Physical Poster + E-Poster Presentation 34th Lorne Cancer Conference 2022

Pinpointing and targeting novel drivers of pancreatic cancer progression and metastasis using TRAP-seq. (#241)

Michael Trpceski 1 2 , Chi Kin (Kenny) Ip 1 2 , Herbert Herzog 1 2 , Kendelle Murphy 1 2 , Tatyana Chtanova 1 2 , Paul Timpson 1 2 , David Herrmann 1 2
  1. The Garvan Institute of Medical Research, Sydney, New South Wales, Australia
  2. St Vincent’s Clinical School, University of New South Wales, Sydney, New South Wales, Australia

Background:

Pancreatic ductal adenocarcinoma (PDAC) has a low 5-year survival rate of 10%1, which can be attributed to its’ rapid metastatic spread and resistance to standard-of-care chemotherapy. Therefore, there is an urgent need to identify novel ‘druggable’ candidates that become de-regulated during PDAC progression and can be co-targeted with standard-of-care chemotherapy to improve patient survival. We aim to use innovative Translating-Ribosome-Affinity-Purification followed by RNA-sequencing (TRAP-seq) to enrich in a cell-type-specific manner for mRNAs, which are actively being translated into proteins in metastatic PDAC and may therefore represent valid ‘druggable’ targets.

 

Methods:

Our genetically engineered mouse models closely mimic the mutational landscape, histopathology and progression of human PDAC. Both models are driven by an initiating Kras mutation and either loss of p53 (poorly metastatic) or a gain-of-function mutation in p53 (highly metastatic)2. These were crossed to the Cre-inducible Rpl10a-GFP mouse to activate expression of the GFP-tagged ribosomal protein Rpl10a specifically in PDAC cells, which can be immunoprecipitated with associated translating mRNAs for downstream RNA-seq analysis.

 

Results:

We isolated tumours for TRAP-seq analysis from end-stage mice of both models (120-220 days of age) and also established primary cell lines, which retain Rpl10a-GFP expression ex vivo. Initial optimisation has shown that TRAP-seq purification results in high-quality, enriched mRNA transcripts. Comparison of translational profiles between poorly metastatic and highly metastatic PDAC tumours (and cancer cells) will be used to specifically tease out drivers of PDAC progression and metastasis.

 

Conclusion:

TRAP-seq technology will be coupled with validation of candidate targets in our libraries of murine and human PDAC samples, as well as functional assessment using our established in vitro and in vivo PDAC models. This will allow us to identify and validate novel ‘druggable’ candidates to target PDAC progression and metastasis in conjunction with standard-of-care chemotherapy in a pre-clinical setting.

  1. Siegel, R.L et al. (2021) Cancer statistics, 2021. CA Cancer J. Cin. 71 (1), 7-33.
  2. Morton, J.P et al. (2010) Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer. PNAS. 107 (1), 246-251.