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. 2021 Feb 5;13(2):e12640.
doi: 10.15252/emmm.202012640. Epub 2020 Dec 17.

Mithramycin induces promoter reprogramming and differentiation of rhabdoid tumor

Maggie H Chasse  1 , Benjamin K Johnson  1 , Elissa A Boguslawski  1 , Katie M Sorensen  1 , Jessica E Rosien  2 , Min H Kang  3 , C Patrick Reynolds  3 , Lyong Heo  1 , Zachary B Madaj  1 , Ian Beddows  1 , Gabrielle E Foxa  1 , Susan M Kitchen-Goosen  1 , Bart O Williams  1 , Timothy J Triche Jr  1 , Patrick J Grohar  1   4   5
Affiliations

Mithramycin induces promoter reprogramming and differentiation of rhabdoid tumor

Maggie H Chasse et al. EMBO Mol Med. .

Abstract

Rhabdoid tumor (RT) is a pediatric cancer characterized by the inactivation of SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex. Although this deletion is the known oncogenic driver, there are limited effective therapeutic options for these patients. Here we use unbiased screening of cell line panels to identify a heightened sensitivity of rhabdoid tumor to mithramycin and the second-generation analogue EC8042. The sensitivity of MMA and EC8042 was superior to traditional DNA damaging agents and linked to the causative mutation of the tumor, SMARCB1 deletion. Mithramycin blocks SMARCB1-deficient SWI/SNF activity and displaces the complex from chromatin to cause an increase in H3K27me3. This triggers chromatin remodeling and enrichment of H3K27ac at chromHMM-defined promoters to restore cellular differentiation. These effects occurred at concentrations not associated with DNA damage and were not due to global chromatin remodeling or widespread gene expression changes. Importantly, a single 3-day infusion of EC8042 caused dramatic regressions of RT xenografts, recapitulated the increase in H3K27me3, and cellular differentiation described in vitro to completely cure three out of eight mice.

Keywords: EC8042; SWI/SNF; epigenetics; mithramycin; pediatric cancer.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Rhabdoid tumor is sensitive to mithramycin, but not general chemotherapeutic agents

  1. Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a screen of 23 pediatric cancer cell lines. Rhabdoid tumor cell lines (red) cluster toward the left of the graph indicating these cell lines are more sensitive to mithramycin. These results confirm a previously published screen (Osgood et al, 2016).

  2. Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a published screen of 445 agents in 62 sarcoma cell lines (Teicher et al, 2015). The rhabdoid tumor cell line, G401 (red), appears on the left side of the graph indicating this cell line is more sensitive to mithramycin.

  3. Dose–response curves of rhabdoid tumor and Ewing sarcoma cell lines. RT cell lines (black) are sensitive to mithramycin treatment with a similar IC50 value as TC32 ES cells (gray). RT cell lines are not sensitive to three broadly active chemotherapeutic agents: etoposide, doxorubicin, or SN38. Data represent mean with standard deviation derived from three independent experiments.

Figure 2
Figure 2. Mithramycin sensitivity is not due to DNA damage and drives divergent phenotypes in rhabdoid tumor
  1. A, B

    Western blot showing concentration‐dependent increase in γH2AX following 8‐h exposure to etoposide in BT12 and G401 RT cells (A). Red bar indicates the measured IC50 (Fig 1C). Despite induction of DNA damage, 15 μM etoposide does not lead to apoptosis as indicated by live cell imaging in the presence of cleaved caspase 3,7 (CC3,7) reagent that fluoresces with caspase activation (B). Scale bar (lower left): 150 μm.

  2. C, D

    Western blot showing concentration‐dependent increase in γH2AX following 8‐h exposure to mithramycin (MMA) in BT12 and G401 RT cells (C). Red bar indicates the measured IC50 (Fig 1C). 100 nM mithramycin induces apoptosis at 8 h as measured by CC3,7 fluorescence without the presence of DNA damage (D). Scale bar (lower left): 150 μm.

  3. E

    BT12 cells treated with 20 nM MMA show a different cellular phenotype than with 100 nM. Beyond 24 h of exposure, there is evidence of mesenchymal differentiation and the appearance of maturing adipocytes. Scale bar (lower left): 150 μm.

  4. F, G

    Western blot showing concentration‐dependent increase in γH2AX following 8‐h exposure to EC8042 in BT12 and G401 RT cells. Red bar indicates the measured IC50 (Fig 1C). In contrast to apoptotic induction, 75 nM EC8042 exhibits evidence of mesenchymal differentiation, similar to 20 nM MMA (E,G). Please note that solvent control for (E) and (G) is the same although different fields are shown. Scale bar (lower left): 150 μm.

