ALBERT

Description: 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.

Description: Pan-histone deacetylase inhibitors, for example, vorinostat and panobinostat (LBH589; Novartis Pharmaceuticals, East Hanover, NJ), have shown clinical efficacy against advanced cutaneous T-cell lymphoma (CTCL). However, the molecular basis of this activity remains unclear. HDAC7, a class IIA histone deacetylase (HDAC), is overexpressed in thymocytes, where it represses expression of the proapoptotic nuclear orphan receptor Nur77. Here, we demonstrate that treatment with panobinostat rapidly inhibits the in vitro and intracellular activity, as well as the mRNA and protein levels of HDAC7, and induces expression and translocation of Nur77 to the mitochondria. There, Nur77 converts death resistance protein Bcl-2 into a killer protein, promoting cell death of cultured and patient-derived human CTCL cells. Treatment with panobinostat improved survival of athymic nude mice implanted with human CTCL cells. Ectopic expression of Nur77 induced apoptosis and sensitized HH cells to panobinostat, whereas combined knockdown of Nur77 and its family member Nor1 was necessary to inhibit panobinostat-induced apoptosis of CTCL cells. Cotreatment with the Bcl-2/Bcl-xL antagonist ABT-737 decreased resistance and synergistically induced apoptosis of human CTCL cells. These findings mechanistically implicate HDAC7 and Nur77 in sensitizing human CTCL cells to panobinostat as well as suggest that cotreatment with an anti–Bcl-2 agent would augment the anti-CTCL activity of panobinostat.

Description: Hydroxamic acid analog pan-histone deacetylase (HDAC) inhibitors (HA-HDIs) have shown preclinical and clinical activity against human acute leukemia. Here we describe HA-HDI–resistant human acute myeloid leukemia (AML) HL-60 (HL-60/LR) cells that are resistant to LAQ824, vorinostat, LBH589, and sodium butyrate. HL-60/LR cells show increased expression of HDACs 1, 2, and 4 but lack HDAC6 expression, with concomitant hyperacetylation of heat shock protein 90 (hsp90). Treatment with HA-HDI failed to further augment hsp90 acetylation, or increase the levels of p21 or reactive oxygen species (ROSs), in HL-60/LR versus HL-60 cells. Although cross-resistant to antileukemia agents (eg, cytarabine, etoposide, and TRAIL), HL-60/LR cells are collaterally sensitive to the hsp90 inhibitor 17-AAG. Treatment with 17-AAG did not induce hsp70 or deplete the hsp90 client proteins AKT and c-Raf. HL-60/LR versus HL-60 cells display a higher growth fraction and shorter doubling time, along with a shorter interval to generation of leukemia and survival in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Thus, resistance of AML cells to HA-HDIs is associated with loss of HDAC6, hyperacetylation of hsp90, aggressive leukemia phenotype, and collateral sensitivity to 17-AAG. These findings suggest that an hsp90 inhibitor-based antileukemia therapy may override de novo or acquired resistance of AML cells to HA-HDIs.

Description: Histone deacetylase 6 (HDAC6) is a heat shock protein 90 (hsp90) deacetylase. Treatment with pan-HDAC inhibitors or depletion of HDAC6 by siRNA induces hyperacetylation and inhibits ATP binding and chaperone function of hsp90. Treatment with 17-allylamino-demothoxy geldanamycin (17-AAG) also inhibits ATP binding and chaperone function of hsp90, resulting in polyubiquitylation and proteasomal degradation of hsp90 client proteins. In this study, we determined the effect of hsp90 hyperacetylation on the anti-hsp90 and antileukemia activity of 17-AAG. Hyperacetylation of hsp90 increased its binding to 17-AAG, as well as enhanced 17-AAG–mediated attenuation of ATP and the cochaperone p23 binding to hsp90. Notably, treatment with 17-AAG alone also reduced HDAC6 binding to hsp90 and induced hyperacetylation of hsp90. This promoted the proteasomal degradation of HDAC6. Cotreatment with 17-AAG and siRNA to HDAC6 induced more inhibition of hsp90 chaperone function and depletion of BCR-ABL and c-Raf than treatment with either agent alone. In addition, cotreatment with 17-AAG and tubacin augmented the loss of survival of K562 cells and viability of primary acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) samples. These findings demonstrate that HDAC6 is an hsp90 client protein and hyperacetylation of hsp90 augments the anti-hsp90 and antileukemia effects of 17-AAG.

