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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.

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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.

Background: Of all subdivisions of stroke, intracerebral hemorrhagic stroke (ICH) only accounts for around 10-15% but is characterized by the highest mortality and morbidity. Lately, understanding immunological and inflammatory responses post-hemorrhagic stroke allows for the development of alternative therapeutic intervention. Toll-like receptors (TLR) are a large family of recognition receptors which have a regulatory pathway that is a key factor in determining the neurological outcomes of patients with ICH. In this study, we took a closer look at understanding the role that the overexpressing the soluble Toll-like receptor 4 (sTLR4) has on neuroprotection after ICH. Methods: Intracerebral injections of autologous blood were used to induce the ICH preclinical model on adeno-associated virus serotype 1 (rAAV1)-mediated FcV5 (control) and FcV5-sTLR4 expression in the brains of mice. Functional outcomes of these mice were assessed using several behavioral testing methods such as rotarod, open field locomotor test, and a neurological deficit score (NDS) at the different time-points of 24h, 48h and 72h. To assess changes to the lesion, hematoma and tissue injury, a histological stain, cresyl violet, was performed. Results: The data reveal a 41.9+/-8.2% smaller lesion volume in sTLR4 overexpressed mice. Further analysis with histological and immunochemical stains shows a trend towards a smaller tissue injury and hematoma volume, unchanged heme oxygenase 1 and iron levels, and unchanged microgliosis and astrogliosis levels. Conclusion: These findings reveal that the overexpression of sTLR4 does improve preliminary ICH outcomes. We will also discuss mechanisms behind the relationship between sTLR4 signaling and ICH along with possibilities of future methods of therapeutic interventions.