To the Editor,

Waldenström Macroglobulinemia (WM) is a rare hematologic malignancy characterized by the accumulation of IgM-secreting lymphoplasmacytic lymphoma cells within a permissive bone marrow microenvironment [1]. Activating mutations in MYD88 are present in 93–97% of WM patients [1], while the tumor suppressor TP53 remains unaffected in most patients and thus susceptible to intervention [2]. Only a few WM patients achieve complete remission with the current standard-of-care treatments, highlighting the need for novel therapies.

G protein-coupled estrogen receptor 1 (GPER1) is a membrane estrogen receptor that regulates cell growth, migration, apoptotic cell death, and other cancer-related biological functions [1, 3,4,5,6,7]. Its pharmacological activation by the selective small-molecule agonist G-1 or its enantiomer LNS8801 is emerging as an attractive therapeutic strategy in human malignancies [5,6,7,8], as increasing GPER1 activity frequently increases p53 expression [9]. Therefore, we investigated GPER1 and its pharmacologic activation in WM.

GPER1 is upregulated in WM

We used RNA-seq to analyze the expression of GPER1 mRNA in CD19+ cells from WM patients (n = 72) and in healthy donor–derived B cells. These latter included CD19+/CD27+ B cells (n = 9), CD19+/CD27+ B cells (n = 9) and CD138+ plasma cells (n = 16). We found a remarkable upregulation of GPER1 in WM (Fig. 1A). Moreover, by analyzing clinically relevant patient subgroups [1], we observed higher GPER1 expression in patients carrying activating mutations in MYD88 and a wild-type CXCR4 gene (Fig. 1B). We further validated GPER1 mRNA upregulation by querying the GSE9656 and GSE61597 datasets of WM patients (Additional file 1: Fig. S1A, B). Moreover, we confirmed the upregulation of GPER1 protein expression by IHC analysis of lymph node samples derived from WM patients compared to healthy donors (Fig. 1C). Finally, we showed that BCWM-1 and MWCL-1 WM cell lines express GPER1 mRNA and protein similarly to the positive control breast cancer cell line MCF7(Additional file 1: Fig. S2A, B).

Fig. 1
figure 1

GPER1 is upregulated in WM, and its pharmacological activation triggers in vitro and in vivo anti-tumor activity. A RNA-seq analysis of GPER1 mRNA expression in CD19 + cells from WM patients (n = 72) and healthy donor (HD)-derived B cells. These latter included CD19 + /CD27 + B cells (n = 9), CD19 + /CD27 + B cells (n = 9) and CD138 + plasma cells (n = 16). B RNA-seq analysis of GPER1 mRNA expression in WM patients carrying MYD88-WT (n = 20) or MYD88-MUT (n = 52), with the latter further divided into CXCR4-WT (n = 32) or CXCR4-MUT (n = 20). C IHC analysis of GPER1 performed in lymph node specimens from WM patients (n = 4) and normal lymph nodes (n = 2), (20× magnification). D Cell viability was assessed by CTG assay 72 h after treatment with G-1 at the indicated doses. E A colony assay was performed in methylcellulose using WM cells treated with 1 μM G-1 for 72 h; representative pictures of colonies (10× magnification) are reported. F BCWM-1 cells were transfected with [100 nM] scrambled siRNAs or two different GPER1-targeting siRNAs (siGPER1#1 and siGPER#2), and then treated with G-1 [1 μM] or DMSO. GPER expression and cell viability were assessed 48 h after transfection by WB and CTG respectively. G Cell viability was assessed in primary CD19 + cells by CTG assay 72 h after treatment with G-1 [1 μM] at the indicated doses. H Average and SD of tumor volume (mm3) from groups of mice (n = 5/group) versus time (days) when tumor was measured. BCMW.1 cells (5 × 106 in 100 mL of serum-free RPMI1640 medium) were implanted in the flank of NOD/SCID mice. After tumor detection, mice were randomized to intraperitoneal treatment with G-1 [1 mg/Kg] or vehicle. Data are mean tumor volume ± SD. Arrows represent treatments. I Kaplan–Meier survival plot showing survival for mice treated with vehicle or G-1. *p < 0.05 from a Wilcoxon rank sum test in A, from a pairwise comparison using Wilcoxon rank sum exact test in B, and from Student’s t-test in other panels. **p < 0.05 from a log-rank test

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Pharmacological activation of GPER1 antagonizes tumor cell growth in WM, both in vitro and in vivo

We next studied the effect of GPER1 pharmacological manipulation using selective agonist (G-1) and antagonist (G-36 and G-15) [10]. We found that low-micromolar G-1 concentrations reduced the growth (Fig. 1D) and clonogenicity (Fig. 1E) of BCWM-1 and MWCL-1 WM cell lines. These effects were abrogated after genetic silencing of GPER1, confirming on-target activity (Fig. 1F). G-1 antagonized the growth of CD19+ cells from three WM patients (Fig. 1G) while sparing B cells from healthy donors (Additional file 1: Fig. S2C). A treatment cycle with G-1 resulted in a significant reduction of tumor growth in a clinically relevant BCWM-1 xenograft model (Fig. 1H), and prolonged animal survival (Fig. 1I). On the other hand, GPER1 antagonists G-36 and G-15 promoted the survival of BCWM-1 and MWCL-1 cells (Additional file 1: Fig. S2D).

