Diptoindonesin G is a middle domain HSP90 modulator for cancer treatment

Abstract

HSP90 inhibitors can target many oncoproteins simultaneously, but none have made it through clinical trials due to dose-limiting toxicity and induction of heat shock response, leading to clinical resistance. We identified diptoindonesin G (dip G) as an HSP90 modulator that can promote degradation of HSP90 clients by binding to the middle domain of HSP90 (K_d = 0.13 ± 0.02 μM) without inducing heat shock response. This is likely because dip G does not interfere with the HSP90-HSF1 interaction like N-terminal inhibitors, maintaining HSF1 in a transcriptionally silent state. We found that binding of dip G to HSP90 promotes degradation of HSP90 client protein estrogen receptor α (ER), a major oncogenic driver protein in most breast cancers. Mutations in the ER ligand-binding domain (LBD) are an established mechanism of endocrine resistance and decrease the binding affinity of mainstay endocrine therapies targeting ER, reducing their ability to promote ER degradation or transcriptionally silence ER. Because dip G binds to HSP90 and does not bind to the LBD of ER, unlike endocrine therapies, it is insensitive to ER LBD mutations that drive endocrine resistance. Additionally, we determined that dip G promoted degradation of WT and mutant ER with similar efficacy, downregulated ER- and mutant ER-regulated gene expression, and inhibited WT and mutant cell proliferation. Our data suggest that dip G is not only a molecular probe to study HSP90 biology and the HSP90 conformation cycle, but also a new therapeutic avenue for various cancers, particularly endocrine-resistant breast cancer harboring ER LBD mutations.

Keywords: ESR1; LBD mutations; breast cancer; estrogen receptor; heat shock factor protein 1; heat shock protein 90; inhibitor.

Figure 1

Dip G promotes ER degradation in MCF7 cells.A, Western blot of ER levels in MCF7 cells treated with DMSO, 10 nM 17β-estradiol (E2), 1 μM 4-hydroxytamoxifen (OHT), 100 nM fulvestrant, 1 μM tanespimycin, or 10 μM dip G for 6 h. MCF7 cells were hormone starved for 3 days prior to treatment. Actin was used as a loading control. Three independent experiments with three biological replicates. One representative blot is shown. B, (Upper) Western blot of ER levels in MCF7 cells treated with DMSO, 1, 5, or 10 μM dip G for 24 h. MCF7 cells were hormone starved for 3 days prior to treatment. Actin was used as a loading control. Three independent experiments with three biological replicates. One representative blot is shown. (Lower) Molecular structure of diptoindonesin G. C, (Upper) Western blot of ER levels in MCF7 cells treated with DMSO, 1, 2.5, 5, or 10 μM deoxy-dip G for 24 h. MCF7 cells were hormone starved for 3 days prior to treatment. Actin was used as a loading control. Three independent experiments with three biological replicates. One representative blot is shown. (Lower) Molecular structure of deoxy-diptoindonesin G. D, ELISA assay of ER levels in MCF7 cells following treatment with DMSO, 1, 10, 50, and 1000 nM of fulvestrant, DMSO, 0.1, 5, 7.5, and 10 μM dip G, or DMSO, 0.25, 0.5, 1, 2, and 4 μM tanespimycin for 24 h. MCF7 cells were hormone starved for 3 days prior to treatment. ER protein levels were normalized to 1 × 10⁷ cells for fulvestrant and dip G and to total protein concentration for tanespimycin. Two independent experiments with three biological replicates and two technical replicates were carried out for fulvestrant and dip G. Three independent experiments with three biological replicates and two technical replicates were carried out for tanespimycin. Individual biological replicates are plotted. Data are represented as mean ± SD. E, RT-qPCR analysis of ER target gene GREB1, PGR, and MYC expression in T47D cells treated with DMSO, 1 nM E2, or 1 nM E2 plus increasing concentrations of fulvestrant (0.5–1024 nM) or dip G (1–32 μM) for 24 h. Cells were hormone starved for 3 days prior to treatment. Expression was normalized to 18s rRNA. Two independent experiments with two biological replicates and three technical replicates were carried out. Individual biological replicates are plotted. Data are represented as mean ± SD. dip G, diptoindonesin G; ER, estrogen receptor.

