NEK2 is a cell cycle-regulated kinase of the never in mitosis A (NIMA) family that is highly enriched at the centrosome. We discovered that NEK2 expression was highly correlated to drug resistance, rapid relapse, and poor outcome in myeloma and multiple other cancers. Given that plasma cells in myeloma are originated from differentiated B cell lineage, we generated a B cell specific NEK2 transgenic mouse line, controlling by a B cell specific CD19 promoter, to explore the functions of NEK2 in B cell development.
Floxed STOP-polyA -NEK2 (Nek2fl) mice generated in our laboratory were back crossed to C57BL/6 for six generations (F6). Since the expression of the NEK2 transgene was blocked by the floxed STOP-polyA until the STOP-polyA was removed by Cre, thus F6 was further crossed to the CD19-Cre knock in mice to generate B-cell-specific NEK2 transgenic mice (NEK2fl/fl CD19-cre heterozygous), termed here as NEK2+/CD19 mice. Single-cell suspensions were prepared from BM and spleen and cell phenotypes were evaluated on a BD FACSCanto II and FlowJo software. Spontaneous Cell proliferation was measured by BrdU incorporation in vivo (1mg per mouse, ip). Mouse spleens were fixed in 10% neutral formalin then processed and embedded in paraffin for sections and H&E stain. Gene expression profiling (GEP) comparison between B cells from NEK2+/CD19 mice and NEK2−/CD19 littermate controls (LMC) was performed using the Affymetrix mouse genome 430 2.0 array.
NEK2+/CD19 mice were healthy with no obvious abnormality in organogenesis. NEK2 transgene did not perturb early B cell development (up to the stage of pro/pre B cells in BM), but impaired B cell maturation after immature B cells egressed from the BM into the spleen. Analysis of B cell surface expression of CD21 and CD23, which discriminates immature B cells (CD21negCD23neg), follicular B cells (CD21intCD23high) and marginal-zone B cells (MZ B) (CD21highCD23high) cells in spleen, showed significantly decreased MZ B in the spleen of NEK2+/CD19 mice compared with that of LMC (7.2 ±1.3 versus 3.18±0.9, p<0.001, n=12). Germinal center (GC), the light zone in spleen, where GC centrocytes undergo specific changes in their transcriptional programs, committed to plasma or memory B cell differentiation. Eight of nine (88%) of unimmunized NEK2+/CD19 mice manifested numerous spontaneous GCs in the spleens, and only one spontaneous GC was observed in unimmunized LMC. BrdU uptake experiment showed that 3.69% of B cells in NEK2+/CD19 BM and 1.04% of B cells in NEK2+/CD19 spleen were proliferating, while no BrdU incorporation was detected in BM or spleen of LMC. Previous study suggested that PP1/AKT and Wnt pathways were involved in NEK2 inducing cancer cell drug resistance, proliferation and chromosomal instability. GEP performed on B cells from NEK2+/CD19 mice and LMC showed that BCAP, a B cell adaptor for PI3K that couples B-cell receptor signaling to PI3K/AKT activation as well as NF-kB signaling, was significantly up-regulated in the NEK2+/CD19 mice. Real-time PCR analysis confirmed the GEP result, showing that BCAP mRNA in NEK2+/CD19 mice was 7 folds higher than that of LMC.
We conclude that NEK2 is required for the normal development of MZ B cells and may also contribute to the development of conventional B cells, particularly to the transition from immature to mature B cell. BCAP may play an important role in NEK2 signaling in regulating B cell development. Future studies will investigate B cells function in the NEK2+/CD19 mice. The new mouse model presented here suggests that specifically manipulating NEK2 expression in B cells may be of therapeutic value in combating myeloma.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.