RBM15 in leukemogenesis: an emerging regulator at the interface of RNA biology and hematopoiesis

Abstract

RNA-binding motif protein 15 (RBM15) is a key regulator of hematopoiesis and leukemogenesis, linking RNA metabolism, epitranscriptomic regulation, and lineage specification. Identified through the RBM15::MKL1 fusion in acute megakaryoblastic leukemia (AMKL), RBM15 functions within the N⁶-methyladenosine (m⁶A) writer complex, where its RNA-recognition motifs and SPOC domain enable site-specific methylation and protein recruitment. In hematopoietic stem and progenitor cells (HSPCs), RBM15 supports stem cell quiescence, self-renewal, and balanced lineage output, with additional roles in B-cell, myeloid, and megakaryocytic differentiation. Mechanistically, RBM15 links m⁶A deposition to chromatin regulation, RNA export, and splicing. This review summarizes RBM15 structure, function, and regulation, and highlights its emerging roles in leukemia, including AMKL and myelodysplastic syndromes, while outlining key questions for future studies.

Introduction:

Cancer remains one of the leading causes of premature mortality worldwide and represents a substantial global health challenge [1]. Leukemia accounts for a significant fraction of pediatric cancer diagnoses [2–4]. Globally, childhood leukemia accounts for 30–35% of all new cancer cases in children aged 0–4 years, with acute myeloid leukemia (AML) (see Glossary) comprising 15–20% childhood leukemia cases [5,6]. Despite advances in clinical management, many subtypes of leukemia remain associated with poor prognosis, underscoring the urgent need to delineate the molecular underpinnings of leukemogenesis and to identify new therapeutic avenues.

RNA-binding protein (RBP) biology has emerged as a critical frontier in cancer research, as RBPs govern essential post-transcriptional gene regulation and cellular fate decisions. Members of the Split-end (SPEN) family of RBPs, including RNA-binding motif protein 15 (RBM15), have attracted particular interest due to their structural features, evolutionary conservation, and diverse functions in transcriptional and post-transcriptional control [7,8]. These proteins typically harbor RNA recognition motifs (RRMs) and conserved SPOC (Spen paralog and ortholog C-terminal) domains, which mediate interactions with RNA and protein cofactors, respectively [9,10].

A central axis in this regulatory landscape is N⁶-methyladenosine (m⁶A), the most abundant epitranscriptomic modification in eukaryotic mRNA [11]. m⁶A exerts profound effects on hematopoiesis by influencing alternative splicing, transcript stability, nuclear export, and translation [12–16]. Dysregulation of m⁶A-modifying enzymes or their cofactors disrupts hematopoietic differentiation, and m⁶A modifications appear to be critical for malignant transformation [17,18]. Given that RBM15 is a known cofactor of the m⁶A methyltransferase complex (MTC), it occupies a unique position at the interface between RNA processing and lineage commitment in the hematopoietic system [19].

Building on its central role in RNA epitranscriptomic modification and transcriptional regulation, RBM15 has emerged as a key determinant of hematopoietic stem and progenitor (HSPC) homeostasis. Through its association with the MTC and its ability to modulate key transcriptional programs, RBM15 influences hematopoietic stem cell (HSC) quiescence, self-renewal, and differentiation [20–22]. Functional studies in both human and murine models have demonstrated that RBM15 loss disrupts normal hematopoietic balance, resulting in aberrant lineage output and impaired regenerative capacity [23,24]. These findings underscore RBM15’s critical role in maintaining hematopoietic integrity and preventing maladaptive signaling events that can precipitate leukemic transformation.

In this review, RBM15 is highlighted as an emerging regulator in leukemogenesis, synthesizing recent discoveries on its structural properties, role in RNA metabolism, and its contributions to normal and malignant hematopoiesis. By consolidating findings across developmental hematology, RNA regulation, and leukemia research, this review aims to provide a framework for understanding how RBM15 functions as a regulator of hematopoietic integrity that is disrupted in leukemia – underscoring its potential as a therapeutic target.

Domain Architecture of RBM15:

RBM15 was first identified as protein One Twenty-Two (OTT) that forms part of the aberrant RBM15::MKL1 fusion gene that arises from the t(1;22)(p13;q13) translocation in infant acute megakaryoblastic leukemia (AMKL) [25,26]. The RBM15 locus is on chromosome 1 at cytogenetic band 1p13.3 and encodes up to six different RBM15 isoforms due to the presence of two 5’-AUG codon start sites coupled with variations in alternative splicing on the 3’-end of the transcript (Figure 1A) [27]. The structure of RBM15, including three N-terminal RNA recognition motifs (RRMs) and the Spen paralog ortholog C-terminal (SPOC) domain (Figure 1A, B), is conserved and no functional differences have been identified between the isoforms (Figure 1B) [27]. This review will refer specifically to the longest RBM15 isoform that contains 977 amino acids, which has been extensively used by other investigators [25,26,28]. Because of the high degree of both sequence and functional similarity among all six isoforms, the molecular and cellular properties discussed in this review are likely to be recapitulated across all RBM15 variants. Consistent with this, all isoforms have been reported to localize to the nucleus [27].

Figure 1. RBM15 protein structure.

(A) Six different RBM15 isoforms are encoded due to two transcriptional start sites (AUG) and three distinct C-termini resulting from alternative splicing events include short (S), long (L), and short including alternative exon (S+AE) variants indicated by white boxes or black bars. There are three RNA recognition motif (RRM in yellow) domains, and the Spen paralog and ortholog C-terminal (SPOC in green) domain. Figure adapted with permission from [25,27]. (B) Predicted structure of full-length RBM15. (C) Inset of RRM1 containing two α-helices (purple) and four β-strands (blue). (D) Inset of the SPOC domain containing four α-helices (purple) and seven β-strands (blue). The predicted structure of RBM15 was generated using AlphaFold3 and visualized using UCSF ChimeraX [98,99].

