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Auranofin (AF) is an oral disease-modifying anti-rheumatic agent. Using a high throughput screening assay of 2,816 FDA-approved drugs against primary tumor cells in vitro, we have previously identified AF as one of five drugs with selective anti-Chronic Lymphocytic Leukemia (CLL) activity. We have shown that AF induces oxidative stress and apoptosis in CLL cells in vitro independent of classic prognostic markers (ASH Meeting Abstract 865, 2012). Here, we evaluated the in vivo effects of AF on CLL using blood samples collected from six patients treated with single agent AF at the NIH as part of a multi-center clinical trial led by the University of Kansas Cancer Center (NCT01419691). Five CLL patients and one patient with Small Lymphocytic Lymphoma (SLL) were enrolled in this open-label phase II study of AF in relapsed/refractory patients. Patients were started on AF 6 mg daily administered orally on 28-day cycles, with a dose escalation to 9 mg after the first cycle if no grade ≥2 toxicity occurred. AF was generally well tolerated. The best response was stable disease. Sequential blood samples were obtained prior to, and during the first cycle on drug. CLL cells were isolated by density gradient centrifugation. To study the in vivo effect of AF on redox balance we used dihydroethidium (DHE) and concomitantly measured cell viability using 3,3′-dihexyloxacarbocyanine iodide (DiOC6) in CD19 gated fresh cells by flow cytometry. Within 24 hours of the first dose AF induced an average 1.8 fold increase in DHE+ CLL cells indicating increased levels of ROS. Concomitantly there was a similar increase in apoptosis as shown by Annexin V staining. The increase in ROS production and apoptosis was transient; by day 7 all these changes had reverted to baseline or were even below baseline in three patients. A concomitant and equally transient decrease in the absolute lymphocyte count (ALC) and lactate dehydrogenase (LDH) level was also observed. To investigate the in vivo effect of AF on tumor biology, total RNA (2.5 μg) from CLL cells of three patients treated with AF was profiled on Human Genome U133 Plus 2.0 arrays (Affymetrix). Three time points were analyzed: baseline (D0), after one dose of AF (D1), and a week later (D7). Compared to baseline, there were 182 genes (29-up, 153-down) whose expression changed >1.5-fold at P Taken together, in vivo AF appears to transiently decrease anti-oxidant defenses regulated by NRF2 concomitant with an increase in cellular ROS levels and induction of some degree of cellular apoptosis. However, in vivo an adaptive response emerged that mobilized potentially compensatory mechanisms, including induction of pathways that may prevent intracellular accumulation of AF and a decrease in cellular processes that generate ROS, such as protein folding. The transient nature of the cellular response in vivo is consistent with the limited clinical activity seen in our patients. Plans are underway to determine the AF maximum tolerated dose in CLL patients. We will continue to evaluate the in vivo effects of AF on CLL cells at higher doses. Supported by the Intramural Research Program of NHLBI and NCATS, NIH; a grant from The Leukemia and Lymphoma Society Therapy Acceleration Program to The Learning Collaborative™, as well as philanthropic support. We thank our patients for participating in these research studies. Disclosures: Off Label Use: Auranofin is not FDA approved for use in CLL.

During DNA damage response (DDR), stability and activity of ATR and CHK1 is essential for the cell cycle arrest and subsequent DNA repair through BRCA1-regulated homologous recombination (HR). We have previously reported that ATR and CHK1 are hsp90 client proteins and treatment with hsp90 inhibitor sensitizes cancer cells to DNA damage. In the present studies, we determined that treatment with pan-HDAC inhibitors vorinostat (VS) and panobinostat (PS) induced hyper-acetylation and inhibition of chaperone function of nuclear hsp90, leading to proteasomal degradation and depletion of ATR, CHK1 and BRCA1. This led to inhibition of DDR and DNA repair following ionizing radiation (IR) through destabilization of ATR-CHK1 and BRCA1 proteins. Specific siRNA-mediated knockdown of HDAC3 but not of HDAC1 or HDAC2 also induced hyper-acetylation of the nuclear hsp90 and depletion of ATR and CHK1, indicating that among the class I HDACs, HDAC3 is the deacetylase for the nuclear hsp90. We next determined whether, by depleting DDR proteins and BRCA1 and inducing ‘BRCAness’, treatment with VS would sensitize the triple negative breast cancer (TNBC) SUM159PT and MB-231 (lacking BRCA1 mutation) as well as BRCA1 mutant HCC1937 cells to the PARP inhibitor ABT888 (veliparib). Indeed, combined treatment with VS or PS and ABT888 (10 to 20 μM) for 48 hours synergistically induced apoptosis (combination indices (CI) by isobologram analysis being < 1.0) of TNBC cells with (HCC1937) or without BRCA1 mutation (MB-231 and SUM159PT cells). As compared to treatment with each agent alone, co-treatment with VS and ABT888 also induced significantly more DNA strand breaks, as demonstrated by higher γ-H2AX levels. Combined treatment also induced markedly greater tail moment, as determined by the comet assay that evaluates the SYBR® Green-stained DNA tails by fluorescent microscopy. Co-treatment with VS and ABT888 also induced more BH3 domain-only pro-death protein BIM, while knockdown of BIM by shRNA significantly reduced the apoptosis induced by co-treatment with VS and ABT888. It is noteworthy that treatment with VS also sensitized the TNBC cells to cisplatin (2.0 to 10 μM)-induced apoptosis. Moreover, co-treatment with VS and ciplatin synergistically induced apoptosis of TNBC cells (CIs < 1.0). These findings indicate that treatment with pan-HDAC inhibitors VS or PS creates ‘BRCAness’, and in combination with a PARP inhibitor or cisplatin synergistically induces apoptosis in human TNBC cells. These findings support a compelling rationale to confirm whether the combination of VS or PS with ABT888 and cisplatin would be a highly active treatment of human TNBC, irrespective of its expression of mutant BRCA1. Citation Format: Warren C. Fiskus, Rekha Rao, Soumyasri Das Gupta, Lata Chauhan, Kapil N. Bhalla. Induction of ‘BRCAness’ by treatment with histone deacetylase inhibitors sensitizes human triple negative breast cancer cells to PARP inhibitor and cisplatinum. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1011. doi:10.1158/1538-7445.AM2013-1011

Raman spectroscopic studies of zircon CeVO4 is carried out at high pressures upto 13 GPa. Further pressurization leads to vanishing of Raman intensity. Changes in the Raman spectra across the zircon to monoclinic transition are reported. In addition, evolution of Raman spectra of the recovered monazite phase is reported.

