Establishment and characterization of a new mantle cell lymphoma cell line with a NOTCH2 mutation, Arbo

Abstract Cell lines represent an essential tool used in preclinical research. Most hematologic malignancies have a wide array of cell lines representing their respective molecular and pathologic spectra. In mantle cell lymphoma (MCL), cell lines become specifically valuable in view of the heterogeneity of this disease. Unfortunately, the number of MCL cell lines that are available for the research community remains small, with only nine cell lines available for purchase through the American Type Culture Collection (ATCC). We have established a novel blastoid MCL cell line, isolated from the malignant pleural effusion of a 69‐year‐old male with refractory MCL. Arbo was fully characterized with cytogenetics, immunophenotyping, whole exome sequencing and drug sensitivity assays. One of the most notable mutations identified in Arbo (but not in normal tissue) was the missense mutation NOTCH2 R2400*, which has been proposed as a clinically significant mutation in MCL seen in 5% of cases. NOTCH2 R2400* results in a truncated Notch2 protein, leading to a more stable and active protein. Using pharmacologic inhibition of Notch2, we showed a dependence of Arbo on NOTCH2 signaling, as well as a link between CD23 expression on Arbo and NOTCH2 activity. Arbo represents a NOTCH2 mutated model that is useful in MCL as well as other lymphomas with such mutation. We plan to deposit Arbo at the ATCC to be available for the research community.

due to limited availability of such samples. Additionally, although cell lines represent a very useful tool for cancer research, there are only a few that have been fully characterized and are currently used in MCL research. A search for MCL cell lines in the Cellosaurus database [2] revealed only 10 cell lines available for purchase through the American Type Culture Collection (JMP-1, JeKo-1, REC-1, JVM-2, JVM-13, Mino, PF-1, MAVER-1, Z-138, and NCEB-1). Unfortunately, this cell line menu is relatively old and does not cover the wide spectrum of MCL molecular and biologic heterogeneity. Recent advances in MCL unveiled roles for novel genetic alterations with potential therapeutic applications. In a recent study, Bea et al. used next generation sequencing on MCL primary samples as well as six MCL cell lines to investigate for recurrent genetic alterations. Most encountered mutations involved ATM, CCND1, and TP53 that have been previously described, as well as mutations in WHSC1, MLL2, BIRC3, MEF2B, TLR2, and NOTCH2.
Some of these mutations such as MLL2 and MEF2B are represented in MCL cell lines (MLL2 in JEKO and JVM2, and MEF2B in REC). However, NOTCH2-activating mutations, namely R2400*, observed in 5.2% of patients and negatively impacting their prognosis, remain without a representative cell line [3].
Notch2 is a transmembrane protein that plays a critical role in B-cell maturation throughout most of its lifespan [4]. Notch2 is activated following cleavage and release of its intracellular domain in the cytoplasm followed by its translocation into the nucleus where it induces target genes transcription. At the end of its life cycle, Notch2 is inactivated by phosphorylation and subsequent ubiquitination leading to degradation by the proteasome. The C-terminal PEST domain is essential for the deactivation process; mutations in this domain, such as R2400*, interfere with this process, thus resulting in enhanced stability (and subsequently activity) of Notch2 [3,4].
Here we present Arbo, a recently established NOTCH2 R2400*- The cells were able to survive multiple freeze/thaw cycles and were maintained in continuous culture for more than 12 months.
To determine whether Epstein Barr Virus (EBV) played a role in Arbo's immortalization, qPCR was performed using EBV-specific primers on DNA extracted from Arbo and primary MCL cells that had been purified from the malignant pleural effusion and selected by CD19+ on-column selection using magnetic beads. Both Arbo and the primary MCL cells were negative for EBV DNA, using appropriate controls.
To determine Arbo's dependence on Notch2 for survival, we cultured Arbo with increasing concentrations of gliotoxin, a Notch2 transactivation inhibitor [6]. Following 48-h incubation with gliotoxin, we detected a dose-dependent decrease in cell viability using a colorimetric assay, resulting in an IC 50 of ∼100 nM.
Classically, MCL cells lack expression of the B-cell differentiation/activation marker CD23, a pattern used to differentiate MCL from chronic lymphocytic leukaemia (CLL). However, a subset of MCL expresses CD23 with ill-defined causes and consequences. Additionally, the expression of CD23 has been highly associated with Notch2 activity [7]. To determine whether CD23 expression on Arbo is controlled by Notch2 signaling, we quantified CD23 expression by flow cytometry following Notch2 inhibition by serial concentrations of gliotoxin. As expected, we observed a dose-dependent downregulation of CD23 expression following Notch2 inhibition confirming the relationship between the two. While in CLL, Notch2 overexpression is linked to in vitro resistance to the Bcl-2 inhibitor venetoclax [8], the association of which in MCL remains unknown. To determine Arbo's sensitivity to Bcl-2 inhibition, we treated Arbo cells with increasing concentrations of venetoclax for 72 h and found that the cells are highly resistant and can withhold concentrations up to 4 µM without significant cell death ( Figure S3).
In contrast, Arbo seems to depend on B-cell receptor (BCR) signaling for survival, as they were sensitive to BTK inhibition with ibrutinib, with a 48-h IC 50 of ∼2 µM ( Figure S3). Dependence on BCR signaling was also confirmed by the detection of pBTK by immunoblot in unstimulated cells ( Figure 1C). In conclusion, Arbo is a new blastoid NOTCH2 mutated MCL cell line that is well characterized, and that can serve as a model for Notch2 signaling in MCL. Arbo is planned to be deposited in ATCC to be available to the research community.

CONFLICTS OF INTEREST
The authors declare they have no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available in the Supporting Information of this article.

ETHICS STATEMENT
The study received the IRB approval and appropriate guidelines were followed.

PATIENT CONSENT STATEMENT
Samples were collected from the patient following written consent.