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. 2020 May;19(5):1183-1196.
doi: 10.1158/1535-7163.MCT-19-0775. Epub 2020 Mar 3.

CDK9 Blockade Exploits Context-dependent Transcriptional Changes to Improve Activity and Limit Toxicity of Mithramycin for Ewing Sarcoma

Guillermo Flores  1   2 , Joel H Everett  3 , Elissa A Boguslawski  1 , Brandon M Oswald  1 , Zachary B Madaj  4 , Ian Beddows  4 , Sergey Dikalov  5 , Marie Adams  4 , Carleen A Klumpp-Thomas  6 , Susan M Kitchen-Goosen  1 , Scott E Martin  6 , Natasha J Caplen  7 , Lee J Helman  8 , Patrick J Grohar  9   8   10   11   12   13
Affiliations

CDK9 Blockade Exploits Context-dependent Transcriptional Changes to Improve Activity and Limit Toxicity of Mithramycin for Ewing Sarcoma

Guillermo Flores et al. Mol Cancer Ther. 2020 May.

Abstract

There is a need to develop novel approaches to improve the balance between efficacy and toxicity for transcription factor-targeted therapies. In this study, we exploit context-dependent differences in RNA polymerase II processivity as an approach to improve the activity and limit the toxicity of the EWS-FLI1-targeted small molecule, mithramycin, for Ewing sarcoma. The clinical activity of mithramycin for Ewing sarcoma is limited by off-target liver toxicity that restricts the serum concentration to levels insufficient to inhibit EWS-FLI1. In this study, we perform an siRNA screen of the druggable genome followed by a matrix drug screen to identify mithramycin potentiators and a synergistic "class" effect with cyclin-dependent kinase 9 (CDK9) inhibitors. These CDK9 inhibitors enhanced the mithramycin-mediated suppression of the EWS-FLI1 transcriptional program leading to a shift in the IC50 and striking regressions of Ewing sarcoma xenografts. To determine whether these compounds may also be liver protective, we performed a qPCR screen of all known liver toxicity genes in HepG2 cells to identify mithramycin-driven transcriptional changes that contribute to the liver toxicity. Mithramycin induces expression of the BTG2 gene in HepG2 but not Ewing sarcoma cells, which leads to a liver-specific accumulation of reactive oxygen species (ROS). siRNA silencing of BTG2 rescues the induction of ROS and the cytotoxicity of mithramycin in these cells. Furthermore, CDK9 inhibition blocked the induction of BTG2 to limit cytotoxicity in HepG2, but not Ewing sarcoma cells. These studies provide the basis for a synergistic and less toxic EWS-FLI1-targeted combination therapy for Ewing sarcoma.

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Conflict of interest statement

Grant Support:

Disclosure of Potential Conflicts of Interest: None to disclose.

