1657P - Understanding the role of SAGA and mediator in the resistance to Pdk1 deletion in PDAC

Background

Pancreatic ductal adenocarcinoma (PDAC) represents the fourth highest rate of cancer-related deaths worldwide. The absence of early symptoms, early metastasis, and inefficient systemic therapies lead to a fatal prognosis for PDAC patients. Currently, surgical resection is the only curative option. However, it is often linked to severe postoperative complications and a considerable tumor recurrence rate. Therefore, identifying the resistance mechanisms of PDAC and developing targeted therapies is crucial.

Methods

Mutant KRAS and its effector Pdk1 play a major role in PDAC carcinogenesis. Previous works showed that Pdk1 deletion causes impairment of PDAC cell proliferation in vitro and in vivo. However, a minor subpopulation of cells continues to proliferate. To investigate this resistance, a genome wide CRISPR/Cas9 knockout screen was performed. Members of SAGA and Mediator complexes were among the top hits of the screen. The CRISPR knockouts of these genes were generated in the bulk PDAC epithelial cell line and in two single cell clones - one resistant and one sensitive to Pdk1 deletion. Clonogenic assays were performed to analyze the proliferation of the knocked-out cells, while RNA sequencing was conducted to investigate the differentially expressed genes.

Results

In the CRISPR/Cas9 knockout screen, members of SAGA and Mediator complexes showed enrichment in Pdk1 deletion resistant clones. Amongst all hits, significant proliferation of the Usp22- and Atxn7L3- as well as Med13- and Med16-knockout in the Pdk1 deleted PDAC cells was observed, with the strongest effect in the resistant clone. Several enhanced or downregulated hallmark pathways were observed in the knocked-out cells, providing insight into possible resistance mechanisms of the PDAC.

Conclusions

The results of the RNA sequencing of both SAGA and Mediator knocked-out cells showed an enhancement of the Hedgehog signalling, a pathway reported in the initiation of a growing number of cancers. The results of this research suggest furthermore, that the loss of the connecting units Atxn7L3 and Med16 promotes an unhinged transcription activity of the enzymatically active SAGA and Mediator complexes and leads to an enhanced PDAC cell proliferation.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

German Research Foundation.

Disclosure

All authors have declared no conflicts of interest.