Poster Presentation 35th Lorne Cancer Conference 2023

Pinpointing and targeting novel drivers of pancreatic cancer progression and metastasis using TRAP-seq (#341)

Michael Trpceski 1 2 , Chi Kin (Kenny) Ip 1 2 , Cecilia Chambers 1 2 , Shona Ritchie 1 2 , Daniel Reed 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

Pancreatic Cancer (PC) has a low 5-year survival rate of 11% [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 gain-of-function mutant p53-driven PC progression and metastasis and can be co-targeted with standard-of-care chemotherapy or immunotherapy to improve patient survival.

Here, 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 PC and may therefore represent valid ‘druggable’ targets.

 

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

 

We isolated primary tumour cells for TRAP-seq analysis from end-stage mice (120-220 days of age) of both models, as well as cells from matched KPC metastasis tumours. All cancer cells retain Rpl10a-GFP expression in vitro and we confirmed the genomic presence of a Kras mutation and either loss of p53 or a gain-of-function mutation in p53. TRAP of collected cells results in high-quality, enriched mRNA transcripts suitable for RNA-sequencing. This technology will be coupled with validation of deregulated candidate targets in our libraries of murine and human PC samples, as well as functional assessment using our established in vitro and in vivo PC models. This will allow us to identify and validate novel ‘druggable’ candidates to target gain-of-function mutant p53-driven PC progression and metastasis in conjunction with standard-of-care chemotherapy or immunotherapy in a pre-clinical setting.

  1. Siegel, R.L., et al., Cancer statistics, 2022. CA: A Cancer Journal for Clinicians, 2022. 72(1): p. 7-33.