13039 Background: HDAC6 is a cytosolic α-tubulin deacetylase, which shuttles misfolded polyubiquitylated proteins into aggresomes. Our studies demonstrated that the hydroxamic acid (HA) analogue pan-HDAC inhibitors (HDIs), e.g., LAQ824 and LBH589, induce α-tubulin and hsp90 acetylation, thereby inhibiting ATP-binding and chaperone function of hsp90. This promotes the polyubiquitylation and degradation of the pro-growth and pro-survival hsp90 client proteins, e.g., Bcr-Abl, mutant FLT-3, c-Raf and AKT in leukemia and Her-2 in breast cancer cells.METHODSHuman CML K562 and breast cancer SKBR-3 and BT-474 cells were exposed to LAQ824 (100 nM) or LBH589 (20 to 50 nM) and/or 17-AAG (1 to 5 μM) and/or BZ (5 to 100 nM). K562 and BT-574 cells were also transfected with siRNA to HDAC6. Immunoprecipitation and/or immunoblot analysis were performed to determine the expression or binding of HDAC6 with hsp90, and of hsp90 acetylation and binding with its co-chaperones and client proteins. Immunofluorescent microscopy was utilized to estimate aggresome formation. Cellular cytotoxicity was determined by % inhibition of colony growth.RESULTSHDAC6 knockdown by its siRNA induced the acetylation of hsp90 and α-tubulin but not of the co-chaperones p23 and hsp40. HDAC6 binds to hsp90. Hsp90 acetylation increased its binding to biotinylated-geldanamycin (GM) and its analogue 17-AAG. Depletion of HDAC6 levels (∼70%) or its activity by treatment with HA-HDI also inhibited the binding of hsp90 to ATP, p23 and hsp90 client proteins, which was augmented by co-treatment with 17-AAG. Knockdown of HDAC6 sensitized the cells to loss of clonogenic survival induced by HA-HDI and/or BZ and/or 17-AAG. Depletion of HDAC6 led to attenuation of Her-2 levels and loss of survival of Her-2 amplified SKBR-3 and BT-474 cells. Co-localization of HDAC6 and polyubiquitylated proteins into aggresomes was enhanced by co-treatment with BZ and/or 17-AAG. This was inhibited by depletion of HDAC6 activity.CONCLUSIONSThese findings support the in vivo testing of HDAC6 inhibitors, which through inhibition of hsp90 and aggresome formation sensitizes cancer cells to 17-AAG and/or BZ induced cellular stress due to misfolded and polyubiquitylated proteins. [Table: see text].

Genetic alterations affecting RAS proteins are commonly found in human cancers. Roughly a fourth of melanoma patients carry activating NRAS mutations, rendering this malignancy particularly challenging to treat. Although the development of targeted as well as immunotherapies led to a substantial improvement in the overall survival of non-NRASmut melanoma patients (e.g. BRAFmut), patients with NRASmut melanomas have an overall poorer prognosis due to the high aggressiveness of RASmut tumors, lack of efficient targeted therapies or rapidly emerging resistance to existing treatments. Understanding how NRAS-driven melanomas develop therapy resistance by maintaining cell cycle progression and survival is crucial to develop more effective and specific treatments for this group of melanoma patients. In this review, we provide an updated summary of currently available therapeutic options for NRASmut melanoma patients with a focus on combined inhibition of MAPK signaling and CDK4/6-driven cell cycle progression and mechanisms of the inevitably developing resistance to these treatments. We conclude with an outlook on the most promising novel therapeutic approaches for melanoma patients with constitutively active NRAS.An estimated 75000 patients are affected by NRASmut melanoma each year and these patients still have a shorter progression-free survival than BRAFmut melanomas. Both intrinsic and acquired resistance occur in NRAS-driven melanomas once treated with single or combined targeted therapies involving MAPK and CDK4/6 inhibitors and/or checkpoint inhibiting immunotherapy. Oncolytic viruses, mRNA-based vaccinations, as well as targeted triple-agent therapy are promising alternatives, which could soon contribute to improved progression-free survival of the NRASmut melanoma patient group.

