Abstract 3160: Establishment and characterization of a new mantle cell lymphoma cell line with a NOTCH2 mutation, ArBo
Authors: Satyavarapu Ishwarya , Safa Firas M. , Rasmussen Terri , Saba Nakhle 
Published: June 2022

Cell lines represent a very useful tool for cancer research. In mantle cell lymphoma (MCL), the number of characterized cell lines remains small. We have recently established a novel blastoid MCL cell line, isolated from a malignant pleural effusion of a patient with MCL.

A sample of the malignant pleural effusion was obtained with informed consent, and mononuclear cells were isolated. Following a few days of in-vitro culture, cells started to grow rapidly resulting in an estimated doubling time of 48h. The cells survived multiple freeze/thaw cycles and have been maintained in culture for the last 8 months. Hence the establishment of a novel blastoid MCL cell line, named Arbo.

Both Arbo and the primary MCL cells obtained from the pleural effusion were negative for EBV DNA by qPCR.

Flow cytometry showed kappa-restricted cells that were positive for CD5, 19, 20, 22, and 23, and negative for CD2, 3, 7, 10, and 71. FMC7 was positive in Arbo and negative in primary cells, as opposed to CD38 that was negative in Arbo and positive in the primary cells. Arbo’s karyotype was complex contrasting with the normal 46XY karyotype found in the primary MCL cells. The presence of t(11;14)(q13;q32) was confirmed by FISH, additionally overexpression of cyclin-D1 was confirmed by western blot.

Whole exome sequencing (WES) was performed on DNA collected from Arbo revealing a total of 148,059 SNPs. Mutations were detected in the ATM, TP53 and NOTCH2(C19W and R2400*) genes. Mutations in NOTCH2 have not been described in other MCL cell lines, while they were identified in 5.2% of MCL patients. Immunoblotting for NOTCH2 confirms the expression of the protein by the cell line. Additionally, expression of CD23, which has been shown to be induced through NOTCH2 signaling, was confirmed by flow cytometry. To further characterize the effect of NOTCH2 on the survival of Arbo cells and CD23 expression, we cultured the cell line with increasing concentrations of the NOTCH2 transactivation inhibitor gliotoxin. We observed a dose-dependent inhibition of cell growth with an IC50 of 100 nM. Additionally, a dose dependent decrease in the expression of CD23 was observed by flow cytometry following treatment with gliotoxin.

To determine the dependence of Arbo on B-cell receptor (BCR) signaling for survival, we cultured the cells with increasing concentrations of the BTK inhibitor ibrutinib for 48h. Inhibition of cell growth was observed in a dose-dependent manner resulting in an IC50 of 0.4 µM.

In conclusion, Arbo is a novel blastoid MCL cell line with a NOTCH2 mutation that is fully characterized and will be made available to the research community.

Supported by a grant from the Ladies Leukemia League, Inc., of the Gulf South Region.

Citation Format: Ishwarya Satyavarapu, Firas M. Safa, Terri Rasmussen, Nakhle Saba. Establishment and characterization of a new mantle cell lymphoma cell line with a NOTCH2 mutation, ArBo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3160.