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[17]. 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 novel explored programmed cell death modality. It could reverse resistance of tumor cells to chemotherapy drugs and as well promote removal of defective cells[18]. Consequently, ferroptosis is potentially a novel mechanism for tumor therapy. Several researches have concentrated on the role of ferroptosis in DLBCL treatment and development[12, 13].
In our study, based on the expression profile of FRGs, consensus clustering analysis divided the DLBCL patients in two clusters. The results showed obvious 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 pathway analyses unveiled the genes 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 tumors and hematological 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[19–21]. Calcium signaling also paticipate the GA101-induced cell death in lymphoma cells[22]. Recent evidence indicates that Ca2+ ions play an important role in cell death mediated by oxidative glutamate toxicity or oxytosis, a form of programmed cell death similar to ferroptosis[23]. The GO enrichment mainly included several iron-related biological processes or molecular functions, such as ion gated channel activity and ion 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[24]. 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[25]. Regulatory T cells and CD4+ T cells, which had a correlation with improved survival in DLBCL has a lower ratio in the C1 subtype[24, 26]. These results confirm that ferroptosis has a regulatory effect on the TME.
Previous studies have proved that ferroptosis-inducer erastin is useful for inducing cell 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 unclear. In this study, we identified the novel risk scoring model constructed by six genes (GCLC, LPCAT3, NFE2L2, ABCC1, SLC1A5, and GOT1) to classify DLBCL patients into two classes and predicted the OS of DLBCL patients independently. Gene markers related to ferroptosis were established, and the expression level of the six genes are not influenced by the variances in the underlying diseases of DLBCL, indicating that the constructed prognosis signature could be well performed in different types of DLBCL patients. Moreover, the hybrid nomogram based on the six-gene signature also provide support to clinicians to develop optimal treatment strategies. By concentrating on the function of the six ferroptotic genes, former researches have showed that the six genes play key roles in the development of cancer cells.
GCLC is a rate-limited enzyme that primarily regulates de novo synthesis of glutathione. It has been revealed that activation of GCLC is related to drug resistance in various tumors, like breast, head, lung, and neck cancers[27–29]. LPCAT3 is an enzyme that primarily maintain systemic homeostasis by transfer lysophosphatidylcholine to phosphatidylcholine in the liver. It also participates in the intestinal stem cell growth and phospholipid remodeling and tumorigenesis[30, 31]. NFE2L2 is a main regulator of the antioxidant response and regulates the activity of several ferroptosis and lipid peroxidation-related proteins[32]. ABCC1 plays a pivotal role in protecting cells by removal a vast array of drugs to sub-lethal levels. In the last decade many studies clarified the mechanisms of function and molecular characteristics of ABCC1 [33]. SLC1A5 is a cell surface solute-carrying transporter that facilitates uptake of neutral amino acids[34]. Blocking SLC1A5 to prevent glutamine uptake successfully restrains tumor cell proliferation in melanoma[35], breast cancer[36], and acute myeloid leukemia[37]. GOT1 plays an active role in ROS balance and energy metabolism in chronic acidosis stress[38]. Few studies on the role of these genes played in DLBCL patients' prognosis have been reported and their influence on the development of ferroptosis needs to be clarified. We further evaluated the protein expression level 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[39, 40], and GCB DLBCL have more favorable outcomes than those with ABC DLBCL when treated with standard immunochemotherapy[41]. 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, ABCC1, SLC1A5 and GOT1 than the GCB DLBCL, while NFE2L2 had the reverse tendency. These results supported that GCLC, LPCAT3, ABCC1, SLC1A5 and GOT1 are inclined to be the adverse prognostic biomarkers, while the NFE2L2 is apt to be a protective factor in DLBCL.