Vibrational properties of delafossite CuLaO2 are investigated using Raman spectroscopy at ambient conditions and at high pressures. Changes are observed in the Raman spectrum at around 1.8 GPa which could be related to a phase transition. The changes observed are reversible from 7 GPa.

During the recent decade, the world has witnessed the rapid growth of MNEs from emerging economies. Their increasing participation in cross-border mergers and acquisitions (M&As) has raised great a...

e13568 Background: The gold-containing compound auranofin (AF) (Ridaura), is a treatment of rheumatoid arthritis. Recently, AF was shown to inhibit thioredoxin reductase (TRR), increase reactive oxygen species (ROS) and induce apoptosis in cancer cells. Methods: We determined the ability of AF (250 to 1000 nM) to induce oxidative, proteotoxic and lethal endoplasmic reticulum (ER) stress in CD19+ primary CLL cells, including those with deletion of chromosome 17p. Results: Treatment with AF induced 20 to 40% increase in ROS levels, decreased TRR activity (mean of 45%), but not its protein expression, in CLL cells. AF-mediated oxidative stress induced NRF2 activity with increase in hemeoxygenase-1 (HO-1) and glutamate cysteine ligase modifier (GCLM) levels. AF induced ER stress, associated with increase in GRP78 and the pro-apoptotic transcription factor CHOP protein levels. AF also induced the pro-apoptotic BH3-only domain protein BIM. Exposure to AF increased the intracellular levels of misfolded polyubiquitylated proteins. This disrupted the binding of heat shock protein (hsp) 90 with histone deacetylase 6 (HDAC6), resulting in hyperacetylation and inhibition of the chaperone function of hsp90. This led to proteasomal degradation of CLL-relevant hsp90 client proteins, including ZAP70, c-Raf and AKT. Nuclear STAT3 levels also declined. Exposure to AF induced significantly more apoptosis (range 40 to 60%) in primary CLL cells, as compared to CD19+ normal B cells and CD34+ human cord blood and bone marrow progenitor cells (< 15% apoptosis) (p < 0.01). AF treatment also induced apoptosis in CD19+ cells from patients with poor prognosis CLL with deletion of 17p or with deletion of 13q. Co-treatment with AF and the proteasome inhibitor carfilzomib or the GCLM antagonist buthionine sulfoximine (BSO) synergistically induced apoptosis in primary CLL cells. A FDA IND-supported phase II clinical trial of AF (6 to 9 mg PO/day) with correlative biomarker analysis (as above) is underway and three patients are enrolled. Conclusions: These findings support the full evaluation of clinical activity and predictive biomarkers of response to the re-purposed AF in patients with CLL.

10549 Background: LSD1 (KDM1A) is FAD-dependent histone H3K4Me2 demethylase. Inhibition of LSD1 increases H3K4Me3-a permissive mark for gene expression, and inhibits growth of pluripotent cancer cells. We have previously noted that treatment with the histone deacetylase (HDAC) inhibitor panobinostat (PS) depletes EZH2 (the catalytic subunit of the polycomb repressive complex 2, PRC2) and disrupts its interaction with the other PRC2 proteins, attenuates LSD1, and de-represses growth inhibitory and pro-apoptosis genes. Methods: In the present studies, we determined the chromatin effects and the pre-clinical in vitro and in vivo anti-AML activity of the novel, non-monoamine oxidase, reversible inhibitor of LSD1, HCI2509 (100 to 500 nM), alone and in combination with PS, utilizing cultured (OCI-AML3 and HL-60) and primary human AML blast progenitor cells. Results: Treatment with HCI2509 dose-dependently increased the levels of H3K4Me3, p16, p27, and CEBPα in cultured AML cells. This correlated with inhibition of cell growth and induction of morphologic differentiation in AML cells. Exposure to HCI2509 disrupted the binding of LSD1 with the co-repressor CoREST and HDAC1. Treatment with PS (10 to 50 nM) dose-dependently depleted not only LSD1 but also EZH2, SUZ12 and BMI1 in AML cells. Co-treatment with PS enhanced the chromatin modifying effects of HCI2509. The combination also synergistically induced loss of viability of cultured and primary AML cells (combination indices, CI

