Understanding and tackling the resistance to BH3-mimetics in diffuse large B-Cell lymphoma
Authors:   , Yildirim Nahide 
Published: January 2025

Lymphoma remains a significant global health challenge, contributing substantially to morbidity and mortality. Among the diverse subtypes of lymphoma, diffuse large B-cell lymphoma (DLBCL), an aggressive form, constitutes 30 to 40% of cases within Non-Hodgkin lymphomas (NHL). The evolving understanding of DLBCL subtypes emphasizes the urgent need for tailored treatment approaches, considering the substantial heterogeneity within this disease. Recognizing this, there is a critical demand for novel strategies and a deeper comprehension of resistance mechanisms against therapy to enhance the overall cure rates for individuals with DLBCL. Furthermore, it was shown that resistance to apoptosis is a common characteristic in cancer, often linked to the overexpression of anti-apoptotic BCL-2 proteins. To counteract this imbalance, the development of BH3-mimetics for inhibiting anti-apoptotic BCL-2 proteins is a promising strategy in cancer treatment. Venetoclax (ABT-199), a selective BCL-2 inhibitor, has demonstrated preclinical effectiveness against tumors and is the first BH3-mimetic approved for clinical use. Unfortunately, also resistance to ABT-199 can occur in patients, resulting in unfavorable treatment outcomes. This underscores the significance of ongoing research and clinical trials focused on elucidating and mitigating resistance mechanisms associated with ABT-199. The ultimate goal is to improve therapeutic strategies and enhance the effectiveness of ABT-199-based treatments. In this thesis our aim was to understand and explain the mechanisms behind the pre-existing resistance and the acquired resistance in DLBCL cell lines.
To investigate mechanisms of pre-existing resistance, we used specific DLBCL cell lines characterized by high BCL-2 to undergo cell death following ABT-199 treatment. These experiments are described under Part 1. To this end, we initially ruled out the possibility that protein expression patterns within the BCL-2 family contribute to resistance, as well as the expression of MDR1, which pumps out the drug from the cell, was not present in the selected cell DLBCL cell lines. Next, we selected two cell lines with high BCL-2 expression that displayed different responses to ABT-199 treatment. Our findings revealed notable variations in the binding patterns of anti-apoptotic and pro-apoptotic proteins upon treatment in the chosen sensitive (RIVA) and resistant (HBL-1) cell line. Upon observing the notable discrepancy in HBL-1 cells, where MCL-1 sequesters free BIM from BCL-2 in response to ABT-199, we hypothesized that MCL-1 was involved in the resistance. This was proven mechanistically by co-treatment with ABT-199 and the MCL-1 inhibitor S63845.
In the second part of the thesis, we investigated the role of the BH3-only protein NOXA in conferring resistance to cell death triggered by ABT-199 in the initially sensitive RIVA cell line. Employing CRISPR/Cas9-mediated knockout, we demonstrated that the absence of NOXA influenced sensitivity to ABT-199, both in vitro and in vivo. The absence of NOXA reinforced the role of MCL-1 as an anti-apoptotic protein, emphasizing its longer half-life and the newly identified interaction partner BIM. This hypothesis was validated through genetic silencing of MCL-1, resulting in the resensitization to ABT-199. Furthermore, in the absence of NOXA, we induced synergistic cell death through the co-treatment of ABT-199/S63845. Notably, we identified variations in the retranslocation of BAX in the presence and absence of NOXA, asserting that NOXA presence is crucial for BAX activation at the MOM, leading to pore formation with BAK.
The final section of the thesis explores the mechanism behind acquired resistance to ABT-199. To this end, RIVA cells were subjected to prolonged exposure to ABT-199. These adapted cells demonstrated the ability to overcome sensitivity to ABT-199 by elevating the expression of alternative anti-apoptotic proteins, specifically BCL-XL. To highlight this reliance, we utilized genetic silencing of BCL-XL, effectively resensitizing the cells to ABT-199. Furthermore, adapted RIVA cells exhibited sensitivity to co-treatment of ABT-199 with the BCL-XL inhibitor A1331852, resulting in synergistic cell death. In essence, our goal was to reveal the resistance mechanisms in order to identify better treatment strategies using BH3-mimetics in DLBCL patients. These findings highlight the importance of adopting a comprehensive strategy to address resistance mechanisms and improve the therapeutic effectiveness of ABT-199 in diverse combinations with other BH3-mimetics in treating DLBCL.