To mediate RNA binding, each of the three RRM domains is composed of four antiparallel β-strands and two α-helices (Figure 1C) that allow RBM15 to bind U-rich RNA motifs [29]. Deletion or mutation of the RRMs severely impairs RBM15’s ability to bind RNAs for targeted m⁶A modification [29,30]. Mechanistically, recent work shows that each RRM is able to bind RNA on its own with variable affinity, and that binding affinity is increased when RRMs are present in tandem, perhaps due to the availability of multiple binding sites or perhaps due to conformational changes induced by intervening amino acid linkers [31]. Specifically, RRM2 and RRM3 together form a “sandwich” to bind long non-coding RNAs that have a stem-loop structure, in a predicted conformation similar to that of the RBM15 homolog SHARP and another RRM-containing protein Nucleolin [31,32]. This conformation puts multiple nucleotides of RNA within very close proximity (<6 Å) to the two RRMs. Although the RRMs are generally thought of as the main drivers of RNA binding, modeling predicts two other regions of RBM15 that may also bind RNA: one upstream of RRM1, and the other just upstream of the SPOC domain [31]. How these regions interact with RNA, particularly in the context of the full RBM15 protein, is unknown.

The SPOC domain is located downstream of RRM3 (Figure 1A, B) and is found in all SPEN family proteins [10]. Structurally, the SPOC domain contains seven β-strands and four α-helices (Figure 1D) [33]. Based on crystallized protein structures, the SPOC domain functions as a phosphoserine-binding module that recognizes phosphorylated proteins, including Wilms’ tumor 1-associating protein (WTAP), a critical protein in the m⁶A methyltransferase complex, as well as the C-terminal domain (CTD) of RNA polymerase II [28]. Crystal structures of the RBM15 SPOC domain identified two conserved surface basic patches comprised of arginine or lysine residues that potentially modulate phosphoserine binding – with a major patch (K795, R834, and K898) and a second minor patch (R847, R848, and K850) located 21 Å away [28]. The SPOC domain structure is conserved across SPEN family members, including SHARP and RBM15B, and across species, including Drosophila and Caenorhabditis [9,10]. Within the SPOC domain, the major basic patch is structurally conserved in other SPOC domain-containing proteins (e.g., PHF3 and DIDO) [28].

An alanine point mutation (R834A) within the conserved basic patch in the RBM15 SPOC domain reduces binding affinity to WTAP [28]. Additionally, knockout of RBM15 or deletion of just the SPOC domain of RBM15 leads to a decrease in m⁶A modified mRNA, consistent with the known role of the RBM15 SPOC domain in mRNA m⁶A modification [28]. While the SPOC domain forms protein-protein interactions with phosphorylated partners such as WTAP and RNA polymerase II, the determinants of this interaction specificity, and how they vary with post-translational modifications or cellular context remain to be elucidated [28]. Six known RBM15 protein isoforms are expressed in hematopoietic cells, yet the functional distinctions, if any, among these variants are poorly defined since most known RBM15-related functions are associated with the longest RBM15 isoform [27]. A detailed understanding of isoform-specific localization, interactors, and regulatory functions could reveal new roles of RBM15 in hematopoiesis.

Located between its structured domains, RBM15 also contains low complexity intrinsically disordered regions (IDRs) that could potentially facilitate protein-protein interactions or allow for multivalent binding and condensate formation [34,35]. Consistent with this, RBM15 and WTAP as well as other m⁶A methyltransferase complex-associated adaptor proteins localize to nuclear speckles in the nucleoplasm [36]. Although the RRMs and the SPOC domain of RBM15 have been structurally characterized, large regions of the protein remain unstudied. These include extensive IDRs that may confer multivalent binding capacity, phase separation potential, or spatial organization within nuclear speckles [37,38]. Interestingly, METTL3, METTL14, and WTAP of the m⁶A methyltransferase complex have been shown to co-localize in nuclear speckles of HeLa cells [39]. Whether the IDRs of RBM15 facilitate compartmentalization of the m⁶A methyltransferase complex to modulate target selectivity remains unknown.

RBM15 Protein-Protein Interactions:

Interactions within the m⁶A methyltransferase complex (MTC)

RBM15 is a well-established component of the m⁶A “writer” complex and is often conceptualized as a bridging factor that recruits or stabilizes components of the MTC on RNA (Figure 2A) [33,40]. RBM15’s interaction with WTAP is particularly critical for forming this bridge since WTAP acts as a scaffold that recruits the METTL3-METTL14 catalytic core of the MTC to RBM15, which can bind RNA – enabling site-specific deposition of m⁶A [41,42]. Within the MTC, METTL3 is responsible for catalyzing the methylation of target adenosines to m⁶A [43–45].

Figure 2. RBM15 protein-protein interactions.

(A) RBM15 is a core member of the m⁶A writer complex that functions to methylate target adenosines in RNA to m⁶A. The cartoon depiction of the m⁶A complex is adapted with permission from its cryo-electron microscopy structure [42]. (B) Outside of the m⁶A complex, RBM15 also binds to NXF1 to regulate nuclear mRNA export into the cytoplasm. (C) RBM15 SPOC domain interacts with SETD1B which forms part of the COMPASS complex that regulates epigenetic histone methylation of H3K4. (D) RBM15 interacts with the splicing factor SF3B1.

Ancillary protein cofactors within the MTC interact directly or indirectly with RBM15 to modulate RNA methylation site choice, localization, and stability. These cofactors include HAKAI, VIRMA, and ZC3H13 (Figure 2A) [46]. HAKAI is a conserved component of the MTC in both Drosophila and humans and interacts indirectly with RBM15 through VIRMA [47,48]. Depletion of HAKAI in HeLa or U2OS cells via dsRNA-mediated RNA interference (RNAi) reduces VIRMA protein levels, without affecting RBM15 levels [47]. Although direct binding of VIRMA to RBM15 has not yet been experimentally confirmed, cryo-EM structures of the human m⁶A MTC complex suggest that VIRMA forms protein-protein interactions with both WTAP and ZC3H13 [42]. Co-immunoprecipitation assays in mouse embryonic stem cells (mESCs) show that Zc3h13 co-immunoprecipitates with Rbm15 and Wtap [41], but direct protein-protein contact has not been confirmed. Thus, RBM15 is known to directly bind WTAP and to be in a tight molecular complex with ZC3H13, HAKAI, and VIRMA of the MTC [28]. However, it is not yet established whether RBM15 primarily serves as a recruitment scaffold for the m⁶A writer complex, an allosteric regulator of the methyltransferase activity, or a temporal coordinator of RNA processing. Mapping the hierarchical order of the m⁶A writer complex assembly and disassembly will be essential for understanding how m⁶A deposition is integrated with transcription and splicing.

