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Oncogenic hijacking of a developmental transcription factor evokes therapeutic vulnerability for ROS-induction in Ewing sarcoma

Aruna Marchetto, Shunya Ohmura, Martin F. Orth, Jing Li, Fabienne S. Wehweck, Maximilian M. L. Knott, Stefanie Stein, David Saucier, Chiara Arrigoni, Julia S. Gerke, Michaela C. Baldauf, Julian Musa, Marlene Dallmayer, Tilman L. B. Hölting, Matteo Moretti, James F. Amatruda, Laura Romero-Pérez, Florencia Cidre-Aranaz, Thomas Kirchner, Giuseppina Sannino, Thomas G. P. Grünewald

Preprint posted on March 14, 2019 https://www.biorxiv.org/content/10.1101/578666v1

Oncogenic activation of the SOX6 developmental transcription program drives Ewing Sarcoma, but exposes its ROS-sensitive and druggable Achille’s heel.

Selected by Hannah Brunsdon

Categories: cancer biology

 

Introduction

Ewing sarcoma (EwS) is the second most common paediatric cancer of bone and soft tissues.  85% of EwS are initiated by a chromosomal translocation generating the oncogenic fusion transcription factor EWSR1-FLI1. EWSR1-FLI1 binds to otherwise inert GGAA-microsatellites to act as an enhancer of nearby genes, thereby driving malignancy by drastically changing gene expression. In theory, the uniqueness of the EWSR1-FLI1 oncogene should make it a perfect target for therapies, but a number of factors have made this difficult so far.

As it is known that EwS probably originates in osteochondrogenic progenitor cells, and EWSR1-FLI1 drives the acquisition of undifferentiated cell fates, Marchetto and colleagues hypothesised that EWSR1-FLI1 might drive tumour malignancy through dysregulation of bone developmental pathways. In this preprint, they show how EWSR1-FLI1 activates the developmental bone transcription factor SOX6 in EwS to drive malignancy, but in doing so increases sensitivity to the anti-cancer drug Elesclomol.  

 

Key findings

Through analysis of previously generated microarray datasets, the authors identified SOX6 to be upregulated in EwS – although to a variable degree – relative to other cancers and healthy tissue. To test if EWSR1-FLI1 activity could be responsible for this specific SOX6 upregulation, the authors introduced Dox-inducible shRNA against EWSR1-FLI1 into EwS cell lines. This caused a strong downregulation of SOX6 both in vitro, and in vivo xenografts. Subsequent investigation of ChIP-seq data from two EwS cell lines identified a prominent EWSR1-FLI1 peak at a GGAA-microsatellite within intron 1 of SOX6, that was reduced upon EWSR1-FLI1 knockdown. This suggested that in EwS, EWSR1-FLI1 directly induces SOX6 expression.

The authors then asked whether this SOX6 upregulation contributes to EwS tumorigenicity. They first generated EwS cell lines harbouring Dox-inducible SOX6 shRNA, and then used Affymetrix Clariom D arrays to explore differential gene expression after Dox treatment. Gene set enrichment analysis showed that SOX6 knockdown caused depletion of proliferation-associated gene signatures. Validating this finding in vitro, SOX6 knockdown caused a reduction in viable EwS cell counts as well as a reduction in 2D clonogenic and 3D sphere formation capacities. Furthermore in vivo, Dox-induced SOX6 knockdown in EwS xenografts caused a significant reduction in tumour growth, suggesting that SOX6 upregulation in EwS contributes to proliferation, clonogenic growth and tumorigenicity.

Next, Marchetto and colleagues investigated whether the SOX6-high transcriptional programme in EwS could be a druggable therapeutic target. They investigated a published EwS gene expression dataset with matching drug response data to find a correlation between SOX6 expression levels and responsiveness to the drug Elesclomol. Elesclomol is known to have a pro-apoptotic effect in cancer as it increases ROS levels above a tolerable threshold. Addition of Elesclomol to EwS cells with high SOX6 expression dramatically decreased their viability relative to low-SOX6 osteosarcoma and mesenchymal stem cell lines. Importantly, mice treated with Elesclomol exhibited reduced tumour growth of EwS xenografts due to increases in apoptotic cells within tumours. This suggests that Elesclomol could be a promising candidate for treating incidences of EwS with high SOX6 levels.

In the final part of the preprint, the authors sought to understand why elevated SOX6 expression confers vulnerability to Elesclomol. The induction of ROS by Elesclomol in EwS cell lines seemed to be dependent on SOX6, as ROS elevation could be rescued by SOX6 knockdown. Returning to the microarray data, the authors identified TXNIP – an inhibitor of the thioredoxin antioxidant system – as being downregulated after SOX6 knockdown, suggesting that SOX6 could be involved in ROS metabolism. Interestingly, knockdown of TXNIP also reduced intracellular ROS levels in EwS cells, suggesting that increased SOX6 expression might elevate ROS in EwS by modulating antioxidant metabolic pathways.

Taking this all together, the authors conclude that the activation of the SOX6 developmental program by EWSR1-FLI1 drives tumorigenicity in EwS, but at the cost of increased oxidative stress, which sensitises it to Elesclomol.

 

Why I chose this preprint

As someone working in a lab that combines aspects of developmental biology with cancer research, I think this preprint is a great example of how knowledge of developmental signalling can provide clues about the mechanisms of tumour initiation in cancer, and also how this might be reversed. It was particularly interesting how knockdown of SOX6 or Elesclomol treatment reduced EwS tumour growth in vivo, and it will be interesting to see if Elesclomol is a viable non-toxic method for treating EwS longer term.

 

Questions for the authors

  1. The EwS cell lines used in this paper were selected as they had the highest SOX6 activity compared to other EwS cell lines. I think these were subsequently compared to non-EwS cell lines for experiments in this paper. Have the authors tried adding Elesclomol to EwS lines with intermediate or lower SOX6 levels to see if these are also more sensitive than non-EwS lines? I wonder if there are other targets of EWSR1-FLI1 in EwS that may also contribute towards Elesclomol sensitivity?
  2. Connected to this, the EwS SOX6-high cell lines display greater signs of oxidative stress when Elesclomol is added. If Elesclomol is not added, do EwS cells still have a higher baseline level of ROS compared to low-SOX6 EwS and non-EwS cells, which predisposes them to Elesclomol sensitivity over non-EwS cancers?

 

 

Posted on: 12th April 2019

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