Přehled o publikaci

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with various somatic mutations, the most frequent of which targeting ATM, TP53, NOTCH1, MYD88 and SF3B1 genes. Their thorough exploration could shed light on the disease etiology, or even lead to discovery of potential novel drug targets. However, patient CLL cells do not proliferate ex vivo, thus generation of isogenic cell lines is needed for extensive experiments. Using CRISPR/Cas9 in CLL-derived HG3 and MEC1 cells, we generated isogenic cell lines carrying disruptive mutations in ATM or TP53. These cell lines show complete loss of the respective proteins and abrogation of downstream signaling pathways.We also used CRISPR/Cas9-based homology directed repair to obtain HG3 cells with recurrent mutations of NOTCH1 (P2514fs), SF3B1 (K700E) and MYD88 (L265P). Selected cell lines were subjected to CRISPR/Cas9 dropout screening to identify genes, whose deletion is lethal to the introduced mutations. In particular, SPDYE1 and LUC7L3 were found to be synthetically lethal with the NOTCH1 mutation, while SNUPN and UQCRC1 were found to be essential for SF3B1-mutated cells. Simultaneously, the cell lines were screened with a library of 859 approved drugs. The screening demonstrated sensitivity of NOTCH1-mutant and SF3B1-mutant cell lines towards inhibitors of various hormone receptors or inhibitors of 20S proteasome. The knockout models were also used for studies of the performance of anti-CD19 CAR T-cells. We observed different effectiveness at eradicating tumor cells in vivo depending on the driver mutation, with TP53 mutations connected to inferior performance of CAR T-cells. In summary, we generated a panel of isogenic cell lines carrying mutations recurring in CLL patients. These models are indispensable for further studies of the mutations’ impact on CLL therapy.