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Heat shock protein (hsp) 90 is an ATP-dependent molecular chaperone that maintains the active conformation of client oncoproteins in cancer cells. An isoform, hsp90alpha, promotes extracellular maturation of matrix metalloproteinase (MMP)-2, involved in tumor invasion and metastasis. Knockdown of histone deacetylase (HDAC) 6, which deacetylates lysine residues in hsp90, induces reversible hyperacetylation and attenuates ATP binding and chaperone function of hsp90. Here, using mass spectrometry, we identified seven lysine residues in hsp90alpha that are hyperacetylated after treatment of eukaryotic cells with a pan-HDAC inhibitor that also inhibits HDAC6. Depending on the specific lysine residue in the middle domain involved, although acetylation affects ATP, cochaperone, and client protein binding to hsp90alpha, acetylation of all seven lysines increased the binding of hsp90alpha to 17-allyl-amino-demethoxy geldanamycin. Notably, after treatment with the pan-HDAC inhibitor panobinostat (LBH589), the extracellular hsp90alpha was hyperacetylated and it bound to MMP-2, which was associated with increased in vitro tumor cell invasiveness. Treatment with antiacetylated hsp90alpha antibody inhibited in vitro invasion by tumor cells. Thus, reversible hyperacetylation modulates the intracellular and extracellular chaperone function of hsp90, and targeting extracellular hyperacetylated hsp90alpha may undermine tumor invasion and metastasis.

Purpose: The molecular chaperone heat shock protein (hsp)-90 maintains estrogen receptor (ER)-alpha in an active conformation, allowing it to bind 17beta-estradiol (E2) and transactivate genes, including progesterone receptor (PR)-beta and the class IIB histone deacetylase HDAC6. By inhibiting HDAC6, the hydroxamic acid analogue pan-HDAC inhibitors (HA-HDI; e.g., LAQ824, LBH589, and vorinostat) induce hyperacetylation of the HDAC6 substrates alpha-tubulin and hsp90. Hyperacetylation of hsp90 inhibits its chaperone function, thereby depleting hsp90 client proteins. Here, we determined the effect of HA-HDIs on the levels and activity of ERalpha, as well as on the survival of ERalpha-expressing, estrogen-responsive human breast cancer MCF-7 and BT-474 cells., Experimental Design: Following exposure to HA-HDIs, hsp90 binding, polyubiquitylation levels, and transcriptional activity of ERalpha, as well as apoptosis and loss of survival, were determined in MCF-7 and BT-474 cells., Results: Treatment with HA-HDI induced hsp90 hyperacetylation, decreased its binding to ERalpha, and increased polyubiquitylation and depletion of ERalpha levels. HA-HDI treatment abrogated E2-induced estrogen response element-luciferase expression and attenuated PRbeta and HDAC6 levels. Exposure to HA-HDI also depleted p-Akt, Akt, c-Raf, and phospho-extracellular signal-regulated kinase-1/2 levels, inhibited growth, and sensitized ERalpha-positive breast cancer cells to tamoxifen., Conclusions: These findings show that treatment with HA-HDI abrogates ERalpha levels and activity and could sensitize ERalpha-positive breast cancers to E2 depletion or ERalpha antagonists.

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.

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-x(L), 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-x(L) 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.

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-beta-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-AAG-mediated 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.

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 co-treatment of SAHA with trastuzumab or docetaxel in human breast cancer BT-474 and SKBR-3 cells containing amplification of Her-2., Experimental Design: The cells were treated with SAHA (1.0-5.0 micromol/L) and/or trastuzumab (5-40 microg/mL) or docetaxel (5-20 nmol/L). Following this, apoptosis and the levels of p21(WAF1), p27(KIP1), 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 G2-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. Co-treatment with SAHA significantly increased trastuzumab or docetaxel-induced apoptosis of BT-474 and SKBR-3 cells. Additionally, median dose-effect analysis revealed that co-treatment 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.

The hydroxamic acid (HAA) analogue pan-histone deacetylase (HDAC) inhibitors (HDIs) LAQ824 and LBH589 have been shown to induce acetylation and inhibit the ATP binding and chaperone function of heat shock protein (HSP) 90. This promotes the polyubiquitylation and degradation of the pro-growth and pro-survival client proteins Bcr-Abl, mutant FLT-3, c-Raf, and AKT in human leukemia cells. HDAC6 is a member of the class IIB HDACs. It is predominantly cytosolic, microtubule-associated alpha-tubulin deacetylase that is also known to promote aggresome inclusion of the misfolded polyubiquitylated proteins. Here we demonstrate that in the Bcr-abl oncogene expressing human leukemia K562 cells, HDAC6 can be co-immunoprecipitated with HSP90, and the knock-down of HDAC6 by its siRNA induced the acetylation of HSP90 and alpha-tubulin. Depletion of HDAC6 levels also inhibited the binding of HSP90 to ATP, reduced the chaperone association of HSP90 with its client proteins, e.g. Bcr-Abl, and induced polyubiquitylation and partial depletion of Bcr-Abl. Conversely, the ectopic overexpression of HDAC6 inhibited LAQ824-induced acetylation of HSP90 and alpha-tubulin and reduced LAQ824-mediated depletion of Bcr-Abl, AKT, and c-Raf. Collectively, these findings indicate that HDAC6 is also an HSP90 deacetylase. Targeted inhibition of HDAC6 leads to acetylation of HSP90 and disruption of its chaperone function, resulting in polyubiquitylation and depletion of pro-growth and pro-survival HSP90 client proteins including Bcr-Abl. Depletion of HDAC6 sensitized human leukemia cells to HAA-HDIs and proteasome inhibitors.