Interactions beyond the MTC

Outside of the m⁶A MTC, RBM15 engages with a range of proteins involved in chromatin modification, RNA processing, splicing, and export – extending its functional impact. For example, RBM15 binds to nuclear export factor 1 (NXF1) which is a key mRNA export receptor and forms a complex with DDX19 to direct mRNA binding and transport to the cytoplasm (Figure 2B) for translation [27,49]. The paralog RBM15B, which has high sequence and domain homology to RBM15, also interacts with NXF1 through its C-terminal region (i.e., SPOC domain) [50]. Thus, it is possible that the SPOC domain of RBM15 similarly binds NXF1 to allow it to function as a nuclear export factor (Figure 2B), though this is not known. Additionally, the SPOC domain of RBM15 directly binds to the LSD (LPDSD motif) domain of SETD1B, the protein that methylates target histone H3 lysine 4 (H3K4) and forms the Complex of proteins associated with Set1 (COMPASS) complex to activate genes (Figure 2C) [51]. The interactions between SETD1B and RBM15 show a potential link between chromatin modification and RNA binding [52].

Within hematopoietic cells, RBM15 has been proposed to participate in RNA splicing through its interaction with the spliceosome component SF3B1. In AMKL cell lines, RBM15 co-immunoprecipitates with SF3B1, and the depletion of RBM15 is associated with alternative exon usage in a subset of transcripts involved in hematopoietic differentiation [53]. This suggests a potential role for RBM15 in splice-site selection, which may regulate hematopoietic differentiation (Figure 2D) [53]. In the same study, methylation of RBM15 by protein arginine methyltransferase 1 (PRMT1) on its IDR residue R578, promotes its ubiquitylation and subsequent degradation, thereby reducing the pool of available RBM15 that can bind to SF3B1 [53]. This is particularly relevant to leukemogenesis, where the overexpression of PRMT1 in AMKL cell lines blocks terminal megakaryocyte differentiation by downregulating RBM15 protein levels via PRMT-mediated RBM15 methylation [53]. The influence of post-translational modifications on RBM15 stability and binding affinity is still emerging, with two main modifications known to date: methylation in its IDR and lactylation in its SPOC domain, leading to RBM15 degradation or stabilization, respectively [53,54]. Although the former of these two findings supports a potential role for RBM15 in RNA splicing, this model currently relies largely on a single study, and the extent to which RBM15 regulates splicing independently, or in concert with, its established functions in m⁶A biology remains unresolved.

RBM15 in Normal Hematopoiesis:

RBM15 regulates HSC function, by modulating HSC quiescence and long-term repopulating potential. Expressed most highly in human and mouse HSPCs (Figure 3), Rbm15 loss in mouse models leads to an increase in the number of LSK cells (Lineage- Sca-1+ c-Kit+ cells, a subpopulation enriched for stem and progenitor cells) and long-term HSCs (LT-HSCs). However, these HSCs cannot function to engraft the hematopoietic system post-transplant due to defects in differentiation [20–22,55]. Rbm15-null LT-HSCs have cell cycle abnormalities featuring a reduced percentage of cells in the G0 resting state and a defect in their differentiation to short-term HSCs [21]. This loss of quiescence is associated with signatures of HSC aging, including increased NF-κB signaling, reactive oxygen species (ROS), and DNA damage [23]. Consequently, RBM15-deficient HSCs are unable to compete with wild-type cells for engraftment and cannot maintain themselves under replicative stress, demonstrating a profound defect in self-renewal [23,24]. This phenotype is partly attributed to the upregulation of the cell adhesion molecules N-cadherin and beta integrin [21]. A summary of these studies and their experimental approaches is shown in Table 1.

Figure 3. RBM15 in hematopoiesis.

RBM15, essential in hematopoiesis, is expressed the highest in human and mouse hematopoietic stem and progenitor cells (HSPC) within the lineage^-Sca-1⁺c-Kit⁺ (LSK) compartment (green). RBM15 is expressed in both myeloid (orange) and lymphoid (pink) lineages of hematopoiesis and plays a role in megakaryocyte and B-cell differentiation, respectively. The classical model of hematopoiesis depicted in this figure has been adapted with permission [100,101]. LT-HSC, long-term hematopoietic stem cell; ST-HSC, short-term hematopoietic stem cell; MPP, multipotent progenitor cell; CMP, common myeloid progenitor cell; CLP, common lymphoid progenitor cell; MEP, megakaryocyte-erythroid progenitor cell; GMP, granulocyte-macrophage progenitor cell.

Table 1.

RBM15 in normal hematopoiesis.

