MEF2D Fusions Drive Oncogenic Pre-BCR Signaling in B-ALL

B acute lymphoblastic leukemia (B-ALL) is a heterogenous disease characterized by recurrent chromosomal alterations that result in chimeric fusions commonly involving molecules implicated in tumor suppression, kinase signaling, chromatin remodeling, and B-cell differentiation. Fusions involving the transcription factor MEF2D are found in approximately 5% of B-ALL cases and are associated with poor outcome (1).

MEF2D belongs to the myocyte enhancer factor (MEF2) family of transcription factors (TF) which includes 4 paralogs in vertebrates (MEF2A–D). MEF2 proteins, originally identified as critical players in muscle and neuronal differentiation, are pleiotropic gene regulators characterized by highly conserved N-terminal dimerization and DNA-binding domains. In a tissue- and context-dependent fashion, MEF2 TFs act in multimeric complexes with transcriptional coactivators/repressors and epigenetic modifiers such as EP300 and HDACs to alter target gene expression. Highlighting a role for MEF2 in normal B-lymphopoiesis, conditional deletion of Mef2c/d in B-cells results in a block at the pre–B-cell stage by impeding activation of normal transcriptional differentiation programs (2).

To date, the MEF2D fusions identified in B-ALL invariably fuse the N-terminus of MEF2D with a range of 3′ partners, the most common of which are BCL9 and HNRNPUL1 (1). Although a gene-specific function for the 3′ partners cannot be excluded, the extreme similarity in gene expression observed in patients with different MEF2D fusions suggests that the central malignant phenotype is driven by the MEF2D TF itself. Studies that examined protein expression of the MEF2D fusions found these were more active and expressed at significantly higher levels than nonrearranged MEF2D, and it is thought that the 3′ partners act largely to stabilize the MEF2D-containing fusion (1, 3). The stability of the MEF2D fusions may be further enhanced by translocation-mediated loss of miRNA sites that negatively regulate MEF2D expression (3). Although large-scale gene expression studies indicate that MEF2D-rearranged B-ALL represents a distinct subtype of B-ALL, several groups have observed a strong overlap with patients harboring the TCF3–PBX1 fusion (1, 4); however, the significance of this was not clear.

To unravel the genome-wide occupancy of MEF2D fusions in B-ALL, Tsuzuki and colleagues (5) utilized CRISPR editing to insert a HA-tag in-frame to the endogenously expressed MEF2D–HNRNPUL1 fusion in Kasumi-7 B-ALL cells. This allowed them to perform fusion-specific chromatin immunoprecipitation sequencing (ChIP-seq) that uncovered 7,077 unique MEF2D–HNRNPUL1 binding sites in proximity (<3 kb) of recognized transcription start sites (TSS) and defined super-enhancer (SE) elements. Strikingly, MEF2D–HNRNPUL1 binding peaks were highly enriched for genes associated with pre–B-cell receptor (pre-BCR) signaling, including PI3KCD, IGLL1, VPREB1, CD79A, and BCL6 (6, 7). Deletion of these pre-BCR components was toxic to MEF2D-rearranged B-ALL cells consistent with a functional dependence on this pathway.

Assembly of the pre-BCR marks the first important checkpoint of B-cell development. Following productive rearrangement of immunoglobulin (Ig) V, D, and J gene segments, expression of a functional immunoglobulin μ-heavy chain paired with the surrogate light chain (SLC) initiates signaling via SYK, PI3K, and MAPK to trigger clonal expansion of pre-B cells. Importantly, the authors demonstrate unmistakable expression of surface pre-BCR components in MEF2D-rearranged B-ALL samples, which was dependent on intact MEF2D–HNRNPUL1-binding sites around the TSS of these elements. Further in line with positive regulation of the pre-BCR itself, enforced expression of MEF2D–BCL9 in murine pro-B cell progenitors gave rise to pre–BCR expressing clones in vivo, which ultimately developed into aggressive pre–B-cell leukemias.

As MEF2 TFs largely function by forming multicomponent regulatory complexes, Tsuzuki and colleagues hypothesized that MEF2D fusions may integrate into a core autoregulatory circuit with B-cell transcription factors. Through detailed functional and mapping experiments, they were able to narrow down a “core regulatory circuit” (CRC) of four key TFs acting in concert with the MEF2D fusion, namely SREBF1, FOS, EGR1, and BCL6. In addition to occupying genomic regions near TSSs of genes involved in pre-BCR signaling, co-occupancy of the MEF2D fusion with the four CRC TFs was confirmed near the TSSs of their respective gene loci, indicating positive self-feedback regulation. Previously, tonic pre-BCR signaling was shown to enhance the transcriptional activity of MEF2C in normal pre-B cells, through ERK-mediated phosphorylation (2). Similarly, in this study, activation of the cAMP response element-binding protein (CREB) downstream of ERK kinases was shown to further promote expression of the MEF2D–HNRNPUL1 fusion in Kasumi-7 cells. Together, these results suggest that MEF2D fusions form part of a coherent feed-forward loop involving a CRC of B-cell TFs and the pre-BCR signaling axis to drive the growth of B-ALL cells (Fig. 1).