Figure 2

CHIP is not required for dip G-mediated ER degradation through the 26S proteasome.A, Western blot of ER and ubiquitin levels in MCF7 cells treated with DMSO, 10 μM dip G, 0.5 μM bortezomib (BORT), or a combination of the two compounds, for 6 h. MCF7 cells were hormone starved for 3 days prior to treatment. Actin was used as a loading control. Three independent experiments with three biological replicates were carried out. One representative blot is shown. B, volcano plot of proteins significantly downregulated (green) and upregulated (red) in MCF7 LCC2 shControl cells in response to 10 μM dip G treatment. Log₂ (fold change) is plotted on the x-axis and significance, or the -log₁₀(p-value) is plotted on the y-axis. Proteins not significantly changed are indicated in black. ESR1 (ER), highlighted in cyan, was significantly decreased by dip G treatment in both cell lines. One independent experiment with three biological replicates and three technical replicates was carried out. C, volcano plot of proteins significantly downregulated (green) and upregulated (red) in MCF7 LCC2 shCHIP cells in response to 10 μM dip G treatment for 24 h. Log₂ (fold change) is plotted on the x-axis and significance, or the -log₁₀(p-value) is plotted on the y-axis. Proteins not significantly changed are indicated in black. ESR1 (ER), highlighted in cyan, was significantly decreased by dip G treatment in both cell lines. One independent experiment with three biological replicates and three technical replicates was carried out. D, Western blot of ER and CHIP levels in MCF7 shControl and MCF7 shCHIP cells treated with DMSO or 10 μM dip G for 24 h. MCF7 cells were hormone starved for 3 days prior to treatment. Actin was used as a loading control. Three independent experiments with three biological replicates were carried out. One representative blot is shown. E, Western blot of CHIP levels in MCF7 CHIP KO clones #1 and #2 cells. Actin was used as a loading control. Three independent experiments with three biological replicates were carried out. One representative blot is shown. F, Western blot of ER and CHIP levels in MCF7 parental and CHIP KO clone #1 and #2 cells treated with DMSO or 10 μM dip G for 24 h. Cells were hormone starved for 3 days prior to treatment. Actin was used as a loading control. Three independent experiments with three biological replicates were carried out. One representative blot is shown. G, quantification of the Western blot shown in (F) individual biological replicates are plotted. Data are represented as the mean ± SD. Significance was determined using an unpaired Welch’s t test. dip G, diptoindonesin G; ER, estrogen receptor.

