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

Using CRISPR activation CROP-seq techniques to mimic signals from the microenvironment that mediate resistance to venetoclax in aggressive lymphomas (#247)

Gaoyuan Wang 1 2 , Johannes Wichmann 1 2 , Yexuan Deng 1 3 , Andrew J Kueh 1 2 , Lin Tai 1 , Sarah T Diepstraten 1 2 , Gemma L Kelly 1 2 , Marco J Herold 1 2
  1. Walter and Eliza Hall Institute of Medical Research, North Melbourne, VIC, Australia
  2. Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia
  3. School of Life Sciences, Nanjing University, Nanjing, Jiangsu, China

Venetoclax is a BCL-2-specific BH3-mimetic drug that is FDA approved for the treatment of Chronic Lymphocytic Leukemia (CLL) and Acute Myeloid Leukemia (AML). While initial treatment success was very high, recent reports suggest relapse of patients being treated with venetoclax. Hence, identifying resistance factors to venetoclax therapy is an area of great clinical relevance. Therefore, our lab has recently established a novel mouse model of aggressive lymphoma that expresses high MYC and BCL-2, resembling double hit lymphoma (DHL). The murine DHLs are highly sensitive to treatment with venetoclax. This model provides an excellent tool to identify venetoclax therapy resistance factors. While a plethora of resistance mechanisms have been described, we are particularly interested in signals provided by the tumour micro-environment that mediate drug resistance of haematopoietic cancers. It is known that the upregulation of BCL-2 family proteins, for example, results from microenvironmental signals that can help cancer cells evade apoptosis. We designed a CRISPR droplet sequencing (CROP-seq) based transcriptional activation library to upregulate genes that mimic signals from the microenvironment (~30 genes; 5sgRNAs/gene). The CROP-seq method allows detection of each cell’s sgRNA along with its single cell transcriptomic landscape, providing gene expression signatures for individual gene perturbations. We have introduced this library into our DHL cell lines, that already express the CRISPR activation machinery, and treated the cells with various doses of venetoclax. To identify the sgRNA mediating survival post-venetoclax treatment, genomic DNA will be isolated and the sgRNA identified by next generation sequencing. In parallel we will optimize protocol to identify the sgRNA and the transcriptomic landscape on a single cell level in venetoclax treated samples. The ultimate goal of this project is to obtain more insight into the pathways mediating venetoclax resistance and use this information to target synergistic pathways increasing the treatment response to venetoclax in blood cancer patients.