Investigating the Drug Resistance Mechanisms of Refractory Diffuse Large B-Cell Lymphoma Based on Proteomic Research

Diffuse large B-cell lymphoma (DLBCL) is the most common histological subtype of non-Hodgkin's lymphoma, more than half of the patients of which can achieve complete remission or even be cured with multidrug immunochemotherapy regimen(R-CHOP). However, for patients with relapsed or refractory DLBCL, there is no effective treatment, while the unfavorable prognosis is related to the primary and acquired resistance to existing therapies. Proteomics is the study of the presence and activity of all proteins in cells. In exploring the mechanisms of DLBCL progression, proteomic technology can identify and quantify changed proteins in DLBCL, reveal the relationship between changes in protein expression levels and different stages of lymphoma occurrence and development, and thus provide information on variation in related proteins and protein-related signaling molecules. Based on proteomic technology research, this study is expected to discover new molecular mechanisms, further clarify resistance mechanisms, and find new therapeutic targets for DLBCL, providing more powerful weapons for better prognosis.

We first collected paired clinical patient samples that met the experimental requirements and selected biopsy specimens from 3 patients who relapsed within 1 year after systemic treatment, including biopsy specimens at initial diagnosis and relapse. Next, paraffin sections and HE-stains were performed on the samples, and target areas were micro dissected using a laser microscope. The dissected samples were collected in specific EP tubes, lysed ultrasonically by adding lysis buffer to each group of samples, and the protein concentration of the supernatant was determined after centrifugation. Equal amounts of protein from each sample were digested with trypsin and the final concentration was controlled. Analysis was then performed using the EASY-nLC 1200 ultra-high performance liquid phase system and the Q Exactive ^TM HF-X mass spectrometer. Finally, bioinformatic analysis was performed to analyze differential proteins and find relevant drug targets or markers.

The number of identified peptides was 36,107, the number of identified proteins was 6,338, and the number of quantifiable proteins was 6,165. The top five differentially upregulated proteins in the volcano plot of differential proteins were HOPX, TSNAX, PTMA, FTH1, and IFI44, respectively; and the top five downregulated differential proteins were KRT9, KRT1, KRT10, KRT2, and PIP, respectively. Through enrichment analysis of differential proteins, we noticed the ferroptosis pathway as a key pathway, and found that FTH1, TF, and GSH were involved in iron death-related processes, while FTH1 was one of the top five upregulated differential proteins. Iron death is an iron-dependent, non-apoptotic form of cell death discovered in recent years, activation of which can lead to tumor cell death and inhibit tumor growth. Increased expression of the iron-binding subunit FTH1 can suppress activation of iron death and reduce iron death in tumor cells. This suggests that by developing targeted drugs that act on increasing FTH1 expression or iron death inducers which are capable of slowing down the process, it may be possible to address the problem of traditional chemo-resistance in DLBCL and provide new directions for overcoming DLBCL resistance.