Description: Hydroxamic acid analogue pan-histone deacetylase (HDAC) inhibitors (HA-HDIs), e.g., vorinostat, LAQ824 and LBH589, induce in vitro growth arrest, differentiation and apoptosis of human acute leukemia cells. Continuous and protracted use of HA-HDI, as currently used in the clinic against hematologic malignancies is likely to result in the emergence of HA-HDI resistance in leukemia cells. By continuous in vitro exposure of the AML HL-60 cells to the cinnamic acid analogue HA-HDI LAQ824, we have generated an in vitro and in vivo model of HA-HDI-resistant HL-60/LR cells, which are capable of growth in high concentrations (200 nM) of LAQ824. HL-60/LR versus the parental HL-60 cells have a shorter doubling time (12 versus 24 hours), increased % of cells in the S phase of the cell cycle (62.4 versus 40.0) and exhibit shorter interval to generation of leukemia and survival in NOD/SCID mice. As compared to HL-60, HL-60/LR cells have a resistance index of 100 for LAQ824, and are cross-resistant to other antileukemia agents exhibiting resistance index for LBH589: 50; trichostatin A: 15; vorinostat: 30; sodium butyrate: 10; etoposide: 5.0; Ara-C: 3.3 and TRAIL: 31.3. As compared to HL-60, HL-60/LR cells express higher levels of Bcl-xL and XIAP but lower levels of MCL-1. HL-60/LR versus HL-60 cells also express markedly reduced levels of Bim and Bak but higher levels of Bax. Although expressing higher levels of the death receptors (DR) 4 and 5 and lower levels of c-FLIP, HL-60/LR cells lack expression of caspase-8 and show barely detectable levels of FADD. Additionally, HL-60/LR versus HL-60 cells have markedly higher levels of AKT, c-RAF, and p-STAT5. Although expressing higher levels of HDAC1, HDAC2, and HDAC4, HL-60/LR cells lack detectable expression of HDAC6, with increased expression of hyper-acetylated hsp90 and α-tubulin- two of the substrates deacetylated by HDAC6. As compared to hsp90 in HL-60 cells, hyper-acetylated hsp90 in HL-60/LR cells exhibits less binding to ATP and p23. Utilizing a polyclonal antibody generated against acetylated hsp90α, confocal immunofluorescence microscopy showed higher and mostly cell surface expression of acetylated hsp90α in HL-60/LR versus HL-60 cells. As compared to HL-60, treatment of HL-60/LR cells with LAQ824 failed to induce p21 and hsp70, or increase the levels of hyper-acetylated hsp90 and α-tubulin. Notably, although cross-resistant to several anti-leukemia drugs, HL-60/LR cells are collaterally sensitive to the hsp90-inhibiting geldanamycin analogues 17-allylamino-demothoxy geldanamycin (17-AAG) and 17-DMAG with a four and five-fold increased sensitivity to 17-AAG and 17-DMAG, respectively. This was associated with a lack of both a 17-AAG mediated induction of hsp70 and a lesser decline in the levels of AKT and c-RAF in HL-60/LR versus HL-60 cells. Taken together, these findings elucidate several notable in vitro and in vivo biologic characteristics and drug-sensitivity profile of the first fully-characterized HA-HDI-resistant human AML cells. Our findings clearly demonstrate that in vitro resistance to HA-HDIs is associated with loss of HDAC6 expression, hyperacetylation of hsp90, aggressive leukemia phenotype, but cross-sensitivity to 17-AAG. These findings also suggest that hsp90 inhibitors should be tested for overriding de novo or acquired HA-HDI resistance in AML.

