Selective Rapid B-Cell Depletion In Vivo by Pharmacologic IKK2 Inhibition.

Studies using genetically deficient mice have revealed that members of the NF-kB family play key roles in B-cell development. IKK2 which activates NF-kB by targeting degradation of IkB, is also required for B-cell development. We have studied the role of IKK2 in hematopoiesis using a chemical specific inhibitor (ML120B). Mice given daily oral dosing of ML120B for 4 days had severe B-cell depletion in spleen and bone marrow. B-cells at all stages (pro-B, pre-B, immature and mature B) were depleted 10 fold in the bone marrow while granulocyte numbers were largely unaffected. IKK2 inhibition in vivo showed selective sensitivity of B-cell progenitors (4 fold decrease) in the marrow compared to myeloid progenitors which were unaffected at an equivalent dose. Foci of cells with an apoptotic morphology were visible in bone marrow and spleen within 6 hours of a single oral dose. Apoptotic cells detected by labeling fragmented DNA were increased within splenic follicles (6 fold) and bone marrow. Also an increase in B220+ / annexin V+ cells and a decrease in pre-B (B220+/IgM−) cells in the marrow were observed. RNA expression studies in the marrow 6 hours after a single oral dose revealed a decrease in IL-7 and increased GM-CSF expression. Image analysis of B220 in spleens within 18 hours of a single dose of an IKK inhibitor revealed decreased follicle size. In order to evaluate hematopoietic progenitor sensitivity to NF-kB inhibition, dose responses to ML120B, panepoxydone (PPD) and proteasome inhibitor Lactacystin (Lcyst) were evaluated in B-cell and myeloid bone marrow colony assays. Inhibitors PPD and Lcyst were more effective at inhibiting B-cell colony growth than myeloid colony growth. In summary, pharmacologic inhibition of IKK2 results in a rapid induction of apoptosis with preferential depletion of B-cells and retention of myeloid cells and progenitors within the bone marrow.