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Background Antibody-drug conjugates (ADCs) have revolutionized cancer treatment by enabling targeted delivery of cytotoxic agents to malignant cells. However, their efficacy is often limited by tumor-intrinsic resistance mechanisms, including impaired antigen processing and lysosomal dysfunction, which reduce intracellular release of drugs and weakens therapeutic efficacy. Inotuzumab ozogamicin (IO), an ADC targeting CD22, is highly active in B-cell acute lymphoblastic leukemia (B-ALL) but not in aggressive B-cell non-Hodgkin lymphoma (B-cell NHL), suggesting the existence of a resistance barrier. Lysosomal dysfunction, frequently observed in the tumor microenvironment, may inhibit ADC processing and limit the activation of cytotoxic agents. We previously demonstrated that mTORC1/2 inhibition restores lysosomal function and sensitizes acute myeloid leukemia cells to gemtuzumab ozogamicin. 1 2 Based on these findings, in this study we investigate whether the combination of IO with an mTORC1/2 inhibitor can enhance antitumor efficacy in B-cell NHL by restoring lysosomal activity.Methods We evaluated the combination of IO and the mTORC1/2 inhibitor AZD2014 in seven B-cell NHL cell lines (KML-1, Daudi, TK, SLVL, Ramos, Raji, RL). Cell death was assessed using flow cytometry with Annexin V/PI staining. The combination index (CI) was used to determine additive (CI = 1), synergistic (CI < 1), or antagonistic (CI > 1) effects. Lysosomal function was measured using Lysotracker Red DND-99. Expression of Bcl-2 and MDR-1 was analyzed by real-time PCR, Western blotting, and apoptosis assays. Additionally, cells isolated from lymph node biopsies of patients with diffuse large B-cell lymphoma (DLBCL) were cultured and tested under similar conditions.Results Synergistic effects between IO and AZD2014 were observed in 4 of 7 B-cell NHL cell lines (KML-1, Daudi, TK, SLVL) ( figure 1). AZD2014 activated lysosomal function in all cell lines tested, but this was especially evident in the cell lines that showed synergy (figure 2). Cell lines that did not show synergy had elevated expression of Bcl-2 or MDR-1, suggesting that these factors contribute to resistance. Only one of the two patient-derived samples showed synergy; however, both samples showed enhanced lysosomal function.Conclusions Our findings suggest that mTORC1/2 inhibition can improve the anti-lymphoma efficacy of IO by enhancing lysosomal activity. However, resistance mediated by Bcl-2 and MDR-1 remains a barrier. Strategies to overcome these resistance mechanisms are needed to optimize ADC-based therapies for B-cell NHL.References Maimaitili Y, Inase A, Miyata Y, Kitao A, Mizutani Y, Kakiuchi S, et al. An mTORC1/2 kinase inhibitor enhances the cytotoxicity of gemtuzumab ozogamicin by activation of lysosomal function. Leuk Res. 2018 Nov;74:68–74.Mizutani Y, Inase A, Maimaitili Y, Miyata Y, Kitao A, Matsumoto H, et al. An mTORC1/2 dual inhibitor, AZD2014, acts as a lysosomal function activator and enhances gemtuzumab ozogamicin-induced apoptosis in primary human leukemia cells. Int J Hematol. 2019 Oct;110(4):490–9.Ethics Approval The project received approval from the ethics committees of Kobe University (#1481).Abstract 689 Figure 1The cytotoxic effect of inotuzumab ozogamicin (IO) was enhanced by the combination of the mTORC1/2 inhibitor AZD2014 in B-cell non-Hodgkin lymphoma (B-cell NHL) cell linesAbstract 689 Figure 2AZD2014, alone or in combination with IO, enhanced lysosomal function in all B-cell NHL cell lines