Description: Poor clinical outcome of therapy of Mantle Cell Lymphoma (MCL) has generated the need to develop and test novel treatments for human MCL. Here we have determined that treatment with hydroxamic acid analogue (HA) pan-histone deacetylase (HDAC) inhibitor (HDI), e.g., LBH589 (Novartis Pharmaceuticals Inc) and vorinostat (Merck Pharmaceuticals), 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, HA-HDI also induced Bax, Bak and Bim, 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. By inhibiting HDAC6, both LBH589 (10 to 50 nM) and vorinostat (0.5 to 2.0 uM) 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. HDAC6 has been shown to shuttle and sequester misfolded and polyubiquitylated proteins into the protective perinuclear aggresome. Present studies demonstrate that inhibition of HDAC6 abrogates formation of the aggresome and augments the ER-based unfolded protein response (UPR). Treatment of MCL cells with the proteasome inhibitor bortezomib (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 the spliced form of XBP1 (XBP1s) and p-eIF2α protein. It was also associated with increased levels of the protective ER chaperone protein GRP78, and increased expression of pro-death proteins, CHOP and Noxa. Treatment with BZ or HA-HDI also increased the expression of the transcriptional repressor, PRDM1. Co-treatment of MCL cells with LBH589 abrogated BZ-induced aggresome formation, but increased the levels of BZ-induced XBP1s and p-eIF2α, indicating increased ER stress response. Concomitantly, higher CHOP and Noxa levels suggested a protracted ER-stress, associated with significantly increased apoptosis of MCL cells (p 〈 0.01). These findings suggest that co-treatment with LBH589 accentuates BZ-induced ER-stress and cell death of MCL cells despite up-regulation of GRP78 levels. Next, we determined the effects of knocking down GRP78 on BZ-induced ER-stress response. As compared to the control siRNA treated cells, knockdown by siRNA to GRP78 markedly increased BZ-induced CHOP and Noxa levels and significantly augmented BZ-induced apoptosis of cultured MCL cells. Collectively, these findings strongly support the in vivo testing of the efficacy of the combination of HA-HDI with BZ in inducing protracted and lethal ER stress in MCL cells. These results also create the rationale to develop targeted knockdown of GRP78 as a novel strategy to augment the lethal ER stress in human MCL cells.

Description: The trafficking and mobilization of normal hematopoietic progenitors and their leukemic counterparts is programmed in part by chemotactic gradients of CXCL12, which are transduced by CXCR4. This mechanism also results in decreased sensitivity to pro-apoptotic and anti-leukemic agents, mediated through CXCR4 activation of Akt and Raf phosphorylation and/or harboring in a microenvironmental niche. Both CXCL12 and CXCR4 are overexpressed in AML and expression of CXCR4 has been associated with a poor prognosis. Moreover, inhibition of CXCR4 with AMD3100 enhanced the sensitivity of leukemic myeloblasts to anti-leukemic agents. We therefore explored therapeutic mechanisms to decrease CXCR4 expression and tested them in combination with AMD3100 as well as a new generation CXCR4 inverse agonist, FC131. Exposure to 10 to 50 nM of the pan-HDAC inhibitor LBH589 (Novartis) depleted mRNA and protein levels of CXCR4 in the cultured human acute leukemia OCI-AML3, HL-60 and Jurkat cells, as well as in primary AML cells in a dose and time-dependent manner. LBH589 depleted CXCR4 levels in the presence or absence of 10 nM of CXCL12 in the culture medium. LBH589 mediated depletion of CXCR4 levels was partly due to decreased CXCR4 mRNA levels (by RT-PCR analysis). LBH589 induced acetylation of heat shock protein (hsp) 90 and attenuated the binding of CXCR4 to hsp90 with subsequent degradation of CXCR4 by the proteasome. LBH589 treatment also increased hsp70 levels and acetylation, as well as its binding to CXCR4, which also resulted in increased extra-cellular, cell surface co-localization of CXCR4 and hsp70. This was markedly inhibited by siRNA-mediated knockdown of hsp70 in HL-60 cells. While exposure of cultured and primary AML cells to CXCL12 markedly increased cytosolic levels of p-AKT and p-ERK1/2, co-treatment with LBH589 markedly attenuated the phosphorylation of AKT and ERK1/2 induced by CXCL12, resulting in apoptosis of up to 50% of cultured and primary AML cells. Treatment with AMD3100 (10 uM for 24 hours) alone decreased levels of CXCR4, p-AKT and p-ERK1/2, without significantly increasing apoptosis of AML cells. Notably, co-treatment with LBH589 and AMD3100 caused greater depletion of CXCR4, p-AKT and p-ERK1/2 levels, and exerted synergistic apoptotic effects against AML cells with combination indices of 〈 1.0 utilizing isobologram and median effect analyses. Co-treatment with LBH589 and FC131 (10 nM for 24 hours), which is a more potent CXCR4 antagonist than AMD3100, also induced synergistic apoptosis of cultured and primary AML cells. Taken together, these findings provide direct evidence that CXCR4 is a novel target depleted by LBH589 in AML cells. Furthermore, our in vitro findings highlight the novel combination of LBH589 and a CXCR4 antagonist, AMD3100 or FC131, exerts a synergistic effect on acute leukemia cells. These findings strongly support the in vivo testing of this synergistic combination in the therapy of human acute leukemias that express CXCR4.