  5. H

    Mesenchymal differentiation confirmed with oil red O staining of lipid deposits following 20 nM MMA or 75 nM EC8042 treatment for 24 and 48 h. Please note that solvent control for 20 nM MMA and 75 nM EC8042 is the same. Scale bar (lower left): 150 μm.

  6. I

    PPARγ mRNA expression is induced following 20 nM mithramycin treatment as measured by qPCR fold change relative to GAPDH (2ddCT). **P = 0.002, ****P = 0.0001. Data represent mean with standard deviation derived from three independent experiments. P‐values were determined by one‐way ANOVA using Dunnett test for multiple comparisons.

Source data are available online for this figure.
Figure EV1
Figure EV1. Rhabdoid tumor cells are sensitive to mithramycin and EC8042, a second‐generation mithramycin analogue
  1. A

    COMET assay confirmation of DNA damage from Fig 2. Solvent and 100 nM mithramycin do not lead to DNA damage after 8 h of exposure while 15 μM etoposide does. Scale bar (lower left): 50 μm.

  2. B–D

    Bar graphs quantify the percent of each cell population in G1, S, or G2 following 1‐h (B), 8‐h (C), or 18‐h (D) mithramycin exposure. Solvent was treated for 18 h. Concentrations represent 0.5× (10 nM), 1× (20 nM), and 2× (40 nM) mithramycin IC50. Values reported in Appendix Table S2. Data represent mean with standard deviation derived from three independent experiments.

  3. E

    Dose–response curve of BT12 (circle) and G401 (triangle) rhabdoid tumor cells treated with EC8042. Both rhabdoid tumor cell lines are sensitive to EC8042. Data represent mean with standard deviation derived from three independent experiments.

  4. F

    SMARCC1 and SMARCE1 mRNA expression does not change following 100 nM mithramycin treatment in G401 cells as measured by qPCR fold change relative to GAPDH (2ddCT). Data represent mean with standard deviation derived from three independent experiments.

  5. G

    Addition of the proteasome inhibitor (bortezomib) rescues the loss of protein expression following 8 and 18‐h mithramycin treatment. G401 cells were treated for 8 and 18 h with solvent (S), mithramycin (M, 100 nM), bortezomib (PI, 2.5 μM), or combination (C).

Source data are available online for this figure.
Figure 3
Figure 3. Mithramycin induced morphological changes are dependent on SWI/SNF eviction and the induction of H3K27me3
  1. A, B

    Mithramycin displaces SMARCC1 and SMARCE1 SWI/SNF subunits from chromatin in a time‐dependent manner in BT12 rhabdoid tumor (A) but not U20S osteosarcoma (B) cells. Western blot analysis showing whole cell lysate (Total), cytoplasmic soluble (CS), nuclear soluble (NS), and chromatin‐bound (Chr) fractions collected after exposure to solvent (S) or 100 nM mithramycin for 8 or 18 h and probed for the SWI/SNF subunits (BRD9, SMARCC1 or SMARCE1) or H3 (chromatin fraction control) and GAPDH (soluble fraction control).

  2. C

    SMARCC1 and SMARCE1 mRNA expression does not change following 100 nM mithramycin treatment as measured by qPCR fold change relative to GAPDH (2ddCT). Data represent mean with standard deviation derived from three independent experiments.

  3. D

    Addition of the proteasome inhibitor (bortezomib) rescues the loss of protein expression following 8‐ or 18‐h mithramycin treatment. BT12 cells were treated for 8 or 18 h with solvent (S), mithramycin (M, 100 nM), bortezomib (PI, 2.5 μM), or combination of 100 nM mithramycin and 2.5 μM bortezomib (C).

  4. E

    Loss of SWI/SNF occupancy at defined loci in the genome as measured by chromatin immunoprecipitation qPCR (ChIP‐qPCR) at previously described SWI/SNF target genes, MYT1 (8 h, ****P = 0.0001; 18 h, ****P = 0.0001) and CCND1 (8 h, ****P = 0.0001; 18 h, ****P = 0.0001). ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represent mean with standard deviation derived from three independent experiments. P‐values were determined by one‐way ANOVA using Dunnett test for multiple comparisons.

  5. F

    Chromatin immunoprecipitation qPCR (ChIP‐qPCR) of H3K27me3 at MYT1 (8 h, ****P = 0.0001; 18 h, ****P = 0.0001) and CCND1 (8 h, P = 0.02; 18 h, ****P = 0.0001). H3K27me3 occupancy is increased in a time‐dependent manner. ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represent mean with standard deviation derived from three independent experiments. P‐values were determined by one‐way ANOVA using Dunnett test for multiple comparisons.

