Oral Presentation 43rd Lorne Genome Conference 2022

Finding critical cancer driving genes using functional genomics screening in vivo. (#41)

Marco Herold 1 2 , Shinsuke Mizutani 1 2 3 , Margaret A Potts 1 2 , Yexuan Deng 1 2 4 , Gaoyuan Wang 1 2 , Göknur Giner 1 2 , Andrew J Kueh 1 2 , Martin Pal 1 2 5 , Geraldine Healey 1 , Lin Tai 1 , Stephen Wilcox 1 2 , Andrew Wei 1 6 , Giovanna Pomilio 1 6 , Yang Liao 1 2 7 , Sarah Diepstraten 1 2 , Tony Papenfuss 1 2 , Gemma L Kelly 1 2 , Wei Shi 1 7 8 9 , Andreas Strasser 1 2
  1. Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic, Australia
  2. Department of Medical Biology, The University of Melbourne, Melbourne, Vic, Australia
  3. Division of Hematology and Oncology, Department of Medicine, Kyoto Prefectural University of Melbourne, Kyoto, Japan
  4. The State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, China
  5. School of Dentistry and Medical Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia
  6. Australian Centre for Blood Diseases, Melbourne, Vic, Australia
  7. Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia
  8. School of Computing and Information Systems, The University of Melbourne, Melbourne, Vic, Australia
  9. School of Cancer Medicine, La Trobe University, Heidelberg, Vic, Australia

Genome sequencing of cancers has enabled identification of genes responsible for the development and growth of cancers. However, DNA sequencing does not distinguish between cancer driver and passenger mutations, which makes it difficult to prioritize hits for further drug development. Functional genetic screening approaches enable the identification of genes involved in the development of cancer. For these studies we utilised a model of MYC-driven B cell lymphoma (Eµ-Myc). We performed genome-wide CRISPR knockout screens in Em-Myc; Cas9 double transgenic haematopoietic stem and progenitor cells (HSPCs) in vivo. We identified members of the mTOR inhibitor complex GATOR1. Specifically we found a critical tumour suppressive role of NPRL3 and DEPDC5 in Em-Myc lymphomagenesis. In parallel we performed in vivo knockout screens using a focused CRISPR library targeting > 700 TRP53 promoter/ enhancer elements. These experiments revealed that deletion of a TRP53 binding site in the NPRL3 promoter accelerated Em-Myc lymphomagenesis to a similar extend as the loss of NPRL3 itself. Excitingly, GATOR1 deleted Em-Myc lymphomas were highly susceptibility to the mTORC1 inhibitor rapamycin due to increased mTORC1 activity identifying a clinical application of our research. These results clearly demonstrate the power of functional genetic screens for identifying cancer drivers and potential prognostic markers for the clinical application in vivo. To further delineate the complex genetic changes found in cancer, we are currently extending our screening toolbox by employing other CRISPR modalities, such as CRISPR activation combined with CROP-seq techniques, in vitro and in vivo.