Abstract 6264: Mechanisms of resistance to doxorubicin in HOS-MNNG cell line
Authors: Tesfaye Robel A. , Lavaud Mélanie , Postec Anaïs , Charrier Céline , Thépault Rose-Anne , Verrecchia Franck , Baud'huin Marc , Lamoureux François , Georges Steven , Ory Benjamin 
Published: June 2022

Abstract
The use of poly-chemotherapy combined with surgical resection has good efficiency and increased the survival rates of osteosarcoma (OS) patients to 70%, however these rates have not evolved since the 1970’s. On the other hand, survival drops to 30% among patients with metastases and/or bad response to treatment. This suggests that metastasis and resistance to treatment are key obstacles to tackle in order to reduce mortality. In this context this study has the aim to identify mechanisms that could participate in the emergence of resistance to doxorubicin, one of the most often used chemotherapeutic molecules to treat OS. For this purpose, HOS-MNNG cells were exposed to increasing concentrations of doxorubicin over a long period of time (over 6 months). Transcriptional and super-enhancer (SE) profiles of highly resistant (IC50 greater by 250 times) cells obtained as such were then compared to naive untreated cells. We hypothesized identification of target genes of resistant-specific super-enhancers, which are regulatory regions that mobilize a large proportion of the cell’s transcriptional apparatus and epigenetic regulators, would allow to unveil key genes in the emergence of resistance. We observed the presence of highly efficient super-enhancers within loci located in close proximity to genes encoding for ATP-Binding Cassette (ABC) transporters only within the high dose doxorubicin resistant cells. The associated genes were among the most highly up-regulated genes in the resistant cells and western blot analysis confirms higher concentration of encoded proteins compared to naive cells. The use of one BETi namely JQ1 - that inhibits recruitment of transcriptional co-factors on regulatory DNA regions such as enhancers and SEs - sensitizes resistant cells to doxorubicin. This suggests that the identified SEs in close proximity could play key role in the emergence of resistance. In order to track emergence of the mechanisms of resistance and to appreciate the transcriptional and epigenetic heterogeneity among resistant cells, we performed single cell 10x multiome (ATAC+RNA) on naive, intermediate and high dose doxorubicin resistant cells. While the high expression of genes encoding for the ABC transporters revealed in bulk analysis seemed ubiquitous among all the clusters of high-dose resistant cells, intermediate-dose resistant cells demonstrate no or very low expression for these genes. This could suggest that the first form of resistance passes through other mechanisms.
In conclusion ABC transporters could be highly involved in the emergence of resistance to high-doses of doxorubicin in OS. A heterogeneous feature could be expected in patients as resistance to intermediate doses do not seem to depend on this mechanism. Finally, the use of BETi could be suggested in patients with bad response to treatment.
This project is supported by Région Pays de la Loire, ANR, la Ligue contre le cancer and SFCE.
Citation Format: Robel A. Tesfaye, Mélanie Lavaud, Anaïs Postec, Céline Charrier, Rose-Anne Thépault, Franck Verrecchia, Marc Baud'huin, François Lamoureux, Steven Georges, Benjamin Ory. Mechanisms of resistance to doxorubicin in HOS-MNNG cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6264.