In this issue of Blood, Mayday and colleagues1 present new insights into the basis of pediatric acute megakaryoblastic leukemia (AMKL) driven by the RBM15-MKL1 fusion protein. Their study reveals that Wnt signaling and METTL3 activity are essential for leukemia and shows that these pathways are novel therapeutic targets in AMKL.
AMKL, a relatively common form of acute myeloid leukemia in children with Down syndrome (DS) but rare in other children, is primarily driven by chromosomal translocations that result in fusion proteins including CBFA2T3-GLIS2 and RBM15-MKL1.2 Treatment outcomes for children with non-DS-associated AMKL are poor, and therefore new insights and novel therapeutic targets are needed.
Several groups have studied the biology of the t(1;22) translocation, which leads to the RBM15-MKL1 (aka OTT-MAL) fusion protein in AMKL. RBM15 binds RNA through 3 RNA recognition motifs and recruits the N6-methyladenosine (m6A) complex through its Spen paralog and ortholog C-terminal (SPOC) domain,3 and MKL1 is a coactivator of serum response factor, which regulates normal megakaryocyte maturation.4 RBM15 alone and in partnership with MKL1 enhances Notch signaling in hematopoietic cells, and this activity promotes abnormal fetal megakaryopoiesis and leukemogenesis.5,6 Combining RBM15-MKL1 expression with an activated form of the thrombopoietin receptor transforms hematopoietic progenitors and is sufficient to drive AMKL in mice.6 Nevertheless, the precise way in which RBM15-MKL1 promotes leukemia and can be therapeutically targeted has remained unclear.
To study the mechanism by which RBM15-MKL1 promotes leukemia, Mayday and colleagues generated hematopoietic cell lines that overexpress epitope tagged RBM15 or the fusion protein. They found that overexpression of the fusion, but not RBM15 alone, blocked megakaryocyte maturation as would be expected for a driver of AMKL. With these cells, they next performed a number of multiomic studies including RNA-sequencing, enhanced crosslinking immunoprecipitation (eCLIP)-sequencing, m6A eCLIP-sequencing, and Time Lapse-sequencing. Integration of gene expression, eCLIP-sequencing, and m6A eCLIP-sequencing identified transcripts that are bound and modified by the m6A methyltransferase complex, referred to as “couplets,” and showed that there is an association between altered expression and altered RNA binding and modification. A deeper analysis that incorporated transcript synthesis and stability further revealed that the genes associated with the RBM15-MKL1 couplets were stabilized. Finally, the authors discovered that these stabilized transcripts were enriched in oncogenic signaling pathways including Wnt signaling, a well-described pathway that leads to stabilization of β-catenin and confers stem cell–like properties and tumor cell growth.7 The observation that RBM15-MKL1 expression is associated with enhanced Wnt signaling in this t(1;22) AMKL was confirmed by analysis of published RNA-seq data. Of note, AMKL samples with the RBM15-MKL1 fusion had higher Wnt scores than other AMKL subtypes, other AMLs, and healthy hematopoietic progenitors. To directly assess the requirement for Wnt signaling in growth of RBM15-MKL1 leukemia, the authors knocked down Frizzled (Fzd) genes, which are critical components of the pathway that are overexpressed in the leukemia. They found that knockdown of Fzd family members inhibited AMKL tumor growth in vitro and in vivo, which was ameliorated by drug-induced activation of downstream β-catenin stabilization.
Finally, given that expression of the Fzd genes is m6A dependent, the authors examined the effect of inhibitors of METTL3, a key m6A writer in acute leukemia.8 They found that METTL3 small molecule inhibitors,9 including STM3675, had potent effects on the growth of AMKL cells with the fusion both in cell culture and animal models, with the drug conferring a significant survival advantage to mice engrafted with RBM15-MKL1 expressing tumor cells. A critical finding was that RBM15-MKL1 tumors are particularly sensitive to METTL3 inhibition, and the degree of sensitivity was associated with the level of expression of FZD2.
Together these studies paint a picture of the way that RBM15-MKL1 promotes AMKL and highlights 2 therapeutic targets, METTL3 and the Wnt pathway. Although this article provides key new insights, there are some limitations. First, the authors did not show preclinical efficacy in patient-derived xenografts due to the paucity of specimens. Second, the rare nature of the leukemia may make an RBM15-MKL1 AMKL clinical study challenging; however, the activation of Wnt in other leukemias, including the CBFA2T3-GLIS2 subtype,10 mitigates this issue. Third, although METTL3 inhibitors are under clinical investigation for other AMLs, development of Wnt pathway inhibitors has been challenging.7 Nevertheless, Mayday and colleagues provide critical new insights into a subtype of leukemia that represents an area of unmet need.
Conflict-of-interest disclosure: J.D.C. is on the scientific advisory board of Alethiomics and receives research funding from Syndax.
