1043 - Regulation of the NF-kB pathway by NEDDylation in chronic lymphocytic leukemia: Beyond IkB

The NF-kB survival pathway is the final effector of many of the alterations found in CLL and is heavily over-activated in CLL. Cells control NF-kB activity by altering the stability of the inhibitor protein of the pathway (IkB) through its phosphorylation and subsequent poly-ubiquitination and degradation at the proteasome and it was assumed that NEDDylation was exclusively modulating the NF-kB pathway through the destabilization of IkB. Indeed, molecules targeting the ubiquitin-proteasome axis have been explored to control CLL.

To better understand the role of NEDDylation on the NF-kB pathway, we analyzed samples from CLL patients and healthy donors and the effect of the NEDDylation inhibitor MLN4924 over these samples. When we did a proteomic screening of ubiquitin-like post-translational modifications (UBL-PTM) in CLL cells, we did not find an elevated ubiquitination of IkB. Moreover, when we blocked the proteasome function with bortezomib, MLN4924 scarcely incremented IkB⍺ stability any further, but strongly increased its phosphorylation. This suggested that NEDDylation was modulating IkB⍺ phosphorylation. Our screening in CLL found alterations affecting many UBL-PTMs of upstream members of the NF-kB pathway. B-CLL tumoral cells showed an elevated modification of the IkB⍺ kinase IKKß at lysine 106 that increased even more under the action of MLN4924. Our experiments showed how modulation of the cellular NEDDylation state through chemical and genetic strategies alter the stability of IKKß and consequently the phosphorylation and stability of IkB⍺. In addition, MLN4924 potentiated the cell death induced by the IKKß inhibitor BAY11-7082. Mutation of IKKß lysine 106 to the non-modificable residue arginine made the protein refractory to destabilization with NEDD8 and affected its kinase activity.

NF-kB pathway activates the transcription of its own inhibitor IkB⍺ in a feedback loop to terminate its signaling. Unexpectedly, NFKBIA mRNA levels (coding for IkB⍺) accumulated in peripheral blood mononuclear cells from CLL patients at a lower level than in those from healthy donors. NFKBIA inhibition through a GSK-3ß-mediated phosphorylation that turns p65/RelA into a transcriptional repressor is a characteristic of senescence cells. We explored whether B-CLL cells may fit into this phenotype. Chemical and genetic inhibition of GSK-3ß reverted the accumulation of NFKBIA in B-CLL cells, leading to an increment in their cell death. GSK-3ß also affected the expression of other inflammatory-related genes, but their pattern of transcription did not support a senescence phenotype of B-CLL cells. We explored whether the general elevated level of UBL-PTMs in B-CLL cells also affected the transcription of NFKBIA. Indeed, in knock out cells for the de-NEDDylation protein NEDP1 that show an increase in basal protein NEDDylation levels, NFKBIA mRNA declined in parallel with a stabilization of GSK-3ß. On the other hand, NEDDylation also modulated the function of p65/RelA, controlling its protein levels, phosphorylation, translocation to the nucleus and DNA binding.

In summary, we provide evidence that widens our knowledge of the regulatory action of NEDDylation over the NF-kB pathway in CLL and may help finding new therapeutic targets. Our data indicate that NEDDylation controls the transcription of the NFKBIA gene and the phosphorylation and stability of IkB and RelA/p65. This demonstrates the complexity of this regulation and warns about the general inhibition of NEDDylation that may have contradictory effects on cell biology.