Figure 3

Dip G is a middle domain HSP90 modulator.A, fluorescence polarization plots measuring the K_d (bottom right corner of each plot) of deoxy-dip G to ER. Fluorescence polarization (in units of mP) is plotted on the y-axis (linear scale) and the protein concentration is plotted on the x-axis (logarithmic scale). The concentration of deoxy-dip G used was 1 μM. B, fluorescence polarization plots measuring the K_d (bottom right corner of each plot) of deoxy-dip G to HSP90. Fluorescence polarization (in units of mP) is plotted on the y-axis (linear scale) and the protein concentration is plotted on the x-axis (logarithmic scale). The concentration of deoxy-dip G used was 1 μM. C, fluorescence polarization plots measuring the K_d (bottom right corner of each plot) of deoxy-dip G to CHIP. Fluorescence polarization (in units of mP) is plotted on the y-axis (linear scale) and the protein concentration is plotted on the x-axis (logarithmic scale). The concentration of deoxy-dip G used was 1 μM. D, fluorescence polarization plots measuring the K_d (bottom right corner of each plot) of deoxy-dip G to the HSP90 M domain. Fluorescence polarization (in units of mP) is plotted on the y-axis (linear scale) and the protein concentration is plotted on the x-axis (logarithmic scale). The concentration of deoxy-dip G used was 1 μM. E, fluorescence polarization plots measuring the K_d (bottom right corner of each plot) of deoxy-dip G to the HSP90 N-terminus. Fluorescence polarization (in units of mP) is plotted on the y-axis (linear scale) and the protein concentration is plotted on the x-axis (logarithmic scale). The concentration of deoxy-dip G used was 1 μM. F, fluorescence polarization plots measuring the K_d (bottom right corner of each plot) of deoxy-dip G to the HSP90 C-terminus. Fluorescence polarization (in units of mP) is plotted on the y-axis (linear scale) and the protein concentration is plotted on the x-axis (logarithmic scale). The concentration of deoxy-dip G used was 1 μM. G, Coomassie-stained SDS-PAGE gel of full length HSP90 (FL-HSP90), purified HSP90 N-domain (N), HSP90 middle domain (M), and HSP90 C domain (C). The expected molecular weights are as follows: FL-HSP90 - 90 KDa, GST-HSP90 N (AA 9–236) - 55 KDa, HSP90 N - 26 KDa, GST-HSP90 M (AA 272–617) -62 KDa, HSP90 M - 41 KDa, GST-HSP90 C (AA 626–732) -38 KDa, HSP90 C -11.91 KDa, GST - 26 KDa. H, RT-qPCR analysis of HSP27, HSP40, HSP70, and HSP90 expression in MCF7 cells treated with DMSO, 10 μM dip G, 2 μM tanespimycin, or 40 μM novobiocin in full medium for 3 hours. Expression was normalized to 18s rRNA. Four independent experiments with four biological replicates and three technical replicates were carried out. For HSP70, only three independent experiments with three biological replicates and three technical replicates were run. Significance was determined using an unpaired t test with Welch’s correction. Individual biological replicates are plotted. Data are represented as mean ± SD. I, Western blot of ER levels in MCF7 cells pretreated with 10 μM dip G or 1 μM tanespimycin for 0.5 h and then treated with DMSO, 10 nM 17β-estradiol (E2), 1 μM 4-hydroxytamoxifen (OHT), 100 nM fulvestrant (Fulv), 1 μM tanespimycin (Tan), or 10 μM dip G for 5.5 h. MCF7 cells were hormone starved for 3 days prior to treatment. Actin was used as a loading control. Three independent experiments with three biological replicates were carried out. One representative blot is shown. J, quantification of the Western blot shown in (I) individual biological replicates are plotted. Data are represented as the mean ± SD. Significance was determined using an unpaired Welch’s t test. K, model of HSP90 showing that tanespimycin binds to the N-terminus, dip G binds to the middle domain, and novobiocin binds to the C-terminus. dip G, diptoindonesin G; ER, estrogen receptor; HSP90, Heat shock protein 90.

Figure 4

Dip G regulates a subset of tanespimycin-affected proteins.A, Western blot of ER in MCF7 cells treated with 10 μM dip G or 2 μM tanespimycin for 24 h. Actin was used as a loading control. Three independent experiments with three biological replicates were carried out. One representative blot is shown. B, volcano plot of proteins significantly downregulated (blue) and upregulated (red) in MCF7 cells treated with 10 μM dip G. Log₂ (fold change) is plotted on the x-axis and significance, and the -log₁₀(p-value) is plotted on the y-axis. Proteins not significantly changed are indicated in black. Three biological replicates with three technical replicates were carried out. C, volcano plot of proteins significantly downregulated (blue) and upregulated (red) in MCF7 cells treated with 2 μM tanespimycin (right). Log₂ (fold change) is plotted on the x-axis and significance, and the -log₁₀(p-value) is plotted on the y-axis. Proteins not significantly changed are indicated in black. Three biological replicates with three technical replicates were carried out. D, Venn diagrams comparing proteins significantly upregulated by both dip G and tanespimycin. Proteins unique to tanespimycin are on the left, and proteins unique to dip G are on the right. Proteins shared by both are shown in the overlapping region. E, Venn diagrams comparing proteins significantly downregulated by both dip G and tanespimycin. Proteins unique to tanespimycin are on the left, and proteins unique to dip G are on the right. Proteins shared by both are shown in the overlapping region. dip G, diptoindonesin G; ER, estrogen receptor.