We have recently reported that in addition to checkpoint kinase 1 (CHK1), ataxia-telangiectasia mutated and Rad3- related (ATR) is chaperoned by hsp90. Treatment of breast and cervical cancer cells with hsp90 inhibitor induces proteasomal degradation and depletion of ATR and CHK1. This results in impairment of the DNA damage response (DDR) and causes sensitization to α-irradiation and replication stress due to hydroxyurea (Mol Cancer Ther 10:1194, 3011). In the present studies, we determined that treatment with pan-histone deacetylase (HDAC) inhibitor panobinostat (20 to 50 nM) or vorinostat (0.5 to 2 μM) induced polyubiquitylation and depletion of ATR and CHK1. Co-treatment with bortezomib, a proteasome inhibitor, restored PS-mediated depletion of ATR and CHK1. Notably, treatment with panobinostat induced hyperacetylation of both nuclear and cytoplasmic hsp90. This inhibited the chaperone association of nuclear hsp90 with ATR and CHK1, thereby promoting their degradation. PS treatment induced γ-H2AX levels, which were inhibited by co-treatment with N-acetylcysteine, suggesting that PS-induced ROS is involved in DNA damage. Moreover, PS treatment further increased α-irradiation induced DNA damage and its repair, characterized by the increase in α-irradiation-induced comet tail moment and the lack of DNA repair-mediated attenuation of the comet tail moment. To determine which class I HDAC is involved in deacetylation of nuclear hsp90, we individually knocked down (KD) HDAC 1, 2 and 3, by utilizing specific shRNA, and determined the effect on nuclear hsp90 hyperacetylation and the levels of ATR and CHK1. Our findings show that HDAC3 binds to hsp90 and KD of HDAC3, but not of HDAC1 or 2, caused hyperacetylation of nuclear hsp90 and depletion of ATR and CHK1. This was also observed in HDAC3 null mouse embryonic fibroblasts (MEFs). Ectopic overexpression of HDAC3 inhibited nuclear hsp90 acetylation in transformed cells. These findings demonstrate that HDAC3 is the nuclear hsp90 lysine deacetylase. They also demonstrate that genetic KD of HDAC3, or its inhibition by pan-HDAC inhibitors, induces hyperacetylation of hsp90, mediates loss of hsp90 chaperone association and depletion of ATR and CHK1, abrogates α-irradiation-induced DDR, and results in sensitization of transformed cells to DNA damage due to α-irradiation or replication stress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4698. doi:1538-7445.AM2012-4698

LSD1 or KDM1A is a FAD-dependent demethylase, with homology to amine oxidases. LSD1 demethylates di- and mono-methylated lysine (K)4 on histone H3, reducing the permissive H3K4Me3 chromatin mark. LSD1 inhibition attenuates growth of pluripotent cancer cells with OCT4 and SOX2 expression. LSD1 complexes with HDAC1/2 and Co-REST, and high LSD1 expression confers poor prognosis in cancers. Previous studies have shown that HDAC inhibitors downregulate LSD1 thru Sp1 inhibition. Inhibition of LSD1 leads to increase in H3K4Me3-a permissive mark for gene expression. HCI2509 is an FAD-binding pocket, non-MAOA and MAOB LSD1 inhibitor. In the present studies, we determined the chromatin-modifying and cytotoxic effects of HCI2509 alone and in combination with the pan-histone deacetylase inhibitor, panobinostat (PS) in cultured (HL-60 and OCI-AML3) and primary human acute myeloid leukemia (AML) cells. Treatment with HCI2509 (100 to 500 nM), dose-dependently increased the levels of H3K4Me2 & Me3, p16 and p27, which was associated with inhibition of cell proliferation as measured by a decrease in Ki-67 expression. Chromatin immunoprecipitation followed by PCR demonstrated that treatment with HCI2509 increased the H3K4Me3 mark on the promoters of KLF4, HMOX1, and CDH1 in AML cells. Treatment with HCI2509 also induced C/EBPα expression and features of morphologic differentiation of cultured and primary AML cells. Treatment with HCI2509 (25 mg/kg B.I.W. via IP injection) significantly improved the survival of NOD/SCID mice bearing OCI-AML3- AML xenografts. We have previously reported that treatment with PS (Novartis Pharma) depleted PRC2 complex proteins EZH2, and SUZ12 but also modestly depleted LSD1 expression in AML cells. Co-treatment with PS enhanced the chromatin modifying effects of HCI2509 on K4 of Histone H3 in AML cells. Co-treatment with HCI2509 and PS synergistically induced apoptosis of the cultured AML cells (combination indices, CI Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1043. doi:1538-7445.AM2012-1043

A first-principles computation of tetragonal distortion, structural stability, and equation of state has been carried out for indium, using the linear muffin-tin orbital electron-band-theory technique. Results on the variation of tetragonal distortion (c/a) with compression show a broad maximum around V/V 0 ≅ 0.8 (19 GPa) in agreement with experimental data. We do not find 5s5p-to-5d electron transfer as speculated to be the cause of the maximum in c/a. However, our results reveal that the core overlap resulting from broadening of the 4d states is the probable reason for the turnover in the c/a ratio. Within the atomic-sphere approximation we find the face-centered orthorhombic phase to be marginally stable beyond 56 GPa in accord with the experiments of Takemura et al.

Structural transitions in diamond at ultrahigh pressures are of great current interest. Theoretical estimates show that the first transition in diamond would be to the bc‐8 structure near 11 Mbar. However, the stability of primitive hexagonal (ph) structure of carbon has not been studied so far though Si and Ge, also group IVB elements, are known to transform to this structure under pressure. Hence we carried out the first principles linear muffin‐tin orbital (LMTO) method total energy calculations for the ph structure (with empty spheres) in comparison with other postulated structures, with the motivation to examine whether diamond would transform to this structure. We find that ph and the simple cubic structures are of comparable stability at megabar pressures, but ph becomes more stable near 17 Mbar. Calculations are also reported for ω structure of carbon, which also occurs in group IVA elements under pressure.

Results of linear muffin‐tin orbital calculations for tetragonal distortion under pressure in indium are compared with the existing experimental data. We find that c/a values as a function of volume show a broad maximum and then show a decrease in agreement with the data. Within atomic sphere approximation we simulate the experimentally observed structural phase transition. The possible cause of turnover in the axial ratio is also discussed.

