Investigating a combinatorial approach of engineered NK cells and small molecule inhibitors for targeting aggressive B-cell non-Hodgkin’s lymphoma

Non-Hodgkin lymphomas (NHLs) are a diverse group of lymphoid malignancies, with aggressive subtypes such as Diffuse Large B-Cell Lymphoma (DLBCL) and Burkitt Lymphoma (BL) posing significant therapeutic challenges due to drug resistance and frequent relapse. While CAR-T cell therapies targeting tumor-associated antigens (e.g., CD19) have improved outcomes in relapsed/refractory B-cell NHLs, they face limitations including high cost, manufacturing delays, immune toxicities, and antigen escape. Natural killer (NK) cells have emerged as a promising alternative for cellular immunotherapy. They offer several advantages: antigen-independent recognition of tumor cells, lower risk of graft-versus-host disease, reduced immune toxicities, and the feasibility of “off-the-shelf” allogeneic use. NK cells can also be engineered with chimeric antigen receptors (CARs) for enhanced specificity. However, their efficacy is often hindered by tumor cell resistance mechanisms, particularly the evasion of apoptosis. This study aimed to enhance NK cell-mediated cytotoxicity against relapsed/refractory B-cell NHL by targeting key apoptosis pathways. An optimized protocol was developed for NK cell isolation, expansion, and cryopreservation to ensure a reliable source of viable and functional NK cells for research. Next, a focused drug screen targeting pro-survival and anti-apoptotic pathways in NHL cells identified cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2) as key mediators of resistance to NK cell killing. Treatment with the SMAC mimetic birinapant (TL32711) at nanomolar concentrations downregulated cIAP1/2 in BL and DLBCL cell lines, sensitizing them to NK-mediated cytotoxicity without impairing NK cell viability or function. Mechanistically, birinapant repressed NF-κB-regulated anti-apoptotic proteins including Bcl-2, Bcl-XL, Mcl-1, and cFLIP. Direct inhibition of NF-κB, Bcl-2, or Mcl-1 similarly enhanced NHL susceptibility to NK-mediated killing, confirming the importance of these pathways. Notably, cell death was primarily driven by death ligand-receptor interactions rather than lytic granule-mediated mechanisms. Building on these findings and prior studies highlighting the role of TRAIL (TNF-related apoptosis-inducing ligand) in hematological malignancies, the study further explored NK cell engineering. TRAIL or receptor-selective TRAIL variants were overexpressed on NK cells to compensate for the limited natural expression of membrane-bound TRAIL. These engineered NK cells exhibited enhanced cytotoxicity against NHL cells. In summary, this study demonstrates that targeting anti-apoptotic pathways particularly via cIAP1/2 and NF-κB inhibition and engineering NK cells to overexpress TRAIL significantly improves NK cell-mediated killing of aggressive B-cell NHL. These strategies offer a promising avenue to overcome resistance and improve the efficacy of NK cell-based immunotherapies in relapsed or refractory NHL.

Funder

Irish Research Council
Blood Cancer Network Ireland
European Union’s Horizon 2020

Publisher

University of Galway

Rights

CC BY-NC-ND

cbn

Collections

University of Galway Theses (PhD Theses)