Figures

Figure 1.
Figure 1.
Mithramycin synergizes with cyclin-dependent kinase 9 inhibitors. A, Schematic of the drug matrix screen. Plates with serial dilutions of mithramycin and different transcriptional inhibitors were stamped onto 384-well plates to test 48 different drug combinations in quadruplicate with at least two independent experiments. TC32 cells were added to the drug combination and viability was determined at 60 hours. The degree of synergy is determined by Bliss independence and represented by the height of the peak and color (red = synergy; blue = antagonism). B, A panel of transcription inhibitors demonstrate limited synergy (amanitin, BS-181, SU9561), diffuse synergy (palbociclib, triptolide, camptothecin) or require high doses of compound (K08361). C, A focus of synergy between mithramycin and CDK9 inhibitors at the clinically achievable concentration of mithramycin with 6 different CDK9 inhibitors.
Figure 2.
Figure 2.
The combination of mithramycin and PHA-767491 reverses the activity of EWS-FLI1. A and B, 100 nM mithramycin for 18 hours blocks the expression of the EWS-FLI1 induced targets EZH2 and NR0B1 while inducing the expression of the repressed target PHLDA. Lower concentration (20 nM for 18 hours) of mithramycin had a minimal impact on expression of EWS-FLI1 induced (NR0B1, EZH2) or repressed targets (PHLDA1) unless combined with 2 μM PHA-767491. Data represents fold change (2ΔΔCT) in expression relative to GAPDH as measured by RT-qPCR in TC32 (n=6), TC252 (n=6), and TC71 (n=3) treated with either media (M), solvent (S), 100 nM mithramycin (100), 20 nM mithramycin (20), 2 μM PHA-767491 (P), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (C) for 18 hours. Each biological replicate had 3 or 4 technical replicates. * = P < 0.05, ** = P < 0.01, *** = P <0.001, **** = P < 0.0001, error bars show standard deviation. C, EWS-FLI1 downstream target proteins are suppressed with high dose mithramycin or the combination of mithramycin and PHA-767491. Immunoblot showing expression of the EWS-FLI1 downstream targets (NR0B1, EZH2) relative to loading control (GAPDH) following 18-hour exposure to medium (M), solvent (S), 100 nM mithramycin (100), 20 nM mithramycin (20), 2 μM PHA-767491 (P), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (C). Immunoblots shown are representative of three independent experiments per cell line. D and E, Reversal of the EWS-FLI1 gene signature requires either high dose mithramycin or combination treatment as demonstrated by RNA sequencing following treatment with medium (M), solvent (S), or 100 nM mithramycin (100 MMA), 20 nM mithramycin (20), 2 μM PHA-767491 (PHA), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (Combo) for 12 hours in TC32 (n=3) and TC252 (n=3) cell lines.
Figure 3.
Figure 3.
2 μM PHA-767491 inhibits CDK9. A, 2 μM PHA-767491 blocks serine-2 phosphorylation independently or in combination with mithramycin. Immunoblot showing RNAPII and RNAPII CTD phosphoserine-2 relative to GAPDH loading control in TC32, TC252, and TC71 cell lines following exposure to medium (M), solvent (S), 100 nM mithramycin (100), 20 nM mithramycin (20), 2 μM PHA-767491 (P), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (C) for 18 hours. Data representative of three independent experiments. B, 2 μM PHA-767491 induces the expression of endogenous retroviral RNA (ERV). Data represents fold change in expression (2ΔΔCT) of ERV-F and ER9–1 relative to GAPDH in TC32 (n=3), TC252 (n=3), and TC71 (n=3) cells following exposure to medium (M), solvent (S), 100 nM mithramycin (100), 20 nM mithramycin (20), 2 μM PHA-767491 (P), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (C) for 18 hours. C, Schematic of nuclear run on assay used to measure RNA processivity. Primer pairs to both a proximal and distal region on the EZH2 locus were used for RT-qPCR. D, Processivity of RNA as measured by qPCR enrichment of mRNA from the proximal (start) vs. distal (end) amplicon of EZH2 relative to solvent after a TC32 nuclear run-on assay. Nuclei were exposed to 2 μM PHA-767491 during the run-on reaction (PHA), 20 nM mithramycin during the run-on reaction (MMA Run on), or cells were pretreated with 20 nM mithramycin for 18 hours before the run-on reaction (MMA Pre). Each biological replicate in the figure had three technical replicates. * = P < 0.05, ** = P < 0.01, *** = P <0.001, **** = P < 0.0001, error bars show standard deviation.
Figure 4.
Figure 4.
The combination of 20 nM mithramycin and 2 μM PHA-767491 decreases Ewing sarcoma cell viability. A, Mithramycin (MMA) concentration response curves alone or in combination 2 μM PHA-767491 (MMA + PHA). Data represents percent viability normalized to medium (MMA) or 2 μM PHA-767491 (MMA + PHA) in TC32, TC252, and CHLA9 Ewing sarcoma cell lines as measured by MTS assay. Best fit lines represent 3rd order polynomial with variable slope and error bars represent standard deviation. B, Proliferation as measured by cell count of GFP labeled nuclei every two hours of TC32 cells incubated with solvent (48 hours), 20 nM mithramycin (48 hours), 2 μM PHA-767491 (48 hours), or 20 nM mithramycin and 2 μM PHA-767491 (combo, 24 hours). *** = P <0.001, shading represents standard deviation. C, Spaghetti plot showing TC32 xenograft growth of individual mice as a function of treatment with vehicle, 1.5 mg/kg mithramycin intraperitoneal as a continuous infusion over 72 hours, 50 mg/kg PHA-767491 oral gavage twice daily for three days or the combination of both on the same schedule. 2 million TC32 cells were implanted into the gastrocnemius muscle of nude athymic female mice (n=11–12) and allowed to establish to a minimum diameter of 0.5 mm prior to starting treatment. Three mice were sacrificed for weight loss of unknown etiology (see Fig. S9)((PHA group, n =1 of 12; combination group (n =2 of 12)). D, Waterfall plot displaying best response as measured as the largest reduction in tumor volume following treatment. Each bar represents an individual animal. E, Same experiment as in C but with 3 mg/kg mithramycin eluted over 72 hours (n=6). Three mice in combination had end-of-infusion toxicity and were found dead in the combination cohort (see Fig. S9).
Figure 5.
Figure 5.
Mithramycin induces BTG2 expression and ROS production in liver cells which is reversed by PHA-767491. A, Heat map showing fold change increase (red) or decrease (blue) in expression of 84 different liver toxicity changes as measured by qPCR (2ΔΔCT) for each gene relative to GAPDH control in HepG2 cells following exposure to 50 nM mithramycin for 6 hours. BTG2 was the gene induced to the greatest degree with treatment (see BTG2 and arrow). B, Expression of BTG2 in HepG2 liver cells and TC32 Ewing sarcoma cells. Fold change was calculated using 2ΔΔCT method using GAPDH as a control. C, Mithramycin induces a dose dependent increase in superoxide that is rescued with siRNA silencing of BTG2. The data represents the amount of superoxide radicals per unit mass of protein as measured by electron paramagnetic resonance following exposure to 20 nM or 50 nM mithramycin. Silencing of BTG2 with siRNA for 30 hours reduces the accumulation of superoxide and rescues the induction caused by 50 nM mithramycin. * = P < 0.05, error bars represent standard error. MMA = mithramycin D, Silencing of BTG2 with siRNA has no impact on viability but mitigates mithramycin cytotoxicity at 3 different concentrations of drug. Data represents viability as determined by MTS assay following 30 hours of silencing for a total of 60 hours in HepG2 cells relative to medium, solvent, a non-targeting siRNA (siNEG), siRNA targeting of BTG2 (siBTG2), a positive control (siDeath) or 50 nM, 75 nM or 100 nM mithramycin (MMA) alone or in combination with silencing of BTG2 (siBTG2 + 50, 75, 100 nM MMA). E, Cellular proliferation of HepG2 cells (n=3) following silencing of BTG2 (siBTG2) alone or in combination with 50 nM mithramycin (MMA) relative to a non-targeting siRNA control (siNeg) as measured by percent confluence on an IncuCyte ZOOM system. * = P < 0.05, ** = P < 0.01, error bars represent standard deviation. F and G, 2 μM PHA-767491 blocks the induction of BTG2 (F) mRNA or protein (G) expression. Data represents qPCR fold change ( 2ΔΔCT) in expression of BTG2 relative to GAPDH as a control following exposure of TC32 cells to medium (M), solvent (S), 100 nM mithramycin (100), 20 nM mithramycin (20), 2 μM PHA-767491 (P), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (C) for 6 hours or immunoblot following identical exposure for 18 hours. Each biological replicate had 3 technical replicates. **** = P < 0.0001, error bars represent standard deviation. Immunoblots shown are representative of three replicates per cell line. GAPDH used as loading control. H, HepG2 growth over time as measured by percent confluence on an IncuCyte ZOOM with exposure to solvent, 100 nM mithramycin (100 nM MMA), 20 nM mithramycin (20 nM MMA), 2 μM PHA-767491, or 20 nM mithramycin and 2 μM PHA-767491 (Combo) for 72 hours and allowed to grow for 110 hours. **** = P < 0.0001, shaded regions represent standard deviation.
Figure 6.
Figure 6.
Summary of therapeutic strategy. Created with BioRender.com. A, EWS-FLI1 suppression by mithramycin requires a clinically unachievable concentration of 50–100 nM. A clinically achievable concentration of drug can be used if combined with sequential targeting of the transcription process. B, Since only Ewing sarcoma cells express EWS-FLI1, this strategy should have minimal effect on non-Ewing sarcoma cells. Furthermore, mithramycin induced liver damage is also transcriptionally regulated as BTG2 induction leads to ROS accumulation and a loss of liver cell viability. CDK9 inhibition abolishes the induction of BTG2 thus mitigating mithramycin induced damage in liver cells.
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References

