Abstract
The 26S proteasome inhibitor bortezomib (BZ), which increases intracellular unfolded protein levels and toxicity through endoplasmic reticulum (ER) stress response, was shown to have a single agent activity in relapsed mantle cell lymphoma (MCL). Here we have determined that treatment with hydroxamic acid analogue (HA) pan-histone deacetylase (HDAC) inhibitor (HDI), e.g., panobinostat (LBH589, Novartis Pharmaceuticals Inc) induces the CDK inhibitors p21 and p27, and attenuates the levels of c-Myc, CDK4 and cyclin D1 in the cultured (Jeko-1, MO-2058 and Granta-519) and in primary patient-derived MCL cells. In a dose-dependent manner, panobinostat also induced Bax and Bak, and attenuated Bcl-xL, XIAP, survivin, AKT and c-Raf levels, resulting in growth inhibition and apoptosis of MCL cells. We have previously demonstrated that HDAC6 deacetylates heat shock protein (hsp) 90, as well as shuttles and sequesters misfolded and polyubiquitylated proteins into the protective perinuclear aggresome.. By inhibiting HDAC6, panobinostat (10 to 50 nM) induced acetylation of hsp90 in MCL cells. This inhibited the ATP binding and co-chaperone association, and abrogated the chaperone function of hsp90 for the MCL- relevant, hsp90 client proteins, e.g., cyclin D1, CDK4, c-Raf and AKT in the cultured and primary MCL cells. Panobinostat mediated inhibition of HDAC6 abrogated formation of the aggresome and augmented endoplasmic reticulum (ER)-based unfolded protein response (UPR). Treatment of MCL cells with BZ induced the formation of aggresome (as detected by confocal immuno-fluorescence microscopy and electron microscopy), as well as induced UPR and ER stress response. The latter was associated with BZ-mediated increased levels of GRP78, the spliced form of XBP1 (XBP1s) and p-eIF2α protein. As compared to the control siRNA treated cells, knockdown of GRP78 by siRNA markedly increased BZ-induced CHOP and Noxa levels and significantly augmented BZ-induced apoptosis of cultured MCL cells. Co-treatment of MCL cells with panobinostat abrogated BZ-induced aggresome formation, decreased the levels of ATF4, XBP1s and p-eIF2α, as well as increased the levels of CHOP, Noxa and GADD34. Ultrastructural analysis of Jeko-1 cells also revealed that co-treatment with panobinostat and BZ showed pronounced ER dilatation compared to panobinostat treatment alone, suggestive of enhanced ER stress. Higher and persistent CHOP and Noxa levels suggested a protracted ER-stress, associated with synergistic increase in apoptosis of MCL but not normal CD34+ bone marrow progenitor cells (p < 0.01). Conversely, knockdown of CHOP levels by siRNA significantly inhibited panobinostat and BZ-induced cell death of MCL cells. Results of ongoing in vivo studies of panobinostat and/or BZ in the NOD/SCID mouse xenograft model of Jeko-1 MCL cells will be presented. These findings strongly support further in vivo evaluation of the efficacy of the combination of panobinostat with BZ against human MCL. Additionally, the findings create the rationale to develop targeted knockdown of GRP78 as a novel strategy to augment lethal ER stress due to panobinostat and BZ and resulting activity against MCL cells.
Endoplasmic Reticulum Stress Promotes iNOS/NO and Influences Inflammation in the Development of Doxorubicin-Induced Cardiomyopathy