Investigating the Metabolic Underpinnings of Venetoclax Resistance in DLBCL

Background: Large B cell lymphoma is a heterogeneous lymphoid malignancy where the expression of anti-apoptotic protein BCL-2 is often dysregulated by chromosomal translocation and/or overexpression. Lymphoma patients with BCL-2 dysregulation have worse prognosis under standard chemoimmunotherapy treatment regimens and represent a clinical unmet need. BCL-2 sequesters to the mitochondrial pore forming with pro-apoptotic, proteins BAX and BAK thereby preventing apoptosis and promoting cell survival. The mechanisms of resistance and/or subsequent relapse to BCL-2 based therapies in DLBCL remain unclear. Mechanisms of resistance to BCL-2 inhibition can arise from mutations in the BCL-2 binding pocket, upregulation of anti-apoptotic proteins BCL-XL and MCL-1 and/or through metabolic reprogramming. the goal of our study is to delineate the metabolic basis of venetoclax resistance to better understand BCL-2 biology and to aid in identifying potential routes for the development of combination therapies by dampening pathways or targeting vulnerabilities that drive venetoclax resistance.

Methods: We generated three venetoclax resistant (VR) DLBCL cell lines (OCI-Ly1, OCI-Ly2 and SUDHL-4) by gradually exposing them to increasing concentrations of venetoclax and analyzed their metabolic phenotypes using an XFe96, Seahorse bioanalyzer. Subsequently, we monitored changes to mitochondrial mass and reactive oxygen species using standard flow cytometry dyes. To determine alterations in mitochondrial protein content between the venetoclax resistant and sensitive cells, we used a mitochondrial enrichment protocol followed by a mass spectrometry based proteomics pipeline.

Results: The VR cell lines were able to proliferate in the presence of 1uM of Ven at 48 hours as compared to their venetoclax sensitive (VS) counterparts which did not. We find that VR cell lines lose expression of BCL-2 entirely while upregulating BCL-XL and downregulating MCL-1. VR cells were observed to have significantly altered bioenergetics as compared to the VS cells. The VR cells have higher oxygen consumption rates (OCR), maximal respiration rates and extracellular acidification rates (ECAR) compared to the VS cell lines. Additionally, VR cells have greater spare respiratory capacity suggesting greater metabolic plasticity to adjust under metabolic stress. We find that mitochondrial, glycolytic and overall ATP production rates are markedly elevated in the VR cells.

In line with the observed mitochondrial phenotype, we find that the VR cells trend towards both increased mitochondrial mass and elevated mitochondrial ROS as determined by flow cytometry. To further understand alterations in the mitochondrial proteome that occur in venetoclax resistant cells, we optimized a mitochondrial enrichment protocol to use in concert with a proteomics pipeline to take inventory of the VS and VR mitochondrial proteomes. We found pathways related to mitochondrial protein degradation/homeostasis, aerobic respiration, the electron transport, citric acid cycle, GTP signalling and mitochondrial translation to be among the top processes that are upregulated in the VR cell lines.

Conclusions: VR cell lines show altered expression of BCL-2 family proteins, increased metabolic plasticity and elevated levels of mitochondrial mass, ROS and OCR. To support the increases in mitochondrial activity, VR cells appear to upregulate pathways involved in mitochondrial protein ubiquitination, protein quality control and oxidative phosphorylation. We interpret these results to suggest that the mitochondria in VR cells, while operating at elevated levels of oxidative phosphorylation and producing high levels of ROS, activate additional pathways for the surveillance and removal of damaged or unfolded proteins. In addition, this also requires the engagement of pathways controlling mitochondrial translation to restore degraded proteins and maintain protein homeostasis.

The findings from this study will guide subsequent investigations on the altered metabolic phenotype of VR cells and how they can be targeted to overcome mechanisms of venetoclax resistance, potentially providing a route to rationale venetoclax combinations for the treatment of lymphoma.