Figure 5

ER Y537S-expressing cells are sensitive to dip G in ER+ cell lines.A, ELISA assay of ER levels in MCF7 Y537S cells following treatment with DMSO, 1, 50, 100, and 1000 nM of fulvestrant, with DMSO, 0.1, 5, 7.5, and 10 μM dip G or DMSO, 0.5, 1, 2, and 4 μM tanespimycin for 24 h. MCF7 cells were hormone starved for 3 days prior to treatment. ER protein levels were normalized to 1 x 10⁷ cells for fulvestrant and dip G and to total protein concentration for tanespimycin. Two independent experiments with three biological replicates and two technical replicates were carried out for fulvestrant and dip G. Three independent experiments with three biological replicates and two technical replicates were carried out for tanespimycin. Individual biological replicates are plotted. Data are represented as mean ± SD. B, hallmark gene set enrichment analysis of significantly changed gene sets (FDR < 25%, NES < -1.3 or NES > 1.3, NOM p-value < 0.05) from RNA sequencing of T47D and T47D Y537S cells treated with DMSO or 10 μM dip G for 24 h. One independent experiment with two biological replicates was carried out. C, GSEA enrichment plot for hallmark gene set estrogen response early and estrogen response late. D, RT-qPCR analysis of ER target gene GREB1, PGR, and MYC expression in T47D Y537S cells treated with DMSO, 1 nM E2, or 1 nM E2 plus increasing concentrations of fulvestrant (0.5–1024 nM) or dip G (1–32 μM) for 24 h. Cells were hormone starved for 3 days prior to treatment. Expression was normalized to 18s rRNA. Two independent experiments with two biological replicates and three technical replicates were carried out. Individual biological replicates are plotted. Data are represented as mean ± SD. E, cell counting data of MCF7 (pink) or MCF7 Y537S (blue) cells treated for 3 days with increasing concentrations of fulvestrant (0.25–512 nM) dip G (0.5–16 μM) or tanespimycin (0.625–20 μM) in full medium, using a BioTek Lionheart automated microscope. Shown are the number of cells present on the final day of treatment plotted in response to the log of the molar concentration of fulvestrant. Three independent experiments with eight biological replicates were carried out. Data are represented as mean ± SD. dip G, diptoindonesin G; ER, estrogen receptor; GSEA, Gene Set Enrichment Analysis; FDR, false discovery rate.

Figure 6

Dip G is effective in a patient-derived xenograft organoid model of ER+ breast cancer.A, phase contrast microscopy images of HCI-011 PDXOs treated for 2 weeks with DMSO, 10 μM dip G, 1 μM tanespimycin, or 1 μM fulvestrant in full medium. The scale bar corresponds to 1000 μm. Three biological replicates were carried out with 12 pictures/replicate. One representative image is shown. B, MTS data of HCI-011 PDXOs treated for 2 weeks with DMSO, 10 μM dip G, 1 μM tanespimycin, or 1 μM fulvestrant in full medium. Three independent experiments with three biological replicates and three technical replicates were carried out. Plotted are individual biological replicates. Data are represented as mean ± SD. Significance was determined using an unpaired Mann Whitney U test. C, RT-qPCR of ER target gene GREB1 in HCI-011 PDXOs treated with DMSO, 10 μM dip G, 1 μM tanespimycin, or 1 μM fulvestrant in full medium. Expression was normalized to 18s rRNA. Three independent experiments with three technical replicates were carried out. Individual biological replicates are plotted. Data are represented as the mean ± SD. Significance was determined using an unpaired Mann Whitney test. D, Western blot of ER in HCI-011 PDXOs treated with DMSO, 10 μM dip G, 1 μM tanespimycin, or 1 μM fulvestrant in full medium for 3 days. Actin was used as a loading control. Four independent experiments with four biological replicates were carried out. One representative blot is shown. E, quantification of the Western blot shown in (D) individual biological replicates are plotted. Data are represented as the mean ± SD. Significance was determined using an unpaired Mann Whitney test. F, summary of key data. Dip G promotes ER degradation through the 26S proteasome. CHIP is not required for dip G–mediated ER degradation. Dip G binds to the M-domain of HSP90, which is required for dip G–mediated ER degradation. Y537S mutants are sensitive to dip G and HSP90 inhibitors, but resistant to fulvestrant. dip G, diptoindonesin G; ER, estrogen receptor; HSP90, Heat shock protein 90; PDXO, patient-derived xenograft organoid.