Harnessing BCL2 inhibition with BTK inhibition in follicular lymphoma: A translational trial with mechanistic insights from single cell analysis

Abstract Background: Follicular lymphoma (FL), the most common indolent non-Hodgkin lymphoma, remains incurable despite initial responsiveness to treatment, with many patients developing relapsed/refractory disease. While FL carries BCL2 translocations and displays elevated B-cell receptor (BCR) signaling, single-agent targeted therapies such as BTK inhibitors (BTKi) and BCL2 inhibitors (BCL2i), have shown limited efficacy. We hypothesized that the tumor microenvironment (TME) contributes to the activation of alternative survival mechanisms and dual inhibition of BTK and BCL2 may overcome TME-mediated resistance and improve therapeutic response. Methods This study investigates the therapeutic potential of combining the BTK inhibitor zanubrutinib (zanu) with the potent BCL2 inhibitor sonrotoclax (sonro) in FL, leveraging a novel ex vivo co-culture model (iNHL-TME) that mimics the lymph node microenvironment. Our system incorporates patient-derived indolent lymphoma cells, bone marrow fibroblasts (BMF) and several B-cell growth factors. Using this platform, we evaluated the effects of zanu (BTKi) and sonro (BCL2i), both alone and in combination. Drug responses are categorized as Highly Sensitive (HS), Sensitive (S), Lowly Sensitive (LS) and Non-Responsive (NR). Results: Initial synergy assays in a FL cell line (FSCCL) demonstrated additive, not synergistic, effects of BTKi and BCL2i. Testing in primary FL tumors, a historically difficult-to-model disease, revealed heterogeneous responses. Among nine FL samples tested, using our predefined response criteria (see Methods), 4 were responsive to zanu (HS and S) and 4 to sonro (HS). Among these 8. one case responded well to both zanu and sonro single agent. Of 9, 7 exhibited enhanced cell killing when both drugs were combined. The remaining two were highly sensitive to sonro alone resulting in very low cell viability, thus no additional effects by zanu can be observed. Overall, all nine responded well to the dual inhibition. To dissect the molecular basis of this enhanced cytotoxicity, we conducted single-cell RNA sequencing on a FL lymph node biopsy treated ex vivo with zanu, sonro, or both (zanu+sonro) as well as the DMSO control. The cells were collected before the ensuing phenotypic cell death following a short period of four hours of drug treatment. Transcriptional profiling at this early time point identified four B-cell states with distinct molecular pathway activities. Tumor B cells clustered with 1) active BCR signaling, 2) ER stress, 3) active NF-kB signaling and 4) Heat-shock response. Analysis of the entire B cell population under the four drug conditions revealed both expected and unexpected findings. As expected, we found that 1) BCR signaling was downregulated by zanu and zanu+sonro, and 2) Apoptosis was upregulated by sonro and zanu+sonro. Surprisingly, sonro and the combination also upregulated cytoprotective NF-kB signaling with the induction of pro-survival genes. This is evident by the dramatic increase of the number of cluster 3 cell population (mentioned above with active NF-kB signaling) under sonro treatment alone and zanu+sonro treatment. Notably, this population increase was not observed with zanu alone. These unexpected findings suggest that a subset of tumor B cells survive through the BCL2-targeted therapy by activating alternative pathways that eventually upregulate cytoprotective NF-kB. Conclusion: This study integrates functional drug testing with high-resolution molecular profiling. Functional phenotypic studies suggest that the combination of BTK and BCL2 inhibitors deliver effective cell killing. The mechanistic study also suggests that most tumor cells are under drug-induced stress and undergo apoptosis. However, a subpopulation of tumor cells survives by upregulating cytoprotective pathways. Further studies are needed to verify these results and to determine if this upregulation would lead to tumor persistence and resistance.