Abstract 733 NPM1 is a nucleolar phosphoprotein that functions as an oligomeric molecular chaperone for both proteins and nucleic acids. NPM1 is mutated in approximately one-third of patients with AML, especially those with the normal karyotype. The mutant (mt) NPM1 contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal, which causes mtNPM1 to be localized in the cytoplasm (NPM1c+). NPM1 has an N-terminal conserved, hydrophobic, oligomerization domain, which is common to all isoforms of NPM1 and critical for its chaperone activity. Recently, NSC348884 was identified as a small molecule inhibitor that disrupts wild-type (WT) and mtNPM1 dimer/oligomer formation, inducing apoptosis of cancer cells. Here, we determined the effects of specifically targeting mtNPM1 in cultured and primary AML cells. Treatment with shRNA specific to mtNPM1 or specific to WT and mtNPM1, as compared to the control non-targeted shRNA, induced p53, p21, p27 and CEBPα, but down regulated the levels of HOXA9 and Meis1 in the AML OCI-AML3 cells that heterozygously expressed mt NPM1. This led to an increase in the % of cell cycle G1 phase and a decline in % S phase cells, as well as marked inhibition of the colony growth of OCI-AML3 cells (p < 0.01). Knockdown of mtNPM1 induced myeloid/monocytic differentiation of OCI-AML3, as detected morphologically (∼30% over control) and induction of CD11b by flow cytometry. Knockdown of mtNPM1 also induced the proteins levels of RARα (two-fold) and sensitized OCI-AML3 cells (but not cultured AML cells with wtNPM1) in vitro to all-trans retinoic acid (ATRA) (0.25 to 2.0 μM)-induced p53, p21 and C/EBPα, as well as ATRA-induced differentiation and apoptosis (p < 0.01). Furthermore, mtNPM1 knockdown sensitized OCI-AML3 cells to cytarbine (0.5 to 2.0 μM)-induced apoptosis. Notably, mice bearing OCI-AML3 cells transduced with NPM1 shRNA and showing knockdown of total NPM1 demonstrated significantly less spleen enlargement and dramatically improved survival, as compared to the control mice (p < 0.01). Exposure to NSC348884 (1.0 to 3.0 μM) dose-dependently induced apoptosis and sensitized OCI-AML3 and primary AML cells expressing NPM1c+ to ATRA-induced apoptosis (p < 0.01). NSC348884 treatment (3 μM) significantly disrupted the dimer (∼64 kDa) formation and increased the monomeric NPM1 levels in OCI-AML3 but not in HL-60 cells, as analyzed by native polyacrylamide gel electrophoresis (PAGE). OCI-AML3 cells transfected with FLT3-ITD lentivirus (OCI-AML3/FLT3-ITD cells) exhibited several-fold higher expression of FLT3-ITD, without causing any change in the levels of NPM1. As compared to the vector control cells, OCI-AML3/FLT3-ITD cells, as well as primary AML cells expressing both mtNPM1 and FLT3-ITD, were less sensitive to treatment with NSC348884 and/or ATRA. Notably, combination with NSC348884 (3.0 μM) and PKC412 (100 to 500 nM), a FLT3 antagonist (Novartis Pharmaceuticals), induced more apoptosis than either agent alone against OCI-AML3/FLT3-ITD and primary AML cells expressing both mtNPM1 and FLT3-ITD (p < 0.01). These findings demonstrate that knocking down of the levels and oligomerization of mtNPM1 and WT NPM1 induces differentiation and growth inhibition, as well as sensitizes AML cells with mtNPM1 to ATRA-induced differentiation and apoptosis. Furthermore our findings support the rationale of combining NPM1 antagonist with FLT3 inhibitor against AML with dual expression of mtNPM1 and FLT3-ITD. Disclosures: No relevant conflicts of interest to declare.

Abstract 929 Auranofin (AF) (Ridaura®) is an oral, FDA-approved, lipophilic, gold-containing compound used for the treatment of rheumatoid arthritis, based upon its anti-inflammatory and immunosuppressive effects. In the present studies, we determined for the first time the lethal activity of AF (250 to 1000 nM) and its underlying mechanism(s) in CD19+ primary CLL cells, including those with del(17p13.1). We demonstrate that treatment with AF induced 20 to 40% increase in reactive oxygen species (ROS) levels (assessed by flow cytometry), decrease in thioredoxin reductase (TRR) activity (mean reduction of 45%) without alteration in the expression of TRR, as determined in the AF treated cell lysates of CLL cells. AF treatment also increased the levels of thioredoxin binding protein 2. These findings were associated with induction of nuclear factor erythroid 2-related factor 2 (NRF2) activity marked by substantial increase in hemeoxygenase-1 (HO-1) levels. Thus, AF treatment perturbed the redox status in CLL cells. Consistent with increased levels of intracellular ROS, AF treatment also induced the markers of ER stress response including the induction of GRP78 and a sustained increase in the levels of the pro-apoptotic transcription factor CHOP (CAAT/enhancer binding protein homologous protein). This was associated with induction of the levels of the pro-death BH3 only domain protein BIM, which is a known mediator of lethal ER stress. Exposure to 1000 nM AF induced significantly more apoptosis (range 40 to 60%) in primary CLL cells, as compared to CD19+ normal B cells and CD34+ human cord blood and bone marrow progenitor cells (< 15% apoptosis) (p < 0.01). AF treatment induced apoptosis to a similar extent in primary CLL cells with del(17p13.1) as compared to other genetic abnormalities. Based on the observations that AF treatment induced oxidative and ER stress, we determined whether exposure to AF also increased the intracellular levels of misfolded and polyubiquitylated proteins, which would disrupt the cytosolic complex consisting of the heat shock protein (hsp) 90, heat shock factor (hsf) 1, histone deacetylase 6 (HDAC6) and p97/VCP, resulting in hyperacetylation and inhibition of the chaperone function of hsp90. Indeed, treatment with AF disrupted the association between hsp90 and HDAC6, resulting in hyperacetylation of hsp90 and depletion of the levels of HDAC6 in CLL cells. AF mediated hyperacetylation of hsp90 and α-tubulin was associated with proteasomal degradation of several of the CLL-relevant hsp90 client proteins, including ZAP70, c-Raf and AKT. Co-treatment of CLL cells with AF and the proteasome inhibitor carfilzomib (20 nM) significantly restored the levels of ZAP70, c-Raf, AKT and HDAC6. Finally, relatively lower levels of AF (250 nM) combined with the pan-HDAC inhibitor panobinostat (20 nM) or carfilzomib (5 nM) induced more cell death of primary CLL cells than each agent alone. These findings indicate that treatment of primary CLL cells with AF results in oxidative and ER stress. AF treatment also promotes the depletion of pro-survival CLL-relevant hsp90 client proteins. Collectively, our data creates a strong rationale for determining the in vivo activity of AF in patients with CLL including those with del(17p13.1). Disclosures: Reyes: Millennium, Sanofi Aventis: Consultancy.

