Liquid-liquid phase separation of ZHX2 protects DLBCL cells against ferroptosis through induction of SLC3A2

Abstract

Diffuse large B-cell lymphoma (DLBCL), the most common B-cell non-Hodgkin lymphoma (B-NHL), is characterized by strong aggression, high heterogeneity, and poor prognosis. Consequently, there is an urgent need to identify crucial therapeutic targets. Here, we found that the transcription factor zinc-finger and homeobox 2 (ZHX2) was highly expressed in DLBCL. Subsequently, ZHX2 was proven to be critical for promoting DLBCL cell proliferation by inhibiting ferroptosis. Mechanistically, ZHX2 bound to the promoter region of the solute carrier family 3-member 2 (SLC3A2) gene through liquid-liquid phase separation (LLPS) and activated its function to negatively regulate ferroptosis. Furthermore, we constructed lipid nanoparticles ZHX2-siRNA@LNP targeting DLBCL, which effectively inhibited the growth of the tumors in vivo. In summary, our study indicated that the LLPS of ZHX2 protected DLBCL against ferroptosis through induction of SLC3A2, and disturbing it with ZHX2-siRNA@LNP could significantly repress DLBCL, providing a promising therapeutic strategy for DLBCL.

Fig. 1. ZHX2 is overexpressed in DLBCL.

A IHC staining revealed ZHX2 expression in GCB DLBCL (n = 30), non-GCB DLBCL tissues (n = 30), and tonsil tissues (n = 30). B ZHX2 mRNA expression levels in GCB DLBCL (n = 30), non-GCB DLBCL (n = 30), and GC B cells from tonsil tissues (n = 30). C Western blot analysis of ZHX2 protein expression in GCB DLBCL (n = 3), non-GCB DLBCL (n = 3), and GC B cells from tonsil tissues (n = 6). For (A), scale bar, 50 μm (upper); 10 μm (lower). Data are mean ± SD values.

Fig. 2. ZHX2 knockdown attenuates the proliferation of DLBCL.

A CCK-8 assay of SU-DHL-4, DB, and U2932 cells (30,000 cells/mL) in control and ZHX2 knockdown group for 0, 24, 48, 72, and 96 h (n = 3). B Colony formation assay of SU-DHL-4, DB, and U2932 cells in control and ZHX2 knockdown group (20,000 cells/mL). After 2 weeks, colonies were counted (n = 3). C Representative image of tumors from SU-DHL-4 cell xenograft models (n = 5 in each group). For (B), scale bar, 2 mm. Data are mean ± SD values.

Fig. 3. ZHX2 knockdown promotes ferroptosis to inhibit the malignant progression of DLBCL.

A TEM images of ZHX2 knockdown and control in DLBCL cells, including SU-DHL-4, DB, and U2932. White arrows indicate mitochondria. B Levels of intracellular Fe²⁺ measured by FerroOrange probes. C Flow cytometry analysis of the ROS levels in control or ZHX2 KD cells. For (A), scale bars, 1 μm. For (B), scale bar, 10 μm. Data are mean ± SD values.

Fig. 4. ZHX2 undergoes LLPS.

A The amino acid sequence of the ZHX2 protein was analyzed via the disorder region prediction tool PONDR (http://www.pondr.com/). B Upon treatment with 300 mM NaCl and 10% PEG 8000, EGFP-ZHX2 (30 μM) formed microdroplets, which disappeared 10% 1, 6-Hex was added for 1 min, while EGFP did not. C HEK293T cells transfected with EGFP or ZHX2-EGFP plasmids (2 μg/mL) for 48 h were treated with 0.1 M mannitol, showing no aggregation in EGFP-transfected cells, while punctate aggregates appeared and then disappeared within 3 min of 10% 1, 6-Hex treatment in ZHX2-EGFP-transfected cells (n = 3). For (B), scale bar, 5 μm. For (C), scale bar, 2 μm.

Fig. 5. ZHX2 inhibits LLPS through the HD3 to regulate cell proliferation in DLBCL.

A No puncta were observed in DLBCL cells transfected with Z-ΔHD3-EGFP plasmid (2 μg/mL) for 48 h, whereas the ZHX2-EGFP group showed puncta (n = 3). B Cell viability of control, ZHX2 with LLPS, and Z-ΔHD3 without LLPS treated DLBCL cells (30,000 cells/mL) at 48 h (n = 3). C Representative photograph of tumors, tumor weights, and volumes of SU-DHL-4 cell xenograft models (n = 5 in each group). For (A), scale bar, 2 μm. Data are mean ± SD values.

Fig. 6. LLPS of ZHX2 represses cell proliferation by activating SLC3A2.

A The IGV diagram showing that phase-separated ZHX2 bound near the TSS of the SLC3A2 gene promoter from ChIP-seq data, while Z-ΔHD3 without LLPS did not. B ZHX2 or Z-ΔHD3 overexpression plasmids (1 μg/mL) were co-transfected into DB cells with 1 μg/mL wild-type or mutant SLC3A2 luciferase reporter vectors. Dual-luciferase reporter assay showed the effects of ZHX2 and Z-ΔHD3 on wild-type and mutant SLC3A2 reporter genes (n = 4). C Cell viability of DB cells (30000 cells/mL) treated with SLC3A2 KD in the control, ZHX2 with LLPS, and Z-ΔHD3 without LLPS groups at 48 h (n = 3). Data are mean ± SD values.

Fig. 7. Nanoparticle tumor-targeted delivery of ZHX2-siRNA attenuates DLBCL progress.

A Schematic of the preparation process of ZHX2-siRNA@LNP. B For the SU-DHL-4 xenograft models, the load and distribution of LNP in tumors and organs were photographed with an IVIS (n = 4 in each group). C Representative HE and Ki-67 images of tumors from SU-DHL-4 cell xenograft models, including the PBS, ZHX2-siCtrl@LNP, and ZHX2-siRNA@LNP groups. For (C), scale bar, 50 μm. Data are mean ± SD values.