Purpose: Histone deacetylase inhibitors can alter gene expression and mediate diverse antitumor activities. Herein, we report the
safety and activity of the histone deacetylase inhibitor panobinostat ...(LBH589) in cutaneous T-cell lymphoma (CTCL) and identify
genes commonly regulated by panobinostat.
Experimental Design: Panobinostat was administered orally to patients with CTCL on Monday, Wednesday, and Friday of each week on a 28-day cycle.
A dose of 30 mg was considered excessively toxic, and subsequent patients were treated at the expanded maximum tolerated dose
of 20 mg. Biopsies from six patients taken 0, 4, 8, and 24 h after administration were subjected to microarray gene expression
profiling and real-time quantitative PCR of selected genes.
Results: Patients attained a complete response ( n = 2), attained a partial response ( n = 4), achieved stable disease with ongoing improvement ( n = 1), and progressed on treatment ( n = 2). Microarray data showed distinct gene expression response profiles over time following panobinostat treatment, with
the majority of genes being repressed. Twenty-three genes were commonly regulated by panobinostat in all patients tested.
Conclusions: Panobinostat is well tolerated and induces clinical responses in CTCL patients. Microarray analyses of tumor samples indicate
that panobinostat induces rapid changes in gene expression, and surprisingly more genes are repressed than are activated.
A unique set of genes that can mediate biological responses such as apoptosis, immune regulation, and angiogenesis were commonly
regulated in response to panobinostat. These genes are potential molecular biomarkers for panobinostat activity and are strong
candidates for the future assessment of their functional role(s) in mediating the antitumor responses of panobinostat.
Histone deacetylase (HDAC) inhibitors (HDI) induce endoplasmic reticulum (ER) stress and apoptosis, while promoting autophagy, which promotes cancer cell survival when apoptosis is compromised. Here, ...we determined the in vitro and in vivo activity of the combination of the pan-HDI panobinostat and the autophagy inhibitor chloroquine against human estrogen/progesterone receptor and HER2 (triple)-negative breast cancer (TNBC) cells. Treatment of MB-231 and SUM159PT cells with panobinostat disrupted the hsp90/histone deacetylase 6/HSF1/p97 complex, resulting in the upregulation of hsp. This was accompanied by the induction of enhanced autophagic flux as evidenced by increased expression of LC3B-II and the degradation of the autophagic substrate p62. Treatment with panobinostat also induced the accumulation and colocalization of p62 with LC3B-II in cytosolic foci as evidenced by immunofluorescent confocal microscopy. Inhibition of panobinostat-induced autophagic flux by chloroquine markedly induced the accumulation of polyubiquitylated proteins and p62, caused synergistic cell death of MB-231 and SUM159PT cells, and inhibited mammosphere formation in MB-231 cells, compared with treatment with each agent alone. Finally, in mouse mammary fat pad xenografts of MB-231 cells, a tumor size-dependent induction of heat shock response, ER stress and autophagy were observed. Cotreatment with panobinostat and chloroquine resulted in reduced tumor burden and increased the survival of MB-231 breast cancer xenografts. Collectively, our findings show that cotreatment with an autophagy inhibitor and pan-HDI, for example, chloroquine and panobinostat results in accumulation of toxic polyubiquitylated proteins, exerts superior inhibitory effects on TNBC cell growth, and increases the survival of TNBC xenografts.
Our previous studies demonstrated that inhibition of histone deacetylases (HDACs) by trichostatin A reactivates estrogen receptor alpha (ER) gene expression in ER-negative breast cancer cells. Here, ...we use the clinically relevant HDAC inhibitor, LBH589 (LBH) to explore the roles of HDAC in ER gene silencing. In the ER-negative human breast cancer lines, MDA-MB-231 and MDA-MB-435, treatment with LBH for 24 hours restored ER mRNA and protein expression without a concomitant demethylation of the CpG island at the ER promoter. The expression of ER mRNA was sustained at least 96 hours after withdrawal of LBH treatment. Restoration of ER expression by LBH enhanced 4-hydroxytamoxifen sensitivity in MDA-MB-231 cells. The molecular mechanisms by which LBH reactivated silenced ER gene in MDA-MB-231 cells were examined with emphasis on chromatin structure reorganization. By chromatin immunoprecipitation analysis, LBH treatment released DNMT1, HDAC1, and the H3 (Lys-9) methyltransferase SUV39H1 from the ER promoter. Such changes were associated with an active chromatin formation manifested as accumulation of acetylated histones H3 and H4, a decrease in methylated H3 lysine 9 (H3-K9), and an impaired binding of heterochromatin protein 1 (HP1α) at the promoter. Our findings suggest that HDAC inhibitors could restore expression of the silenced ER gene by reorganizing the heterochromatin-associated proteins without alteration in promoter DNA hypermethylation.
