Identify and understand the deregulated pathways that control the development and growth of therapy-resistant cancer.

• Applicant: Dr. Christina König
• Subject Area: Hematology, Oncology ; Immunology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 442680333

Neoplastic diseases result from the unrestricted growth of cells that have been transformed into a malignant state. Genetic defects – mainly caused by single nucleotide variants (SNPs) in tumour suppressor genes or in proto-oncogenes – allow cancer cells to acquire essential biological properties and deregulate several cellular processes, ensuring their survival and efficient growth. In one of our aims, we employ the application of the recently described CRISPR base editing (BE) technology to model these SNPs. We establish experimental procedures in vitro using the CRISPR BE technology together with single guide (sg)RNA libraries specifically designed to introduce the top mutations in human cancer-causing genes into the mouse genome. Hence, we transduce EµMYC cells with lentiviral BE plasmids able to introduce C-to-T (CBE) or A-to-G (ABE) base changes and a respective sgRNA library for targeting the individual mutations in the genes of interest. We then treat this pool of engineered cells with diverse chemotherapeutic drugs, such as DNA-damaging agents or BCL-2 family inhibitors. In a second approach, we will isolate haematopoietic stem and progenitor cells (HSPCs) from newly developed EµMYC/CBE double-transgenic animals (model of B cell lymphoma) and transduce them with BE sgRNA libraries to reconstitute lethally irradiated recipient mice. Tumours that arise at an accelerated pace will be isolated and the tumour promoting sgRNA and genetic mutation identified. In a second aim we focus on a very aggressive subtype of Diffuse Large B Cell Lymphoma (DLBCL) called Double Hit Lymphoma (DHL). This lymphoma type expresses high levels of the pro-survival protein BCL-2 and cMYC. The standard of care for patients with relapsed or refractory DLBCL is CD19 CAR-T cell therapy. Yet, many patients do not respond, and the resistance mechanisms are not well understood. Therefore, we use mouse DHL cells generated by the unique CRISPR activator (dCas9A) technology in CD19 CAR-T cell mediated killing assays to identify novel resistance factors of DHL cells. To this end, we transduce dCas9A+DHL cells with genome wide lentiviral CRISPRa sgRNA libraries that in combination with dCas9A are able to induce robust expression of all genes in the genome. While co-culturing the manipulated DHL cells with CD19 CAR-T cells will kill most of the tumour cells the upregulation of certain genes will inhibit CAR-T cell mediated killing. The sgRNA responsible for the resistance inducing gene will then be identified by next generation sequencing. Findings of these aims will reveal crucial tumour suppressor pathways and oncogenes involved in the transformation of haematological malignancies, enhance our understanding of tumour resistance mechanisms of hard-to-treat cancers, and will provide us with novel targets for multiple anti-cancer therapies.

• DFG Programme: Research Fellowships
• International Connection: Australia
• Host: Professor Dr. Marco Herold, Ph.D.