  6. G–I

    Western blot showing concentration‐dependent increase in H3K27me3 following exposure to 100, 50, 25 nM mithramycin for 18 h in BT12 (G) and G401 (H) cells relative to loading control (H3). Re‐expression of SMARCB1 following doxycycline treatment inhibits the mithramycin‐dependent effects on H3K27me3 amplification (I).

Source data are available online for this figure.
Figure EV2
Figure EV2. Mithramycin sensitivity in rhabdoid tumor is not due to reprogramming of housekeeping genes or incomplete loss of siRNA knockdown
  1. A, B

    Chromatin immunoprecipitation of IgG, SMARCC1 (A), or H3K27me3 (B) at the control locus, GAPDH. Data represent mean with standard deviation derived from three independent experiments.

  2. C

    Time course of 100 nM mithramycin exposure in G401 cells. Cells were treated with 100 nM MMA for the indicated times followed by a replacement of drug‐free media. After 8 h (red) of mithramycin exposure, the cells have an irreversible suppression of proliferation compared to solvent control. Data from A, B represent mean with standard deviation derived from three independent experiments. Data in C are mean with standard deviation of 3 biological replicates and representative of three independent experiments.

  3. D, E

    Western blot confirming efficient protein knockdown of EZH2 in BT12 rhabdoid tumor cells (D) and SMARCB1 in U2OS osteosarcoma cells (E) compared with media and siNegative controls indicative of silencing in Fig 4E and F.

Source data are available online for this figure.
Figure 4
Figure 4. H3K27me3 amplification and SMARCB1 deletion drives mithramycin sensitivity in rhabdoid tumor
  1. A, B

    Mithramycin displaces BRD9 and SMARCE1 SWI/SNF subunits from chromatin in a time‐dependent manner in G401 (A) but only BRD9 with SMARCB1 re‐expression in G401 (B) cells. Western blot analysis showing whole cell lysate (Total), cytoplasmic soluble (CS), nuclear soluble (NS), and chromatin‐bound (Chr) fractions collected after exposure to solvent (S) or 100 nM mithramycin for 8 or 18 h and probed for the SWI/SNF subunits (BRD9, SMARCB1 or SMARCE1) or H3 (chromatin fraction control) and GAPDH (soluble fraction control).

  2. C

    BT12 cells show a threshold of exposure that leads to irreversible growth inhibition. The cells were exposed to 100 nM MMA for the indicated times followed by replacement with drug‐free medium. Beyond 8 h (red) of mithramycin exposure, the cells do not recover proliferative potential and exhibit a phenotype consistent with cell death. Data represent mean with standard deviation derived from three independent experiments.

  3. D

    Mithramycin leads to H3K27me3 amplification in a time‐dependent manner that precedes the induction of apoptosis as measured by the cleavage of PARP. Western blot lysates collected at 2, 4, 6, 8, 12, 18, and 24 h of continuous 100 nM mithramycin treatment.

  4. E

    Suppression of EZH2 expression and activity antagonizes mithramycin activity in BT12 rhabdoid tumor cells. Data represent dose–response curves of mithramycin in BT12 cells following a 48‐h exposure in the presence of siRNA silencing of the PRC2 subunit EZH2 or treatment with the EZH2 inhibitor EPZ‐6438 relative to mithramycin alone (media) or a non‐targeting siRNA (siNeg). Data represent mean with standard deviation derived from three independent experiments.

  5. F

    Suppression of SMARCB1 sensitizes U2OS osteosarcoma cells (wild‐type SWI/SNF) to mithramycin. Data represent dose–response curves of mithramycin in BT12 cells following a 48‐h exposure in the presence of siRNA silencing of the SWI/SNF subunit SMARCB1 relative to a non‐targeting siRNA (siNeg). Data represent mean with standard deviation derived from three independent experiments.

  6. G

    Western blot showing concentration‐dependent increase in H3K27me3 following exposure to 100, 50, 25 nM mithramycin for 18 h in SMARCB1‐silenced U2OS cells relative to loading control (H3). Knockdown of SMARCB1 following siRNA suppression triggers mithramycin‐dependent H3K27me3 amplification, while siNegative (control) does not have an effect on H3K27me3 following mithramycin exposure.