Purpose: Angiogenesis is required for tumor progression and represents a rational target for therapeutic intervention. Histone deacetylase
(HDAC) inhibitors have been shown to have activity against ...various tumor cell types by inhibiting proliferation and inducing
apoptosis both in vitro and in vivo . HDAC inhibitors have also been reported to inhibit angiogenesis. The goal of this study was to characterize the antiangiogenic
and antitumor activity of a recently developed HDAC inhibitor, the hydroxamic derivative LBH589.
Materials and Methods: To evaluate the antiangiogenesis activity of LBH589, we did cell cycle analysis, cell proliferation, tube formation, invasion
assays in vitro , and Matrigel plug assay in vivo . To determine the antitumor activity of LBH589, we established human prostate carcinoma cell PC-3 xenografts in vivo . To evaluate the effect of LBH589 on endothelial signaling pathways, gene expression, and protein acetylation, we did Western
blots and reverse transcription-PCR in human umbilical vein endothelial cells (HUVEC). Immunohistochemical analysis was done
to evaluate new blood vessel formation in vivo .
Results: LBH589 induced acetylation of histone H3 and α-tubulin protein in HUVECs. Histone and nonhistone protein acetylation correlated
with induction of G 2 -M cell cycle arrest, inhibition of HUVEC proliferation, and viability. Noncytotoxic concentrations of LBH589 inhibited endothelial
tube formation, Matrigel invasion, AKT, extracellular signal-regulated kinase 1/2 phosphorylation, and chemokine receptor
CXCR4 expression. In vivo dosing of mice with LBH589 (10 mg/kg/d) reduced angiogenesis and PC-3 tumor growth.
Conclusion: This study provides evidence that LBH589 induces a wide range of effects on endothelial cells that lead to inhibition of
tumor angiogenesis. These results support the role of HDAC inhibitors as a therapeutic strategy to target both the tumor and
endothelial compartment and warrant the clinical development of these agents in combination with angiogenesis inhibitors.
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.
Histone deacetylases (HDAC) have been identified as therapeutic targets due to their regulatory function in DNA structure and organization. LBH589 is a novel inhibitor of class I and II HDACs. We ...studied the effect of LBH589 and ionizing radiation (IR) on DNA repair in two human non-small cell lung cancer (NSCLC) cell lines (H23 and H460). gamma-H2AX foci present at DNA double-strand breaks (DSBs) were detected in the nuclei following 3 Gy irradiation for up to 6 hours. LBH589 administered before irradiation increased the duration of gamma-H2AX foci beyond 24 hours. Furthermore, radiation alone induced translocation of HDAC4 to the nucleus. In contrast, treatment with LBH589 followed by irradiation resulted in HDAC4 confinement to the cytoplasm, indicating that HDAC inhibition affects the nuclear localization of HDAC4. The findings that LBH589 confines HDAC4 to the cytoplasm and increases the duration of gamma-H2AX foci in irradiated cell lines suggest that HDAC4 participates in DNA damage signaling following IR. Annexin-propidium iodide flow cytometry assays, cell morphology studies, and cleaved caspase-3 Western blot analysis revealed a synergistic effect of LBH589 with IR in inducing apoptosis. Clonogenic survival showed a greater than additive effect when LBH589 was administered before irradiation compared with irradiation alone. In vivo tumor volume studies showed a growth delay of 20 days with combined treatment compared with 4 and 2 days for radiation or LBH589 alone. This study identifies HDAC4 as a biomarker of LBH589 activity and recognizes the ability of LBH589 to sensitize human NSCLC to radiation-induced DNA DSBs.
Histone deacetylases (HDAC) play a critical role in chromatin modification and gene expression. Recent evidence indicates
that HDACs can also regulate functions of nonhistone proteins by catalyzing ...the removal of acetylated lysine residues. Here,
we show that the HDAC inhibitor LBH589 down-regulates DNA methyltransferase 1 (DNMT1) protein expression in the nucleus of
human breast cancer cells. Cotreatment with the proteasomal inhibitor MG-132 abolishes the ability of LBH589 to reduce DNMT1,
suggesting that the proteasomal pathway mediates DNMT1 degradation on HDAC inhibition. Deletion of the NH 2 -terminal 120 amino acids of DNMT1 diminishes LBH589-induced ubiquitination, indicating that this domain is essential for
its proteasomal degradation. DNMT1 recruits the molecular chaperone heat shock protein 90 (Hsp90) to form a chaperone complex.
Treatment with LBH589 induces hyperacetylation of Hsp90, thereby inhibiting the association of DNMT1 with Hsp90 and promoting
ubiquitination of DNMT1. In addition, inactivation of HDAC1 activity by small interfering RNA and MS-275 is associated with
Hsp90 acetylation in conjunction with reduction of DNMT1 protein expression. We conclude that the stability of DNMT1 is maintained
in part through its association with Hsp90. Disruption of Hsp90 function by HDAC inhibition is a unique mechanism that mediates
the ubiquitin-proteasome pathway for DNMT1 degradation. Our studies suggest a new role for HDAC1 and identify a novel mechanism
of action for the HDAC inhibitors as down-regulators of DNMT1. (Mol Cancer Res 2008;6(5):873–83)
Combinations of drug treatments based on bortezomib or lenalidomide plus steroids have resulted in very high response rates in multiple myeloma. However, most patients still relapse, indicating the ...need for novel combination partners to increase duration of response or to treat relapsed disease. We explored the antimyeloma activity of triple combinations of these well-established schemes with panobinostat, a novel deacetylase inhibitor with a multi-targeted profile.
