Poster Presentation 49th Lorne Conference on Protein Structure and Function 2024

Deletion of a deubiquitylating (DUB) enzyme sensitises KRAS-mutant lung adenocarcinoma to T cell mediated killing (#403)

Mara Zeissig 1 2 , Jackson McDonald 1 2 , Danielle Chen 1 2 , Sarah Diepstraten 1 2 , Leanne Scott 1 2 , Stephen Wilcox 1 2 , Sarah MacRaild 1 2 , Lauren Ashwood 3 4 , Olga Kondrashova 3 4 , Marco Herold 5 , Pieter Eichhorn 6 , David Komander 1 2 , Kate Sutherland 1 2
  1. The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia
  2. Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia
  3. QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia
  4. The University of Queensland, Brisbane, Queensland, Australia
  5. Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia
  6. Curtin Health Innovation Research Institute and Faculty of Health Sciences, Curtin University, Bentley , Western Australia, Australia

Immunotherapy has revolutionised the treatment of lung adenocarcinoma patients, however, response rates are variable. Critically, lung adenocarcinomas that harbour concurrent mutations in KRAS, STK11(LKB1) and KEAP1 (KLK tumours) fail to respond to immunotherapy. Therefore, identifying the mechanisms for how KLK tumours evade immune detection is required to enhance therapeutic outcomes.

Here, we have used an unbiased whole-genome CRISPR knockout co-culture screening approach to identify gene targets that sensitise lung cancer cells to T cell mediated killing. Briefly, Keap1 was targeted in cell lines generated from lung tumours that arose in KrasLSL-G12D/+;Lkb1f/f (KL) mice using CRISPR-Cas9 gene editing. KL sgKeap1 cells were then transduced with a whole-genome CRISPR-Cas9 library, followed by serial application of CD8+ T cells. Excitingly, a deubiquitylating (DUB) enzyme was identified as the top candidate gene, with loss of the candidate DUB sensitising KLK tumour cells to T cell killing. In vitro validation of the CRISPR screen (using CRISPR-Cas9 and PROTAC methodologies) demonstrated that deletion of the candidate DUB in KLK tumour cells significantly increased T cell killing of KLK cells, as assessed by crystal violet assays and flow cytometry. Critically, re-expression of the candidate DUB in knockout cell lines rescued the phenotype, decreasing the sensitivity of these cell lines to T cell killing in vitro. Consistent with this, loss of candidate DUB in KLK cells decreased tumour burden in immunocompetent C57BL/6 compared with KLK control cells. In contrast, KLK cells lacking the candidate DUB showed similar growth rates to control cells when injected in immunocompromised NSG mice that lack T, B and NK cells. Finally, analysis of publicly available lung cancer patient datasets revealed an association between expression of the candidate DUB and poor prognosis.

Our studies demonstrate that deletion of a candidate DUB in tumour cells sensitises KLK tumours to T cell mediated killing and decreases tumour growth in immunocompetent mice.  These results demonstrate the power of whole genome CRISPR screens in identifying candidate genes that may serve as therapeutic targets to improve treatment responses in KRAS/STK11/KEAP1 mutant lung cancer patients.