Reference	Perturbation	Methods and Readout	Model or Cell Line	Main Results
[22]	Conditional deletion (Mx1Cre inducible)	Mx1-Cre pIpC induction to delete Rbm15; flow cytometry and colony-forming unit assays	Six- to twelve-week-old adult mouse bone marrow and spleen cells	Rbm15 loss led to loss of peripheral B cells (block in pro/pre-B cells), expansion of myeloid and megakaryocytic compartments, increased LSK (HSPC) fraction, and skewing towards granulocyte fate.
[23] and [24]	Conditional deletion (Mx1Cre inducible)	Mx1-Cre pIpC induction to delete Rbm15; flow cytometry, thrombopoietin (TPO) response assays, and transplantation assays	Twelve-week-old adult mouse bone marrow and E14.5 fetal livers	Rbm15 regulates alternative splicing of c-Mpl to produce a dominant-negative truncated (TR) Mpl-TR isoform. Rbm15 loss alters TPO signaling, impairs HSC engraftment, and perturbs megakaryopoiesis resulting in abnormally low ploidy.
[21]	Conditional deletion (Mx1Cre inducible)	Mx1-Cre pIpC induction to delete Rbm15; flow cytometry, gene expression profiling, transplantation assays, colony-forming assays, and megakaryocyte culture assays	Eight- to ten-week-old adult mouse bone marrow and spleen cells	Rbm15 loss leads to the expansion of long-term HSCs (LT-HSCs) due to a defect in differentiation from LT-HSC to ST (short term) HSC. Rbm15-KO LT-HSCs had both serial transplantation and competitive reconstitution defects with abnormally small and low-ploidy megakaryocytes. Rbm15 loss also resulted in abnormal HSC niche interactions due to increased N-cadherin and β1 integrin expression.
[52]	Knockdown (shRNA mediated)	Lentiviral transduction of RBM15-specific shRNA; flow cytometry and intracellular immunostaining	Human umbilical cord blood CD34+ cells induced to differentiate into megakaryocytes	RBM15 knockdown decreased CD41 and CD42 cell surface markers indicating fewer mature megakaryocytes.
[20]	Knockdown (shRNA mediated) and enforced expression	Retroviral transduction of Rbm15-specific shRNA and transient transfection for enforced expression; Northern blot, flow cytometry, and cell proliferation assays	Murine cell lines: erythroid myeloid lymphoid (EML) and 32Dwt18.	In EML cells, Rbm15 expression decreased within 24–120 hours of differentiation; in 32Dwt18 cells, Rbm15 expression decreased within 72 hours of differentiation. Rbm15 knockdown in murine 32Dwt18 cells promoted myeloid differentiation (Mac-1 myeloid cell surface marker) while its enforced expression inhibited differentiation.

Beyond its role in the stem cell compartment, RBM15 plays a role in directing hematopoietic lineage commitment. Its absence creates significant shifts in blood cell production, most notably a block in B-cell development at the pro/pre-B cell stage [22]. In contrast, RBM15 loss after commitment to myelopoiesis promotes both myeloid and megakaryocytic commitment [21,22]. This results in an expansion of myeloid progenitors, particularly granulocyte-monocyte progenitors (GMPs), and an increase in myeloid cells and megakaryocytes in both the bone marrow and spleen [22]. However, this skew toward megakaryopoiesis is dysfunctional; the resulting megakaryocytes are abnormally small with low ploidy [21,22]. Consistent with this finding in mice, shRNA-mediated knockdown of RBM15 in human cord blood-derived CD34⁺ cells leads to production of less mature megakaryocytes, as measured by the loss of mature megakaryocyte markers CD41 and CD42 [56]. In vitro studies corroborate this role; enforced RBM15 expression inhibits myeloid differentiation, while knockdown promotes it [20]. Together, these findings illustrate that RBM15 exerts cell type-specific functions; it is required to maintain quiescence within the LSK stem/progenitor compartment, and it promotes myeloid differentiation and terminal megakaryocyte maturation.

The hematopoietic consequences of RBM15 loss partially overlap with those reported following the disruption of other proteins in the m⁶A MTC. A similar combination of increased phenotypic HSC abundance and diminished functional reconstitution is observed following Mettl3 deletion in the adult hematopoietic system, where loss of m⁶A disrupts HSC identity and differentiation [13,57]. The disruption of the Mettl3-Mettl14 heterodimerization has likewise been associated with increased HSC recycling and reduced repopulating activity, suggesting that RBM15 and the catalytic writer complex may converge in maintaining HSC quiescence and differentiation competence [45]. Interestingly, loss of MYC expression has been identified as a downstream effect in both Rbm15- and Mettl3- deficient HSCs, suggesting a potential mechanistic point of convergence despite the distinct molecular functions of these proteins [57–59]. However, the phenotypes are not completely identical. Rbm15 deficiency also produces lineage-specific abnormalities, including altered thrombopoietin responsiveness and the expansion of small, low-ploidy megakaryocytes, while Mettl3 disruption has additionally been associated with impaired erythropoiesis [58,60,61]. Hematopoietic-specific loss-of-functional studies of other m⁶A MTC components, including WTAP, ZC3H13, and VIRMA, remain limited and will be needed to distinguish shared writer-complex functions from RBM15-specific activities.

RBM15 in Leukemia:

RBM15’s role in m⁶A modifications and in hematopoiesis has been investigated in the context of cancer and leukemogenesis, where dysregulation of the m⁶A modification process has been linked to oncogenesis [62,63]. As part of the m⁶A writer complex, RBM15 affects a variety of processes in blood cancers.

RBM15::MKL1 in acute megakaryoblastic leukemia

The role of RBM15 in cancer is perhaps most directly apparent in the t(1;22) translocation of AMKL, in which RBM15 is fused to MKL1 producing the RBM15::MKL1 (RM) fusion protein. Like RBM15, MKL1 (Megakaryoblastic Leukemia 1 gene, also MRTFA) as a wildtype protein is well described – it is a transcriptional cofactor important for hematopoiesis and specifically megakaryopoiesis [64–66]. The t(1;22) translocation contains all putative functional domains of RBM15, where the translocation occurs in intron 1 of RBM15, downstream of the C-terminal SPOC domain [25]. Similarly, though there are two translocation sites within the MKL1 gene, most functional domains of MKL1 are retained in the encoded fusion protein [26,64]. Thus, RM fusion protein-induced AMKL (RM-AMKL) offers an interesting opportunity to explore the role of RBM15 in oncogenesis. Recent work has demonstrated that the fusion protein maintains the m⁶A function of wildtype RBM15 while also having novel targets [67]. RM binds WTAP and shares both binding and modification targets of RM as well as wildtype RBM15 (Figures 4A, B). Notably, m⁶A-modified transcripts uniquely targeted by RM have increased stability compared to those regulated by wildtype RBM15, suggesting that the fusion protein enhances the stability of specific target RNAs thereby altering the downstream transcriptional program (Figure 4B) [67]. Additionally, STM3675, a METTL3 inhibitor, induces differentiation and apoptosis of murine RM-AMKL cells and prolongs survival of mice in a syngeneic transplant model suggesting that RM-mediated m⁶A modifications are critical for the maintenance of leukemia cells [67]. Together, this demonstrates a reliance of RM-AMKL on RBM15-mediated m⁶A and additionally suggests a mechanism whereby RM coopts the m⁶A modification function of RBM15 to increase stability and expression of genes important to leukemogenesis. Thus, dysregulation of RBM15 via its fusion with MKL1 has downstream epitranscriptomic consequences that directly contribute to leukemia progression.

