AML is the most prevalent form of leukemia in adults with extreme malignancy [37]. Current treatment for AML relies on intense chemotherapy but these patients continue to be plagued by extremely poor prognosis and high mortality rates. Treatment options for AML have changed significantly over the past decade, particularly with the advent of the concept of ferroptosis [38, 39]. Ferroptosis is a regulated form of death and its activation can be used to treat cancer, particularly malignancies resistant to conventional therapies. However, studies on ferroptosis-related genes as targets for AML diagnosis and treatment are scanty. We used LASSO analysis to establish a predictive model using ferroptosis-related genes for AML. We assessed AML typing and the level of risk factors in each type by analyzing single-cell sequencing data. Finally, we verified that β-sitosterol and hydrastinine exerted synergistic inhibition on AML cells. Our findings offer a novel predictive model for the diagnosis of AML using ferroptosis-related genes, while also suggesting targets and potential treatment options for its treatment.
Ferroptosis is a non-apoptotic cell death form. It occurs due to the accumulation of intracellular iron leading to lipid peroxidation and increased concentration of intracellularly generated ROS at lethal levels [40, 41]. Growing evidence suggests that multiple inducers of ferroptosis have great cancer therapeutic potential [22, 42]. Daolin Tang et al. suggest that erastin, a inducer of ferroptosis, selectively enhances the sensitivity of HL-60 cells to chemotherapeutic agents in a Ras-independent manner [14]. Fei-hu Chen et al. show that ferroptosis participates in the action of ATPR-induced differentiation of AML cells [16]. We clustered the expression of 259 ferroptosis-related genes in AML, conducted differential analysis and COX univariate analysis between the two groups of patients, and trained the genes obtained by LASSO to obtain a risk score calculation formula for the model based on ferroptosis-related genes to predict the prognoses of patients. In this risk model, interestingly, 11 signature genes were associated with various cancers. For instance, PPIF, a peptidyl-prolyl cis-trans isomerase, is privy to the regulation of mPTP in mitochondria and affects cell death. It can act as a suitable therapeutic target for different diseases [43]. Trigger receptor-2 (TREM2) expressed on myeloid cells is a crucial autoimmune negative regulator. It binds to various tissue damage-related ligands and has anti-inflammatory and macrophage fusion functions. It plays an essential role in the tumor environment [44]. TCF15 is involved in early mesoderm transcriptional regulation and inhibits Id protein, rapidly downregulates Nanog, and accelerates somatic cell lineage differentiation, associated with the tumor prognosis [45]. CPNE8 belongs to the copine family and encodes a calcium-dependent protein, which is intimately linked to the occurrence of various types of tumors; it inhibits the proliferation of AML cancer cells [46]. CBR1, G protein-coupled receptor, is a subtype of the CBR family, known to exert anti-tumor effects [47]. DDIT4, a DNA damage response 1 or stress-triggered protein, can be activated under various stress conditions, and dysregulated dit4 expression has been observed in many malignancies [48]. UTF1 is expressed in early embryonic development and primordial germ cells and maintains spermatogonial stemness. It is highly expressed in cancer cells and associated with the transcriptional regulation [49]. PTP4A3, a phosphatase associated with tumor metastasis, is consistently highly expressed in tumor cells and regulates the adhesion of tumor cells to the extracellular matrix [50]. SH3BGR is localized in the matrix and participates in various life activities like protein assembly; its role in the pathogenesis of cancer, however, is unclear [51]. CCDC167 is related to cell proliferation and migration signaling and is aberrantly activated in several tumors [52]. Interestingly, interleukin-2 receptor subunit alpha (IL2RA) is a transmembrane protein, found mainly in the spleen, lymph nodes, and other tissues, Treg cells, basophils, activated T cells, and other cell surfaces [53]. IL2RA participates in the regulation of immune tolerance by affecting the activity of Treg cells. It is highly expressed in activated Treg cells, and specific antibodies kill Treg cells to relieve immunosuppression, crucial in anti-tumor therapy [54]. IL2RA promotes the proliferation of AML cells and affects chemoresistance and poor prognosis of these patients [55]. However, the clinical potential of anti-IL2RA therapy warrants further investigation. We aggregated the results from the training and test sets, performed KM survival analysis, and calculated the AUC values, which verified that the risk coefficient obtained from our prediction model was highly consistent for accurate prognoses of these patients. In summary, we verified the utility and credibility of the model in both the training and test sets through several methods.
