Implicating a functional domain (SPOC) of RBM15 in the pathogenesis of RBM15::MKL1 fusion protein-induced acute megakaryoblastic leukemia

Abstract In acute megakaryoblastic leukemia (AMKL), immature megakaryoblasts accumulate due to excessive proliferation and failure to terminally differentiate. AMKL represents roughly 10% of pediatric AML and carries a poor prognosis despite aggressive treatment. A recurrent cytogenetic alteration found in a subset of AMKL cases is the t(1;22) translocation, which produces the RBM15::MKL1 (RM) fusion protein by joining RBM15, a regulator of N6-methyladenosine (m6A) RNA modification, with MKL1, a transcriptional coactivator. RM retains all functional domains from both wild-type proteins and is considered the primary oncogenic driver of this leukemia, often initiating in utero. RM binds RNA and has been associated with elevated m6A deposition and increased transcript stability. However, the mechanism by which RM engages the m6A methyltransferase complex (MTC), and whether this interaction is targetable, remains poorly defined. To investigate RM function, cellular models have been developed, including the 6133 murine AMKL cell line derived from a knock-in mouse expressing murine Rbm15 fused to human MKL1, and human erythroleukemia (HEL) cells engineered to express RM in a doxycycline-inducible manner. We have demonstrated that RM is essential for the proliferation and survival of 6133 cells and inhibits terminal megakaryocyte (Mk) differentiation of TPA-treated HEL cells (mean fluorescence intensity of TPA-induced differentiation antigens decreased by 80-100%, p0.05, n=3), recapitulating key features of pediatric AMKL. RBM15 is a core component of the MTC and has been implicated in modulating m6A deposition on nascent mRNA and affecting transcript stability, splicing, and translation. Additionally, m6A is the most abundant internal epitranscriptomic modification in eukaryotic mRNA and plays a pivotal role in post-transcriptional gene regulation. Co-immunoprecipitation (co-IP) assays revealed that the deletion or truncation of RBM15's SPOC domain abrogates its interaction with WTAP (p0.05, densitometry of co-IP blots, n=3), another critical MTC component. Since RM retains RBM15's functional motifs, including the SPOC domain, we are testing the hypothesis that RM's interaction with WTAP is essential for RM-mediated leukemogenesis. Based on published data and AlphaFold3 structure predictions, we have focused on two conserved basic patches in the RBM15 SPOC domain that may mediate WTAP binding. Alanine-scanning mutagenesis of conserved positively charged residues within these regions revealed that three amino acids in Patch 1, but not Patch 2, are required for WTAP binding (p0.05, densitometry of co-IP blots, n=3). To evaluate the functional consequences of mutating the SPOC domain to abrogate RM binding to WTAP in the MTC, we have established HEL cells with doxycycline-inducible RM or mutant (∆) RM expression to assess its impact on maturation. Upon TPA treatment, RM expression impedes hallmark differentiation processes including polyploidization, upregulation of Mk surface markers CD49b and CD49f, and downregulation of erythroid marker CD235a (p0.05, mean fluorescence intensities, n=3). Morphological changes observed by cytospin confirmed reduced cell size and decreased membrane blebbing, consistent with impaired differentiation. In contrast, induction of RM∆SPOCPatch1 expression only partially blocks TPA-induced differentiation processes indicating that the SPOC domain is required for the block in Mk maturation. We are also assessing the effects of RM∆SPOCPatch1 on growth and survival of 6133 AMKL cells, a system in which RM knockdown significantly impairs viability. We have created these cells and predict that Patch 1-mutant RM will be unable to rescue proliferation, mimicking the effects of RM depletion. Collectively, this research aims to define how the RM fusion protein hijacks m6A epitranscriptomic machinery via its interaction with WTAP to promote AMKL. By defining the molecular underpinnings of RM-driven proliferation and differentiation blockade, these studies may identify novel therapeutic targets that exploit vulnerabilities in mRNA modification pathways in pediatric leukemia.