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K. Ohki et al.
ment of an early diagnostic method and a new therapeutic strategy are necessary for this type of B-ALL. As ex vivo sen- sitivity of xenografted leukemic cells harboring MEF2D fusions to HDAC inhibitors has been recently reported,10 it may offer a plausible therapeutic option for this type of B- ALL. In relation to diagnosis, we have already established a qPCR based detection assay for the frequent MEF2D fusions, which may be useful for rapid diagnosis in combi- nation with FISH.
Another interesting observation is that B-ALL cases with MEF2D fusions have a characteristic immunophenotype, most typically presenting as CD5- and cytoplasmic μ chain- positive. Although it was previously reported that their immunophenotypes were characterized by weak or absent expression of CD10 and high expression of CD38,10 we found that CD10 expression was not significantly lower than B-other-ALL, and that CD38 expression level was sim- ilar to that of TCF3-PBX1-positive cases. Therefore, B-ALL patients with MEF2D fusions could be more effectively diagnosed by combining genetic testing with immunophe- notyping.
As shown in Table 1, MEF2D fusion cases were mutually exclusive of the known risk stratifying chromosomal translocations, at least at the cytogenetic level. Among addi- tional genetic alterations in MEF2D fusion cases, we identi- fied a significantly higher frequency of CDKN2A/ CDKN2B deletions. At initial diagnosis, 7/8 (87.5%) relapsed patients had CDKN2A/CDKN2B deletions, while they were seen in only 2/7 (28.6%) non-relapse patients. Thus, the prognostic impact of CDKN2A/CDKN2B deletions in MEF2D fusion B-ALL needs to be assessed in larger patient cohorts. Interestingly, we also identified that MEF2D fusion patients had mutations in genes known to be recurrent in T-ALL; in particular, there was an unexpectedly high frequency of mutations in PHF6 (8/16). PHF6 encodes a plant home- odomain (PHD) factor with a proposed role in gene expres- sion control.30 As PHF6 has been proposed to play a role as a tumor suppressor gene,24 it may participate in the patho- genesis of MEF2D fusion B-ALL. Thus, further investigation is needed to clarify this role. Additionally, mutations of genes in the RAS pathway (NRAS, KRAS, NF1, and PTPN11) or IKZF1 alterations were not observed in our cases, in conflict with previous reports.
Our data also showed that MEF2D fusion B-ALL has a gene expression signature significantly distinct from other genetic subtypes of B-ALL. Supervised analysis led to sepa- ration of MEF2D fusion cases into isolated clusters both in PCA and hierarchical clustering, and these clusters were the closest neighbors to TCF3-PBX1-positive cases. This obser- vation is consistent with the knowledge that both types of B-ALL tend to show a pre-B ALL immunophenotype, and high-level expression of pre-BCR components, including IGHG/IGHV5-78 and IGLL1, were commonly observed in both subtypes.31 However, when we examined the gene expression signatures closely, there were significant differ- ences between them.
It has recently been shown that MEF2 family proteins, including MEF2C and MEF2D, play a critical role in early B- cell differentiation. It was demonstrated that B-cell develop- ment was blocked at the pre-B-cell stage in Mef2c/d-defi- cient mice, indicating that they are essential for progression of B-cell precursors (large to small pre-B-cell transition).32 It was further shown that, upon activation via pre-B-cell receptor signaling, Mef2c/d induces target genes, including interferon regulatory factor 4 (Irf4; stimulates the expression of
Ikzf1/3) and histone deacetylase 5/9 (Hdac5/9; encoding the protein known to repress Mef2c activity) as we have sum- marized schematically in Online Supplementary Figure S4. On the other hand, MEF2D fusion B-ALL cases exhibited elevated expression of IRF4 and HDAC9 with down-regu- lation of MEF2C, although it has been suggested that dereg- ulated expression of the N terminus of MEF2D should induce the up-regulation of the target genes of MEF2C/D, whereas subsequent excess of HDAC9 activity represses MEF2C and its downstream gene expression cascade.10
Our existing data further revealed that up-regulation of GATA3 is another gene expression characteristic of MEF2D fusion ALL. GATA3 is a critical transcription factor in early T-cell development33-35 and its transcriptional repression is essential for early B-cell commitment,36-37 while it has also been reported that GATA3 exhibits myeloid-inducing activ- ity in committed B-lymphocytes under the defect of PAX5 function.38-41 Furthermore, significantly increased GATA3 mRNA levels were associated with ZNF384 fusion-positive cases and a higher risk of relapse in childhood B-ALL with the Ph-like phenotype.42-43 As our data also indicated down- regulation of GATA3 expression in TCF3-PBX1-positive cases, ectopic overexpression of GATA3 appears to partici- pate in establishment of a characteristic gene expression sig- nature as well as the biological signature of MEF2D fusion ALL distinct from that of other genetic subtypes of B-ALL.
The roles of the MEF2D fusion partners in terms of their biological effects within the fusion molecules of MEF2D fusion ALL are largely unknown. The genes known to be fused to MEF2D in B-ALL have a variety of biological func- tions. For example, BCL9 is known to be related to WNT/β- catenin signaling, whereas the MEF2D-BCL9 fusion retains only the last one or two exons of the BCL9 gene, thus lack- ing the functional domains required for WNT/β-catenin sig- naling, suggesting a role other than deregulation of WNT/β- catenin signaling.10 HNRNPUL1 encodes a nuclear RNA- binding protein of the heterogeneous nuclear ribonucleo- protein family that may play a role in nucleocytoplasmic RNA transport and DNA repair,18 whereas its role as a por- tion of the fusion molecule in the pathogenesis of B-ALL remains unclear. As HNRNPH1 is a member of the same protein family as HNRNPUL1, both MEF2D-NRNPUL1 and MEF2D-HNRNPH1 likely share the same unknown function.
In conclusion, we have shown that ALL patients harbor- ing MEF2D fusion genes possess a characteristic immunophenotype and gene expression signature as well as distinct clinical features, defining them as a distinct genetic subtype among B-other-ALL. Although additional studies are required to elucidate the biological function of the MEF2D fusion protein in leukemogenesis, our data has allowed improved characterization of this new B-ALL sub- type.
Acknowledgments
The authors would like to thank K. Itagaki, H. Yagi, Y. Katayama, A. Tamura, K. Takeda, K. Hayashi, and the staff of the Laboratory for Genotyping Development, Riken Center for Integrative Medical Sciences for their excellent data management and experimental assistance. We thank all members of the Committees of ALL and of Research and Diagnosis of TCCSG.
Funding
This work was supported in part by a Health and Labour Sciences Research Grant (3rd-term comprehensive 10-year
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haematologica | 2019; 104(1)