Present studies show that LBH589, a novel cinnamic hydroxamic acid analog histone deacetylase inhibitor, induces acetylation of histone H3 and H4 and of heat shock protein 90 (hsp90), increases p21 levels, as well as induces cell-cycle G(1) phase accumulation and apoptosis of the human chronic myeloid leukemia blast crisis (CML-BC) K562 cells and acute leukemia MV4-11 cells with the activating length mutation of FLT-3. In MV4-11 cells, this was associated with marked attenuation of the protein levels of p-FLT-3, FLT-3, p-AKT, and p-ERK1/2. In K562 cells, exposure to LBH589 attenuated Bcr-Abl, p-AKT, and p-ERK1/2. Treatment with LBH589 inhibited the DNA binding activity of signal transducers and activators of transcription 5 (STAT5) in both K562 and MV4-11 cells. The hsp90 inhibitor 17-allyl-amino-demethoxy geldanamycin (17-AAG) also induced polyubiquitylation and proteasomal degradation of FLT-3 and Bcr-Abl by reducing their chaperone association with hsp90. Cotreatment with LBH589 and 17-AAG exerted synergistic apoptosis of MV4-11 and K562 cells. In the imatinib mesylate (IM)-refractory leukemia cells expressing Bcr-Abl with the T315I mutation, treatment with the combination attenuated the levels of the mutant Bcr-Abl and induced apoptosis. Finally, cotreatment with LBH589 and 17-AAG also induced more apoptosis of IM-resistant primary CML-BC and acute myeloid leukemia (AML) cells (with activating mutation of FLT-3) than treatment with either agent alone.

Bcr-Abl-expressing primary or cultured leukemia cells display high levels of the antiapoptotic heat shock protein (hsp) 70 and are resistant to cytarabine (Ara-C), etoposide, or Apo-2L/TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis. Conversely, a stable expression of the cDNA of hsp70 in the reverse orientation attenuated not only hsp70 but also signal transducers and activators of transcription 5 (STAT5) and Bcl-x(L) levels. This increased apoptosis induced by cytarabine, etoposide, or Apo-2L/TRAIL. Ectopic expression of hsp70 in HL-60 cells (HL-60/hsp70) inhibited Ara-C and etoposide-induced Bax conformation change and translocation to the mitochondria; attenuated the accumulation of cytochrome c, Smac, and Omi/HtrA2 in the cytosol; and inhibited the processing and activity of caspase-9 and caspase-3. Hsp70 was bound to death receptors 4 and 5 (DR4 and DR5) and inhibited Apo-2L/TRAIL-induced assembly and activity of the death-inducing signaling complex (DISC). HL-60/hsp70 cells exhibited increased levels and DNA binding activity of STAT5, which was associated with high levels of Pim-2 and Bcl-x(L) and resistance to apoptosis. Expression of the dominant negative (DN) STAT5 resensitized HL-60/hsp70 cells to cytarabine, etoposide, and Apo-2L/TRAIL-induced apoptosis. Collectively, these findings suggest that hsp70 inhibits apoptosis upstream and downstream of the mitochondria and is a promising therapeutic target for reversing drug-resistance in chronic myeloid leukemia-blast crisis and acute myeloid leukemia cells.

Purpose: Mutant FLT-3 receptor tyrosine kinase is a client protein of the molecular chaperone heat shock protein 90 and is commonly present and contributes to the leukemia phenotype in acute myelogenous leukemia (AML). LAQ824, a cinnamyl hydroxamate histone deacetylase inhibitor, is known to induce acetylation and inhibition of heat shock protein 90. Here, we determined the effects of LAQ824 and/or PKC412 (a FLT-3 kinase inhibitor) on the levels of mutant FLT-3 and its downstream signaling, as well as growth arrest and cell-death of cultured and primary human AML cells., Experimental Design: The effect of LAQ824 and/or PKC412 treatment was determined on the levels of FLT-3 and phosphorylated (p)-FLT-3, on downstream pro-growth and pro-survival effectors, e.g., p-STAT5, p-AKT, and p-extracellular signal-regulated kinase (ERK) 1/2, and on the cell cycle status and apoptosis in the cultured MV4-11 and primary AML cells with mutant FLT-3., Results: Treatment with LAQ824 promoted proteasomal degradation and attenuation of the levels of FLT-3 and p-FLT-3, associated with cell cycle G(1)-phase accumulation and apoptosis of MV4-11 cells. This was accompanied by attenuation of p-STAT5, p-AKT, and p-ERK1/2 levels. STAT-5 DNA-binding activity and the levels of c-Myc and oncostatin M were also down-regulated. Cotreatment with LAQ824 and PKC412 synergistically induced apoptosis of MV4-11 cells and induced more apoptosis of the primary AML cells expressing mutant FLT-3. This was also associated with more attenuation of p-FLT-3, p-AKT, p-ERK1/2, and p-STAT5., Conclusions: The combination of LAQ824 and PKC412 is highly active against human AML cells with mutant FLT-3, which merits in vivo studies of the combination against human AML.

