NFKBIE-mutated CLL cells reshape the immune microenvironment and display selective resistance to BTK inhibitor treatment

Inactivating mutations in the NF-kB inhibitor NFKBIE are frequent in chronic lymphocytic leukemia (CLL) and have been associated with accelerated disease progression and inferior responses to chemotherapy. To further understand the role of NFKBIE mutations in CLL, we disrupted by CRISPR/Cas9 editing the NFKBIE gene in CLL cells derived from the Eµ-TCL1 transgenic mouse model and investigated how this will affect CLL growth and response to B cell receptor inhibitor treatment. In vitro and adoptive transfer experiments showed that NFKBIE-mutated cells have a growth advantage over NFKBIE-wild type cells when exposed to microenvironmental signals that activate the canonical NF-kB pathway and can induce alterations within the tumor microenvironment that may allow for escape from immune surveillance, including the expansion of CD8+ T cells with an exhausted phenotype and increased expression of PD-L1 on the malignant B cells. Consistent with these findings, significantly greater expression of the exhaustion markers PD1 and TIGIT was observed on T cells from CLL patients with NFKBIE-mutated compared to NFKBIE-wild type leukemia. In addition, in vitro and in vivo experiments showed that NFKBIE-mutated murine CLL cells are selectively resistant to BTK inhibitor treatment while remaining sensitive to treatment with a PI3K or SYK inhibitor. Reduced sensitivity to BTK inhibitor treatment was also observed in a series of 229 ibrutinib-treated CLL patients showing inferior outcomes for the NFKBIE-mutated cases. These findings provide evidence that NFKBIE-mutated CLL cells reshape and are selected by the tumor microenvironment and may account for suboptimal ibrutinib responses. Overall design: In the study presented here, the RNA-seq profile was analyzed in NFKBIE-wild type and NFKBIE-mutated TCL1-355 TKO cells. These were first separately propagated for 21 days in two C57BL/6 mice and then transplanted via intraperitoneal injection in two groups of C57/BL6 recipient mice (5 mice per group). The leukemic cells were recovered after 10 days from the spleen and peritoneal cavitiy of the transplanted mice and 4 samples from each group were analyzed by RNA sequencing. We also analyzed NFKBIE-mutated and NFKBIE-wild type TCL1-355 TKO cells that were isolated from spleens of three different mice and separately cultured for two weeks prior to RNA isolation.