Source data are available online for this figure.
Figure 5
Figure 5. Mithramycin does not lead to general transcription inhibition and instead favors pro‐survival pathways
  1. A–C

    Volcano plot showing gene expression trends at 8‐h MMA treatment. Dashed lines represent a 2 log2FC (drug over control) (A) or 1.5 log2FC (drug over control) (B) and 10e‐7 q‐value threshold. Quantification of induced and repressed genes in (C). Labels indicate names of genes that meet the logFC and q‐value threshold.

  2. D–F

    Volcano plot showing gene expression trends at 18‐h MMA treatment. Dashed lines represent a 2 log2FC (drug over control) (D) or 1.5 log2FC (drug over control) (E) and 10e‐7 q‐value threshold. Quantification of induced and repressed genes in (F). Labels indicate names of genes that meet the logFC and q‐value threshold.

  3. G

    SP1 protein expression is reduced following mithramycin exposure. Western blot showing suppression of SP1 expression compared to loading control (GAPDH) after 100 nM mithramycin exposure for 1, 4, 8, 12, and 18 h.

  4. H, I

    The loss of SP1 expression is associated with a decrease in SWI/SNF occupancy of the SP1 promoter and an increase in H3K27me3. Data represent ChIP‐qPCR analysis following 8 or 18 h of 100 nM mithramycin exposure and immunoprecipitation of SMARCC1 (8 h, ***P = 0.0006; 18 h, ****P = 0.0001) (H) and H3K27me3 (8 h, **P = 0.009; 18 h, ****P = 0.0001) (I). ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represent mean with standard deviation derived from three independent experiments. P‐values were determined by one‐way ANOVA using Dunnett test for multiple comparisons.

  5. J

    fgsea analysis of gene ontology terms upregulated in primary AT/RT tumors as described in (Wang et al, 2017) following 8 h of mithramycin exposure. Mithramycin downregulates the expression of these pathways. P‐values derived from the fgsea analysis package.

  6. K, L

    fgsea analysis of hallmark pathways downregulated after 8 h of MMA exposure (K) or upregulated after 18‐h MMA exposure (L). P‐values derived from the fgsea analysis package.

Source data are available online for this figure.
Figure EV3
Figure EV3. SP1 expression is lost following mithramycin treatment but does not affect cell proliferation

  1. SP1 mRNA expression is reduced following 100 nM mithramycin treatment as measured by qPCR fold change relative to GAPDH (2ddCT) (8 h, P = 0.0001; 18 h, P = 0.0001). Data represent mean with standard deviation derived from three independent experiments. P‐values were determined by one‐way ANOVA using Dunnett test for multiple comparisons.

  2. SP1 expression is dependent on SWI/SNF. siRNA silencing of SMARCC1 and SMARCA4 subunits is associated with a similar loss of SP1 expression as direct silencing of SP1 as measured by qPCR relative to GAPDH (2ddCT). ****P = 0.0001. Data represent mean with standard deviation derived from three independent experiments. P‐values were determined by one‐way ANOVA using Dunnett test for multiple comparisons.

  3. Knockdown of SP1 (black) does not affect BT12 rhabdoid tumor cell proliferation compared with a siNeg (gray, solid line) negative control as measured by live cell imaging. siDeath (gray, dotted line) is a positive control for knockdown efficiency. Data represent mean with standard deviation derived from three independent experiments.

Figure 6
Figure 6. Mithramycin reprograms rhabdoid tumor promoters to trigger a change in cellular state and favor a differentiation phenotype

  1. Heatmaps depicting ATAC‐seq global chromatin structure following 8 h (middle) and 18 h (right) 100 nM MMA treatment. A 2 kb window is centered on the TSS. Chromatin accessibility clusters at the TSS and does not change globally relative to solvent. Quantification of genes that gain accessibility or reduce accessibility on the right.

  2. CTCF is the top downregulated motif from the ATAC‐seq analysis (P = 1e‐58).

  3. Schematic for the 18 state chromHMM model built for MRTs and collapsed into six super states. Chromatin states were called if the state was present in at least 50% of samples. ATAC‐seq and H3K27ac ChIP‐seq peaks were queried against the six super states. P‐values derived from the homer motif analysis package.

  4. Donut plots representing the percentage of each chromatin super state across treatment time (from 8 to 18 h) that increased 1.5‐fold (left) or decreased 1.5‐fold (right). Below, Motif analysis of the top upregulated motifs from the H3K27ac ChIP‐seq gene lists that pass a 1.5 logFC. P‐values derived from the homer motif analysis package using a binomial algorithm.

