene c-MYC and the key components of PI3K/AKT oncogenic pathway, PI3K p110β, p110α (PIK3CA), and AKT2 are significantly downregulated but the tumor suppressor p53 was significantly up-regulated upon the combination treatment compared to single drug control in both mRNA and protein level (Fig. 1E, 1F). Expression of PIK3CA (PI3K p110α) and AKT2 were further analyzed in two independent Ph+ALL patient cohorts from our institute and the GEO database. Results showed that PIK3CA and AKT2 were significantly over-expressed in patients with Ph+ALL compared with normal controls in both cohorts (P<0.05) (Fig. 1G). These data indicated that the combination-induced cell proliferation arrest and apoptosis are mediated through targeting the PI3K/AKT oncogenic signaling. c-MYC and p53 are critical transcription factors in cancer, and they interact in a negative feedback manner. Both c-MYC and p53 are reported to be involved in the transcriptional regulation of PI3K/AKT genes. The combination of CHI with FLU suppresses c-MYC but enhances the expression of the p53, which results in the suppression of the PI3K/AKT signaling to exert the anti-leukemia effect. The mechanism model of the synergy is summarized in (Fig. 1H).

Conclusions The combination of CHI with FLU has a synergistic anti-leukemia effect on cell proliferation arrest and apoptosis in Ph+ALL cells by targeting PI3K/AKT signaling through the p53/c-MYC axis. Our data provide experimental evidence for a new potential combination of CHI with FLU in the therapy of Ph+ALL, and the in vivo preclinical studies will further highlight the future clinical trial of the combination in clinic therapy of ALL.

No relevant conflicts of interest to declare.