Identify and understand the deregulated pathways that control the development and growth of therapy-resistant cancer.
Immunology
Final Report Abstract
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 tumor 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 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 isolate hematopoietic stem and progenitor cells (HSPCs) from newly developed EµMYC/CBE doubletransgenic animals (model of B cell lymphoma) and transduce them with BE sgRNA libraries to reconstitute lethally irradiated recipient mice. Tumors that arise at an accelerated pace will be isolated and the tumor promoting sgRNA and genetic mutation identified. In a second aim we focus on CAR-T therapy which is a new immunotherapy that has revolutionised treatments of some blood cancers such as acute lymphoblastic leukaemia (ALL) and diffuse large B cell lymphoma (DLBCL). CAR-T therapy has increased cure rates of historically incurable cancers. Yet, a crucial limitation of the therapy is that only a minority of patients with aggressive lymphomas achieve long term benefit (e.g. <30% at 2 years for double/triple hit lymphoma) due to relapsing disease. To address this, we use mouse DHL cells generated by the unique CRISPR activator (dCas9A) technology in CD19CAR-T cell mediated killing assays to identify novel resistance factors of DHL cells. Therefore, we transduce dCas9A+DHL cells with genome wide lentiviral CRISPRa sgRNA libraries able to induce robust expression of genes when being in contact with dCas9A, and co-culture them with CD19CAR-T cells for different time periods, thus, enabling the individual focus on novel resistance factors towards either contact– or cytokine–dependent killing. We then identify resistance-promoting sgRNAs in the surviving DHL cells and validate top candidates in vitro and in vivo. Hence, we will transplant dCas9A+DHL cells containing these sgRNAs into immunocompromised RAG1-/- animals, alongside CD19CAR-T cells. Findings upon completion of these aims will reveal crucial tumour suppressor pathways and oncogenes involved in the transformation of hematologic malignancies, enhance our understanding of tumor resistance mechanisms of hard-to-treat cancers, and will provide us with novel targets for multiple anticancer therapies.
Publications
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Using CRISPR activation applications to identify novel tumour resistance mechanisms of aggressive lymphomas in CAR-T cell therapy. Annual Scientific Meeting of the Australian and New Zealand Society for Immunology, Melbourne Australia 2022
Koenig C. et al.
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Using CRISPR base editing applications to accurately identify tumour promoting mutations in lymphomagenesis. ECDO, Bonn Germany 2022
Koenig C. et al.
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Using CRISPR activation applications to identify novel tumour resistance mechanisms of aggressive lymphomas in CAR-T cell therapy. Asia-Pacific Vaccine and Immunotherapy Congress, Brisbane Australia 2023
Koenig C. et al.
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Using CRISPR activation applications to identify novel tumour resistance mechanisms of aggressive lymphomas in CAR-T cell therapy. CIMT meeting, Mainz Germany 2024
Koenig C. et al.
