Analogous phenomena are observed when leukaemia cells are transfected with an IjBa super repressor bearing mutations of S32/S36 that prevent IjBa phosphorylation and degradation. Notably, both approaches result in a significant increase of HDACI lethality, associated with down TCR Pathway regulation of NF jB dependent anti apoptotic proteins , as well as NF jB inhibition related activation of the stress related SAPK/JNK pathway . In this context, proteasome inhibitors such as bortezomib, by directly blocking IjBa degradation , may act through a similar mechanism. In the present study, coadministration of bortezomib attenuated belinostat mediated RelA/p65 K310 acetylation, an event associated with accumulation of the S32/S36 phosphorylated form of IjBa, in both continuously cultured cell lines and primary acute leukaemia blasts.
Moreover, Pimobendan these events were accompanied by inhibition of RelA/p65 DNA binding activity and NF jB luciferase reporter activity, as well as diminished expression of multiple NF jB dependent anti apoptotic proteins , although the latter events appear to be cell type dependent. Together, these results support the notion that interruption of the canonical NF jB pathway may contribute to potentiation of belinostat lethality by bortezomib in acute myeloid and lymphoid leukaemia cells. In addition to contributions to the regulation of the canonical NF jB pathway, proteasome mediated mechanisms may also play an important regulatory role in the noncanonical NF jB pathway via processing of the precursor p100 into its active form p52 .
It is noteworthy that co administration of bortezomib, particularly in the presence of belinostat, resulted in accumulation of p100 and reduced expression of p52, indicating that the bortezomib/ belinostat farriers regimen may also interrupt the non canonical NFjB pathway in acute myeloid and lymphoid leukaemia cells. The non canonical pathway has been implicated in the survival of certain malignant B cells , and disorders such as multiple myeloma are characterized by frequent aberrations in genes related to this pathway . However, although NF jB activation via the canonical pathway may influence the survival of AML cells , including AML initiating cells , as well as certain ALL sub types , the functional significance of disrupting the non canonical NF jB pathway in AML or ALL cells by the belinostat/bortezomib regimen remains to be more clearly defined.
Exposure to belinostat resulted in K40 acetylation of a tubulin in continuously cultured cell lines of both AML and ALL, as well as in primary leukaemic blasts. Lysine hyperacetylation of a tubulin occurs in response to agents that inhibit the class IIb HDAC6, including both HDAC6 specific inhibitors and pan HDACIs . By inhibiting HDAC6, such agents,when combined with proteasome inhibitors, also disrupt aggresome formation in response to misfolded proteininduced stress by preventing recruitment of misfolded proteins to dynein motors for transport to aggresomes. This leads to amplification of proteotoxic stress, potentially contributing to enhanced lethality of concomitant HDAC/proteasome inhibition in transformed cells e.g. myeloma or leukaemia cells . Consequently, although co administration of bortezomib did not enhance belinostatmediated .