CATALASE BLOCKING INDUCES APOPTOSIS AND POTENTIATES VENETOCLAX-INDUCED CELL DEATH IN CHRONIC LYMPHOCYTIC LEUKEMIA CELLS

Chronic lymphocytic leukemia (CLL) is an incurable disease char- acterized by a highly variable clinical behavior. Although targeted ther- apies, such as the BCL-2 inhibitor Venetoclax, have high initial response rates, relapse remains a challenge. We have recently shown that low lev- els of the catalase antioxidant enzyme identify an indolent CLL, leading us to hypothesize that catalase downregulation could induce escalated levels of reactive oxygen species (ROS) that promote antitumor signals such as cell death, thus accounting for less aggressive behavior and higher therapy sensitivity of cancer cells. To verify our hypothesis, we manipulated catalase activity using the specific inhibitor 3-amino-1,2,4- triazole (ATZ) in MEC1 CLL cell line and primary cells from CLL pa- tients. Moreover, in MEC1 cells we targeted catalase using siRNA technology. We analyzed ROS levels using CM-H2DCFDA fluorescent probe while we measured apoptosis with PI-Annexin V staining and flow cytometry. First, we observed that in vitro spontaneous apoptosis of pri- mary CLL cells was associated with a significant increased accumulation of ROS (Pearson r=0.7552; p=0.0186), thus suggesting a functional role of ROS in inducing apoptosis in CLL cells. Importantly, blocking cata- lase either with ATZ or siRNA induced escalated ROS levels and in- creased apoptosis in leukemic cells (Figure 1A). To extend our findings to a therapeutic setting, we analyzed the ability of catalase to modulate cell survival in vitro in the presence of Venetoclax. Remarkably, inhibit- ing catalase with ATZ in combination with Venetoclax significantly po- tentiated apoptosis in CLL cells compared to those induced by each single agent (Figure 1B). These data support the hypothesis that catalase might play a critical role in regulating susceptibility to spontaneous as well as BCL-2 inhibitor-induced apoptosis in CLL, thus influencing the behavior of cancer cells and contributing to resistance. Moreover, this study provides the rationale for developing novel precision therapies tar- geting redox pathways that, alone or in combination with currently used therapies, could overcome resistance in CLL and other B-cell malignan- cies where BCL-2 inhibitor is used, such as mantle cells lymphoma. We thank the Excellence Project 2023-2027 of the DNBM of the University of Verona funded by MUR; European Union, MUR and MSAL (PNRR PNC-E3-2022-23683266 PNC-HLS-DA, INNOVA and PNRR-TR1- 2023-12378287) for funding support.