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Repurposing proscillaridin A in combination with decitabine against embryonal rhabdomyosarcoma RD cells

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Abstract

Purpose

Embryonal rhabdomyosarcoma (eRMS) is the most common type of rhabdomyosarcoma in children. eRMS is characterized by malignant skeletal muscle cells driven by hyperactivation of several oncogenic pathways including the MYC pathway. Targeting MYC in cancer has been extremely challenging. Recently, we have demonstrated that the heart failure drug, proscillaridin A, produced anticancer effects with specificity toward MYC expressing leukemia cells. We also reported that decitabine, a hypomethylating drug, synergizes with proscillaridin A in colon cancer cells. Here, we investigated whether proscillaridin A exhibits epigenetic and anticancer activity against eRMS RD cells, overexpressing MYC oncogene, and its combination with decitabine.

Methods

We investigated the anticancer effects of proscillaridin A in eRMS RD cells in vitro. In response to drug treatment, we measured growth inhibition, cell cycle arrest, loss of clonogenicity and self-renewal capacity. We further evaluated the impact of proscillaridin A on MYC expression and its downstream transcriptomic effects by RNA sequencing. Then, we measured protein expression of epigenetic regulators and their associated chromatin post-translational modifications in response to drug treatment. Chromatin immunoprecipitation sequencing data sets were coupled with transcriptomic results to pinpoint the impact of proscillaridin A on gene pathways associated with specific chromatin modifications. Lastly, we evaluated the effect of the combination of proscillaridin A and the DNA demethylating drug decitabine on eRMS RD cell growth and clonogenic potential.

Results

Clinically relevant concentration of proscillaridin A (5 nM) produced growth inhibition, cell cycle arrest and loss of clonogenicity in eRMS RD cells. Proscillaridin A produced a significant downregulation of MYC protein expression and inhibition of oncogenic transcriptional programs controlled by MYC, involved in cell replication. Interestingly, significant reduction in total histone 3 acetylation and on specific lysine residues (lysine 9, 14, 18, and 27 on histone 3) was associated with significant protein downregulation of a series of lysine acetyltransferases (KAT3A, KAT3B, KAT2A, KAT2B, and KAT5). In addition, proscillaridin A produced synergistic growth inhibition and loss of clonogenicity when combined with the approved DNA demethylating drug decitabine.

Conclusion

Proscillaridin A produces anticancer and epigenetic effects in the low nanomolar range and its combination with decitabine warrants further investigation for the treatment of eRMS.

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Funding

This work was supported by the Charles-Bruneau Foundation and Upcycle grant from the Cancer Research Society to N J-M R. N J-M R holds a research scholar Junior 2 award from the Fonds de Recherche du Québec en Santé.

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Authors and Affiliations

  1. Département de Pharmacologie et Physiologie, Université de Montréal, Montréal, QC, Canada

    Marielle Huot, Rahinatou Djibo, Rafael Najmanovich & Noël J. M. Raynal

  2. Sainte-Justine University Hospital Research Center, 3175, Chemin de la Côte-Sainte-Catherine, Montréal, QC, H3T 1C5, Canada

    Marielle Huot, Maxime Caron, Chantal Richer, Rahinatou Djibo, Pascal St-Onge, Daniel Sinnett & Noël J. M. Raynal

  3. Département de Pédiatrie, Université de Montréal, Montréal, QC, Canada

    Daniel Sinnett

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  1. Marielle Huot
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Contributions

NJ-MR designed the study. CR, MH, RD, MC and PSt-O, performed experimental studies and bioinformatic analyses. All authors participated in the writing of the manuscript. The datasets generated during the current study are available from the corresponding author on reasonable request.

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Correspondence to Noël J. M. Raynal.

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Supplementary figure S1

: Gene expression data (RPKM) from RNA-sequencing of RD cells (untreated and treated with proscillaridin A, 5nM, 48h, n=3) of a series of genes involved in cell proliferation (A) and differentiation (B) in RMS cells. Genes showed a significant difference in gene expression between untreated (Untr.) and treated conditions. Log2 fold changes are indicated in the figure. A letter ‘’a’’ indicates p<0.05 (n=3) (TIF 21 KB)

Supplementary figure S2: Levels of KAT6A (MOZ), KAT7 (HBO1) and histone 4 acetylation after proscillaridin A treatment in RD cells

. (A-B) Measurement of KAT6A and KAT7 expression levels after a time-course of proscillaridin A treatment (5 nM, 8h to 96h) in RD cells. (A) KAT6A (35 kDa) and KAT7 (70 kDa) expression levels were assessed by western blotting in RD cells. ACTIN was used as loading control. (B) KAT6A and KAT7 expression levels were quantified and expressed as a percentage of untreated cells (n=3). (C-D) Measurement of histone 4 acetylation (H4) levels after a time-course proscillaridin A treatment (5 nM, 8h to 96h) in RD cells. (C) H4 acetylation levels were assessed using antibodies against H4K5ac, H4K8ac, H4K16ac, H4K20ac, and total H4 pan-acetylation (PanAc). H4 was used as loading control (8 kDa). (D) H4 acetylation levels were quantified and expressed as a percentage of untreated cells (n=3) (TIF 2595 KB)

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Huot, M., Caron, M., Richer, C. et al. Repurposing proscillaridin A in combination with decitabine against embryonal rhabdomyosarcoma RD cells. Cancer Chemother Pharmacol 88, 845–856 (2021). https://doi.org/10.1007/s00280-021-04339-6

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  • DOI: https://doi.org/10.1007/s00280-021-04339-6

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