Understanding mechanisms of resistance to precision medicines in diffuse large B cell lymphoma (DLBCL) models.

Around 30% of DLBCL patients are either non-responsive to immuno-chemotherapy or relapse and require additional therapy. Precision medicines, including BCL2 and BTK inhibitors, have significant clinical effects in many B cell malignancies but not in DLBCL. The causes of such inherent resistance in DLBCL remain largely unknown. In my thesis, I investigated alterations in reactive oxygen species (ROS) levels and the consequences of phenotypic and genetic heterogeneity in a panel of well-characterised DLBCL cell line models. Firstly, the levels of ROS and the capacity to mitigate the excessive ROS varied considerably between eight DLBCL cell lines and their BH3 mimetics or BTKi-acquired resistant cell lines. Unexpectedly, venetoclax induced 3 to 6-fold ROS accumulation in mitochondria and nucleus and further caused DNA damage within venetoclax-sensitive and derived cell lines with venetoclax resistance. In contrast, venetoclax did not alter the ROS level of the naturally resistant cell lines. In contrast, BTK inhibition did not alter ROS levels.

Regarding cellular heterogeneity, DLBCL typically displays monoclonality, with little evidence for DLBCL "stem cells". My findings revealed that U-2932, OCI-Ly-19, and TMD8 DLBCL cell lines stably harboured multiple populations across 30 generations. 50% of OCI-Ly19 cells expressed CD10 and this population demonstrated enhanced sensitivity to venetoclax compared to CD10- cells. However, heterogeneity in U2932 showed no connection to "stem cells" and BH3 mimetics resistance. In the TMD8 cell line, EBF1 A99V and SMARCAL1 R617W mutations were stably present at only low levels (<5% in the parental population) but were selectively enriched during BTK inhibitor resistance acquisition. However, their functional role was not confirmed due to a lack of mutation enrichment post-acute BTKi exposure, suggesting potential "passenger" status. My data revealed unanticipated heterogeneity in DLBCL regarding ROS, and the induction of ROS by Venetoclax was an unexpected finding that may have clinical implications.