Figure 4. RBM15 in Acute Megakaryoblastic Leukemia (AMKL).

Upper panels (blue) indicate mechanisms underlying normal hematopoiesis. Lower panels (red) implicate RBM15 in AMKL. (A) Wildtype RBM15 is part of the m⁶A methyltransferase complex (MTC) that converts target adenosines into m⁶A in mRNA transcripts. These m⁶A modifications on mRNA are associated with changes in mRNA destabilization and transcriptional repression. RBM15 binding partners outside the MTC include HDAC3 and RBFOX2, the latter being a regulator of splicing. (B) The aberrant RBM15::MKL1 (RM) fusion protein also binds components of the MTC, resulting in differentially m⁶A-modified but stabilized transcripts. The METTL3 inhibitor STM3675 has anti-leukemic effects resulting in increased AMKL cell differentiation and apoptosis as well as increased in vivo survival in AMKL mice. Figure adapted with permission from [67]. (C) In normal hematopoiesis, PRMT1 binds RMB15 leading to its methylation and subsequent degradation via the ubiquitin-proteasome pathway. (D) In AMKL, PRMT overexpression leads to loss of RBM15 resulting in a block in terminal megakaryocyte differentiation and aberrant accumulation of megakaryoblasts.

PRMT1 methylates RBM15 (Figure 4C), leading to its degradation; this downregulation of wildtype RBM15 results in impaired megakaryocytic differentiation due to alternative splicing normally mediated by RBM15 (Figure 4D) [53]. This suggests that, in addition to its presence in the fusion protein, dysregulation of wildtype RBM15 could also affect the progression of RM-AMKL through its degradation by increased levels of PRMT1. In the RM-AMKL “6133” cell line, aberrantly high expression of PRMT1 promotes progression of the leukemia whereas inhibition of PRMT1 stalls the disease and significantly extends survival of mice in a syngeneic leukemia transplant model, likely due to the decrease of RBM15 and corresponding dysregulation of RBM15-mediated RNA splicing of key transcription factors (e.g., RUNX1 and GATA1) [53,68,69]. Interestingly, in a human erythroleukemia (HEL) cell model, enforced expression of the RM fusion protein decreases endogenous RNA and protein levels of wildtype RBM15 without affecting PRMT mRNA levels [52,67].

RBM15 overexpression may inhibit myeloid differentiation of HSPCs via activation of Notch signaling [20]. Genes of the Notch pathway are upregulated in RM-AMKL leukemia compared to non-RM-AMKLs (particularly Down Syndrome-associated AMKL), with the RM fusion protein activating Notch-related genes [68]. Interestingly, crosslinking and immunoprecipitation (CLIP) data in the HEL cell model show that genes including Notch2 are not differentially upregulated by RM but are differentially bound and modified by RBM15 and are inconsistently expressed in murine RM-AMKLs (e.g., 6133 and CAOM cell lines) compared to related progenitor populations (e.g., pre-megakaryocyte/erythroid progenitor, megakaryocyte progenitor, and erythroid progenitor cells) [67]. This discrepancy could reflect differences in cellular context, suggesting that the effects of RBM15 mediated m⁶A modification on transcript fate, including RNA stability, are context dependent.

In the context of RM-AMKL, leukemogenesis relies on both m⁶A-dependent transcript regulation and Wnt pathway activation [67]. Therefore, combination strategies (e.g., METTL3 inhibitor or degrader coupled with Wnt pathway inhibition or a Frizzled blockade) are rational options to pursue in a preclinical setting. Specifically, METTL3 inhibition can downregulate Frizzled expression in RM models, providing a mechanistic rationale for synergy. Identifying pathways aberrantly regulated by m⁶A/RBM15 in other malignancies may provide additional opportunities for context-specific combination therapies. The identification of the RM fusion protein in AMKL underscores RBM15’s oncogenic potential, yet the mechanistic relationship between wildtype RBM15 activity and fusion-driven leukemogenesis remains incompletely understood. Although both wildtype RBM15 and RM regulate m⁶A-dependent gene expression, the RM fusion may alter mRNA transcript stability alongside chromatin engagement due to its fusion to MKL1 which plays a role in regulating chromatin remodeling [70].

In contrast to METTL3 and METTL14, for which conditional genetic studies have demonstrated essential roles in both leukemia initiation and maintenance, comparable genetic evidence for RBM15 remains limited [71–73]. Nevertheless, functional studies indicate that RBM15 is not merely involved in leukemia initiation, but could also contribute to disease maintenance. Specifically, in murine RM-AMKL cells, the suppression of the RM fusion oncogene impairs leukemic cell proliferation and survival, while the pharmacologic inhibition of Mettl3 promotes differentiation and apoptosis, demonstrating a continued dependence on the RBM15-m⁶A signaling axis [67]. Together these findings support a critical role of RBM15 in sustaining leukemic phenotype when fused to MKL1, however, definitive conditional genetic studies analogous to those performed for METTL3 and METTL14 have yet to be reported.

