Triptolide and its prodrug Minnelide target high-risk MYC-amplified medulloblastoma in preclinical models
Jezabel Rodriguez-Blanco, April D. Salvador, Robert K. Suter, Marzena Swiderska-Syn, Isabel Palomo-Caturla, Valentin Kliebe, Pritika Shahani, Kendell Peterson, Maria Turos-Cabal, Megan E. Vieira, Daniel T. Wynn, Ashley J. Howell, Fan Yang, Yuguang Ban, Heather J. McCrea, Frederique Zindy, Etienne Danis, Rajeev Vibhakar, Anna Jermakowicz, Vanesa Martin, Christopher C. Coss, Brent T. Harris, Aguirre de Cubas, X. Steven Chen, Thibaut Barnoud, Martine F. Roussel, Nagi G. Ayad, David J. Robbins
Jezabel Rodriguez-Blanco, April D. Salvador, Robert K. Suter, Marzena Swiderska-Syn, Isabel Palomo-Caturla, Valentin Kliebe, Pritika Shahani, Kendell Peterson, Maria Turos-Cabal, Megan E. Vieira, Daniel T. Wynn, Ashley J. Howell, Fan Yang, Yuguang Ban, Heather J. McCrea, Frederique Zindy, Etienne Danis, Rajeev Vibhakar, Anna Jermakowicz, Vanesa Martin, Christopher C. Coss, Brent T. Harris, Aguirre de Cubas, X. Steven Chen, Thibaut Barnoud, Martine F. Roussel, Nagi G. Ayad, David J. Robbins
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Research Article Oncology

Triptolide and its prodrug Minnelide target high-risk MYC-amplified medulloblastoma in preclinical models

Abstract

Most children with medulloblastoma (MB) achieve remission, but some face very aggressive metastatic tumors. Their dismal outcome highlights the critical need to advance therapeutic approaches that benefit such high-risk patients. Minnelide, a clinically relevant analog of the natural product triptolide, has oncostatic activity in both preclinical and early clinical settings. Despite its efficacy and tolerable toxicity, this compound has not been evaluated in MB. Utilizing a bioinformatic data set that integrates cellular drug response data with gene expression, we predicted that Group 3 (G3) MB, which has a poor 5-year survival, would be sensitive to triptolide/Minnelide. We subsequently showed that both triptolide and Minnelide attenuate the viability of G3 MB cells ex vivo. Transcriptomic analyses identified MYC signaling, a pathologically relevant driver of G3 MB, as a downstream target of this class of drugs. We validated this MYC dependency in G3 MB cells and showed that triptolide exerts its efficacy by reducing both MYC transcription and MYC protein stability. Importantly, Minnelide acted on MYC to reduce tumor growth and leptomeningeal spread, which resulted in improved survival of G3 MB animal models. Moreover, Minnelide improved the efficacy of adjuvant chemotherapy, further highlighting its potential for the treatment of MYC-driven G3 MB.

Authors

Jezabel Rodriguez-Blanco, April D. Salvador, Robert K. Suter, Marzena Swiderska-Syn, Isabel Palomo-Caturla, Valentin Kliebe, Pritika Shahani, Kendell Peterson, Maria Turos-Cabal, Megan E. Vieira, Daniel T. Wynn, Ashley J. Howell, Fan Yang, Yuguang Ban, Heather J. McCrea, Frederique Zindy, Etienne Danis, Rajeev Vibhakar, Anna Jermakowicz, Vanesa Martin, Christopher C. Coss, Brent T. Harris, Aguirre de Cubas, X. Steven Chen, Thibaut Barnoud, Martine F. Roussel, Nagi G. Ayad, David J. Robbins

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Figure 4

Triptolide decreases MYC levels through transcriptional and posttranslational mechanisms.

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Triptolide decreases MYC levels through transcriptional and posttranslat...
(A) MYC expression in HD:MB03 cells treated with 50 nM triptolide was measured by RT-qPCR (n = 3) and analyzed by 1-way ANOVA followed by Dunnett’s post hoc test. (B) Lysates from HD:MB03 cells treated with 50 nM triptolide were immunoblotted for indicated proteins. (C) HD:MB03 cells were treated with BS-181 (10 μM) alone or with 50 nM triptolide for 6 hours, before determining MYC expression by RT-qPCR (n = 4). Data were analyzed using unpaired, 1-tailed Student’s t test. (D) HD:MB03 cells were treated similarly for 48 hours. Cell viability was assessed by MTT reduction (n = 4), and analyzed using an unpaired, 1-tailed Student’s t test. (E) HD:MB03 cells were treated with MG-132 (10 μM) for 1 hour before adding 50 nM triptolide. RPB1 levels were immunoblotted 4 hours later. (F) HD:MB03 cultures were similarly treated and MYC expression was determined by RT-qPCR 4 hours later (n = 3). (G) Cell viability was assessed by MTT reduction 48 hours after similar treatment (n = 3). (H) HD:MB03 cells were exposed to 50 nM triptolide for 2 hours before adding 25 μM CHX. MYC half-life was calculated using nonlinear regression analyses (n = 3). (I) HD:MB03 cells were treated with 50 nM triptolide. Levels of MYC and its phosphorylated forms were determined by immunoblotting (n = 3), and analyzed by 1-way ANOVA followed by Dunnett’s post hoc test. (J) HD:MB03 cells were exposed to triptolide for 4 hours before immunoprecipitating MYC. Immunoprecipitates and their input extract were immunoblotted for the indicated proteins. (K) HD:MB03 cells were treated with 10 μM MG-132 for 1 hour before adding 50 nM triptolide. MYC levels were immunoblotted 4 hours later. (L) Schematic suggesting triptolide’s mechanism of action on G3 MB. Representative immunoblots are shown. Unless otherwise indicated, statistical significance was assessed by 1-way ANOVA followed by Newman-Keuls post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.