Abstract 2429 PRC (polycomb repressive complex) 2 contains three core protein components, i.e., EZH2, SUZ12 and EED, of which EZH2 has the SET domain with its intrinsic histone methyltransferase activity that mediates the trimethylation (Me3) of lysine (K) 27 on histone (H) 3-a repressive chromatin mark for gene expression. We have previously reported that treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep as well as treatment with the pan-histone deacetylase inhibitor panobinostat (PS, Novartis Pharma) deplete PRC2 complex proteins. LSD1 (KDM1A) is a demethylase of H3K4Me2, and inhibiton of LSD1 leads to increase in H3K4Me3-a permissive mark for gene expression. In the present studies, we determined the chromatin-modifying and cytotoxic effects of LSD1 inhibition alone and in combination with PS or DZNep in cultured (JeKo-1 and Z138C) and primary human Mantle Cell Lymphoma (MCL) cells. Treatment with the non-amine oxidase reversible inhibitor of LSD1 CIT0665 (250 to 1000 nM), or the more potent analogue HCI2509 (20 to 250 nM), dose-dependently increased the levels of H3K4Me2 & Me3, p21 and p27, while decreasing the levels of cyclin D1, which was associated with inhibition of cell proliferation and accumulation of the MCL cells in the G1 phase of the cell cycle. Abrogation of LSD1 by a specific shRNA treatment also induced similar chromatin, cell cycle and growth inhibitory effects. Exposure to CIT0665 or HCI2509 disrupted the binding of LSD1 with the co-repressor CoREST and HDAC1, without affecting the levels of these proteins. As noted above, treatment with PS (10 to 50 nM) dose-dependently depleted the levels of not only EZH2, SUZ12 and the PRC1 complex protein BMI1, but also of LSD1 in MCL cells. PS treatment alone also depleted the levels of AKT, cRAF, CDK4 and cyclin D1, as well as induced cell cycle growth inhibition and apoptosis of MCL cells. Co-treatment with PS enhanced the chromatin modifying effects of CIT0665 or HCI2509. The combination synergistically induced apoptosis of the cultured MCL cells (combination indices, CI Disclosures: Sharma: Novartis: Research Funding.

Presence of aneuploidy increases the intracellular levels of misfolded proteins and proteotoxic stress phenotype in cancer cells, which is exploited and further accentuated by the therapeutic use of a proteasome inhibitor, e.g., the novel, orally bioavailable carfilzomib (CF). This results in the disruption of the cytosolic complex of the histone deacetylase 6 (HDAC6), the ATPase and segregase p97 and the heat shock factor 1 (HSF1)-a transcription factor involved in the transactivation of heat shock proteins (hsps). In the present studies, we demonstrate that treatment of the pancreatic cancer Hs766T and Panc1 cells with CF increased the levels of misfolded polyubiquitylated proteins, which disrupts the repressive HSF1/hsp90/HDAC6/Vp97 complex. This induces the phosphorylation, nuclear localization and activation of HSF1, which transactivated hsps (e.g., hsp70, hsp40 and hsp27) as an adaptive response to CF-induced proteotoxic stress. Treatment with CF also induced endoplasmic reticulum (ER) stress, represented by upregulation of GRP78 and the pro-apoptotic transcription factor CHOP, which induced the BH3 only pro-apoptotic protein Bim. Treatment with CF also dose-dependently decreased the viability of Hs766T and Panc1 cells. To further elucidate the role of HSF1 in the adaptive, protective response to CF-induced proteotoxic stress, we stably knocked down HSF1 in Hs766T cells. As compared to the control cells, following heat shock, Hs766T cells with knockdown (KD) of HSF1 displayed reduced expression of hsps. In the HSF1-KD versus the control Hs766T cells, treatment with CF resulted in a dose-dependent induction of polyubiquitylated proteins, GRP78, CHOP, and Bim, as well as induced significantly more apoptosis. HSF1-KD versus the control Hs766T cells exhibited markedly lower levels of HDAC6 and, consequently, displayed reduced CF-induced perinuclear protective aggresome formation, as determined by confocal microscopy. Additionally, as compared to the control cells, Hs766T cells with HSF1-KD demonstrated higher levels of p-AMPK and CF (5 to 20 nM) or the pan-HDAC inhibitor panobinostat (PS, 50 nM)-induced autophagy, as demonstrated by the accumulation of LC3-II (by immunoblot analysis) and LC3 puncta by immunofluorescent staining. Importantly, co-treatment with the autophagy inhibitor hydroxy-chloroquine (HCQ) induced more CF-induced cell death of HSF1-KD versus the control Hs766T cells. Using an acid-sensitive mCherry-GFP-LC3 reporter, we determined that the induction of LC3-II in the Hs766T HSF1-KD cells is the result of enhanced autophagic flux and not due to inefficient fusion of autophagosomes with lysosomes. Collectively, these findings indicate that inhibition of the transcriptional activity of HSF1 sensitizes pancreatic cancer cells to CF or PS-induced cells death, which is augmented by co-treatment with an autophagy inhibitor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2076. doi:10.1158/1538-7445.AM2011-2076