RBM15 in other hematologic malignancies

RBM15 and m⁶A have been implicated in many cancers, including both solid tumors and hematologic malignancies. DepMap (https://depmap.org/portal) shows that among reported cell lines, those from myeloid and lymphoid lineages have the highest expression of RBM15, and those from myeloid lineages, particularly AMLs, have the highest dependency on RBM15 [74]. RBM15 is upregulated in blast-phase chronic myelogenous leukemia (CML) where it is linked to upregulation of Notch signaling via RBPJk (Figure 5A) [75]. In B-cell acute lymphoblastic leukemia (B-ALL), expression of the RBM15 paralog RBM15B is significantly increased in relapsed compared to naïve samples and correlates with poor survival rates; other m⁶A-related genes (e.g., HNRNPC and FTO) have similar trends (Figure 5B) [76]. In myelodysplastic syndrome (MDS), mutant forms of splicing factors have been shown to bind with RBM15 more strongly than wildtype, resulting in aberrant splicing that contributes to disease phenotypes (Figure 5C) [77]. This points to RBM15 as a key regulator of not only hematopoiesis but also leukemogenesis and that lineage-specific cancers may be particularly sensitive to potential therapies targeting RBM15 and its downstream effects.

Figure 5. RBM15 in Other Hematologic Malignancies.

(A) In chronic myelogenous leukemia, RBM15 gene and protein expression are upregulated in blast crisis. This increased RBM15 is associated with increased clonogenicity, Notch signaling, and decreased apoptosis. Knockdown (KD) via shRBM15 reverses these effects and promotes myeloid differentiation. (B) In B-cell acute lymphoblastic leukemia, expression of the RBM15 paralog RBM15B is significantly increased in relapsed patient samples compared to diagnostic samples. Increased RBM15B expression correlates with decreased survival rates. Figure adapted with permission [102]. (C) In myelodysplastic syndrome, RBM15 binds more strongly to mutant splicing factor SF3B1^K700E compared to its wildtype counterpart. Alternative splicing of the pre-mRNA transcript of TAL1 is dysregulated by SF3B1^K700E, resulting in a truncated (TAL1s) splice variant associated with an alternative start site (ATG) and MDS disease phenotype compared to full-length (TAL1 fl) transcript.

AMLs are reported to have aberrant expression of multiple m⁶A-related genes, and expression of these genes (including RBM15 as well as other writers, readers, and erasers) is associated with poor overall survival [78]. When patients with AML are clustered into three groups based on the expression of 22 m⁶A regulator genes the prognostic risk score that is derived from the expression of six of those m⁶A regulators, including RBM15 is associated both with poor overall survival and increased immune infiltration [78]. These findings suggest that monitoring m⁶A-related genes in AML may help to guide treatment decisions particularly regarding immunotherapy.

Other leukemogenic roles of RBM15

In addition to its role in the epitranscriptome, RBM15 has also been reported to indirectly affect transcription. By interacting with RBFOX2 (Figure 4A), RBM15 recruits components of the MTC to methylate chromatin-associated, promoter-associated RNAs [79]. RBM15 also recruits the m⁶A reader YTHDC1, which in turn engages PRC2 to mediate transcriptional repression [79]. Reducing RBFOX2 levels reduces cell growth and promotes differentiation of several myeloid leukemia cell lines including chronic myeloid leukemia (K562) and acute promyelocytic leukemia (NB4) as well as an AML patient-derived xenograft (PDX) model and a leukemia-initiating cell model (MLL::AF9) [79].

RBM15 also affects the epigenome, where it interacts with histone deacetylase HDAC3, canonically associated with transcriptional repression (Figure 4A), and with histone methyltransferase Setd1b (Figure 2C), a marker of active genes [52,80]. In AMKL, the RM fusion protein loses interactions with HDAC3, perhaps enabling aberrant upregulation of normally repressed genes [80]. The interaction with Setd1b, mediated by the SPOC domain, is maintained in the RM fusion protein and is essential for survival of RM-AMKL 6133 cells [52]. This suggests that in addition to its role in the m⁶A epitranscriptome, RBM15 may contribute to leukemia via interactions with chromatin and the epigenome.

RBM15 is also implicated in alternative splicing of genes. For example, RBM15 directly binds MPL transcripts at intronic regions and additionally interacts with the MPL gene, regulating chromatin acetylation and methylation [24,53]. Both interactions contribute to alternative splicing of MPL, affecting normal and abnormal hematopoiesis. Additionally, RBM15 regulates the alternative splicing of other genes related to megakaryopoiesis and/or erythropoiesis (e.g., RUNX1, TAL1/TAL1s, CDC42, GATA1, and GATA1s), which may be facilitated by recruitment of the splicing factor SF3B1 [53].

RBM15 dysregulation beyond hematologic malignancies

Although this review focuses on the functions of RBM15 in hematopoiesis and leukemogenesis, emerging evidence indicates that its dysregulation also contributes to the pathogenesis of several solid tumors, where its oncogenic effects are similarly mediated through aberrant m⁶A-dependent mechanisms. For example, in hepatocellular carcinoma (HCC), RBM15 is overexpressed and associated with poorer outcomes; mechanistically, it promotes tumor progression by facilitating m⁶A modification of the YES1 transcript, which promotes IGF2BP1-dependent YES1 mRNA stabilization thereby activating downstream MAPK signaling [81]. In triple-negative breast cancer (TNBC), RBM15 is expressed at higher levels than in normal breast tissue, and promotes tumor growth by binding and inducing m⁶A modifications on genes involved in glycine and serine metabolism, resulting in their upregulated expression [30]. In a separate study, increased RBM15 expression in TNBC upregulated TNFSF9, contributing to increased drug resistance in tumor-associated macrophages [82]. Together, these findings suggest that RBM15’s oncogenic activity extends beyond hematologic malignancies and that its role as a modulator of RNA methylation represents a conserved mechanism underlying tumorigenesis in other tissue types.

