HNRNPA2B1 promotes multiple myeloma progression by increasing AKT3 expression via m6A-dependent stabilization of ILF3 mRNA

To the Editor

N6-methyladenosine (m6A) modification is the most frequent RNA modifications in eukaryotic RNAs affecting gene expression, which is seldom investigated in MM [1‐3]. Therefore, we checked the m6A genes in MM compared to normal plasma cells and the correlation of these genes with patient outcome including HNRNPA2B1, Mettl3, Mettl14, Wtap, etc., in MM patient cohorts. Interestingly, HNRNPA2B1 was the exclusive gene, which was not only increased in MM samples but also associated with poor outcome in APEX, TT2 and HOVON65 patient cohorts (Fig. 1a, b, Additional file 1: Fig. S1a–h). HNRNPA2B1, RNA binding protein heterogeneous nuclear ribonucleoprotein A2B1, is a nuclear reader of m6A [4] and highly expressed in several cancers regulating the progression of cancer [5, 6] through multiple processes of mRNAs metabolism [7] including alternative splicing [8], cytoplasmic RNA trafficking [9], transcription and translation [10]. Here, we aimed to explore the potential functions and regulatory mechanism of HNRNPA2B1 in MM.

Initially, we performed MTT assay that demonstrated the cellular proliferation was significantly increased in HNRNPA2B1 overexpression (HNRNPA2B1^OE) cells and decreased in HNRNPA2B1 knockdown (HNRNPA2B1^KD) cells (Fig. 1c, d, Additional file 1: Fig. S1i). Flow cytometry and WB analyses illustrated that knockdown of HNRNPA2B1 promoted cellular apoptosis (Fig. 1e, f). The nude mouse xenograft model by subcutaneous injection of HNRNPA2B1^OE cells showed overexpression of HNRNPA2B1 accelerating tumor growth in vivo (Fig. 1g and Additional file 1: Fig. S1j, k, l). These data indicated that HNRNPA2B1 promoted MM cellular growth in vitro and in vivo.

Next, m6A IP and RNA-seq analyses (MeRIP-Seq) found that ILF3 was downregulated (P < 0.05) of both m6A and transcription in HNRNPA2B1^KD cells compared with controls (Fig. 1h & Additional file 1: Fig. S2). The m6A consensus sequence (RRACH) motif [11] is shown in Fig. 1i, in which the m6A modification of ILF3 was enriched in 3′-noncoding regions (Fig. 1j). Consistently, ILF3 expression was reduced in HNRNPA2B1^KD cells (Fig. 1k, Additional file 1: Fig. S3a) while increased in HNRNPA2B1^OE cells (Additional file 1: Fig. S3b, c). RNA immunoprecipitation (RIP)-qPCR assay revealed that ILF3 mRNA was enriched in the precipitates of HNRNPA2B1 antibody and silencing of HNRNPA2B1 decreased the abundance of the ILF3 transcript binding to HNRNPA2B1 (Fig. 1l). To further verify the role of m6A in regulation of ILF3 expression, methylation inhibitor cyclolencine was used that induced remarkable reduction of ILF3 in MM cells (Fig. 1m, Fig. S3d, e). Therefore, we can conclude that HNRNPA2B1 may stabilize ILF3 mRNA to play an important role. As expected, the stability of ILF3 was decreased in HNRNPA2B1^KD cells (Fig. 1n, Additional file 1: Fig. S3f). The biological effects of HNRNPA2B1 might be related to its nucleocytoplasmic localization, as HNRNPA2B1 was distributed in both nuclear and cytoplasmic along with increased cytoplasmic localization of ILF3 (Fig. 1o, Additional file 1: Fig. S3g). The above results illustrated that HNRNPA2B1-induced expression of ILF3 was due to the enhanced stability of ILF3 mRNA transcripts upon recognition and bound of the m6A sites to HNRNPA2B1.

Notably, ILF3 expression was significantly elevated in plasma cells from MM patients (Fig. 2a) and associated with poor survival (Fig. 2b, Additional file 1: Fig. S4a, b). We established the ILF3 knockdown (ILF3^KD) cells (Fig. 2c), which displayed decreased cell growth rate after induction compared to the non-induced cells (Fig. 2d). Apoptosis assay showed that ILF3 inhibited MM cellular apoptosis (Additional file 1: Fig. S4c, d, e). In addition, MTT result demonstrated that ILF3 knockdown by siRNA could reverse the cellular proliferation induced by increased HNRNPA2B1 suggesting that ILF3 is one of the most important m6A/HNRNPA2B1 targets in MM (Additional file 1: Fig. S4f, g).

Further RNA immunoprecipitation-sequencing (RIP-seq) analysis indicated that MAPK pathway was enriched (Fig. 2e) and AKT3 mRNA was significantly enriched by anti-ILF3 antibody in ARP1 cells. The ILF3 binding sites represented by peaks were enriched in AKT3 mRNA transcripts (Fig. 2f). In agreement with above results, decreased AKT3 was observed in ILF3^KD cells (Fig. 2g, h) and RIP-qPCR using anti-ILF3 antibody confirmed a significantly reduced affinity of ILF3 to AKT3 mRNA in ARP1 ILF3^KD cells compared to control (Fig. 2i). RNA decay assay showed a relatively lower stability of AKT3 transcripts in ILF3^KD cells (Fig. 2j, Additional file 1: Fig S5a) correspondingly. These data suggested that ILF3 promoted MM progression through stabilization of AKT3 transcripts.

Finally, we verified the effect of HNRNPA2B1 on AKT3. The expression of AKT3 was decreased in HNRNPA2B1^KD cells (Fig. 2k, l), whereas the elevated expression of AKT3 was observed in HNRNPA2B1^OE cells (Additional file 1: Fig. S5b, c). While AKT3 was interfered with siRNA (Fig. 2m), the cellular proliferation induced by HNRNPA2B1 was attenuated (Fig. 2n). Intriguingly, immunohistochemistry correlation analysis showed that HNRNPA2B1, ILF3 and AKT3 were highly increased in MM patients with statistical-correlated expression trend significantly compared to normal controls (Fig. 2o).

In summary, we demonstrate the m6A-dependent effect of HNRNPA2B1 on regulating AKT signaling pathway and the correlation between HNRNPA2B1 and MM cell growth. It is disclosed that the HNRNPA2B1/m6A/ILF3/AKT3 axis plays a key role in MM progression.