Abstract 906: Development of novel preclinical models of secondary resistance downstream B cell receptor in marginal zone lymphoma
Authors: Arribas Alberto J. , Gaudio Eugenio , Cascione Luciano , Tarantelli Chiara , Zucchetto Antonella , Rossi Francesca , Rinaldi Andrea , Kwee Ivo , Stathis Anastasios , Gattei Valter , Zucca Emanuele , Rossi Davide , Bertoni Francesco 
Published: August 2018

Abstract
Background. Targeting downstream signaling to B-cell receptor (BCR) is one of the promising therapeutic approaches in lymphoma. PI3Kδ-inhibitor (i) idelalisib (IDEL) and BTK-i ibrutinib (IBRU) have been approved by FDA for different indications and are currently used to treat indolent lymphomas including marginal zone lymphoma (MZL). However, a subset of patients relapses due to acquired resistance. A better understanding of resistance mechanisms could help to design improved therapies. With this aim, we generated MZL cell lines resistant to either IDEL or IBRU.
Materials and Methods. VL51 and Karpas1718 cell lines were exposed to IC90 concentration of IDEL or IBRU for several months until they acquired specific drug resistance (RES). In parallel, lines were cultured upon similar conditions in the absence of drug (parental, PAR). Proliferation of acquired stable resistance was tested by MTT assay (72 hrs) in RES and PAR after 2-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out. RES and PAR cells underwent transcriptome profiling by RNA-Seq, whole exome sequencing and were exposed to IDE or IBRU alone or in combination with a library of 348 compounds. Protein expression of p-AKT, p-BTK, p-PLCG2, p-mTOR and p-ERK was determined by FACS.
Results. Six lines with IC50s 5-10 fold times higher than PAR counterparts were generated: two VL51 and two Karpas1718 IDEL-RES and two VL51 IBRU-RES. IDEL-RES-VL51 showed mutations in AKT-mTOR regulators, DNA damage genes and Ca2+/Calmodulin partners while IDEL-RES-K1718 exhibited variants affecting kinases and chromatin remodeling genes. IBRU-RES-VL51 harbored mutations involving kinases and cell cycle genes. IDEL-RES-VL51 showed enrichment in RNA-Seq signatures related to BCR and RAS-RAF signaling while IDEL-RES-K1718 was enriched in cell cycle and HDAC targets suggesting different mechanisms of resistance. IBRU-RES-VL51 exhibited overexpression of PI3K signaling and proliferation signatures. Consistent with RNA-Seq, upon 1µM IDEL, IDEL-RES showed increased p-AKT and p-BTK, while upon 1µM IBRU, IBRU-RES showed enhanced p-PLCG2. Single treatment with DOTL1-i, JAK-STAT-i and TGFB-i decreased cell viability in IDEL-RES. IDEL-combo with tyrosine kinase-i (ABL/SRC, cKit), EZH2-i and RAF-i overcame resistance in IDEL-RES. IBRU-combo with cell cycle modulators and VEGFR-i exhibited synergism in IBRU-RES-VL51, which showed sensitivity to PI3K/mTOR-i, JAK/STAT-i and NFKB-i.
Conclusions. We created three novel models, derived from MZL, of secondary resistance: two to the PI3Kδ-i idelalisib, and one to BTK-i ibrutinib. We also identified potential active treatments for IDEL-RES or IBRU-RES that are worth of further investigations. These models, apparently driven by different biologic processes, will help in clarifying mechanisms of resistance to the drug and to evaluate alternative therapeutic approaches.
Citation Format: Alberto J. Arribas, Eugenio Gaudio, Luciano Cascione, Chiara Tarantelli, Antonella Zucchetto, Francesca Rossi, Andrea Rinaldi, Ivo Kwee, Anastasios Stathis, Valter Gattei, Emanuele Zucca, Davide Rossi, Francesco Bertoni. Development of novel preclinical models of secondary resistance downstream B cell receptor in marginal zone lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 906.