NPM1 is an oligomeric, nucleolar phosphoprotein that functions as a molecular chaperone for both proteins and nucleic acids. NPM1 plays multiple roles in cell growth and proliferation by participating in diverse biological processes, including ribosome biogenesis and transport, centrosome duplication, DNA repair, transcriptional regulation and histone chaperoning. NPM1 has an N-terminal conserved, hydrophobic, oligomerization domain (residues, 1-110), which is common to all isoforms of NPM1 and critical for its chaperone activity. Recently, NSC348884 was identified as a small molecule inhibitor that disrupts NPM1 dimer/oligomer formation, inducing apoptosis of cancer cells. NPM1 is mutated in approximately one-third of patients with AML. The mutant NPM1c+ contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal (NES), which causes NPM1c+ to be aberrantly localized in the cytoplasm. Here, we determined the effects of targeting NPM1 in cultured and primary AML cells. Treatment with siRNA to NPM1, which depleted both NPM1c+ and un-mutated NPM1, significantly induced p21, decreased the % of cells in S-phase of the cell cycle, as well as induced differentiation of the AML OCI-AML3 cells that express both NPMc+ and un-mutated NPM1. This was associated with up-regulation of C/EBPα but attenuation of the levels of HOXA9 and MEIS1. Notably, treatment with NPM1 siRNA sensitized OCI-AML3 more than HL-60 cells (expressing only wild-type NPM1) to all-trans retinoic acid (ATRA) and cytarabine. Moreover, inhibition of NPM1 oligomerization by NSC348884-induced apoptosis and sensitized OCI-AML3 and primary AML cells expressing NPM1c+, but not AML or normal CD34+ progenitor cells expressing wild-type NPM1, to ATRA and cytarbine. NSC348884 was also able to sensitize primary AML cells that expressed NPM1c+ as well as FLT3-ITD to ATRA and cytarbine. Addtionally, co-treatment with NSC384884 sensitized these primary AML cells to the lethal effects of the FLT3 kinase inhibitor PKC412 (Novartis Pharma). These results show that attenuating levels or oligomerization of NPM1 selectively induces apoptosis and sensitizes AML cells expressing NPM1c+ alone or with FLT3-ITD to treatment with ATRA, cytarabine and FLT3 kinase inhibitor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3539. doi:10.1158/1538-7445.AM2011-3539

Gene silencing is mediated by multi-protein complexes PRC (polycomb repressive complexes) 1 and 2. Of the three core protein components of PRC2, i.e., EZH2, SUZ12 and EED, EZH2 has the SET domain with its intrinsic histone methyltransferase activity. This induces the trimethylation (Me3) of lysine (K) 27 on histone (H) 3-a repressive chromatin mark for gene repression. The PRC1 components include BMI1, RING1 and RING2, which are responsible for the ubiquitylation (Ub) of K119 on H2A and compaction of the chromatin at PRC2 target genes. We have previously reported that treatment with the pan-histone deacetylase inhibitor panobinostat (PS, Novartis Pharma) depletes EZH2, SUZ12 and the DNA methyltransferase (DNMT) 1. We also showed that co-treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep, further depleted PRC2 complex proteins and, in combination with PS, induced synergistic apoptosis of cultured and primary AML cells. In the present studies we determined that DZNep dose-dependently depleted EZH2, SUZ12 and BMI1 expression as well as inhibited K27Me3 on H3. DZNep treatment also induced p21, p27 and FBXO32, while depleting the levels of cyclin D1 in the cultured MCL JeKo-1 and MO2058 cells. Similar induction of p21, p27 and FBXO32 were also observed, following siRNA knockdown of EZH2. Notably, DZNep also induced similar perturbations in primary, patient-derived MCL cells. Treatment with PS alone attenuated EZH2, SUZ12 and DNMT1, as well as depleted the protein expression of BMI1, RING2 and MEL18 in the cultured MCL cells. This was associated with attenuation of H3K27Me3 and H2K119Ub, and augmentation of H3K4Me3 chromatin marks. Depletion of BMI with shRNA also reduced the levels of H2K119 Ub, which was associated with growth inhibition of MCL cells. PS treatment also decreased the levels of LSD1, a demethylase of H3K4Me2, which led to increase in H3K4Me3. Treatment with the LSD1 inhibitor CIT0665, or knockdown of LSD1 by shRNA, increased H3K4Me2 &Me3 levels, as well as enhanced PS mediated growth inhibition and apoptosis of MCL cells. PS treatment also induced heat shock protein (hsp) 90 acetylation, and depleted the levels of hsp90 client proteins in JeKo-1, including CDK4, c-RAF and AKT. As compared to treatment with each agent alone, co-treatment with DZNep and PS caused greater depletion of EZH2, SUZ12 and BMI1, accompanied with greater induction of p21 and p27 but attenuation of cyclin D1 expression. Co-treatment with DZNep and PS also induced cell cycle growth arrest and synergistically induced apoptosis of JeKo-1, MO2058, and primary MCL cells derived from 3 patients with MCL (combination indices Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2016. doi:10.1158/1538-7445.AM2011-2016

