1042 - LOSS OF IκBε ACCELERATES DISEASE DEVELOPMENT IN CHRONIC LYMPHOCYTIC LEUKEMIA

Background
The NF-κB pathway is particularly relevant in the pathophysiology of Chronic lymphocytic leukemia (CLL), as it is activated in patients with CLL as a result of intrinsic and microenvironmental stimuli. Among the few NF-κB pathway genes found altered in CLL, the NFKBIE gene, which encodes the inhibitor IκBε, is mutated in 3-7% of CLL patients. The presence of mutations correlates with an adverse prognosis and an advanced clinical stage, being particularly enriched in clinically aggressive cases and in patients relapsing under the BTK inhibitor ibrutinib treatment, suggesting that a reduced expression of NFKBIE may contribute to CLL aggressiveness through constitutive NF-κB activation and be an alternative pathway of ibrutinib resistance.
In this study, we investigated the role of IκBε in controlling NF-κB activity and disease expansion in CLL, inactivating the gene by CRISPR-Cas9 both in vitro and in vivo disease models.

Materials and Methods
We engineered the MEC-1 CLL cell and obtained cell lines with a partial (KO1) or total (KO2) ablation of NFKBIE gene. We studied in vitro cell proliferation and migration, gene expression, BcR signaling pathway, and response to treatment with the BTK inhibitor ibrutinib.
In vivo, we performed xenograft experiments by injecting MEC-1 control and KO cells into RAG2-/-γc-/- recipient mice. We also reproduced the most frequent mutation observed in patients with CLL by generating Nfkbie4bpdel/4bpdel mutated mice and crossed them with E-TCL1 mice to obtain immunocompetent Eμ-TCL1-Nfkbie4bpdel/4bpdel. To thoroughly confirm that NFKBIE mutations play a cell-autonomous role in leukemic cell expansion, we performed adoptive transfer (AT) of Eμ-TCL1 or Eμ-TCL1-Nfkbie4bpdel/4bpdel leukemic cells. In mice, we monitored disease development, dissemination, and ibrutinib sensitivity by following CD19+CD5+ leukemic cells in peripheral blood (PB) at 3, 6, and 9 months of age and in the spleen (SP) and bone marrow (BM) at necropsy. In addition, we performed RNA-sequencing analysis in a cohort of 50 patients with CLL (13 carrying NFKBIE mutations and 37 NFKBIE wildtype CLL cases).

Results
Biochemical analyses in KO cells revealed increased cRel and p50 protein levels and increased p65 phosphorylation, all NF-κB transcription factors, indicating unabated cellular activation. Moreover, NFKBIE KO cells showed higher proliferative and migratory capacity than control MEC-1 cells at a magnitude directly related to the NFKBIE ablation rate.
The effect of NFKBIE manipulation was even more striking evident in vivo. Xenografts, generated with MEC-1 NFKBIE KO cells, showed an increased disease expansion in the SP and BM and shorter survival than mice injected with control MEC-1 cells. E-TCL1 mice carrying the 4bp deletion in the Nfkbie gene showed an increased amount of leukemic cells in the PB than E-TCL1 control mice at 3, 6, and 9 months of age. By necropsy at 6 months of age, we also observed the increase in malignant cells in SP, BM, and PB of Eμ-TCL1 Nfkbie mutated mice compared to Eμ-TCL1 mice at a magnitude directly related to the ploidy of the mutated allele. Adoptive transfer experiments confirmed the cell-autonomous effect of Nfkbie inactivation.

In vitro, the proliferation of KO cells was less impacted by ibrutinib than MEC-1 control cells, an event particularly evident in the competition experiments in which KO showed an increased advantage over control cells upon ibrutinib exposure. In vivo, ibrutinib treatment reduced disease expansion in SP of MEC-1 xenografts but not in KO xenografts mice. Interestingly, RNA-sequencing analysis of primary CLL samples revealed that functional ablation of NFKBIE is associated with distinct changes in signaling pathways, including activation of the MAPK pathway. This is particularly relevant as it well associates with the increased cell proliferation observed in our models. In addition, it fits with the observation that the activation of the MAPK pathway is associated with the occurrence of resistance to the drug.

Conclusion
Loss of NFKBIE function is associated with more aggressive disease in all CLL models tested, both in vitro and in vivo, accelerating disease progression under pro-tumorigenic CLL conditions. Furthermore, the association with ibrutinib resistance and upregulation of the MAPK pathway highlights the multimodal relevance of the NF-κB pathway in CLL and paves the way for new potential therapeutic strategies with MAPK pathway inhibitors to overcome or prevent drug resistance.