Therapeutic Implications and Targeting Strategies:

Leukemias that involve the dysregulation of RBM15 either through overexpression, fusion to MKL1, or other alterations (i.e., post-translational RBM15 modifications) share common dependencies that can potentially be therapeutically exploited. The current or preclinical strategies are outlined in several categories below:

Targeting the m⁶A “writer” complex (METTL3/METTL14/WTAP axis)

METTL3 inhibition through the first-in-class small molecule STM2457 selectively targets the catalytic site of METTL3 in the MTC [83]. STM2457 co-crystallizes with the METTL3/METTL14 heterodimer and this binding leads to a reduction in overall m⁶A levels [84]. STM2457 impairs leukemic stem cell function and extends survival by promoting myeloid differentiation and apoptosis in murine and human AML models [84]. Additionally, in vivo pharmacological inhibition of METTL3 results in a marked decrease in AML cells in patient-derived xenografts by impairing AML stem cell growth [84]. A similar, but more bioavailable compound, STM3675 is also effective in mice against RM-AMKL models [67].

Another option for inhibition of METTL3, direct METTL3 degradation via proteolysis-targeting chimeras (PROTACs), also reduces overall mRNA m⁶A levels [85]. WD6305 is a PROTAC that targets METTL3 for proteasomal degradation to suppress m⁶A modification, which decreases the proliferation of AML cells by promoting apoptosis [86,87]. ZW27941 is another METTL3-specific PROTAC that has demonstrated synergistic additive effects when combined with standard AML therapeutics (e.g., cytarabine and venetoclax) [85,88].

Wnt pathway inhibition

RM-AMKL relies on m⁶A-mediated Wnt pathway activation [67]. This suggests that inhibiting the Wnt pathway could be effective in stalling RM-mediated leukemogenesis. The use of pharmacological agents like Porcupine (PORCN) inhibitors such as WNT974 (LGK974) can block Wnt ligand palmitoylation and secretion [89]. Additionally, antibodies that target the Frizzled proteins (i.e., Wnt receptors) are already in clinical trials or in preclinical use in solid tumors, opening further possibilities for drug repurposing in the context of leukemia [90–92]. Although anti-Frizzled antibodies have been investigated in multiple solid tumor models and show anti-cancer activity, human trials have been limited by off-target effects including bone fractures [90,91].

Disrupting RBM15- or RM-specific protein interactions

The C-terminal SPOC domain of RBM15 mediates its interactions with phosphorylated partners (e.g., WTAP and RNA Polymerase II) and chromatin regulators (e.g., Setd1b) [28,52]. The identification of a key residue (R834) in the SPOC domain of RBM15 suggests its role in binding WTAP [28]. Because the RM fusion protein retains the C-terminal SPOC domain of RBM15, there is a possibility that selectively targeting the R834 interface in the context of RM could disrupt essential binding partners that are required for RM-AMKL leukemogenesis while potentially sparing the physiological functions of wildtype RBM15 [25]. Additionally, predictive modeling (e.g., AlphaFold3) alongside high-throughput screening could identify small molecules, peptides, or oligonucleotides that may selectively disrupt fusion-unique SPOC-mediated interactions, but with only minimal effects on wildtype SPOC domain in endogenous RBM15 [93].

The therapeutic landscape surrounding RBM15 and the m⁶A machinery is still in its infancy. Small-molecule inhibitors that target METTL3, such as STM2457 and STM3675, demonstrate anti-leukemic efficacy, but also raise concerns about toxicity in hematopoietic progenitors [67,84]. Developing therapeutic agents with cell type-specific selectivity remains a major hurdle. The emerging class of PROTACs against METTL3 offers improved potency and durability. No RBM15-specific degraders currently exist [87,94]. One design challenge in targeting RBM15 is achieving high sensitivity to avoid the RBM15B paralog that potentially has distinct biological roles [50]. Additionally, the unique structural junction between RBM15 and MKL1 in RM may provide a more specific target but drugging intrinsically disordered regions or basic interfaces in this junction poses more difficulty than aiming for a structural domain [95–97].

Concluding Remarks and Future Perspectives:

RBM15 is a multifaceted regulator of RNA processing and hematopoietic lineage specification. Through its interactions with both core components of the m⁶A methyltransferase/writer complex (e.g., METTL3, METTL14, and WTAP), and other factors outside the writer complex, including transcriptional repressors, splicing regulators, and chromatin modifiers, RBM15 coordinates a wide spectrum of epigenetic and post-transcriptional processes [29,36]. Within hematopoiesis, RBM15 functions to maintain stem cell quiescence, modulate lineage commitment, and regulate megakaryocytic maturation – underscoring its essential role in preserving hematopoietic balance [21,25]. Dysregulation of RBM15’s normal functions, whether through altered expression levels, degradation, or chromosomal fusion disrupts these networks and contributes to malignant transformation [26].

The RM fusion exemplifies how aberrant coupling of mRNA and chromatin regulation can drive leukemogenesis. In AMKL, this fusion protein integrates RBM15-mediated m⁶A modification with MKL1-dependent transcriptional activation to sustain leukemic proliferation and block in terminal megakaryocyte maturation. Recent evidence that RM promotes Wnt signaling and depends on RBM15-linked m⁶A pathways highlights potential therapeutic vulnerabilities [67]. Pharmacologic inhibition of METTL3 or the disruption on the Wnt pathway activity could potentially attenuate these leukemogenic dependencies [84]. Moreover, selective targeting of RBM15’s SPOC-mediated interactions or modulations of its post-translational regulation may offer new opportunities for therapeutic intervention.

Despite significant advances, key mechanistic and structural questions remain unsolved (see Outstanding questions). The extensive IDRs of RBM15, which may facilitate its assembly into nuclear condensates or speckle compartments, remain uncharacterized. Whether these regions coordinate methyltransferase complex compartmentalization or regulate substrate accessibility is unclear. Likewise, the biological relevance of RBM15’s multiple isoforms have yet to be systematically explored. Defining isoform-specific functions could clarify how RBM15 exerts context-dependent effects on normal versus malignant hematopoiesis.

Outstanding Questions:

• How do the intrinsically disordered regions of RBM15 contribute to phase separation, multivalent interactions, and nuclear speckle organization in both normal hematopoiesis and leukemogenesis?

• How do post-translational modifications like methylation and lactylation dynamically regulate RBM15 stability, localization, and binding affinity?

• What are the functional differences among the RBM15 isoforms, and do they confer context-specific roles in hematopoiesis?