ABSTRACT We describe the effects of polyethylene glycol-conjugated adenosine deaminase (ADA) replacement therapy on lymphocyte counts, activation, apoptosis, proliferation, and cytokine secretion in a 14-month-old girl with “delayed-onset” ADA deficiency and marked immunodysregulation. Pretreatment lymphopenia affected T cells (CD4, 150/μl; CD8, 459/μl), B cells (16/μl), and NK cells (55/μl). T cells were uniformly activated and largely apoptotic (CD4, 59%; CD8, 82%); and T-cell-dependent cytokine levels in plasma were elevated, including the levels of interleukin 2 (IL-2; 26 pg/ml), IL-4 (81 pg/ml), IL-5 (46 pg/ml), gamma interferon (1,430 pg/ml), tumor necrosis factor alpha (210 pg/ml), and IL-10 (168 pg/ml). Mitogen-stimulated peripheral blood mononuclear cells show reduced IL-2 secretion and proliferation. During the first 5 months of therapy there was clinical improvement and partial immune reconstitution, with nearly normal lymphocyte subset numbers, reduced T-cell activation and CD4-cell apoptosis, and decreased plasma cytokine levels. In parallel, IL-2 secretion and the lymphocyte mitogenic response improved. Between 4 and 7 months, immunoglobulin G antibodies to bovine ADA developed and resulted in the complete reversal of immune recovery.

Androgen receptor plays a critical role in the development of primary as well as advanced hormone-refractory prostate cancer. Therefore, ablation of androgen receptor from prostate cancer cells is an interesting concept for developing a new therapy not only for androgen-dependent prostate cancer but also for metastatic hormone-refractory prostate cancer, for which there is no effective treatment available. We report here that LAQ824, a cinnamyl hydroxamatic acid histone deacetylase inhibitor currently in human clinical trials, effectively depleted androgen receptor in prostate cancer cells at nanomolar concentrations. LAQ824 seemed capable of depleting both the mutant and wild-type androgen receptors in either androgen-dependent and androgen-independent prostate cancer cells. Although LAQ824 may exert its effect through multiple mechanisms, several lines of evidence suggest that inactivation of the heat shock protein-90 (Hsp90) molecular chaperone is involved in LAQ824-induced androgen receptor depletion. Besides androgen receptor, LAQ824 reduced the level of Hsp90 client proteins HER-2 (ErbB2), Akt/PKB, and Raf-1 in LNCaP cells. Another Hsp90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), also induced androgen receptor diminution. LAQ824 induced Hsp90 acetylation in LNCaP cells, which resulted in inhibition of its ATP-binding activity, dissociation of Hsp90-androgen receptor complex, and proteasome-mediated degradation of androgen receptor. Consequently, LAQ824 blocked androgen-induced prostate-specific antigen production in LNCaP cells. LAQ824 effectively inhibited cell proliferation and induced apoptosis of these prostate cancer cells. These results reveal that LAQ824 is a potent agent for depletion of androgen receptor and a potential new drug for prostate 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 α-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 α-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 α-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.

Riboflavin carrier protein (RCP) is a phosphoglycoprotein (37 kDa) that is well studied in chicken. An immunologically cross-reacting protein was identified in mammals and active immunization of male rats and bonnet monkeys with chicken RCP lead to an approximately 80% reduction in fertility. However, the physiological mechanism responsible for inhibition of male fertility has not been investigated. Moreover, information on the cell type-specific localization and the origin of immunoreactive RCP during spermatogenesis is extremely limited. Hence, studies were carried out to determine the pattern of expression of immunoreactive RCP during spermatogenesis and its role in sperm function in the golden hamster. Immunoreactive RCP was germ cell-specific, found to be associated with the acrosome-organizing region of early spermatids and showed interesting patterns of immunolocalization during late stages of spermiogenesis. Mature spermatozoa exhibited acrosome-specific localization, mainly in the peri-acrosomal membrane. The immunoreactive protein was undetectable in (non)gonadal somatic cells tested. The protein had a molecular mass of 45-55 kDa and was biosynthesized by round spermatids. The acrosome-specific localization of immunoreactive RCP was unchanged during capacitation, but it was substantially lost during acrosome reaction. Functional studies indicated that treatment of spermatozoa with anti-RCP antibodies did not have any effect on either capacitation or acrosome reaction, but markedly reduced the rate of sperm penetration into zona-free hamster oocytes. These results show the existence of male germ cell-specific immunoreactive RCP, having a potential role in sperm-egg interaction in hamsters. Also the pattern of immunoreactive-RCP localization makes it an ideal marker to monitor development of acrosome in mammalian spermatozoa.

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.

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.