  5. IGV tracks of rhabdoid tumor genes previously identified to be occupied by non‐canonical SWI/SNF (Michel et al, 2018). ID3 and JUND3 decrease in H3K27ac occupancy and chromatin accessibility following exposure to mithramycin for 8 or 18 h.

  6. IGV tracks of rhabdoid tumor genes previously identified to gain H3K27me3 upon SWI/SNF loss (Erkek et al, 2019). CDK6 and CDK2 decrease in H3K27ac occupancy and chromatin accessibility following exposure to 8‐ and 18‐h mithramycin treatment.

  7. IGV tracks of genes identified to have an increase in accessibility, H3K27ac and gene expression following mithramycin exposure. BMP1 and ADIPOR1 play crucial roles in bone and adipogenic differentiation, respectively.

Figure EV4
Figure EV4. IGV tracks of genes that gain accessibility following mithramycin treatment and SMARCB1 complementation
IGV tracks of genes that increased in accessibility and H3K27ac occupancy after 18 h of 100 nM MMA exposure that correlated with previously published dataset of genes that increase in chromatin accessibility following SMARCB1 complementation in G401 rhabdoid tumor cells. Gray bars indicate peaks called from Weissmiller et al (2019).
Figure EV5
Figure EV5. In vivo analysis of rhabdoid tumor xenografts following mithramycin and EC8042 treatment

  1. Spaghetti plot showing tumor volumes of individual tumors in mice bearing G401 xenografts treated with 2.4 mg/kg of mithramycin (red) or vehicle control (gray) administered continuously intraperitoneal over 72 h. Most mice experienced a suppression or regression of tumor volume that persisted for more than 2 weeks following treatment. The shaded box indicates the duration of treatment.

  2. Bioluminescence imaging of G401 rhabdoid tumor xenografts correlates with caliper measurements in Fig 7A and B. Two mice per treatment group were imaged (left) and quantified in the bar graph (right). Error bars represent mean with SD. Scale bar indicates RFU intensity from (5.0e5–3.5e6, top; 1.0e6–4.0e6 bottom).

  3. Mice treated with EC8042 have reversible body mass loss during treatment with the 3‐day infusion compared to vehicle. However, body weight recovers once treatment ends.

  4. Immunohistochemistry analysis of G401 xenograft tumors on 7 days (day 8) after treatment with vehicle or 3‐day infusion of EC8042. 20× magnification of H&E shows osteoblasts and imbedded osteocytes in the trabecular architecture of treated xenograft tissue. Scale bar (lower left): 50 μm.

  5. PCA analysis of mithramycin‐treated BT12 cells with normal skull. Mithramycin‐treated cells cluster more with skull compared with solvent.

Figure 7
Figure 7. EC8042 leads to durable tumor regression in a rhabdoid tumor xenograft model

  1. Immunohistochemistry analysis recapitulates the biochemistry described in vitro for mithramycin. G401 tumor sections at 20X magnification stained with H&E, cleaved caspase 3 (CC3; apoptosis), or H3K27me3. A marked increase in CC3 that correlates with H3K27me3 staining is seen only in mice treated with the continuous infusion schedule but not vehicle. Scale bar (lower left): 50 μm.

  2. Prolonged durable response and cure of mice bearing G401 xenografts treated with 30 mg/kg EC8042 administered continuously over 72 h. Treatment duration indicated by gray shaded box. Asterisk indicates an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text).

  3. Kaplan–Meier survival curves indicating extended survival for mice bearing established G401 xenografts treated with the 3‐day continuous infusions of EC8042 in (B). The shaded box indicates the duration of treatment. Asterisk indicates an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text).

  4. 20X image of section of G401 treated tumors stained for H3K27me3, cleaved caspase 3, and γH2AX. The sections compare vehicle to treatment started on day 1 with 30 mg/kg of EC8042 administered continuously for 72 h (3‐day pump). H3K27me3 increases and correlates with apoptosis (CC3); however, induction of CC3 is modest. γH2AX staining does not increase with treatment indicating DNA damage is not responsible for these effects. Scale bar (lower left): 50 μm.

Figure 8
Figure 8. EC8042 induces mesenchymal differentiation in rhabdoid tumor xenografts
Immunohistochemistry analysis of H&E stains from G401 xenograft tumors on 1, 3, and 7 days after treatment with vehicle or 3‐day EC8042 infusion. EC8042‐treated xenograft tumors exhibit evidence of mesenchymal differentiation compared to vehicle. micro‐CT analysis of xenograft tumors on 7 days after treatment exhibit enhanced calcification compared to vehicle. IHC scale bar (lower left): 100 μm.
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