Abstract 2856 The proteasome inhibitor bortezpmib has been shown to markedly increase the intracellular levels of misfolded proteins, induce aggresome formation and cause endoplasmic reticulum (ER) stress, resulting in apoptosis of human Mantle Cell Lymphoma (MCL) cells. Consistent with this, Bortezomib displays clinical efficacy in patients with relapsed and refractory MCL. We have recently reported that the pan-histone deacetylase (HDAC) inhibitor panobinostat, by also inhibiting HDAC6, abrogates aggresome formation and induces Endoplasmic Stress (ER) stress, as well as potentiates bortezomib-induced apoptosis of MCL cells. Here, we determined the anti-MCL cell activity of an HDAC6-specific inhibitor, WT-161 alone and in combination with the novel, orally bio-available, proteasome inhibitor carfilzomib (Proteolix Inc.) against human, cultured and primary, patient-derived MCL cells. Treatment with WT-161 (0.1 to 1.0 uM) resulted in a dose-dependent increase in the acetylation of alpha-tubulin and heat shock protein (hsp) 90, without any appreciable increase in the levels of acetylated histone (H) 3. Consistent with WT-161 mediated hyperacetylation and inhibition of hsp90 chaperone function, treatment with WT-161 increased the intracellular levels of polyubiuitylated proteins in the cultured MCL JeKo-1 and Z138 cells. WT-161 was also noted to dose-dependently deplete the levels of cyclin D1 in the cultured MCL cells. Treatment with WT-161 also induced ER stress response in the MCL cells, demonstrated by increase in the protein levels of Glucose regulated protein (GRP) 78, phosphorylated eIF2 (eukaryotic initation factor 2) α, and induction of the pro-apoptotic transcription factor CHOP (CAAT/Enhancer Binding Protein Homologous Protein). We next determined the effects of co-treatment with WT-161 on carfilzomib-induced aggresome formation, ER stress response and apoptosis of the cultured and primary MCL cells. Co-treatment with WT-161 (0.25 uM) abrogated carfilzomib-induced aggresome formation in MCL cells, as evidenced by confocal immunofluorescent staining of aggresomes with anti-HDAC6 and anti-ubiquitin antibodies. Compared to each agent alone, co-treatment with WT-161 and carfilzomib induced more intracellular polyubiquitylated proteins and induced higher levels of CHOP in the cultured MCL cells. Co-treatment with WT-161 and carfilzomib also synergistically induced apoptosis of the cultured MCL cells (combination indices < 1.0). Notably, co-treatment with WT-161 and carfilzomib also synergistically induced apoptosis of primary MCL cells (combination indices < 1.0). These findings strongly support the in vivo testing of the combination of an HDAC6-specific inhibitor such as WT-161 with the proteasome inhibitor carfilzomib against human MCL cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 798 The mutant JAK2-V617F tyrosine kinase (TK) is present in the majority of patients with BCR-ABL negative myeloproliferative neoplasms (MPNs). JAK2-V617F activates downstream signaling through the STAT, RAS/MAPK and PI3/AKT pathways, conferring proliferative and survival advantages in the MPN hematopoietic progenitor cells (HPCs). We have previously reported that pan-histone deacetylase (HDAC) inhibitors e.g. panobinostat (PS) (Novartis Pharmaceuticals), depleted mRNA expression of JAK2-V617F, and disrupted the chaperone association of with hsp90 with JAK2-V617F, thereby promoting the degradation of JAK2-V617F by the proteasome. This led to attenuation of the levels and downstream transcriptional activity of STAT3 and STAT5, resulting in growth arrest and apoptosis of MPN HPCs. Additionally, co-treatment with PS and a JAK2 kinase inhibitor, TG101209, further depleted JAK/STAT signaling and synergistically induced apoptosis of JAK2-V617F expressing HEL92.1.7 and Ba/F3-JAK2V617F cells, as well as exerted greater lethality against primary CD34+CD38-Lin- MPN versus normal CD34+ HPCs. In the present studies, we determined the cytotoxic effects of inhibiting JAK2-STAT3/5 in conjunction with pharmacologic targeting of the collateral, pro-growth and pro-survival signaling through PI3K/AKT, RAF-MEK or PIM1 kinases in MPN cells. For this, the cytotoxic effects of co-treatment with TG101209 and the MEK inhibitor (AZD6244, AstraZenaca), dual PI3K/mTOR inhibitor (BEZ235, Novartis) or the PIM1 kinase inhibitor (SGI-1776, SuperGen) were evaluated in HEL92.1.7, Ba/F3-JAK2V617 and primary human MPN cells. Treatment with BEZ235 dose-dependently attenuated the levels of p-JAK2, p-STAT5, p-STAT3, p-AKT, p-ERK1/2 and p-4EBP1. Co-treatment with BEZ235 and TG101209 was synergistically lethal against the cultured MPN and primary CD34+ MF-MPN cells (combination indices < 1.0). Co-treatment with AZD6244 and TG101209 also induced synergistic apoptosis of cultured MPN cells (combination indices of < 1.0). This was associated with greater attenuation of the levels of p-AKT and p-ERK1/2. PIM1 is a cytoplasmic serine/threonine kinase that serves as a downstream effector of several cytokine signaling pathways promoting cell survival and proliferation. PIM1 collaborates in Myc-induced transformation and known to phosphorylate 4EBP1 and eIF4B, thereby promoting protein translation. Co-treatment with TG101209 and the PIM1 kinase inhibitor, SGI-1776 also induced synergistic apoptosis of HEL92.1.7 cells and Ba/F3-JAK2V617F cells (combination indices < 1.0) but not of Ba/F3-hEpoR cells. PIM kinase mediates PRAS40 phosphorylation and induces mTORC1 activity in phosphorylating 4EBP1. Consistent with this, co-treatment with SGI-1776 and TG-101209 inhibited p-PRAS40 and p-4EBP1 levels in cultured MPN but not in normal progenitor cells. These findings demonstrate for the first time that combined treatment with a MEK inhibitor, PIM1 kinase inhibitor or dual PI3K/mTOR inhibitor enhances the anti-JAK2-V617F activity of TG101209 in cultured and primary human MPN cells. Our findings support the rationale to determine the in vivo activity of TG101209 in combination with inhibitors of MEK, PIM1 or PI3K/mTOR kinase against human MPN cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 2488 Lysine specific histone methylation and deacetylation are chromatin modifications that, along with DNA methylation, are involved in the epigenetic silencing of tumor suppressor genes (TSGs). This silencing is mediated by multi-protein complexes PRC (polycomb repressive complexes) 1 and 2. Of the three core protein components of PRC2, i.e., EZH2, SUZ12 and EED, EZH2 has the SET domain with its intrinsic histone methyltransferase activity, which induces the trimethylation (Me3) of lysine (K) 27 on histone (H) 3-a repressive histone modification mediating gene repression. The PRC1 components include BMI1, MEL18, RING1 and RING2, and it serves to further compact the chromatin at PRC2 target genes. The RING1 and RING2 proteins are responsible for the ubiquitylation of K119 on H2A. We have previously reported that treatment with the pan-histone deacetylase inhibitor panobinostat (PS, Novartis Pharmaceutical Corp) depletes PRC2 complex proteins EZH2 and SUZ12 and the DNA methyltransferase (DNMT) 1. We also showed that co-treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep, further depleted PRC2 complex proteins and, in combination with PS, induced synergistic apoptosis of cultured and primary AML cells (Blood 2009; 114: 2733–43). In the present studies we determined the effects of DZNep and/or PS on the expression of PRC1 and PRC2 proteins in human Mantle Cell Lymphoma (MCL) cells. Treatment with DZNep dose-dependently depleted EZH2, SUZ12 and BMI1 expression as well as inhibited K27Me3, while inducing K27 acetylation on H3. DZNep treatment also induced p21, p27 and FBXO32, while depleting the levels of cyclin D1 in the cultured MCL JeKo-1 and MO2058 cells. Similar induction of p21, p27 and FBXO32 were also observed, following siRNA knockdown of EZH2 in the cultured MCL cells. Notably, DZNep also induced similar perturbations in primary, patient-derived MCL cells. Treatment with PS alone attenuated EZH2, SUZ12 and DNMT1, as well as depleted the protein expression of BMI1, RING2 and MEL18 in the cultured MCL cells. This was associated with attenuation of H3K27Me3 and augmentation of H3K4Me3 chromatin marks. PS treatment also induced heat shock protein (hsp) 90 acetylation, and depleted the levels of hsp90 client proteins in the MCL cells, including CDK4, c-RAF and AKT. As compared to treatment with each agent alone, co-treatment with DZNep and PS caused greater depletion of EZH2, SUZ12 and BMI1, accompanied with greater induction of p21 and p27 but attenuation of cyclin D1 expression. Co-treatment with DZNep and PS also induced cell cycle growth arrest and synergistically induced apoptosis of JeKo-1 and MO2058, as well as of primary MCL cells derived from 3 patients with MCL (combination indices Disclosures: Atadja: Novartis: Employment.

