HMGB3 As a Cargo Protein for XPO1: Implications for Myelodysplastic Syndromes Prognosis and Treatment

Background: In the preliminary research, we verified the activity of azacytidine sequential Selinexor in Myelodysplastic syndromes. However, the underlying mechanism remains to be explored. The family of high mobility group box (HMGB) proteins is involved in various biological processes including immunity and cancer development and progression. Unlike HMGB1 and HMGB2, HMGB3 is primarily expressed in bone marrow, yet its function remains largely unknown. In this study, we found that HMGB3, serves as a tumor suppressor gene in MDS, is a novel cargo protein for XPO1 and holds a pivotal position in the mechanism of azacitidine sequential selinexor therapy.

Method and results: Firstly, we collected nuclear proteins from cell lines treated with azacitidine sequential selinexor. Using XPO1 antibodies for immunoprecipitation (IP) and mass spectrometry analysis, we identified 312 potential XPO1 interacting proteins. Based on the GSE58831 dataset, expression and survival analyses revealed that HMGB3 is significantly downregulated in high-risk myelodysplastic syndromes (MDS) (P<0.001), and low HMGB3 expression is associated with poorer prognosis (P<0.05), suggesting that candidate XPO1 cargo protein HMGB3 might be a key effector protein. Co-immunoprecipitation (Co-IP) confirmed the interaction between XPO1 and HMGB3.

Gene methylation often impacts protein expression, we hypothesized that the low expression of HMGB3 might be related to its methylation levels. Methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) confirmed high methylation levels in the HMGB3 promoter region. Further analysis using real-time quantitative PCR (RT-PCR) and Western Blot demonstrated that HMGB3 mRNA and protein levels were significantly upregulated after azacitidine treatment. Following azacitidine and selinexor treatment, HMGB3 protein levels in the nucleus increased significantly, indicating that HMGB3's localization is crucial for the synergistic effects of the combined treatment.

To further validate the tumor-suppressive role of HMGB3, we performed HMGB3 overexpression experiments in MOLM-13 and SKM-1 cell lines. Functional assays showed that HMGB3 overexpression inhibited cell proliferation and clonogenicity and caused cell cycle arrest, but did not significantly affect apoptosis levels. This indicates that high HMGB3 expression effectively suppresses tumor cell proliferation.

RNA sequencing analysis revealed that HMGB3 overexpression activates the cytoplasmic DNA sensing pathway. We hypothesize that HMGB3 may activate this pathway by increasing intracellular double-stranded DNA (dsDNA) through DNA damage. Immunofluorescence and Western Blot experiments confirmed this hypothesis as cytoplasmic dsDNA accumilation and Elevated expression of H2AX. Previous studies have shown that activation of the DNA sensing signaling pathway can enhance interferon-related innate immune responses, and selinexor has been reported to induce panoptosis under immune activation conditions. Our study found that HMGB3 overexpression in combination with selinexor effectively induces panoptosis in cells, further confirming the role of HMGB3 in this process.

Conclusion：In summary, we identified HMGB3 as a novel cargo protein for XPO1 and demonstrated that HMGB3 acts as a tumor suppressor gene in MDS. HMGB3 induces panoptosis in synergy with selinexor by activating the cytoplasmic DNA sensing signaling pathway, thereby inhibiting the progression of high-risk MDS.