The activity of these combinations was explored in vitro in cell lines by using MTT and annex-in V, ex vivo by flow cytometry, and in vivo using two different murine models of human myeloma: one bearing a subcutaneous plasmacytoma and another with a disseminated myeloma. Moreover, gene expression profiling and immunohistochemical studies were performed.
The addition of panobinostat (LBH589) to dexamethasone and either bortezomib or lenalidomide resulted in clear potentiation in multiple myeloma cell lines, freshly isolated plasma cells, and murine models of multiple myeloma. The quantification of the potency of these combinations by using the Chou-Talalay method showed synergistic combination indices for all of them. This effect derived from the deregulation of a cluster of genes that was completely different from the sum of genes affected by the single agents (895 and 1323 genes exclusively deregulated by panobinostat and dexamethasone plus bortezomib or lenalidomide, respectively). Functional experiments, such as annexin V staining, cell cycle analysis, and immunohistochemical studies also supported this potentiation. Anti-myeloma efficacy was confirmed in an extramedullary plasmacytoma model and a disseminated luciferized model, in which panobinostat also provided a marked benefit in bone disease.
The potent activity, together with the exclusive mechanistic profile, provides the rationale for the clinical evaluation of these drug combinations in multiple myeloma.
Histone deacetylase inhibitors (HDACi) may engage host immunity as one basis for their antitumor effects. Herein, we demonstrate an application of this concept using the HDACi panobinostat to augment ...the antitumor efficacy of trastuzumab (anti-HER2) therapy, through both tumor cell autonomous and nonautonomous mechanisms. In HER2
tumors that are inherently sensitive to the cytostatic effects of trastuzumab, cotreatment with panobinostat abrogated AKT signaling and triggered tumor regression in mice that lacked innate and/or adaptive immune effector cells. However, the cooperative ability of panobinostat and trastuzumab to harness host anticancer immune defenses was essential for their curative activity in trastuzumab-refractory HER2
tumors. In trastuzumab-resistant HER2
AU565
xenografts and BT474 tumors expressing constitutively active AKT, panobinostat enhanced the antibody-dependent cell-mediated cytotoxicity function of trastuzumab. IFNγ-mediated, CXCR3-dependent increases in tumor-associated NK cells underpinned the combined curative activity of panobinostat and trastuzumab in these tumors. These data highlight the immune-enhancing effects of panobinostat and provide compelling evidence that this HDACi can license trastuzumab to evoke NK-cell-mediated responses capable of eradicating trastuzumab-refractory HER2
tumors.
.
Activating mutations in the epidermal growth factor receptor (EGFR) selectively activate signal transducers and activators
of transcription (STAT) and Akt survival signaling pathways important in ...lung cancer cell growth and survival. Many kinases,
such as EGFR, rely on heat shock protein 90 (Hsp90) chaperone function for conformational maturation and proper function.
Histone deacetylase inhibitors (HDACi) have been suggested to regulate signaling protein interactions via modulation of protein
chaperone function through Hsp90. For these reasons, we evaluated the effect of a HDACi in lung cancer cells with defined
EGFR status. Cell lines with defined EGFR status and sensitivity to EGFR tyrosine kinase inhibitors were exposed to the HDACi
LBH589, and the effects on cell survival, proliferation, and downstream signaling were evaluated. LBH589 resulted in increased
acetylation of Hsp90 and reduced association of Hsp90 with EGFR, Akt, and STAT3. LBH589 selectively depleted proteins important
in signaling cascades in cell lines harboring EGFR kinase mutations, such as EGFR, STAT3, and Akt, and these cells underwent
apoptosis following exposure to LBH589. In addition, we found depletion of STAT3-dependent survival proteins, including Bcl-xL,
Mcl-1, and Bcl-2. Conversely, LBH589 had little effect on apoptosis in cells not dependent on EGFR for survival, no changes
were identified in the expression of EGFR or other survival proteins, and the predominant effect was cell cycle arrest rather
than apoptosis. A 10-fold increase in LBH589 was necessary to observe durable depletion of EGFR and Akt in cells not harboring
EGFR mutation. Treatment of cells with erlotinib and LBH589 resulted in synergistic effects on lung cancer cells dependent
on EGFR for growth and/or survival. Based on these results, LBH589 can acetylate Hsp90, deplete EGFR and other key survival
signaling proteins, and trigger apoptosis only in lung cancer cells harboring EGFR mutations. Therefore, EGFR mutation status
may be predictive of outcome with LBH589 and possibly other HDACi. Mol Cancer Ther 2007;6(9):2515–24
PDF
