Lenalidomide in Combination with Etoposide Induced Ferroptotic Cell Death in DLBCL
Authors: Li Jia , Guo Wei , Zhao Yangzhi , Wang Xingtong , Wan Xin , Bai Ou 
Published: December 2023

Background ： Relapse/refractory(R/R) DLBCL after first-line immunochemotherapy have unfavorable outcomes, treatment of which remains challenging. The combination of multiple drugs in the second-line regimen improves efficacy but also increases toxicity, often leading to treatment-related adverse events (AEs) or dose reduction/interruption. This study aims to explore the effects of lenalidomide in combination with ibrutinib in diffuse large B-cell lymphoma and investigate its potential mechanisms.

Methods: 1. From February 2019 to June 2023, patients aged 60 and above who received R 2-ICE regimen were analyzed for the efficacy and AE of the second-line therapy. 2. Human DLBCL cell lines SU-DHL-4 and U2932 were screened using the CCK-8 assay to demonstrate the synergistic cytotoxic effect of different drugs on DLBCL cells. Subsequently, various experiments were conducted to investigate whether oxidative damage occurred and explore the underlying mechanisms . These experiments included measurement of intracellular iron levels, MDA assay, detection of H 2O 2 and ·OH levels, ROS assay, assessment of mitochondrial membrane potential, and Hoechst staining. 3. SCID mouse model with subcutaneous xenografts of SU-DHL-4 cells in the right thigh was established. Different chemotherapy regimens, including PBS, R 2, R 2-ICE, and R 2-E, were administered to the mice, and the tumor-inhibitory effects and in vivo toxicity (evaluated based on body weight) of these treatments were observed.

Results: 1. A total of 19 patients aged 60 years and above with relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) received the R2-ICE regimen as second-line chemotherapy. They completed at least 2 treatment cycles, and the therapeutic efficacy was evaluated. Short-term efficacy: Overall Response Rate (ORR) was 81% (43.75% Complete Response [CR], 37.5% Partial Response [PR]). Long-term efficacy: Median follow-up of 30 months, with 1-year Progression-Free Survival (PFS) of 42.9% and 2-year PFS of 32.1%. One-year Overall Survival (OS) was 75%, and 2-year OS was 37.5%（Figure1A-B）. 2. Through CCK-8 cell proliferation assay, it was observed that lenalidomide，carboplatin and etoposide exerted time- and dose-dependent inhibitory effects on SU-DHL-4 and U2932 cells. Different IC50 concentrations of these drugs were chosen after 48 hours of treatment, and lenalidomide was administered in combination with etoposide or carboplatin. By calculating the CCK-8 cell survival rate and drug synergy formula, it was determined that lenalidomide in combination with etoposide had a synergistic inhibitory effect on DLBCL cell lines. Further CCK-8 experiments showed that ferroptosis inhibitor Fer-1-treated cells partially resisted the cytotoxic effect of lenalidomide in combination with etoposide on DLBCL cells (all differences were statistically significant, Figure 1. C-E). These findings indicate that lenalidomide in combination with etoposide suppresses DLBCL proliferation via the ferroptosis pathway. 3. Experiments of SU-DHL-4 cells were divided into four groups：control，len, eto，len+eto. By performing intracellular iron content detection, MDA assay, H2O2 and ·OH level detection, ROS experiment, mitochondrial membrane potential detection, and Hoechst staining, it was demonstrated that lenalidomide in combination with etoposide induces intracellular accumulation of ROS through an iron-dependent Fenton reaction, leading to cell membrane and DNA damage and DLBCL cell death(Figure 2). 4. SU-DHL-4 pretreated with Fer-1, and the experiments were divided into four groups: control, control + Fer-1, len+eto, len+eto+ Fer-1. By performing experiment mentioned above demonstrated that Fer-1 could reverse the oxidative damage induced by lenalidomide in combination with etoposide（Figure 3）. 5.In in vivo experiments, chemotherapy administered with PBS, R 2, R 2-E, and R 2-ICE regimens showed that R 2-E and R 2-ICE had similar tumor-suppressive effects and R 2-E were lower toxicity（Figure 4）.

Conclusion: Lenalidomide combined with etoposide enhances intracellular reactive oxygenlevels through the ferroptosis pathway, resulting in synergistic killing of DLBCL cells through lipid and DNA oxidation damage. In vivo experiments confirmed that R 2-E showed similar antitumor effects with R 2-ICE and had lower toxicity.