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. 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.

核因子κB（NF-κB）抑制剂NFKBIE的失活突变在慢性淋巴细胞白血病（chronic lymphocytic leukemia, CLL）中发生率较高，且与疾病进展加速及化疗应答不佳密切相关。为进一步阐明NFKBIE突变在CLL中的作用，我们通过CRISPR/Cas9基因编辑技术，在源自Eμ-TCL1转基因小鼠模型的CLL细胞中敲除NFKBIE基因，并探究其对CLL增殖以及B细胞受体抑制剂治疗应答的影响。体外实验与过继转移实验结果显示，当暴露于激活经典NF-κB通路的微环境信号时，携带NFKBIE突变的细胞相较野生型细胞具有生长优势；此类细胞还可重塑肿瘤微环境，进而实现免疫逃逸，具体包括诱导呈现耗竭表型的CD8+ T细胞扩增，以及恶性B细胞表面程序性死亡受体配体1（PD-L1）表达上调。与上述发现一致，相较于携带NFKBIE野生型白血病的CLL患者，携带NFKBIE突变患者的T细胞上，耗竭标志物PD1与TIGIT的表达水平显著升高。此外，体外与体内实验均证实，携带NFKBIE突变的小鼠CLL细胞对BTK抑制剂具有选择性耐药性，但仍对PI3K抑制剂或SYK抑制剂敏感。在对229例接受依鲁替尼（ibrutinib）治疗的CLL患者队列分析中，同样观察到NFKBIE突变患者对BTK抑制剂的敏感性降低，且预后更差。上述研究结果表明，携带NFKBIE突变的CLL细胞可重塑肿瘤微环境并受到其筛选，这或许是依鲁替尼治疗应答不佳的潜在原因。本研究中，我们对NFKBIE野生型与NFKBIE突变型TCL1-355敲除（TKO）细胞的RNA测序（RNA-seq）谱进行了分析。具体实验流程为：首先将两类细胞分别接种于2只C57BL/6小鼠中增殖21天，随后经腹腔注射移植至两组C57/BL6受体小鼠（每组5只）。移植10天后，从受体小鼠的脾脏与腹腔中分离白血病细胞，每组收集4份样本进行RNA测序分析。我们同时分析了从3只不同小鼠脾脏中分离得到的NFKBIE突变型与野生型TCL1-355 TKO细胞，此类细胞在RNA提取前均单独培养了2周。