DLBCL is known as a clinical and molecular heterogeneous malignant hematological tumor. Various classification methods, and molecular markers have been established to characterize this disease[15]. To develop practical molecular markers related to DLBCL prognosis, we have identified a signature of six FRGs by using high-throughput expression analysis. Ferroptosis is a recently recognized programmed cell death modality. It can overcome resistance of malignant cells to chemotherapy and as well facilitate removal of defective cells [16]. Consequently, it is potentially a novel approach for tumor treatment. Current studies mainly focus on the role of iron metabolism in DLBCL development and treatment [12, 13], as far as we know, this is the first attempt to identify the role of ferroptosis in DLBCL prognosis.
In this study, based the expression pattern of FRGs, two DLBCL subgroups were identified by consensus clustering analysis. The results revealed significant differences in OS between the two clusters. C1 patients had a much worse prognosis than C2. Further analyses uncovered DEGs between the two clusters. KEGG analyses revealed the genes mainly participated in PI3K-Akt pathway and the calcium signaling pathway. Both of these pathways participate in the pathogenesis of lymphoma. The PI3K-Akt pathway is frequently activated in a variety of solid tumours and haematological malignancies, hence, PI3K was considered as an attractive therapeutic target in oncology. Currently, two PI3K inhibitors, copanlisib and idelalisib, have been approved for use in the leukaemias and B cell lymphoma[17–19]. Calcium signaling also plays a key role in GA101-induced cell death in lymphoma cells [20]. Recent evidence hints that Ca2 + ions play a fundamental role in cell death mediated by oxidative glutamate toxicity or oxytosis, a form of programmed cell death similar and possibly identical to ferroptosis[21]. The GO enrichment mainly included several iron-related biological processes or molecular functions, such as ion channel activity and ion gated channel activity. We speculated that ferroptosis was related to ion transport in DLBCL pathogenesis. In this study, we also found the C1 group have a smaller ratio of the stromal components. MCPcounter analysis showed a larger percentage of CD8 T cells, B lineage, NK cells, and neutrophils in C1 than C2, and ssgsea analysis presented a larger ratio of activated B cell, effector memory CD8 T cell, activated dendritic cell, MDSC, NK T cell, and plasmacytoid dendritic cell scores in TME. Among these immune cell types, a higher proportion of NK cell was correlated with poorer DLBCL outcome, but dendritic cells might contribute to longer OS[22]. Since the main feature of MDSC is their potent immune suppressive activity, the stronger immunosuppressive effect of MDSC might contribute to the poor prognosis of the high-risk group[23]. Regulatory T cells and CD4 + T cells, which had a correlation with improved survival in DLBCL has a lower ratio in the C1 subtype[22, 24]. These results confirm that ferroptosis has a regulatory effect on the TME.
Previous studies have confirmed that ferroptosis-inducer erastin is effective for inducing death in 114 DLBCL cell lines [12]. Ferroptosis might also be induced by dimethyl fumarate which is a promising novel therapeutic option in the treatment of GCB DLBCL, but the correlation between ferroptosis and DLBCL patients' OS remains largely unknown. In this study, we identified, for the first time, the novel risk scoring model constructed by six genes (GCLC, LPCAT3, NFE2L2, ABCC1, SLC1A5, and GOT1) to classify DLBCL patients into two classes and independently predicted the OS of patients with superior prediction performance. Gene markers related to ferroptosis were established, and the expression characteristics of the six genes are not affected by the differences in the underlying diseases of DLBCL, suggesting that the constructed prognosis model can be applied to various types of DLBCL patients. Moreover, the corresponding nomogram based on the six-gene model also helps clinicians make better clinical decisions and develop treatment strategies. By focusing on the specific function of the six ferroptotic genes, previous studies have demonstrated that most of these genes play a pivotal role in cancer cells.
GCLC (glutamate-cysteine ligase catalytic subunit) is a rate-limited enzyme that primarily regulates de novo synthesis of glutathione. It has been revealed that GCLC activation is associated with anti-tumor drug resistance in breast, lung, liver, head, and neck cancers[25–27]. LPCAT3 is an enzyme that converts lysophosphatidylcholine to phosphatidylcholine in the liver in order to maintain systemic homeostasis. It also participates in the phospholipid remodeling and intestinal stem cell growth and tumorigenesis [28, 29]. NFE2L2 is a master regulator of the antioxidant response and regulates the activity of several ferroptosis and lipid peroxidation-related proteins [30]. ABCC1 plays an active role in protecting cells by its ability to efflux a vast array of drugs to sub-lethal levels. There has been much effort in elucidating the mechanisms of action, structure, substrates, and substrate binding sites of ABCC1 in the last decade [31]. SLC1A5 is a cell surface solute-carrying transporter that mediates uptake of neutral amino acids, including glutamine[32]. Blocking SLC1A5 to prevent glutamine uptake successfully prevents tumor cell proliferation in melanoma[33], breast cancer[34], and acute myeloid leukemia[35]. GOT1 plays an important role in energy metabolism and ROS balance in chronic acidosis stress[36]. Few studies on the role these genes play in DLBCL patients' prognosis have been reported and their influence on the process of ferroptosis remains to be elucidated. We further evaluated the protein expression of the six genes in the DLBCL clinical samples with different molecular types. Double-hit lymphoma, double-expressor lymphoma and DLBCL with TP53 mutant were all known for poor outcome, long term survivors are rare, and GCB DLBCL have more favorable outcomes than those with ABC DLBCL when treated with standard immunochemotherapy. In our results, the DLBCL with adverse molecular factor, which include DH, DE, TP53 and ABC type, manifested a more positive expression of GCLC, LPCAT3, NFE2L2, SLC1A5 and GOT1 than the GCB DLBCL, while ABCC1 had the reverse tendency. These results confirmed that GCLC, LPCAT3, NFE2L2, SLC1A5 and GOT1 are inclinded to be the adverse prognostic biomarkers, while the ABCC1 is apt to be a protective factor in DLBCL.