Pharmacological activation of GPER1 triggers the TP53 pathway in WM

In WM cells treated with G-1, a gene set enrichment analysis (GSEA) found activation of the TP53 (p53) pathway (Fig. 2A), which was further confirmed using a reporter assay measuring p53 transcriptional activity (Fig. 2B). Consistently, G-1 increased the protein expression of p53 and its targets p21, BAX, BAD, and PUMA in BCWM-1 cells (Additional file 1: Fig. S2A) and CD19+ cells from a WM patient (Fig. 2C). Increased p53 protein expression was also observed in tumors retrieved from a SCID/NOD mouse treated with G-1 (Fig. 2D). Importantly, genetic silencing of p53 significantly antagonized the growth inhibitory effects of G-1 in BCWM-1 cells (Fig. 2E).

Fig. 2
figure 2

GPER1 pharmacological activation inhibits cell cycle progression and triggers apoptosis via inducing the p53 pathway. A GSEA performed 48 h after treatment with 1 μM G-1. B A p53 luminometric reporter assay was used to evaluate p53 transcriptional activity in G-1-treated BCWM-1 cells. C WB analysis of p53, p21, BAX, and PUMA in primary CD19+ WM cells treated with G-1 for 24 h. D Immunohistochemical staining for p53 (×20) in tumors sectioned on day 21 from vehicle- or G-1 [1 mg/kg] treated mice. Photographs are representative of one mouse receiving each treatment. E BCWM-1 cells were transfected with scrambled siRNAs (siCNT) or p53 targeting siRNAs and, after 24 h, were treated with vehicle or 1 μM G-1 for an additional 24 h and assessed for cell viability by CTG assay. WB analysis reports p53 knock-down in siP53-transfected cells. F FACS analysis of cell cycle phases of BCWM-1 cells 24 h after treatment with vehicle or G-1. G Annexin V staining of BCWM-1 cells 24 h after treatment with vehicle or G-1. H TEM analysis of BCWM-1 cells treated with G-1 (1 μM) or DMSO (NC). Control cells appear well-preserved with intact mitochondria, orderly chromatin folding and a clear nuclear membrane. Apoptotic cells become pyknotic with many electron-transparent vacuoles (V), chromatin (arrowhead) and cytoplasm condensation (increase in electron density of cytoplasmic matrix and organelles) and formation of apoptotic bodies (A). I Immunohistochemical staining for caspase 3 (×20) in tumors sectioned on day 21 from vehicle- or G-1 [1 mg/kg] treated mice. J Table showing combination indexes resulting from combinatorial treatments of BCWM-1 with G-1 and bortezomib (24 h time point). K WB analysis of p53, p21 and caspase 3 in BCWM-1 cells treated with [0.5 μM] G-1 and 10 nM bortezomib (BZ)

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Pharmacological activation of GPER1 induces cell cycle arrest and apoptosis in WM

G-1 promoted a dose-dependent accumulation of BCWM-1 cells in the G2/M phase (Fig. 2F), with a concomitant increase in the expression of mitotic protein cyclin B1 (Additional file 1: Fig. S3B). G-1 also increased annexin V binding (Fig. 2G) and caspase 3/7 activity (Additional file 1: Fig. S3C), which are markers of apoptotic cell death. Apoptosis was further confirmed by WB analysis of cleaved PARP, caspase 3 and 7 (Additional file 1: Fig. S3D), and by transmission electron microscopy (TEM) revealing the appearance of typical apoptotic features (Fig. 2H). IHC analysis highlighted an increase in the expression of caspase 3 in BCWM-1 xenografts retrieved from mice treated with G-1 (Fig. 2I). The anti-WM activity of G-1 was maintained even in the presence of protective bone marrow stromal cells (Additional file 1: Fig. S3E). Combining G-1 with the proteasome inhibitor bortezomib, a clinically active compound that activates p53 in tumor cells [11, 12], led to synergistic anti-proliferative activity (Fig. 2J), along with a synergistic activation of p53 target p21 and cleaved caspase 3 (Fig. 2K).

Conclusion

This study shows GPER1 is a novel actionable target in WM, providing the framework for translation of G-1 to clinical trials.