Fto Promotes Extramedullary Progression of Multiple Myeloma By Regulation of HSF1 through m⁶a RNA Methylation

Introduction:Extramedullary myeloma (EMM), defined by the presence of clonal cells outside the bone marrow, has extraordinarily heterogeneous biological and clinical features and is associated with an adverse prognosis. Recent studies have shown the upregulation of heat shock transcription factor 1 (HSF1) in EMM (Heimberger et al, Brit J Haematol 2013), but little is known about its role and mechanisms underlying the regulation. There is growing evidence that dysregulation of N⁶-methyladenosine (m⁶A) RNA methylation plays an important role in tumor invasion and metastasis (Pan et al, J Hematol Oncol 2018) and the m⁶A RNA modifications are involved in the heat shock response. This study was designed to reveal that the fat mass and obesity-associate protein (FTO), as a major m⁶A demethylase, plays a critical oncogenic role in extramedullary metastasis in multiple myeloma (MM) through regulating HSF1 and heat shock proteins (HSPs).

Methods and results: Using transcriptome sequencing, we determined that FTO was significantly upregulated in plasma cells (PCs) from EMM and MM patients, compared to healthy donors. We then evaluated the expression levels of FTO and HSF1 in plasma cells (PCs) from EMM patients by qRT-PCR and western blot analysis. The results verified that FTO and HSF1 were markedly upregulated in PCs from newly diagnosed MM patients compared to healthy donors, and it was further upregulated in PCs from patients with EMM. And the expression levels of FTO was positively correlated with HSF1. Measured by colorimetric assay, we found that m⁶A RNA methylation level of EMM patients was lower than the level of MM and healthy donors, which was consistent with the expression of FTO in EMM patients. Importantly, enforced expression or knockdown expression of FTO via lentivirus transfection, dramatically influenced the proliferation, migration and invasion in MM cells.

m⁶A RNA methylation has been reported to been associated with heat shock response (Zhou et al, Nature 2015). We therefore examined whether FTO can regulate HSF1 in EMM. Enforced expression of FTO dramatically elevated the level of HSF1 and HSPs in MM.1R and RPMI8226 cells, while inhibition of FTO reduced the level of HSF1 and HSPs. Through m⁶A sequencing and RNA sequencing, we confirmed that HSF1 was a novel target of FTO. Indeed, the demethylase FTO could positively regulate the expression of HSF1 at both the mRNA and protein levels in MM cells. In addition, knockdown of HSF1 by siRNA markedly reduced the ability of migration and invasion of MM cells overexpressing FTO. Thus, these results suggest that HSF1 is a key target gene of demethylase FTO in EMM. Moreover, pharmacological inhibitors of FTO, MA and MA2, could significantly enhance the effect of bortezomib in MM cells.

Recent studies have suggested that for m⁶A modification to exert its biological functions, it must first be recognized by m⁶A reader proteins. Here, we evaluated whether demethylase FTO could regulate the level of HSF1 via YTHDF2, a crucial m⁶A reader protein. We confirmed with colorimetric assay that overexpression of FTO could reduce m⁶A levels in MM cells, and knockdown of FTO could enhance the m⁶A levels. Using RNA immuno-precipitation sequence (RIP-seq), we identified direct binding between the 3' end of the HSF1 transcript and the m⁶A reader protein YTHDF2. Consistently, we showed that silencing of YTHDF2 with siRNA strongly promoted HSF1 expression in MM cells with knockdown of FTO. In addition, by RNA stability analysis, we found that the YTHDF2 could regulate the expression of HSF1 through affecting the stability of HSF1.

Next, we evaluated whether FTO could regulate extramedullary metastasis of MM in vivo. NCG mice were injected via tail vein with RPMI-8226-luc cells to establish EMM xenografts. Mice were assessed by in vivo bioluminescent imaging and PET-CT. The results showed that combined treatment with MA2 plus bortezomib could remarkably reduce the extramedullary metastasis in MM.

Conclusions: Our datas reveal that FTO is frequently up-regulated in EMM and contributes to MM progression. FTO promotes HSF1 expression through inhibiting the degradation effect of YTHDF2 by reducing m⁶A levels in these mRNA transcripts. Therefore, FTO-YTHDF2-HSF1 regulatory axis may potentially be applied in EMM treatment.