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17-Allylamino-demethoxy geldanamycin (17-AAG) inhibits the chaperone association of heat shock protein 90 (hsp90) with the heat shock factor-1 (HSF-1), which induces the mRNA and protein levels of hsp70. Increased hsp70 levels inhibit death receptor and mitochondria-initiated signaling for apoptosis. Here, we show that ectopic overexpression of hsp70 in human acute myelogenous leukemia HL-60 cells (HL-60/hsp70) and high endogenous hsp70 levels in Bcr-Abl-expressing cultured CML-BC K562 cells confers resistance to 17-AAG-induced apoptosis. In HL-60/hsp70 cells, hsp70 was bound to Bax, inhibited 17-AAG-mediated Bax conformation change and mitochondrial localization, thereby inhibiting the mitochondria-initiated events of apoptosis. Treatment with 17-AAG attenuated the levels of phospho-AKT, AKT, and c-Raf but increased hsp70 levels to a similar extent in the control HL-60/ Neo and HL-60/hsp70 cells. Pretreatment with 17-AAG, which induced hsp70, inhibited 1-B-D-arabinofuranosylcytosine or etoposide-induced apoptosis in HL-60 cells. Stable transfection of a small interfering RNA (siRNA) to hsp70 completely abrogated the endogenous levels of hsp70 and blocked 17-AAG-mediated hsp70 induction, resulting in sensitizing K562/siRNA-hsp70 cells to 17-AAG-induced apoptosis. This was associated with decreased binding of Bax to hsp70 and increased 17-AAG-induced Bax conformation change. 17-AAGmediated decline in the levels of AKT, c-Raf, and Bcr-Abl was similar in K562 and K562/siRNA-hsp70 cells. Cotreatment with KNK437, a benzylidine lactam inhibitor of hsp70 induction and thermotolerance, attenuated 17-AAG-mediated hsp70 induction and increased 17-AAG-induced apoptosis and loss of clonogenic survival of HL-60 cells. Collectively, these data indicate that induction of hsp70 attenuates the apoptotic effects of 17-AAG, and abrogation of hsp70 induction significantly enhances the antileukemia activity of 17-AAG. (Cancer Res 2005; 65(22): 10536-44)

Purpose: We determined the effects of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on hsp90 and its client proteins Her-2, AKT, and c-Raf, as well as evaluated the cytotoxic effects of cotreatment of SAHA with trastuzumab or docetaxel in human breast cancer BT-474 and SKBR-3 cells containing amplification of Her-2. , AKT, c-Raf, and Her-2, as well as of the key regulators of apoptosis were determined. Synergistic interaction between drugs was evaluated by median dose-effect analysis. Results: Treatment with SAHA up-regulated p21 WAF1 and p27 KIP1 levels, increased the percentage of cells in G 2 -M phase of the cell cycle, as well as induced apoptosis in a dose-dependent manner. This was associated with up-regulation of the pro-death Bak and Bim, as well as with attenuation of the levels of Her-2 and XIAP, survivin, Bcl-2, and Bcl-x L proteins. SAHA treatment induced acetylation of hsp90. This reduced the chaperone association of Her-2 with hsp90, promoting polyubiquitylation and degradation of Her-2. SAHA also attenuated the levels of c-Raf and AKT. Cotreatment with SAHA significantly increased trastuzumab or docetaxel-induced apoptosis of BT-474 and SKBR-3 cells. Additionally, median dose-effect analysis revealed that cotreatment with SAHA and trastuzumab or docetaxel induced synergistic cytotoxic effects against the breast cancer cells. Conclusions: These preclinical findings support the development of SAHA in combination with docetaxel and/or trastuzumab against Her-2-amplified breast cancer.

