Abstract 534: Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2

Abstract Background: Copanlisib is a PI3K inhibitor with selectivity against PI3Kδ and PI3Kα and is approved for treating patients with follicular lymphoma and marginal zone lymphoma (MZL). Preclinical studies have demonstrated strong synergism of copanlisib and BCL2 inhibitor venetoclax in different lymphoma models, and the combination is under clinical evaluation. Understanding the resistance mechanisms could help the design of improved therapies. Hence, we generated MZL cell lines resistant to the copanlisib/venetoclax combination. Methods: All cells were tested for cell identity and Mycoplasma status. Resistance was confirmed stable after 3-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out by confirming sensitivity to vincristine. Targeted agents already in the clinics were combined with copanlisib/venetoclax. Cells underwent transcriptome and methylation profiling, targeted DNA sequencing, and immunophenotypic and immunoblotting analyses. Results: We developed SSK41-derived models with resistance to copanlisib (n.=1) or venetoclax (n.=1) as single agents and to copanlisib/venetoclax (n.=2) by long IC50 or by upfront IC90 exposure. The single-drug-resistant cells showed only partial resistance to copanlisib/venetoclax. In contrast, the cells that developed exposing cells to copanlisib/venetoclax IC90 exhibited a more substantial decrease in sensitivity to the combination. Copanlisib/venetoclax induced apoptosis and cell cycle arrest in PAR but not in resistant cells. Sensitivity to other PI3K, BCL2, and BTK inhibitors was decreased in all resistant lines. The response to MCL1-i, mTOR-i, and epigenetic agents (decitabine, HDAC, BET, and EZH2 inhibitors) was maintained. Triple combinations of copanlisib/venetoclax with the AURKA inhibitor alisertib, PLK inhibitor rigosertib, or the multikinase inhibitor amcasertib were active in both parental and resistant cells. No acquired mutations were observed via targeted DNA sequencing designed to cover various coding genomic regions known to be recurrently mutated in mature B-cell neoplasms, including BCL2, nor via RNA-Seq for PIK3CA/B/G/D genes. Integration of RNA and methylation profiles showed deregulation of genes involved in type-I IFN, chemokines (CCL25, CXCL13), apoptosis regulation, and JAK-STAT signaling. Resistant cells upregulated CD19 expression compared to parental cells. Immunoblotting showed the inability of copanlisib/venetoclax to block BCR signaling in resistant cells. Conclusions: We created four novel MZL-derived models of secondary resistance to PI3K and BCL2 inhibitors, which will help clarify possible modalities to overcome resistance. Novel potential targets were already identified, such as AURKA or PLK, to be further exploited. Citation Format: Alberto J. Arribas, Luciano Cascione, Eleonora Cannas, Helen Bellerjeau, Federica Fuzio, Aleix Noguera, Andrea Rinaldi, Manel Esteller, Emanuele Zucca, Andrea Alimonti, Davide Rossi, Anastasios Stathis, Francesco Bertoni. Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 534.