High prevalence of MEF2D fusion in human B‐cell precursor acute lymphoblastic leukemia cell lines

The myocyte enhancer factor 2D (MEF2D) fusion oncogene is a recently identified cytogenetic alternation in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). MEF2D fusion is detected in approximately 2% to 4% and 7% of pediatric and adult BCP-ALL cases, respectively. Of clinical importance, MEF2D fusion-positive (MEF2D fusion+) ALL is associated with poor therapeutic outcome. Thus, to improve the prognosis of MEF2D fusion+ ALL patients, the development is required of an in vitro model system that mimics the cell biology of MEF2D fusion+ ALL. In our laboratory, we developed a cell line bank consisting of 83 human BCP-ALL cell lines (76 cell lines were established from Japanese patients) (Data S1: supplementary information) after approval of the use of human cell lines for a research purpose by the ethics committee of the University of Yamanashi. As indicated in Figure 1A, TCF3-PBX1 (n = 16), BCR-ABL1 (n = 15), and KMT2A fusion (n = 14) were the three major oncogenic fusions. ETV6-RUNX1 and TCF3-HLF were additionally observed in six and four cell lines, respectively, while hyperdiploid was not observed. As a result, 28 cell lines were categorized into the so-called “B-others” ALL, which lacks known chromosomal abnormalities that are detectable by conventional cytogenetic analyses. To investigate oncogenic fusions in these B-others ALL cell lines, we performed an RNA capture sequencing analysis. Of note, MEF2D fusions were detected in 19 (68%) of the 28 B-others ALL cell lines, indicating that majority of “B-others” ALL cell lines were established from MEF2D fusion+ ALL. Among 19 MEF2D fusion+ ALL cell lines, 11 (58%), 7 (37%), and 1 (5%) cell lines harbored MEF2DBCL9, MEF2D-HNRNPUL1, and MEF2D-DAZAP1, respectively (Figure S1). Thus, MEF2D fusion was the most common oncogenic fusion in our series of BCP-ALL cell lines (19/83; 23%). Since the incidence of MEF2D fusions was reported to be 2% to 4% in childhood BCP-ALL and 7% in adult BCP-ALL, the incidence of MEF2D fusions in our BCP-ALL cell lines was approximately five times higher than that in clinical settings. In our series of cell lines, we intended to establish and collect cell lines with previously well-known oncogenic fusions, such as TCF3-PBX1, BCR-ABL1, and KMT2A fusions. In contrast, there was no selection bias in the case of MEF2D fusion+ ALL cell lines. Nevertheless, MEF2D fusion was the most common oncogenic fusion in our series of BCP-ALL cell lines. These observations suggested that MEF2D fusion+ ALL cells have a growth advantage in an in vitro culture system. Consistently, although statistically insignificant, probably due to a limited number of the cell lines with the detailed record, duration of cell line establishment tended to be shorter in two MEF2D fusion-positive ALL cell lines (median 81 days) than in six MEF2D fusion-negative BCP-ALL cell lines (median 118 days) (Figure S2). Moreover, in 73 BCP-ALL cell lines, with basic record, the cell lines established from the samples at diagnosis tended to be more common in MEF2D fusion-positive ALL cell lines (10/16 cell lines; 63%) than in MEF2D fusion-negative BCP-ALL cell lines (20/57 cell lines; 35%) (P = .083 in Fisher's exact test; Figures S3 and S4). Since samples from BCP-ALL patients at diagnosis showed a lower growth potential in vitro than those at relapse, this observation seemed to be consistent with a notion that MEF2D fusion+ ALL cells have a growth advantage in vitro. Previous clinical studies demonstrated that MEF2D fusion+ ALL develops in older children and young adults. Consistently, MEF2D fusion+ ALL cell lines were exclusively established from patients aged 5 years or older (Figures 1B and S3). Immunophenotypic analyses in the samples from MEF2D fusion+ ALL patients previously revealed significant features: an aberrant expression of CD5, a frequent expression of cytoplasmic μ chain, and a weak or absent expression of CD10. Consistently, most of MEF2D fusion+ ALL cell lines (14/19; 74%) were positive (>20%) for cell surface expression of CD5, while only one of 64 MEF2D fusion-negative BCP-ALL cell lines was positive (Figure 1C). In addition, 15 (79%) out of 19 MEF2D fusion+ ALL cell lines were positive (>40%) for cytoplasmic μ chain expression (Figure S5A). Finally, CD10 expression levels in MEF2D fusion+ ALL cell lines tended to be lower in comparison with those in MEF2D fusionnegative ALL cell lines except for KMT2A rearrangement-positive ALL cell lines (Figure S5B). In addition to immunophenotypic analyses, genetic landscape in the samples, from MEF2D fusion+ ALL patients, previously revealed significant features: a frequent CDKN2A/CDKN2B deletion and no IKZF1 deletion. Similar to the clinical samples, in our previous multiplex ligation-dependent probe amplification analysis, CDKN2A/CDKN2B genes were deleted in 17 (89%) of 19 MEF2D fusion+ ALL cell lines (Figures 1D and S3), while no IKZF1 deletion was detected in all of MEF2D fusion+ ALL cell lines (Figures 1E and S3). Since CDKN2A and CDKN2B are well-known tumor suppressor genes, frequent CDKN2A/CDKN2B deletions may be associated with a growth advantage under in vitro culture condition. Consistently, the percentage of the cells in G0/G1 phase was significantly lower in MEF2D fusion+ ALL cell lines (median 59%) than in MEF2D fusionnegative BCP-ALL cell lines (median 64%) (P = .0081 in Mann-Whitney U test) (Figure S6A). Moreover, a higher proportion of dormant cells (G0/G1 > 66.7%) was significantly rare in MEF2D fusion+ ALL cell lines (1/19 cell lines; 5%) than in MEF2D fusion-negative BCP-ALL cell lines (23/64 cell lines; 36%) (P = . 0092 in Fisher's exact test; Received: 27 February 2020 Revised: 28 May 2020 Accepted: 29 May 2020