Presence of the activating length mutation (LM) in the juxtamembrane domain or point mutation in the kinase domain of FMS-like tyrosine kinase-3 (FLT-3) mediates ligand-independent progrowth and prosurvival signaling in approximately one-third of acute myelogenous leukemia (AML). PKC412, an inhibitor of FLT-3 kinase activity, is being clinically evaluated in AML. Present studies demonstrate that treatment of human acute leukemia MV4-11 cells (containing a FLT-3 LM) with the heat shock protein 90 inhibitor 17-allylamino-demethoxy geldanamycin (17-AAG) attenuated the levels of FLT-3 by inhibiting its chaperone association with heat shock protein 90, which induced the poly-ubiquitylation and proteasomal degradation of FLT-3. Treatment with 17-AAG induced cell cycle G(1) phase accumulation and apoptosis of MV4-11 cells. 17-AAG-mediated attenuation of FLT-3 and p-FLT-3 in MV4-11 cells was associated with decrease in the levels of p-AKT, p-ERK1/2, and p-STAT5, as well as attenuation of the DNA binding activity of STAT-5. Treatment with 17-AAG, downstream of STAT5, reduced the levels of c-Myc and oncostatin M, which are transactivated by STAT5. Cotreatment with 17-AAG and PKC412 markedly down-regulated the levels of FLT-3, p-FLT-3, p-AKT, p-ERK1/2, and p-STAT5, as well as induced more apoptosis of MV4-11 cells than either agent alone. Furthermore, the combination of 17-AAG and PKC412 exerted synergistic cytotoxic effects against MV4-11 cells. Importantly, 17-AAG and PKC412 induced more loss of cell viability of primary AML blasts containing FLT-3 LM, as compared with those that contained wild-type FLT-3. Collectively, these in vitro findings indicate that the combination of 17-AAG and PKC412 has high level of activity against AML cells with FLT-3 mutations.

Homologues of the cell division protein FtsZ were previously identified in Bartonella bacilliformis and Bartonella henselae. We report herein that ftsZ is located at the distal end of an operon that includes ddlB, ftsQ, and ftsA. These genes code for homologues of D-alanine D-alanine ligase, an enzyme involved in cell wall biosynthesis, and FtsQ, and FtsA, which are involved in cell division. The DdlB, FtsQ, and FtsA proteins from Bartonella species are most homologous to proteins in closely related species from the Order Rhizobiales, such as Brucella sp., Agrobacterium tumefaciens, and M. loti. The organization of the genes within the ddlB-ftsZ operon of B. bacilliformis and B. henselae (5'ddlB-ftsQ-ftsA-ftsZ 3') is similar to that of Mesorhizobium loti and Escherichia coli. We report the localization of three promoter regions within the ddlB-ftsA sequence of B. bacilliformis that may enhance the transcription of ftsZ mRNA. A promoter region was also identified upstream of the ddlB gene.

The NlpD/LppB homolog of the human pathogen, Bartonella bacilliformis, is an immunogenic 43-kDa protein that is encoded by a 1206-bp open reading frame (ORF-401). The regions flanking the nlpD/lppB gene of B. bacilliformis were sequenced to determine if it is located within the rpoS operon, as it is in most bacteria. We report that the B. bacilliformis nlpD/lppB gene is located immediately downstream of pcm, a gene encoding a 25-kDa protein, L-isoaspartyl protein carboxyl methyltransferase, that is a component of the rpoS operon in other bacteria. However, the genomic organization downstream of the B. bacilliformis nlpD/lppB gene appears to be distinct. In other bacteria, the third gene in the operon is rpoS, a gene that codes for an alternative sigma factor of RNA polymerase. In B. bacilliformis, an open reading frame encoding a protein homologous to the immunodominant YajC protein is located directly downstream of the nlpD/lppB gene. We show that Bartonella henselae, a close relative of B. bacilliformis, also shares this unusual organizational feature. Thus, the genomic organization of the nlpD/lppB genes of B. bacilliformis, and B. henselae appears to be unique among all bacteria for which the sequence of this region has been reported.