Infiltrating immune cells are essential in the tumor microenvironment (TME) of AML. Zhen Cai et al. found that immune-related genes in the TME were linked to AML prognosis [56]. The level of immune infiltration of naive B cells, macrophages.M2, CD4 resting memory T cells, and monocytes showed a sharp rise in the cancer group compared to the normal group. Monocytes also showed significant infiltration in the high-risk group. Naive B cells, i.e., B cells that are not stimulated by antigens, initiate the formation of adaptive immunity and are associated with an active immune tumor environment and patient prognosis [57]. Macrophage M2 is a type of macrophage which is alternatively activated. It activates immune cells to respond to pathogens. Macrophages are important in the initiation and regression phases of the inflammatory processes [58]. Resting memory T cells are a type of T cell affecting secondary immune responses. Its main surface marker is CD4, which regulates or "helps" other lymphocytes in their function [59]. Monocytes originate from hematopoietic stem cells in the bone marrow, where they develop and mature. It is an essential component of the immune system [60]. Monocytes are involved in the immune responses and induce a specific immune response of lymphocytes through antigen presentation [61]. Monocytes promote tumor cell metastasis by influencing the microenvironment [62]. Taken together, we not only could predict clinical prognosis but also reflect the immune cell infiltration status in the TME. Further investigations are warranted to assess whether the prognostic model can be used as a judgment standard for immunotherapy.
Chemotherapeutic drug sensitivity analysis showed a positive correlation between risk fraction-related genes and drug-inductive sensibility of several commonly used chemotherapeutic drugs, suggesting that our prognostic model could be used to predict chemotherapy regimens. A combination of molecular docking and network pharmacology analyses showed that β-sitosterol in triptolide targeted the AML oncogenes, CASP9 and PON1. To further expand the clinical value of our study, we selected the drugs showing the highest sensitivity, hydrastinine and β-sitosterol, for verification at the cellular level. Hydrastinine and β-sitosterol showed a synergistic effect on inhibiting AML. Since 2017, many new targeted drugs have gradually entered the clinical treatment stage for AML. The treatment plan is no longer limited to the standard "3 + 7 protocol" and allogeneic hematopoietic stem cell transplantation [63]. The novel small molecule targeted drugs, antibody drugs, immunotherapy drugs, and CAR-T immunotherapy have broad application prospects in treating AML [64]. Currently, the research and development of AML-targeted therapy mainly include three directions. The first is several small molecule targeted drugs, like IDH1/2 inhibitors, FLT3 inhibitors, BCL2 inhibitors, and XPO-1 inhibitors. The second is CAR-T therapy. The current development and application of CAR-T therapy in AML is lagging behind lymphatic and plasma cell tumors. Finally, it is the antibody drugs, including monoclonal antibodies, double antibodies, and triple antibodies [65, 66]. Notably, 30% of AML patients carry FLT3 mutations, and drugs that target FLT3 can improve the four-year survival in these patients by 16% [67]. Nevertheless, the long-term prognosis for the majority of AML patients remains poor, and the precise treatment of the disease and monitoring of immunity is incomplete. The research on analyzing the pathogenesis of AML must continue for suggesting novel treatment strategies. In recent years, the natural compounds hydrastinine and β-sitosterol extracted from Hydrastis canadensis and LGT, respectively, have played an essential role in the treatment of diseases. Research has demonstrated that β-sitosterol inhibits the growth of cancer cells in the colon, breast, prostate, liver, and mouse fibrosarcoma but its role in AML is unclear [33]. The role of hydrastinine in tumors is largely unclear. In this study, we found that hydrastinine and β-sitosterol affected the expression of IL2RA, thereby synergistically inhibiting the occurrence and development of AML. Moreover, the combinated use of the two drugs improved the drug sensitivity of AML cells to ferroptosis inducers. Taken together, our findings provide a potent combination of TCM treatment options against AML.