• Does RBM15 primarily function as a scaffold, allosteric regulator, or temporal coordinator of m⁶A deposition and RNA processing?

• Can RBM15-specific degraders like PROTACs be developed with sufficient selectivity and tolerability?

• What strategies can achieve paralog-specific targeting of RBM15 over RBM15B?

Future work should also focus on elucidating the hierarchical organization and temporal dynamics of RBM15’s interactions within the m⁶A writer complex and with noncanonical binding partners. Whether RBM15 primarily functions as a structural scaffold, allosteric modulator, or spatiotemporal coordinator of methyltransferase activity remains an open question. In addition, the regulatory impact of RBM15’s post-translational modifications (e.g., methylation and lactylation) on its stability, subnuclear localization, and binding affinity warrants systematic investigation [53,54]. Integrating biochemical, structural, and omics-based approaches, including CLIP-seq, proteomics, and single-cell transcriptomics, will be essential to construct a comprehensive map of RBM15’s interaction networks across hematopoietic states and disease contexts.

Therapeutically, targeting RBM15-associated pathways presents both opportunities and challenges. Strategies that inhibit METTL3 or disrupt other key RBM5-protein interactions may suppress leukemic self-renewal but require precision to avoid perturbing normal hematopoiesis [42,84]. Rational drug combinations that simultaneously modulate m⁶A-dependent RNA processing and transcriptional programs could contribute to enhanced selectivity and efficacy. Ultimately, a deeper understanding or RBM15’s domain architecture, isoform-specific roles, and post-translational regulation will refine our understanding of how RNA- and chromatin-based mechanisms cooperate to maintain hematopoietic homeostasis and how their disruption could be potentially leukemogenic.

Highlights:

Recent studies establish RBM15 as a central regulator of epitranscriptomic control in hematopoiesis and leukemia, functioning as an RNA-binding component of the N6-methyladenosine (m⁶A) writer complex.

Structural and mechanistic insights into the RBM15 SPOC domain reveal phosphoserine-dependent interactions that recruit the m⁶A writer complex, thereby coupling transcription with site-specific m⁶A deposition.

Emerging evidence shows that the RBM15::MKL1 fusion protein exploits the m⁶A machinery to enhance m⁶A-mediated stabilization of leukemogenic transcripts and activate pathways such as WNT signaling.

Advances in transcriptome-wide mapping approaches like m⁶A enhanced crosslinking and immunoprecipitation (eCLIP) refine RBM15-directed m⁶A landscapes and highlight therapeutic opportunities targeting METTL3 or RBM15-WTAP interactions as promising vulnerabilities in acute myeloid leukemia.

Glossary:

Acute megakaryoblastic leukemia (AMKL)

subtype of AML characterized by malignant megakaryocyte precursors

Acute myeloid leukemia (AML)

cancer of immature myeloid cells in the bone marrow and peripheral blood

Alternative splicing

process of generating multiple mRNA isoforms from a single gene

B-cell acute lymphoblastic leukemia (B-ALL)

aggressive B-cell malignancy in which the bone marrow produces too many immature B-cell lymphocytes known as lymphoblasts

Chronic myelogenous leukemia (CML)

slow-growing blood cancer that originates from hematopoietic stem and progenitor cells leading to the overproduction of white blood cells

Complex of proteins associated with Set1 (COMPASS) complex

multi-protein histone methyltransferase complex that catalyzes the methylation of histone H3 on lysine 4 (H3K4), a chromatin mark associated with active gene transcription

Epitranscriptomics

study of covalent RNA modifications that regulate RNA processing, stability, localization, and translation without altering the underlying DNA sequence.

Hematopoiesis

process of blood cell formation from HSPCs in the bone marrow

Hematopoietic stem and progenitor cells (HSPCs)

bone marrow cells including stem cells, which self-renew and can give rise to all blood cells, and progenitor cells with limited self-renewal and variable differentiation potential

Intrinsically disordered region (IDR)

segment of a protein that lacks a stable 3D structure under physiological conditions but remains functionally active, often enabling flexible binding, multivalent interactions, and roles in phase separation

Lineage-negative, Sca-1-positive, and c-Kit-positive (LSK) cells

subset of murine HSPCs that are enriched for HSC and progenitors capable of differentiation into all mature blood lineages

m⁶A enhanced crosslinking and immunoprecipitation (m⁶A eCLIP)

sequencing method that maps m⁶A sites at RNA fragments, enabling transcriptome-wide identification of m⁶A-modified regions

m⁶A methyltransferase/writer complex (MTC)

multi-protein complex that includes WTAP and the METTL3-METTL14 heterodimer, which “writes” (catalyzes) m⁶A deposition on RNA

N⁶-methyladenosine (m⁶A)

most abundant methylation on adenosine in RNA that influences RNA stability, splicing, and translation

Myelodysplastic syndrome (MDS)

group of rare cancers where immature blood cells in the bone marrow fail to properly mature, leading to low blood cell counts that can progress to AML

Nuclear RNA export factor 1 (NXF1)

primary protein receptor in eukaryotes responsible for exporting mature mRNA from the nucleus into the cytoplasm through nuclear pore complexes

Patient-derived xenograft (PDX)

preclinical research model where human tumor tissue is implanted in immunodeficient mice

Proteolysis-targeting chimera (PROTAC)

bifunctional small molecule drug that induces selective degradation of a target protein by hijacking the cell’s ubiquitin-proteasome system

RBM15::MKL1 (RM) fusion protein

oncogenic protein that drives leukemogenesis in infant AMKL

RNA-binding motif protein 15 (RBM15)

RNA-binding protein that recruits the m⁶A writer complex to specific transcripts and regulates hematopoiesis

Spen paralog ortholog C-terminal (SPOC) domain

highly conserved domain in RBM15 mediating phosphoserine-dependent protein interactions like WTAP binding

WNT signaling pathway

cell signaling pathway controlling development, proliferation, and stem cell fate

Wilms’ tumor 1-associating protein (WTAP)

ubiquitous nuclear protein that binds RBM15 and is a regulatory subunit of the m⁶A writer complex