The variable splicing factor RBM17 regulates MDS cell proliferation through H3K27la Free

Myelodysplastic syndrome is characterized by reduced production of all three lineages of blood cells, primarily affecting elderly individuals. Hematopoietic stem cell transplantation is the only curative treatment for MDS, but only a minority of patients can benefit from it as a result of the presence of complications or older age. Current first-line treatments are limited, and it is imperative to identify new therapeutic targets. Our previous study showed that the expression of the splicing factor RBM17 was decreased in HSPCs from MDS patients. We further found that RBM17 regulated MDS cell proliferation by repressing H3K27la.

In order to find new therapeutic targets for MDS patients, we collected bone marrow cells from 3 healthy volunteers and 3 newly diagnosed MDS patients who were not treated for single-cell sequencing. MDS patients had a dysregulation of HSPC cell subpopulations, and MDS-HSPC differentiation into myeloid and lymphoid lineages was also impaired. Moreover, in HSPC cells, the expression of genes related to the spliceosome signaling pathway was dysregulated. Alternative splicing is an important post-transcriptional regulatory mechanism that impacts the development of various tumors. Splicing factors also play a role in hemat malignancies, such as high RBM17 expression maintains leukemia (AML) stemness. By using single-cell sequencing data and other MDS datasets (GSE58831, GSE43399), we found that RBM17 was down-regulated in CD34+ cells from MDS patients. We further divided the MDS patients in the GSE58831 dataset into those with high or low expression of RBM17 based on their mean values. Kaplan-Meier analysis showed that the survival period of MDS patients with low expression of RBM17 was significantly reduced.

To further validate the impact of RBM17 on MDS cell proliferation, we overexpressed RBM17 in MDS cell lines (MUTZ1, SKM1). CCK8 and cell cycle assays showed that RBM17 overexpression inhibited MDS cell proliferation. Moreover, the construction of MDS cell subcutaneous tumor model showed that compared with the control group, RBM17 overexpression reduced the volume of MDS subcutaneous tumors. These results suggest that RBM17 overexpression inhibits MDS cell proliferation.

Next, we further analyzed the possible mechanism of RBM17 in MDS. To further analyze the possible mechanisms of RBM17 in MDS, we performed RNA-seq on SKM1 cells overexpressing RBM17 and SKM1 cells not overexpressing RBM17. Through GSEA analysis, it was found that RBM17 overexpression inhibited glycolysis in SKM1 cells. Current studies had shown that aerobic glycolysis is an important energy source for tumor cells and could regulate the intracellular histone lactylation of tumor cells through the glycolytic product lactic acid, thereby regulating cell proliferation. These results suggested that RBM17 overexpression may inhibit cell proliferation by regulating glycolysis and histone lactylation in MDS cells. Our experimental results also confirmed that RBM17 overexpression inhibited lactic acid production in SKM1 cells and suppressed general protein lactylation in cells. In addition, LC-MS/MS analysis found that H3K27 was lactylated in SKM1 cells. The Western blot experiment also showed that overexpression of RBM17 decreased H3K27la in SKM1 cells. H3K27la could be increased by treating with Nala, and partially reversed the inhibition of SKM1 cell proliferation by RBM17 overexpression. While treatment with 2-DG significantly inhibited SKM1 cell proliferation. We also found that RBM17 overexpression significantly suppressed the expression of KAT2A in SKM1 cells. These results suggest that RBM17 may not only regulate H3K27la in MDS cells through glycolysis, but also reduce H3K27la by inhibiting the expression of Lactyltransferase.

Our study showed that RBM17 was downregulated in HSPCs from MDS patients and significantly associated with poor patient prognosis. RBM17 reduced H3K27la by suppressing glycolysis and lactate production, thereby inhibiting MDS cell proliferation.