We have previously reported that agents that inhibit ATP binding and chaperone function of hsp90 are highly active against wild type and mutant Bcr-Abl and mutant FLT-3 containing human acute leukemia cells. In the present studies, we determined the effects of a more soluble and potent geldanamycin analogue, DMAG (Kosan Biosciences Inc.), and/or hydroxamate histone deacetylase inhibitor (HHDI), vorinostat (Merck & Co., Inc.), against human MCL Jeko1 and MO2058 cells. These cells contain the characteristic MCL-associated chromosomal translocation t(11; 14)(q13;q32), which results in the overexpression of cyclin D1. Recently, HHDIs, such as vorinostat, have been shown to inhibit HDAC6, which results in the acetylation of hsp90 and inhibition of its ATP binding and chaperone function. Treatment with vorinostat (0.5 to 2.0 μM) induced the accumulation of the cells in the G1 and DMAG (0.1 to 0.5 μM) in the G2/M phase of the cell cycle. Both agents induced apoptosis in a dose-dependent manner (up to 50%). While vorinostat induced both p21 and p27 levels, DMAG only increased the intracellular levels of p21. Treatment with either agent depleted the intracellular levels of c-Myc, c-Raf, Akt and cdk4 in a dose dependent manner. It is well established that the chaperone association with hsp90 maintains Akt, c-Raf, cyclin D1 and cdk4 in the native and active conformation, and inhibition of hsp90 promotes their polyubiquitylation and proteasomal degradation. Notably, co-treatment with DMAG (e.g., 0.25 μM) and vorinostat (e.g., 2.0 μM), more than either agent alone, markedly attenuated the levels of cyclin D1 and cdk4, as well as the levels of c-Myc, c-Raf and Akt. The combination of DMAG and vorinostat also induced significantly more apoptosis of Jeko1 and MO2058 cells, as compared to the treatment with either agent alone (p < 0.01). These findings demonstrate that the combined treatment with vorinostat and DMAG is highly active against human MCL cells, and support the rationale to determine the in vivo efficacy and safety of the combination against human MCL.

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.

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.Experimental Design:The cells were treated with SAHA (1.0-5.0 mu mol/L) and/or trastuzumab (5-40 mu g/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 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.

Nerve growth factor (NGF) mediates the phosphorylation and signaling through the receptor tyrosine kinase TrkA, which has been shown to be expressed and active in the early hemopoietic progenitor cells, as well as in the K562 and TF1 leukemia cell lines and AML-ETO-expressing human acute leukemia cells. In AML, a 75-amino acid deletion mutant of TrkA (ΔTrkA) has also been demonstrated to be constitutively active as a pro-growth and pro-survival protein through ERK1/2 and Akt activation. We have previously reported that that the ATP bound molecular chaperone hsp90 binds the leukemia associated Bcr-Abl and FLT-3 tyrosine kinases as client proteins, maintaining them in a properly folded and active conformation, and that geldanamycin analogue hsp90 inhibitors disrupt this chaperone association, resulting in polyubiquitylation and proteasomal degradation of the client proteins. In the present studies, we investigated a) whether TrkA is a client protein of hsp90 and b) the effect of the novel and highly soluble hsp90 inhibitor DMAG (Kosan Biosciences Inc.) on TrkA levels and activity in mouse myeloid 32D cells with or without the ectopic expression of ΔTrkA (32D/ΔTrkA cells), as well as on endogenous levels of wild-type (WT) TrkA in K562 and TF1 cells. Exposure to 0.25 or 1.0 μM DMAG attenuated the levels of WT TrkA in K562, TF1 and 32D, as well as ΔTrkA in 32D/ΔTrkA cells. Co-treatment with the proteasome inhibitor bortezomib (100 nM) restored DMAG mediated depletion of WT TrkA in K562 cells, suggesting that DMAG induced the polyubiquitylation and degradation of TrkA by the 26S proteasome. In K562 cells, immunoprecipitation (IP) with monoclonal anti-TrkA antibody followed by immunoblot (IB) analyses with anti-hsp90 antibody (or IP with anti-hsp90 followed by IB with anti-TrkA antibody) showed that TrkA binds to hsp90, which is inhibited by treatment with DMAG. Following suspension of K562 cells in a serum free medium containing 100 ng/ml of NGF, the levels of pTrkA, pERK1/2 and pAkt significantly increased within 5 to 10 minutes. Co-treatment with 1.0 μM DMAG inhibited pTrkA and pERK1/2 induction, suggesting that hsp90 chaperone function may be required for TrkA activity. Exposure to DMAG also depleted the levels of the other hsp90 client proteins, including c-Raf, Akt and Bcr-Abl in K562 cells, which was associated with growth arrest and apoptosis in a dose-dependent manner. These findings demonstrate that TrkA may be an hsp90 client protein, and hsp90 inhibition by treatment with DMAG would deplete WT or mutant TrkA levels and activity in human leukemia cells. These findings suggest that hsp90 inhibitors may be effective against human acute leukemia cells that may depend on the activity of mutant or WT TrkA for growth and survival.

