In-person Oral Presentation 34th Lorne Cancer Conference 2022

Genome wide CRISPR/Cas9 knockout screens in vivo identify GATOR1 components as tumour suppressors in Eμ-Myc lymphoma development      (#19)

Margaret A Potts 1 2 , Shinsuke Mizutani 1 2 3 , Yexuan Deng 1 2 4 , Goknur Giner 1 2 , Andrew Kueh 1 2 , Geraldine Healey 2 , Lin Tai 2 , Stephen Wilcox 1 2 , Andrew Wei 2 5 , Giovanna Pomilio 2 5 , Yang Liao 6 , Wei Shi 6 , Tony Papenfuss 1 2 , Andreas Strasser 1 2 , Marco Herold 1 2
  1. Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia
  2. Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
  3. Division of Hematology and Oncology, Department of Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan
  4. The State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, China
  5. Australian Centre for Blood Diseases, Division of Blood Cancers, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Australia
  6. Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia

A fundamental goal in cancer research is identifying cancer driving genes and understanding how they promote tumourigenesis so we can develop novel therapeutics. While several tumour driving genes have been identified in targeted studies, deep sequencing data of primary tumours suggests that many more tumour promoting alterations exist.

We aimed to identify and characterise novel tumour suppressor genes (TSGs) through a functional genomic knockout screen using CRISPR/Cas9 in the Eµ-Myc mouse model. These mice develop B cell lymphomas (mean 100 days) due to overexpression of the oncogene c-MYC, which is highly expressed in 70% of human cancers. We proposed that additional deletion of a TSG would accelerate onset of lymphomagenesis. To this end, we transduced Cas9 expressing Eµ-Myc haematopoietic stem and progenitor cells with a whole genome sgRNA library and transplanted these cells into lethally irradiated recipient mice.

While several known tumour suppressors were identified by sequencing tumours arising at an accelerated pace, several novel candidates emerged including the Gator1 complex subunits, Nprl3 and Depdc5, involved in metabolism via negative regulation of the mTORC1 pathway. Independent deletion of Nprl3 and Depdc5 significantly accelerated lymphomagenesis to a median latency of 30.5 and 34.5 days respectively, compared to 88 days in negative control recipient mice, validating these hits as TSGs. Furthermore, we demonstrated that Gator1 deficient Eµ-Myc lymphoma cells were highly sensitive to therapeutic inhibition of mTORC1 both in vitro and in vivo due to a dependency on hyperactive mTORC1 signalling, indicated by elevated phospho-S6K expression analysed by intracellular flow cytometry.

These findings demonstrate the power of functional genomic screens to identify novel TSGs, which can be employed in other cancer models. Additionally, we plan to explore whether elevated phospho-S6K expression could serve as a novel biomarker for mTOR dependency in human cancers, as we demonstrated in our Gator1 deficient Eµ-Myc lymphomas.