AMN107 (Novartis Pharmaceuticals, Basel, Switzerland) has potent in vitro and in vivo activity against the unmutated and most common mutant forms of Bcr-Abl. Treatment with the histone deacetylase inhibitor LBH589 (Novartis) depletes Bcr-Abl levels. We determined the effects of AMN107 and/or LBH589 in Bcr-Abl–expressing human K562 and LAMA-84 cells, as well as in primary chronic myelogenous leukemia (CML) cells. AMN107 was more potent than imatinib mesylate (IM) in inhibiting Bcr-Abl tyrosine kinase (TK) activity and attenuating p-STAT5, p-AKT, Bcl-xL, and c-Myc levels in K562 and LAMA-84 cells. Cotreatment with LBH589 and AMN107 exerted synergistic apoptotic effects with more attenuation of p-STAT5, p-ERK1/2, c-Myc, and Bcl-xL and increases in p27 and Bim levels. LBH589 attenuated Bcr-Abl levels and induced apoptosis of mouse pro-B BaF3 cells containing ectopic expression of Bcr-Abl or the IM-resistant, point-mutant Bcr-AblT315I and Bcr-AblE255K. Treatment with LBH589 also depleted Bcr-Abl levels and induced apoptosis of IM-resistant primary human CML cells, including those with expression of Bcr-AblT315I. As compared with either agent alone, cotreatment with AMN107 and LBH589 induced more loss of cell viability of primary IM-resistant CML cells. Thus, cotreatment with LBH589 and AMN107 is active against cultured or primary IM-resistant CML cells, including those with expression of Bcr-AblT315I.

Human enhancer of zeste 2 (EZH2) protein belongs to the multiprotein polycomb repressive complex 2, which also includes suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). The polycomb repressive complex 2 complex possesses histone methyltransferase activity mediated by the Su(var)3-9, enhancer of zeste, and trithorax domain of EZH2, which methylates histone H3 on lysine (K)-27 (H3K27). In the present studies, we determined that treatment with the hydroxamate histone deacetylase inhibitor LBH589 or LAQ824 depleted the protein levels of EZH2, SUZ12, and EED in the cultured (K562, U937, and HL-60) and primary human acute leukemia cells. This was associated with decreased levels of trimethylated and dimethylated H3K27, with concomitant depletion of the homeobox domain containing HOXA9 and of MEIS1 transcription factors. Knockdown of EZH2 by EZH2 small interfering RNA also depleted SUZ12 and EED, inhibited histone methyltransferase activity, and reduced trimethylated and dimethylated H3K27 levels, with a concomitant loss of clonogenic survival of the cultured acute myelogenous leukemia (AML) cells. EZH2 small interfering RNA sensitized the AML cells to LBH589-mediated depletion of EZH2, SUZ12, and EED; loss of clonogenic survival; and LBH589-induced differentiation of the AML cells. These findings support the rationale to test anti-EZH2 treatment combined with hydroxamate histone deacetylase inhibitors as an antileukemia epigenetic therapy, especially against AML with coexpression of EZH2, HOXA9, and MEIS1 genes.

Purpose: We determined the effects of vorinostat [suberoylanilide hydroxamic acid (SAHA)] and/or dasatinib, a dual Abl/Src kinase (tyrosine kinase) inhibitor, on the cultured human (K562 and LAMA-84) or primary chronic myelogenous leukemia (CML) cells, as well as on the murine pro-B BaF3 cells with ectopic expression of the unmutated and kinase domain-mutant forms of Bcr-Abl. Experimental Design: Following exposure to dasatinib and/or vorinostat, apoptosis, loss of clonogenic survival, as well as the activity and levels of Bcr-Abl and its downstream signaling proteins were determined. Results: Treatment with dasatinib attenuated the levels of autophosphorylated Bcr-Abl, p-CrkL, phospho-signal transducer and activator of transcription 5 (p-STAT5), p-c-Src, and p-Lyn; inhibited the activity of Lyn and c-Src; and induced apoptosis of the cultured CML cells. Combined treatment of cultured human CML and BaF3 cells with vorinostat and dasatinib induced more apoptosis than either agent alone, as well as synergistically induced loss of clonogenic survival, which was associated with greater depletion of Bcr-Abl, p-CrkL, and p-STAT5 levels. Cotreatment with dasatinib and vorinostat also attenuated the levels of Bcr-AblE255K and Bcr-AblT315I and induced apoptosis of BaF3 cells with ectopic expression of the mutant forms of Bcr-Abl. Finally, cotreatment of the primary CML cells with vorinostat and dasatinib induced more loss of cell viability and depleted Bcr-Abl or Bcr-AblT315I, p-STAT5, and p-CrkL levels than either agent alone. Conclusions: As shown here, the preclinical in vitro activity of vorinostat and dasatinib against cultured and primary CML cells supports the in vivo testing of the combination in imatinib mesylate^sensitive and imatinib mesylate^resistant CML cells.