Human enhancer of Zeste homolog (Ezh2) protein belongs to Polycomb Repressive Complex (PRC) 2, which also includes Eed and Suz12. Ezh2 has been shown to promote cellular transformation, and increased Ezh2 expression has been strongly correlated with the invasiveness of prostate and breast cancers. The enzymatically-active Ezh2-containing, PRC2 complex possesses histone methyl transferase (HMTase) activity mediated by the SET domain of Ezh2, which is involved in the methylation of histone (H) 3, lysine (K)-27 and −9. Through this, the PRC2 complex regulates the expression of homeobox domain containing HOX family of transcription factors including HOXA9 and MEIS1, which have been shown to be involved in human leukemogenesis. Co-expression of HOXA9 and MEIS1 is common in acute myeloid leukemia and collaborates in the leukemogenesis. In the present studies, we determined the effect of hydroxamate histone deacetylase inhibitors LBH589 and LAQ824 on Ezh2 and PRC2 complex proteins and their activity in the cultured (K562, LAMA-84, U937 and HL-60) and primary human AML cells. Exposure to 10 to 100 nM of LBH589 or LAQ824 for 24 hours, in a dose dependent manner depleted the protein levels of Ezh2, as well as reduced Suz12 and Eed levels in the cultured and primary leukemia cells. This was associated with decreased levels of the tri- and dimethylated K27 and increased acetylation of K27, both on H3. Correspondingly, these H3 modifications were associated with a significant decline in the levels of HOXA9 and MEIS1. As has been previously reported, treatment with LBH589 and LAQ824 induced p21, as well as caused cell cycle growth arrest in the G1 phase and apoptosis of the cultured and primary AML cells. We next determined whether knockdown of Ezh2 by siRNA to Ezh2 also induces growth inhibitory and cytotoxic effects against the leukemia cells. In the cultured and primary acute leukemia cells, knockdown of Ezh2 expression (by ~80%) by Ezh2 siRNA, but not by the control siRNA, was associated with depletion of Suz12 levels and increased H3K27 acetylation. This was associated with increase in the % of cells in the G1 phase of the cell cycle and significant inhibition of their clonogenic survival in colony growth assays. Co-treatment with LBH589 and siRNA to Ezh2 caused further decline in the Ezh2 levels and increased loss of clonogenic survival of the leukemia cells. These findings suggest that down modulation of Ezh2 and PRC2 complex and its HMTase activity may inhibit clonogenic survival of human acute myeloid leukemia cells. Additionally, combined effect of the knockdown of Ezh2 and its activity along with treatment with LBH589 or LAQ824 may have an improved anti-leukemia efficacy, especially against human AML where HOXA9 and MEIS1 genes are deregulated.

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.

