NFkB p50 (Nfkb1) Contributes to Disease in the Eu-TCL1 Mouse Model of Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is a disease of mature B-cells that are resistant to apoptosis and accumulate in the blood over time. Due to the slowly progressing nature of this disease, studies to determine the molecular events leading to defective apoptosis are challenging. Our group has previously reported that during disease progression in the Eμ-TCL1 CLL mouse model, genes become silenced in a progressive manner over the course of the disease. This silencing is due in part to a high degree of methylation at the promoters of key tumor suppressors. However, even though a high degree of methylation is observed in both this mouse model and in CLL patient samples, agents targeting methylation such as decitabine have proven ineffective in CLL therapy. Therefore other novel methods to reverse gene silencing in CLL are an attractive therapeutic option. In our previous study, we observed early transcriptional mechanisms of gene silencing (occurring prior to methylation) involving NF-κB p50 homodimers. NF-κB is a family of transcription factors which is known to play an important role in the progression of CLL. Advances in next generation sequencing have recently identified loss of function mutations in NFKBIE, a negative regulator of NF-κB signaling, in CLL. These mutations contribute to increased nuclear localization (and hence increased activation) of NF-κB subunits. In addition, a mutagenesis screen in the Eμ-TCL1 mouse found that several of the most frequently occurring mutations that contribute to disease progression occur in genes related to the NF-κB family, particularly in the p50 (Nfkb1) gene.

In the present study, we have generated a new mouse model to further study the role of p50 in CLL pathogenesis. The Eμ-TCL1 mouse was crossed to the p50-/- mouse. The p50-/- mice are fertile with normal growth and development under sterile conditions; however they do exhibit a defective response to infection and decreased antibody production. As such, animals in this study are housed under pathogen free conditions. The initial cross resulted in mice that were all heterozygous for p50 (p50+/-), and either positive or negative for the TCL1 transgene. The p50+/-; TCL1+ animals were subsequently crossed with one another to generate three genotypes: p50+/+. p50+/- and p50-/-. Animals are monitored for disease by monthly flow cytometry analysis of CD19 and CD5 in whole blood, and leukemia is defined as >10% double positive cells. The p50-/- mice have a significantly lower incidence of leukemia compared to p50+/+ mice (Chi-square p <0.001), and p50+/- mice show an intermediate phenotype (p=0.024 compared to p50-/- mice). Despite pathogen free conditions, some mice within the p50-/- group die at an early age with no evidence of disease. Therefore, competing risks regression was performed to take into account the mice that die without leukemia via the CIF (cumulative incidence function) based on the Fine and Gray model. Differences in time to leukemia between the p50+/+ and p50-/- mice remained significant (Subdistribution Hazard Ratio; SHR = 8.45; 95% CI: 1.86 – 38.47; p=0.006). The p50+/- mice still exhibit an intermediate phenotype, with more separation between the p50+/- and p50-/- (SHR = 4.39; 95% CI: 1.00 – 19.29; p=0.050) than the p50+/- and p50+/+ groups (SHR = 0.52; 95% CI: 0.26 – 1.06; p=0.069). Finally, spleens from p50-/- mice are notably smaller than the p50+/+ littermates (average spleen score of 0-1 for p50-/- versus 2-3 for p50+/+, using a 0-4 scale), indicating that disease in the secondary lymphoid tissues is also affected by the loss of p50 in this model.

In conclusion, these data genetically demonstrate the significant contribution of the p50 (Nfkb1) gene to disease progression in the Eµ-TCL1 mouse model. These studies highlight the importance of the NF-κB family in CLL, and suggest that p50 is a promising therapeutic target in this disease.