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Therapeutic activation of G protein-coupled estrogen receptor 1 in Waldenström Macroglobulinemia
Experimental Hematology & Oncology volume 11, Article number: 54 (2022)
Activating G protein-coupled estrogen receptor 1 (GPER1) is an attractive therapeutic strategy for treating a variety of human diseases including cancer. Here, we show that GPER1 is significantly upregulated in tumor cells from different cohorts of Waldenström Macroglobulinemia (WM) patients compared to normal B cells. Using the clinically applicable GPER1-selective small-molecule agonist G-1 (also named Tespria), we found that pharmacological activation of GPER1 leads to G2/M cell cycle arrest and apoptosis both in vitro and in vivo in animal models, even in the context of the protective bone marrow milieu. Activation of GPER1 triggered the TP53 pathway, which remains actionable during WM progression. Thus, this study identifies a novel therapeutic target in WM and paves the way for the clinical development of the GPER1 agonist G-1.
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 . Activating mutations in MYD88 are present in 93–97% of WM patients , while the tumor suppressor TP53 remains unaffected in most patients and thus susceptible to intervention . 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 . 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 , 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).
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) . 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).
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).
This study shows GPER1 is a novel actionable target in WM, providing the framework for translation of G-1 to clinical trials.
Availability of data and materials
The authors declare that all data supporting the findings of this study are available within the article and its Additional Information. Files or reagents are available from the corresponding authors on request.
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We gratefully acknowledge the members of our laboratories for technical advice and critical discussions. We thank Dr. Christina Usher (Dana-Farber Cancer Institute) for editing the manuscript and insightful comments.
This work was mainly supported by funds from AIRC, the Italian Association for Cancer Research (IG24449, PI: N.A.; IG21588; PI:P.T.; IG24365; PI: A.N.). E.M. is supported by a Brian D. Novis Junior Grant from the International Myeloma Foundation, by a Career Enhancement Award from Dana Farber/Harvard Cancer Center SPORE in Multiple Myeloma (SPOREP50CA100707), by a Special Fellow grant from The Leukemia & Lymphoma Society, and by a Scholar Award from the American Society of Hematology. A.G. is supported by a Fellow grant from The Leukemia & Lymphoma Society and by a Scholar Award from the American Society of Hematology. N.C.M. is supported by an NIH/NCI P01 (CA155258-10), by a Department of Veterans Affairs I01 (BX001584-09), and by a NIH/NCI R01 (CA207237-05). A.M.R. is supported by grants from the European Hematology Association and the Italian Association for Cancer Research (Fondazione AIRC).
Ethics approval and consent to participate
Patient samples were collected following informed consent approved by our Institutional Review Board at the Dana-Farber Cancer Institute. Animal studies were performed after approval by the Animal Ethics Committee of the DFCI and performed using institutional guidelines.
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All authors have read and approved the final manuscript.
N.C.M. serves on advisory boards or is a consultant to Takeda, BMS, Celgene, Janssen, Amgen, AbbVie, Oncopep, Karyopharm, Adaptive Biotechnology, and Novartis and holds equity ownership in Oncopep. A.M.R. serves on advisory board of Amgen, Celgene, Janssen, and Takeda. No potential conflicts of interest were disclosed by the other authors.
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Additional file 1: Figure S1.
Analysis of GPER1 mRNA in public datasets GSE9656 (A) and GSE61597 (B). GSE9656: we analyzed GPER1 mRNA in CD19-selected peripheral blood B cells (pBCs; n = 7), bone marrow B cells from WM (WM-BCs; n = 12), bone marrow plasma cells from healthy donors (BM-PCs; n = 10), and WM plasma cells (WM-PCs, n = 9). GSE61597: we analyzed GPER1 mRNA in normal bone marrow CD25+ (n = 7) and CD25– (n = 9) B cells, clonal B cells from newly diagnosed patients with IgM MGUS (n=22), smoldering (n = 17), and symptomatic WM (n = 10). Figure S2. A. qRT-PCR analysis of GPER1 mRNA in MCF7 breast cancer cell line (positive control), MWCL-1 and BCWM-1 WM cell lines, and CD19+ primary cells from four WM patients. B. WB analysis of GPER1 protein in a panel of six cancer cell lines (MCF7, MWCL-1, BCWM-1, DAUDI, RAJI, and MEC1). GAPDH was used as a loading control. C. CTG viability assay in BCWM-1 cells treated with indicated concentrations of GPER1 antagonists G15 and G-36. *Indicates p < 0.05 from a Student’s t-test. Ns indicates p > 0.05 from a Student’s t-test. Figure S3. A. WB analysis of p53, p21, BAX, and PUMA in primary CD19+ WM cells treated with G1 for 24 h. B. Wb analysis of Cyclin B1 in WM cell lines treated with indicated concentrations of G1. GAPDH was used as a loading control. C. Caspase 3/7 activity assay in WM cell lines treated with the indicated concentrations of G-1. Activity is represented relative to untreated cells. D. WB analysis of PARP, cleaved PARP, Caspase-3, and cleaved Caspase-3 in WM cell lines treated with the indicated concentrations of G-1. E. CTG viability assay and Caspase 3/7 activity assay in BCWM-1 cells, co-cultured for 48h with patient-derived bone marrow stromal cells and treated with G-1 [1 µM] or control. *Indicates p < 0.05 from a Student’s t-test.
Additional file 2.
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Morelli, E., Hunter, Z.R., Fulciniti, M. et al. Therapeutic activation of G protein-coupled estrogen receptor 1 in Waldenström Macroglobulinemia. Exp Hematol Oncol 11, 54 (2022). https://doi.org/10.1186/s40164-022-00305-x