NF-κB/TNF-α Positive Feedback Loop with Active Proteasome Machinery Supports Myeloid Leukemia Initiating Cell Capacity

Abstract 654

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy arising from leukemia initiating cells (LIC), and is comprised of highly heterogeneous groups with different cytogenetic and molecular abnormalities, which makes it difficult to establish a broadly effective therapeutic strategy. Since constitutive NF-kB pathway activation has been reported in different types of AML cells, it is one of the promising candidates which are universally involved in the LIC phenotype. However, the mechanism of activation and its significance in leukemia progression have not been studied well. In this study, we explored NF-kB pathway activity and its role in LICs using various myeloid leukemia mouse models including MLL-ENL, MOZ-TIF2, and BCR-ABL/Nup98-HoxA9 leukemias. A number of NF-kB target genes showed elevated expression in LIC of each model: leukemic granulocyte-monocyte progenitors (L-GMP) in MLL-ENL or MOZ-TIF2 model and lineage- Sca-1+ fraction in BCR-ABL/Nup98-HoxA9 model, compared with normal hematopoietic stem cells (HSC) and GMP. Moreover, in immunofluorescence staining, each type of LIC displayed prominent nuclear translocation of NF-kB subunit p65. On the other hand, p65 was localized mainly in the cytoplasm in normal cells and, interestingly, non-LIC fraction in the bone marrow cells of the leukemia mice. To our surprise, LIC retained NF-kB activity even after serum-free culture, indicating that NF-kB pathway is prevalently activated in LIC in an autonomous fashion. To study the mechanism of activation, we analyzed gene expression profiles of LIC and normal HSC in murine and human AMLs and found that LIC showed distinctly elevated expression of TNF-a, one of the major activators of the NF-kB pathway. Consistent with these results, the culture media conditioned by LIC had higher TNF-a levels than those of normal cells. In all of the three types of leukemia mice, bone marrow extracellular fluid included higher TNF-a than that of control mice. Importantly, TNF-a blockage by the neutralizing antibody significantly attenuated p65 nuclear localization in LIC, and NF-kB inhibition by expression of a super repressor form of IkBa (IkB-SR) suppressed the TNF-a expression in LIC, indicating that LIC maintains its NF-kB activity by autocrine NF-kB/TNF-a positive feedback loop. Disruption of this loop by induction of IkB-SR or shRNA-mediated knockdown of TNF-a significantly reduced colony-forming abilities of leukemia cells and prolonged survival of leukemic mice in all the three models. In contrast, transduction of IkB-SR into normal HSC exerted no influence on their colony-forming ability. These results suggested that the NF-kB/TNF-a positive feedback loop plays a vital role for LIC propagation. We also addressed the mechanism of the difference in NF-kB activity between LIC and non-LIC. Notably, LIC had decreased protein levels of IkBa compared with non-LIC in spite of the same mRNA expression levels between them. In addition, basal 20S proteasome activity and the expression levels of proteasome subunit genes in LIC were higher than those in non-LIC, indicating that enhanced proteasomal degradation of IkBa could lead to selectively high NF-kB activity of LIC. The same propensity was seen in human AML CD34+CD38- cells versus CD34- cells in the analysis of microarray expression data. Proteasome inhibition by bortezomib diminished the differences of IkBa protein level. Moreover, its administration to leukemic mice selectively killed LIC fraction and prolonged survival in the in vivo transplantation model. Finally, forcible maintenance of NF-kB activity in LIC by shRNA-mediated knockdown of IkBa significantly enhanced its self-renewal activity as determined by surface marker profiles after in vitro culture. In the analysis of mice reconstituted with the IkBa-downregulated leukemic cells, bone marrow mononuclear cells had increased colony-forming cell ability and enhanced LIC frequency as determined by in vivo limiting dilution serial transplantation assay. These results indicated that the transition from LIC to non-LIC might be associated with the attenuation of NF-kB activity due to inefficient degradation of IkBa. In summary, these findings elucidate that NF-kB/TNF-a signaling in LIC, under support of the proteasome activity, has a critical role for both maintenance and propagation of LIC and provide a widely applicable approach for targeting LIC in myeloid leukemias.