560 Background: Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase (HDAC) inhibitor (HDI), which inhibits class I and II HDACs, induces hyperacetylation of the core nucleosomal histones, increases p21WAF1, and induces growth arrest and apoptosis of human breast cancer cells. In the present studies, we determined the effects of SAHA on Her-2, AKT and c-Raf (all hsp90 client proteins) levels, as well as on the cytotoxic effects of docetaxel and trastuzumab on human breast cancer BT-474 and SKBR-3 cells containing Her-2 amplification. Methods: The cells were treated with SAHA (1.0 to 5.0 μM) and/or trastuzumab (10 to 20 μg/ml) or docetaxel (5 to 20 nM) for 24 to 48 hours. Following this, % of apoptotic cells was determined or Western blot analyses of p21, p27, acetylated histones H3 and H4, AKT, c-Raf, and Her-2, as well as of Bcl-2, Bcl-xL, Bak, Bax, Bim, XIAP and survivin were performed. Attenuation of HDAC6 levels was achieved by siRNA to HDAC6 and estimated by immunoblot analysis. Synergistic interaction between drugs was evaluated by median dose-effect and isobologram analysis. Results: Treatment with SAHA upregulated p21 and p27 levels, increased the % of cells in G2M phase of the cell cycle, as well as induced apoptosis of the cells. This was associated with upregulation of pro-death Bak and Bim, as well as attenuation of the levels of pro-survival XIAP, survivin, Bcl-2, Bcl-xL and Her-2 proteins. SAHA treatment induced acetylation of hsp90. Attenuation of HDAC6 levels by siRNA to HDAC6 also caused acetylation of hsp90, indicating the mechanistic role of HDAC6 in SAHA-mediated acetylation of hsp90. This reduced the chaperone association of Her-2 with hsp90, which promoted the degradation of Her-2 by the proteasome. Co-treatment with SAHA and docetaxel or trastuzumab exerted synergistic apoptotic effects. Co-treatment with SAHA and trastuzumab caused greater attenuation of Her-2, p-AKT, as well as greater induction of p27 and Bim. Conclusions: These pre-clinical findings support the development of SAHA in combination with docetaxel and/or trastuzumab against Her-2 amplified breast cancer. No significant financial relationships to disclose.

Hsp90 is an ATP-dependent molecular chaperone, which helps in folding its client proteins, e.g., Bcr-Abl, FLT-3, c-Raf and Akt, into active conformation. Geldanamycin analogue, 17-AAG (Kosan Biosciences Inc., Hayward, CA) inhibits the chaperone function of hsp90, which promotes polyubiquitylation and proteasomal degradation of the misfolded hsp90 client proteins. We recently reported that, by inhibiting the activity of histone deacetylase 6, the hydroxamate HDIs such as vorinostat (Merck & Co., Inc.) induce acetylation and inhibition of hsp90, thus also causing the depletion of its client proteins. In the present studies, we determined the anti-leukemia effects of the novel, highly soluble, hsp90 antagonist IPI504 (Infinity Pharmaceuticals), which, in vitro and in vivo, interconverts with 17-AAG, ± vorinostat, against human cultured or primary, wild type or mutant Bcr-Abl or mutant FLT-3 containing acute leukemia cells. Treatment with IPI504 (0.5 to 2.0 μM) for 24 to 48 hours, in a dose dependent manner, induced apoptosis of WT Bcr-Abl-expressing K562 and LAMA-84 cells. This was associated with attenuation of the levels of Bcr-Abl, pCrkL, pSTAT5, c-Raf and pAkt. In a dose dependent manner (50 to 500 nM for 48 hours), IPI504 also induced apoptosis of FLT-3 internal tandem duplication (ITD)-containing human acute leukemia MV4-11 cells, which was associated with attenuation of the levels of FLT-3, pAkt, pSTAT5, pERK1/2. Notably, treatment with IPI504 induced similar level of apoptosis of mouse bone marrow BaF3 cells, which had been transformed and rendered IL-3 independent for growth by ectopic expression of WT Bcr-Abl, its P-loop (Bcr-Abl-E255K) or highly imatinib mesylate (IM) resistant, contact-inhibition (Bcr-Abl-T315I) point mutant. This was also associated with attenuation of the levels of WT and mutant Bcr-Abl-E255K or Bcr-Abl-T315I. In previous studies we had demonstrated that treatment with vorinostat depletes WT and mutant Bcr-Abl levels and induces apoptosis of expressing human leukemia cells. Therefore, we determined the effect of the co-treatment of IPI504 (1.0 μM) and vorinostat (1.0 μM) against cultured or primary human CML cells. Co-treatment with IPI504 and vorinostat induced significantly more apoptosis of K562 and MV4-11 cells, which was associated with more depletion of WT-Bcr-Abl and FLT-3-ITD levels in K562 and MV4-11 cells, respectively. Notably, co-treatment with IPI504 and vorinostat, versus treatment with either agent alone, also induced more apoptosis of primary CML cells (4 samples) derived from patients with IM-resistant CML, including a sample of cells documented to have Bcr-Abl-T315I mutation. Additionally, as compared to treatment with either agent alone, the combination of IPI504 and vorinostat also induced more apoptosis of primary AML cells (4 samples), including two samples that contained FLT-3-ITD. These findings demonstrate that the combination of IPI504 with vorinostat exerts a high level of in vitro activity against FLT-3-ITD-containing acute leukemia, as well as against highly IM-resistant mutant Bcr-Abl-expressing leukemia cells.