Establishment of a Novel Waldenström's Macroglobulinemia (WM) Cell Line Bcwm.2 with Del6q and Unique MYD88^S243N, LYN^I297N, SPI1^Q227E Mutations and That Shows Robust In Vivo Engraftment in SCID NOD Mice

Background:

Waldenström's macroglobulinemia (WM) is an indolent B-cell lymphoproliferative disorder characterized by bone marrow (BM) infiltration with lymphoplasmacytic lymphoma and monoclonal immunoglobulin M (IgM) production. Mutations in MYD88 are present in 95-97% of WM patients. Deletions involving chromosome 6q (del6q) occur in up to 50% of WM patients, impacting genes that regulate MYD88, BTK, BCL2, and apoptosis. Cell lines are crucial for studying WM biology and developing treatments. No del6q WM cell line currently exists for functional studies.

Patients and Methods:

We developed and extensively characterized a novel cell line (BCWM.2) from CD19-selected BM lymphoplasmacytic cells from a symptomatic, treatment-naive WM patient. Whole genome sequencing (WGS) was performed on BCWM.2 cells. Since no previous genomic sequencing of WM cell lines has been reported, we also performed for comparative purposes WGS of three other WM cell lines currently in active research use for WM: BCWM.1, MWCL-1, and RPCI-WM1. An in vivo xenograft mouse model of BCWM.2 was developed in NOD SCID mice, and tumor origin in xenografted tumors was confirmed by whole exome sequencing and immunohistochemistry.

Results:

BCWM.2 cells exhibited characteristics of lymphoplasmacytic cells expressing CD19, CD20, CD23, CD38, CD45RA, CD52, surface IgM, and surface immunoglobulin light chain λ. A small subset expressed CD138 and surface IgD; CD3, CD5, CD10, CD11c, and CD117 were absent. WGS revealed BCWM.2 had trisomy of chromosomes 3 and 12, deletion of 6q, and amplification of 6p. BCWM.1 showed no amplifications or deletions, MWCL-1 had deletions in 17p and 17q and amplifications in 11q, and RPCI-WM1 exhibited multiple deletions and amplifications, including uniparental disomies on 17p, 18q, and X. Unlike the other lines, which were heterozygous for the MYD88^L265P mutation, BCWM.2 carried a heterozygous MYD88^S243N mutation. All four cell lines were CXCR4 wild-type. BCWM.2 also had a novel LYN^I297N mutation in the regulatory hinge region. Additional mutations in BCWM.2 included AKAP9^R1276*, HDAC5^H236P, RUNX3^V141L, and SPI1^Q227E, the later representing a recurring mutation in WM that affects the binding of the ETS factor SPI1 and enhances proliferation (Roos-Weil et al, Cancer Discovery 2019). BCWM.2 showed restrained responses to BTK inhibitors but was highly sensitive to the BCL2 inhibitor venetoclax and the JAK2/SRC/IRAK1 inhibitor pacritinib, highlighting its utility for pharmacological screenings. BCWM.2 cells also showed robust engraftment in NOD-SCID mice producing subcutaneous tumors (~500 mm^3) four weeks post-injection of 2 x 10^6 cells/mouse. Human IgM levels in mouse sera increased post-engraftment. Immunohistochemistry confirmed BCWM.2 engraftment, with tumors showing intermediate lymphoid cell morphology, CD20 and MUM1 expression, lambda light chain and IgM heavy chain clonality, and 60% KI67 staining. Whole exome sequencing of engrafted tumors confirmed the presence of MYD88 and LYN mutations.

Conclusions:

We established and extensively characterized a novel WM cell line with MYD88^S243N, LYN^I297N, and SPI1^Q227E mutations with a deletion of 6q, permitting functional studies of this chromosomal aberration that is present in up to 50% of WM patients and impacts genes that regulate MYD88, BTK, BCL2, and apoptosis. BCWM.2 showed robust engraftment in NOD-SCID mice. Our findings provide a novel model for in vitro and in vivo studies of WM pathogenesis, and the development of targeted therapies for MYD88 mutated lymphomas.

Sarosiek:BeiGene: Honoraria, Research Funding; ADC Therapeutics: Research Funding; Cellectar Biosciences: Honoraria, Research Funding. Castillo:AbbVie: Consultancy, Research Funding; Mustang Bio: Consultancy; Pharmacyclics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy; AstraZeneca: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Janssen: Consultancy; LOXO: Consultancy, Research Funding; Cellectar Biosciences: Consultancy, Research Funding. Treon:AbbVie/Pharmacyclics: Honoraria, Research Funding; Eli Lilly: Research Funding; Janssen: Honoraria, Research Funding; Parexel: Honoraria, Research Funding; BeiGene, Inc.: Honoraria, Research Funding.