    1. May WA, Gishizky ML, Lessnick SL, Lunsford LB, Lewis BC, Delattre O, et al.Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation. Proceedings of the National Academy of Sciences of the United States of America 1993;90(12):5752–6. - PMC - PubMed
    1. Riggi N, Suva ML, Suva D, Cironi L, Provero P, Tercier S, et al.EWS-FLI-1 expression triggers a Ewing’s sarcoma initiation program in primary human mesenchymal stem cells. Cancer research 2008;68(7):2176–85 doi 10.1158/0008-5472.can-07-1761. - DOI - PubMed
    1. Hu-Lieskovan S, Heidel JD, Bartlett DW, Davis ME, Triche TJ. Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing’s sarcoma. Cancer research 2005;65(19):8984–92 doi 10.1158/0008-5472.can-05-0565. - DOI - PubMed
    1. Grohar PJ, Woldemichael GM, Griffin LB, Mendoza A, Chen QR, Yeung C, et al.Identification of an inhibitor of the EWS-FLI1 oncogenic transcription factor by high-throughput screening. Journal of the National Cancer Institute 2011;103(12):962–78 doi 10.1093/jnci/djr156. - DOI - PMC - PubMed
    1. Mendiola M, Carrillo J, Garcia E, Lalli E, Hernandez T, de Alava E, et al.The orphan nuclear receptor DAX1 is up-regulated by the EWS/FLI1 oncoprotein and is highly expressed in Ewing tumors. Int J Cancer 2006;118(6):1381–9 doi 10.1002/ijc.21578. - DOI - PubMed

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