Although B-cell precursors depend on survival signals from a functional pre-BCR, in the majority of B-ALL cases these signals are delivered by oncogenic mimics of this pathway (e.g., BCR–ABL1; ref. 8). Active pre-BCR activity is found in only approximately 13.5% B-ALLs (defined as pre-BCR⁺ B-ALL), including cases with the TCF3–PBX1 fusion (7). In a similar way to MEF2D-rearranged B-ALL, TCF3–PBX1 B-ALLs have increased expression pre-BCR components (e.g., PI3KCD, IGLL1, VPREB1, CD79A) most of which are direct targets of the TCF3–PBX1 fusion (7), and would explain the genetic similarities observed for these two groups of patients. A common feature to both normal pre-B cells and to pre-BCR⁺ B-ALL is the high expression of BCL6 (6, 7), which acts as a critical survival factor downstream of tonic pre-BCR signaling. Interestingly, however, unlike MEF2D fusions, ChIP experiments indicate that BCL6 is not likely a direct target of TCF3–PBX1 (7). On the basis of the current identification of BCL6 in the CRC of MEF2D fusions, it would be interesting to determine whether MEF2 TFs may contribute to the high expression of BCL6 in TCF3–PBX1 ALL.

In addition to serving as a powerful survival factor at the pre-BCR checkpoint, BCL6 represents a classic proto-oncogene in germinal center (GC)–derived B-cell lymphomas, which are dependent on tonic signaling from the BCR. Strikingly, recurrent mutations in the MEF2B gene have been identified in patients with GC-derived B-cell lymphoma (9). In DLBCL, the mutations in MEF2B were found to enhance its transcriptional activity and were directly linked to increased expression of BCL6 (10). In this way, it is possible that genetic aberrations affecting MEF2 TFs may result in common transcriptional programs in early (pre-B) and mature (GC-derived) B-cells to invoke expression of factors required for (pre-) BCR signaling and promote malignant B-cell survival.

The development of targeted inhibitors in B-ALL has shown remarkable scope for fusions involving tyrosine kinases (e.g., dasatinib for ABL1 and PDGFR fusions; ruxolitinib for JAK2 lesions), but has proven more complex for oncogenic fusions involving TFs. However, consistent with activation of pre-BCR signaling in TCF3–PBX1 B-ALL, a screen of 51 kinase inhibitors currently in clinical use showed these samples were selectively sensitive to inhibitors of the pre-BCR pathway, including inhibitors targeting the pre-BCR proximal kinases SYK, SRC, and BTK (7). In line with the new classification of MEF2D-rearranged B-ALL as pre-BCR⁺, this study found that, compared with pre-BCR⁻ cells, MEF2D-rearranged B-ALL cell lines and patient-derived B-ALL cells were also significantly more sensitive to inhibition of SYK and SRC kinases. To further determine whether destabilization of the MEF2D fusion at its core could serve as an additional targetable node, the authors focused on one CRC TF, SREBF1. SREBF1 activity depends on cellular lipid levels and hence is susceptible to drug inhibition. SREBF1 is normally anchored to the endoplasmic reticulum membrane in the shape of a precursor protein, and, in response to low levels of lipids such as cholesterol and fatty acids, translocates to the Golgi body where it is cleaved to release a mature functional TF. Remarkably, treatment of MEF2D ALL cells with SREBF1 inhibitors such as fatostatin and FGH10019 reduced the amount of cleaved SREBF1, MEF2D fusion expression, and also expression of the remaining CRC TFs (BCL6, FOS, and EGR1) with subsequent induction of cell death, reflecting the tight intradependence of the CRC TFs required for maintaining survival of MEF2D-rearranged B-ALL cells. SREBF1 has not been implicated in the phenotype of TCF3–PBX1 ALL, and accordingly SREBF1 inhibitors were not effective on these cells, suggesting that unlike targeting of the pre-BCR itself, inhibition of SREBF1 is a unique vulnerability of MEF2D-rearranged B-ALL.

Although the efficacy of pre-BCR and SREBF1 inhibitors in MEF2D-rearranged B-ALL remains to be tested in a clinical setting, the pioneering new findings by Tsuzuki and colleagues have defined MEF2D-rearranged B-ALL as a new subtype of pre-BCR⁺ B-ALL, and highlight the benefit of understanding transcriptional networks modified by aberrant TFs as potential sources for therapeutic intervention.

No potential conflicts of interest were disclosed.