Abstract 394: ERBB4-mediated signaling is a mediator of resistance to BTK and PI3K inhibitors in B cell lymphoid neoplasms
Authors: Arribas Alberto J. , Napoli Sara , Cascione Luciano , Barnabei Laura , Sartori Giulio , Cannas Eleonora , Gaudio Eugenio , Tarantelli Chiara , Mensah Afua A. , Spriano Filippo , Zucchetto Antonella , Rossi Francesca M. , Rinaldi Andrea , de Moura Manuel Castro , Stathis Anastasios , Stussi Georg , Gattei Valter , Brown Jennifer R. , Esteller Manel , Zucca Emanuele , Rossi Davide , Bertoni Francesco 
Published: April 2023

Abstract
Background: Marginal zone lymphoma (MZL) is an indolent yet incurable B cell malignancy. Two BTK inhibitors, ibrutinib and zanubrutinib, are FDA approved for relapsed/refractory MZL patients. PI3K inhibitors have also shown clinical activity. The identification of the mechanisms of resistance can provide useful information to optimize the use of the agents. We previously reported an IL6 driven MZL model of PI3K inhibitors resistance developed by prolonged exposure to the PI3Kδ inhibitor idelalisib (Arribas, Haematologica 2022). Here, we present the detailed characterization of a second model with resistance to both BTK and PI3K inhibitors.
Methods: MTT assay. RNA-Seq, whole exome sequencing, miRNA and methylation profiling. FACS and ELISA analyses.
Results: Resistant cells, developed by continuous exposure of the cell line Karpas1718 to idelalisib, showed resistance to various inhibitors of BTK (ibrutinib, zanubrutinib, acalabrutinib and pirtobrutinib) and PI3K (idelalisib, duvelisib, copanlisib and umbralisib). No mutations affecting BTK, PLCG2 or CXCR4 were identified in resistant cells, which had higher expression of genes involved in ERBB signaling (HBEGF, NRG2, ERBB4), cell proliferation (PBK, MKI67, TCL1A) and DNA recombination (RAG1, RAG2) than parental cells. We confirmed cell surface ERBB4 up-regulation, and the cytoplasmatic expression and secretion of its ligand HBEGF in resistant cells, which led to increased levels of p-AKT and p-ERK. The miRNAs miR-29c and let-7c, known negative regulators of the HBEGF-ERBB axis, were fully methylated and down-regulated in resistant compared to parental cells. ERBB4 genetic silencing improved sensitivity to PI3Kδ inhibitor, and exposure to let-7c or miR-29c mimics decreased secreted HBEGF and recovered sensitivity to PI3K inhibitors in resistant cells. Addition of recombinant HBEGF (rHBEGF) induced resistance to BTK and to PI3K inhibitors in parental cells and in other lymphoma models including mantle cell lymphomas and diffuse large B cell lymphomas (DLBCL). The rHBEGF induced resistance was reverted adding the ERBB inhibitor lapatinib. To extend our findings to the clinical context, using two MZL and one DLBCL expression datasets, we showed HBEGF and ERBB4 expression in clinical specimens. Finally, HBEGF levels appeared elevated in the serum of CLL patients with primary or acquired resistance to PI3Kδ or to BTK inhibitors, compared to patients responding to the drugs and paired for similar clinical features.
Conclusions: We characterized a novel B cell lymphoma model of secondary resistance to BTK and PI3K inhibitors. Our results indicate that epigenetic plasticity led to the activation of HBEGF-ERBB signaling sustaining resistance to BTK/PI3K inhibitors, which can be overcome using epigenetic agents and ERBB inhibitors. These therapeutics approaches could be tested in novel clinical trials. AJA, SN: equally contributed.
Citation Format: Alberto J. Arribas, Sara Napoli, Luciano Cascione, Laura Barnabei, Giulio Sartori, Eleonora Cannas, Eugenio Gaudio, Chiara Tarantelli, Afua A. Mensah, Filippo Spriano, Antonella Zucchetto, Francesca M. Rossi, Andrea Rinaldi, Manuel Castro de Moura, Anastasios Stathis, Georg Stussi, Valter Gattei, Jennifer R. Brown, Manel Esteller, Emanuele Zucca, Davide Rossi, Francesco Bertoni. ERBB4-mediated signaling is a mediator of resistance to BTK and PI3K inhibitors in B cell lymphoid neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 394.