The primary objective of AML therapy is to eliminate minimal residual disease, with patients achieving complete remission (CR) after initial therapy followed by consolidation and/or maintenance therapy. However, 10–40% of newly diagnosed patients fail to attain CR after conventional chemotherapy, and approximately 50% of those who initially achieve CR will subsequently develop relapsed AML(Thol & Ganser, 2020). Overcoming drug resistance remains a prominent challenge in AML treatment.
FTO, known as an obesity risk-associated gene and the first identified m6A eraser, has been found related to cancer drug resistance. Its high expression often leads to increased resistance and poor prognoses, with the Wnt/β-catenin pathway, NF-κB pathway, and STAT3 signaling being activated by FTO, thereby promoting tumor resistance to chemotherapy, radiation therapy, and anti-PD1 therapy(Takeshi Fukumoto et al., 2019; Yang et al., 2019; Zhou et al., 2018). Additionally, FTO can enhance resistance by reducing m6A levels in mRNA transcripts(T. Fukumoto et al.; Lin et al.; Ou et al.). In contrast, Takeshi Fukumoto found that downregulation of FTO and ALKBH5 was sufficient to increase FZD10 mRNA m6A modification and reduce PARPi sensitivity, which correlated with an increase in homologous recombination activity(T. Fukumoto et al.). Our study demonstrates the critical oncogenic role of FTO in leukemogenesis and chemotherapy resistance. We observed that compared to normal patients, higher expression of FTO in the bone marrow of AML patients, particularly those with relapsed/refractory AML, and these results were further confirmed in AML cell lines.
The conventional cytotoxic chemotherapy-based treatment regimen in AML is associated with numerous adverse reactions, and approximately 50% of patients experience recurrence. Therefore, there is an urgent need to develop new targeted therapy drugs with low toxicity and high efficacy for the treatment of relapsed AML patients. Rhein, a small molecule inhibitor of FTO, effectively reduces the proliferation of AML cells in a dose- and time-dependent manner. We observed that the IC50 of Rhein in both parental and resistant AML cells was close, While adriamycin-resistant cell HL60-ADR were about 400-fold more resistant to azacitidine than the parental cell HL60. The resistant AML cells are characterized by high expression of the first member of the ABC transport family ABCB1, that exhibits a broad substrate spectrum and can transport various drug molecules(Holohan, Van Schaeybroeck, Longley, & Johnston, 2013). We speculate that this may be due to the competitive binding of Rhein to the active site of FTO, which prevents ABCB1 from pumping out Rhein like it does with other drugs. Furthermore, FTO has been shown to be correlated with the expression of ABCC10(Xiao et al.). Our study demonstrates the benefits of Rhein in relapsed and refractory AML, which further supports its potential use in the treatment of relapsed and refractory AML.
In our investigation of drug-resistant AML cells, we observed rapid migration of these cells within the bone marrow following drug administration. Recent studies have confirmed that drug-resistant cells "run fast rather than hide", this finding challenges the prevailing theory that drug-resistant cells remain hidden in the bone marrow, suggesting that migration was closely increased in drug-resistant cell (Hawkins et al.). We confirm the migration is inhibited by Rhein with a dose-dependent manner in parent and resistant AML cells, this may be a novel target for reversing drug resistance. Furthermore, recent reports have indicated the upregulation of AKT/mTOR in AML, highlighting its potential as a therapeutic target. Activation of the mTOR pathway has been shown to enhance the invasive ability of AML cells(Deng et al., 2016) and reduce apoptosis(Rahmani et al., 2018; Wang et al., 2023). Therefore, we performed migration experiments and discovered that Rhein inhibits migration and increases apoptosis in leukemia cells, including MDR-AML cells.
AZA is commonly used for the treatment of relapsed, refractory, and elderly AML patients(Thol et al., 2015). Combining AZA with histone deacetylase inhibitors can potentially improve treatment efficacy by modulating the methylation and acetylation states of silenced genes(Topper et al., 2017). Our findings indicate that once leukemia cells become resistant to one drug, they are likely to develop multidrug resistance. For instance, HL60-ADR cells, which are resistant to adriamycin, were also found to be resistant to the DNA demethylation drug AZA. The treatment of AML is a complex process, and combination therapy has been shown to offer greater benefits to patients. Our study examined the combination of Rhein and a DNA methylation drug and found that it had a similar inhibitory effect on both HL60 and HL60-ADR cells. By using Rhein at low concentrations, it can enhance the anti-tumor effect of azacitidine. Furthermore, our results indicate that Rhein is effective even in resistant cells, suggesting its potential as an ideal therapeutic option for AML patients, especially for patient who can’t benefit from intensive regimen therapy.
Our findings suggest that Rhein is a potent inhibitor for studying the effects of RNA methylation on cancer progression and multidrug resistance. In the future, Rhein may be further modified or combined with other chemotherapy drugs to enhance its efficacy. Furthermore, preclinical primary safety evaluations and pharmacokinetic studies of Rhein are urgently needed. Combination therapy using targeted FTO inhibitors and other AML chemotherapy drugs may also hold great promise for the treatment of AML, and is thus a direction worth exploring.