8-Chloro-Adenosine Combined with Homoharringtonine Induces Ferroptosis in Acute Myeloid Leukemia
Authors: Xu Liping , Zhuang Haihui , Li Fenglin , Jin Jie , Lu Ying 
Published: December 2024

Background: Acute myeloid leukemia (AML) is the most common hematological malignancy in adults, characterized by strong heterogeneity, rapid progression and poor prognosis. 8-chloro-adenosine（8-Cl-Ado）, a nucleoside analogue, can induce DNA damage inhibit tumor cell proliferation in FLT3-ITD AML. Inhibition of mTORC1 signaling pathway is one of its anti-tumor mechanisms. However, long-term inhibition of mTORC1 will lead to compensatory activation of PI3K/AKT signaling pathway, resulting in drug resistance. One of the anti-leukemia mechanisms of Homoharringtonine (HHT) is the inhibition of PI3K/AKT pathway, which is also an effective treatment for FLT3-ITD AML. However, whether the combination of 8-Cl-Ado and HHT has a synergistic effect on FLT3-ITD AML is unclear.

Objective: To explore whether 8-Cl-Ado combined with HHT has a synergistic effect in the treatment of acute myeloid leukemia and the potential synergistic mechanism.

Methods: After treating with 8-Cl-Ado and HHT alone or in combination, the viability of AML cell lines and primary tumor cells from patients was detected by MTT method, and the combination index was calculated by Calcusyn software to evaluate the synergistic effect. Apoptosis and cell cycle were detected by flow cytometry. Differentially expressed genes and enrichment of related signaling pathways in different drug treatment groups were analyzed by RNA sequencing; Ferrous ion, malondialdehyde (MDA), and glutathione (GSH) assays were used to assess ferroptosis in AML cells; The activation level of PI3K/AKT signaling related molecules and the protein levels associated with ferroptosis were detected by western blot.

Results: 8-Cl-Ado and HHT alone inhibited the activity of AML cells in a dose-dependent and time-dependent manner, and were more sensitive to FLT3-ITD AML cells. 8-Cl-Ado combined with HHT had synergistic killing effect on FLT3-ITD AML cell lines and primary cells.

Flow cytometry verified that 8-Cl-Ado combined with HHT significantly promoted AML cell apoptosis, arrested AML cell cycle at the G1/G0 phase. The results of RNA sequencing suggested that the up-regulated genes in the combination of 8-Cl-Ado and HHT were mainly concentrated in the ferroptosis pathway. HMOX-1, a key gene in ferroptosis, was significantly up-regulated. By promoting MDA Fe2+ accumulation, and GSH suppression, 8-Cl-Ado combined with HHT triggers ferroptosis in FLT3-ITD AML cell lines. Western blot verified that the combination of the two drugs inactivated the PI3K/AKT signaling pathway and targeted the down-regulation of HMOX-1.

Conclusion: Both 8-Cl-Ado and HHT had killing effects on AML cell lines, and the combination of two drugs had a synergistic killing effect on FLT3-ITD AML. And 8-Cl-Ado combined with HHT inhibited FLT3-ITD AML cell proliferation, promoted cell apoptosis, and arrested cell at the G1/G0 phase. The underlying mechanism of this synergistic effect was that the combination of the two drugs could inactivat the PI3K/AKT signaling pathway and target the down-regulation of HMOX-1 to induce ferroptosis.