Establishment of CRISPR/Cas9-based murine models to study the impact of NF-kB pathway mutations on chronic lymphocytic leukemia growth, survival and resistance to treatment

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in western countries. The disease is caused by the malignant transformation of a subset of autoreactive B lymphocytes with a particular immunophenotype. Strong evidence exists that the disease is driven by the combined effects of signals generated by B cell receptor (BCR) and other microenvironmental stimuli that interact with various recurrent genetic lesions. Inactivating mutations in NF-kB pathway genes, such as the NF-kB inhibitor NFKBIE, are among the more frequent genetic lesions in CLL. However, the role of these genetic lesions in CLL pathogenesis and treatment resistance is still largely unknown. To this end we generated transplantable murine leukemias with inactivating NFKBIE mutations and we investigated their impact on leukemia growth and response to treatment. The NFKBIE mutations were introduced by CRISPR/Cas9 editing in two leukemia lines derived from the Eμ-TCL1 murine CLL model. These cell lines were recently established by our group (Chakraborty et al, Blood 2021) and are characterized by spontaneous in vitro proliferation that is BCR-dependent but independent of other microenvironmental signals. Here, we performed in vitro competition experiments with mixed cells with wild-type and mutated NFKBIE. Interestingly, we observed no change in the mutant allele frequency (MAF) in culture, suggesting that NFKBIE mutations do not affect the spontaneous in vitro growth of these leukemia cells. However, repeated stimulation with CpG-DNA, TNFa, anti-IgM or autoantigen resulted in a significant increase in MAF, suggesting that NFKBIE mutations provide a growth advantage to certain microenvironmental signals. Remarkably, using in vivo competition experiments performed in wild- type mouse recipients, we demonstrated that NFKBIE-mutated cells are preferentially selected in the spleen, implying different interactions of the malignant cells with the tumor microenvironment in different anatomical compartments. As mentioned, the mechanism(s) underlying treatment resistance in CLL are still partially understood. In particular, the BCR inhibitor ibrutinib (IBR), which has emerged as the most effective therapeutic option in CLL, induces clinical responses in the majority of patients, but a substantial proportion subsequently shows disease progression. Because mutations in other NF-kB pathway genes have been associated with resistance to IBR in mantle cell lymphoma, we investigated whether NFKBIE mutations can also affect the response to IBR treatment. Interestingly, we noted that in vitro IBR inhibits proliferation of NFKBIE-mutated cells significantly less compared to wild-type NFKBIE cells while, in vivo, we observed positive selection of NFKBIE-mutated cells in mice treated with IBR 6 compared to controls. To validate these findings in the clinical setting, we investigated the impact of NFKBIE mutations in a cohort of 229 IBR- treated CLL patients, among which 13.5% had mutated NFKBIE. Kaplan-Meier analysis showed a trend towards reduced progression-free survival and significantly reduced overall survival for the NFKBIE-mutated cases, further suggesting that NFKBIE- mutations reduce the sensitivity to BTK inhibitor treatment. Finally, we observed that NFKBIE-mutated cells remain sensitive to other BCR inhibitors, such as the PI3K inhibitor idelalisib and the SYK inhibitor fostamatinib. Collectively, these data demonstrate that NFKBIE mutations can reduce the response to IBR treatment and suggest that such cases may benefit more from treatment with a PI3K or SYK inhibitor.