Hsp70 is an ATP-dependent molecular chaperone that assists in the folding of native proteins into active conformation and prevents aggregation of misfolded and mutated abnormal proteins. In normal non-transformed cells, the expression of hsp70 is low and largely stress-inducible due to misfolded and denatured proteins. Recent studies in our laboratory have demonstrated that human acute leukemia cells abundantly express hsp70, which exerts strong antiapoptotic effects upstream and downstream of the mitochondria. Additionally, as compared to the controls, the mouse myeloid 32D or BaF3 cells transformed by Bcr-Abl or FLT-3 also show increased expression of hsp70. To further elucidate the pro-survival and pro-growth effects of hsp70 and its role in the leukemia transformation, we created stable hsp70 transfectants of the IL-3-dependent 32D and BaF3 (normally maintained in culture in IL-3 containing 10% WEHI medium), i.e., 32D/hsp70 and BaF3/hsp70 cells. These cells displayed 3 to 5 fold higher levels of hsp70, as compared to the control 32D or BaF3 cells. Both 32D/hsp70 and BaF3/hsp70 cells showed significantly improved growth and survival supported by 10% WEHI medium. Following culture in 0%, and less so in 1%, WEHI medium for 24 hours, 32D and BaF3 cells undergo cell cycle G1 phase accumulation, with corresponding decline in the % of cells in the S phase. Following this exposure, they also show markedly increased apoptosis and loss of clonogenic survival, as determined by the colony growth assays in methylcellulose. In contrast, under similar conditions of exposure to reduced % of WEHI conditioned medium, 32D/hsp70 and BaF3/hsp70 cells displayed significantly less accumulation in G1 phase, as well as reduced loss of clonogenic survival and apoptosis (p<0.05). This was also associated with reduced loss of mitochondrial membrane potential and increased accumulation of reactive oxygen species. Notably, following IL-3 withdrawal, exposure to 10 ng/ml of G-CSF for 72 hours induced significantly less differentiation of 32D/hsp70 versus 32D cells, as determined by increase in the % of cells expressing CD11b and GR1 (determined by specific antibody staining and flow cytometry) or by evaluation of the morphologic features of differentiation (p<0.05). Western blot analyses demonstrated that both 32D/hsp70 and BaF3/hsp70 cells, compared to their controls, possessed significantly higher expression of IL-3β receptor (R) and pSTAT5. Importantly, the supernatants of hsp70 overexpressing cells, compared to their controls, also showed higher levels of IL-3, as detected by an ELISA. In addition, BaF3/hsp70, versus the control cells, showed increased DNA binding activity and transactivation by the AP1 transcription factor, utilizing a protein/DNA binding array assay (Panomics, Redwood City, CA) and AP-1-luciferase cis-reporting analysis (Stratagene, La Jolla, CA), respectively. These findings strongly suggest that increased hsp70 levels confer a growth and survival advantage through an IL3- IL-3βR-STAT5-dependent mechanism in the marrow progenitor cells, which may contribute to the transformation induced by leukemia associated oncoproteins.

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