High pressure behavior of the compound Bi12SiO20 is investigated using in situ Raman spectroscopic and synchrotron-based angle dispersive x-ray diffraction techniques. Results indicate that the compound remains stable in the ambient pressure cubic structure up to 26 GPa. From the structural studies, bulk modulus B0, and its pressure derivative B′ of Bi12SiO20 are evaluated to be 36 GPa and 16.7 GPa, respectively. Mode Gruneissen parameters of various Raman active modes of Bi12SiO20 are also reported. The stability of Bi12SiO20 at high pressure is discussed in the light of the pressure-induced amorphization reported in bismuth-orthosilicate (Bi4Si3O12) and -orthogermanate. Comparison of the observed phonon behavior with that reported for Bi4Si3O12 reveals that two of the Raman modes in Bi4Si3O12 have negative pressure dependencies clearly indicating dynamic instability while Bi12SiO20 does not show any signatures of zone-center instabilities.

The stress phenotype of transformed cells is associated with increased levels of heat shock proteins (hsp) and autophagy to avoid cell death. Autophagy is a conserved catabolic pathway in which cytoplasmic macromolecules and organelles are sequestered in autophagosomes, which fuse with lysosomes for protein degradation and amino acid recycling. The molecular cascade initiating autophagosome formation is triggered by a lipid kinase (PI3KC3) signaling complex (Vps34, Vps15, ATG6 and ATG14) that mediates phagophore nucleation. We have previously reported that treatment with panobinostat (PS), a pan-histone deacetylase (HDAC) inhibitor induced hyperacetylation of hsp70 and hsp90. In the present study we demonstrate that treatment of MB231 cells with PS increased the association of hyperacetylated hsp70 with the PI3KC3 VPS34, ATG6 (Beclin-1) and KAP1 (an E3 ligase for sumoylation). PS treatment also induced the levels of ATG7, ATG5 and LC3-II (a phosphatidylethanolamine conjugated ATG8) in MB-231 cells. Treatment with 2-phenylethynesulfonamide (PES), a small molecule inhibitor of hsp70 function resulted in the abrogation of PS-induced interaction of hsp70 with hsp40, VPS34 and (the lysosome-associated membrane protein A (LAMP-2A), a protein involved in chaperone mediated autophagy. Consequently, as compared to treatment with each agent alone, co-treatment with PS and PES resulted in significantly more cell death in MB-231 and SUM159PT cells (p < 0.05). Co-treatment with PS also significantly enhanced lethality of breast cancer MB-231 and MCF-7 cells due to autophagy inhibitor 3-methyl-adenosine (3-MA, inhibitor of Vps34), or hydroxychloroquine (inhibitor of lysosome acidification). In orthotopically implanted MB-231 xenografts in the mammary fat pads of NOD/SCID mice, tumor size increase to 100, 200, 500, 1000 and 2000 cu mm demonstrated tumor size-dependent increase in the intracellular levels of hsp90 and hsp70, hyperacetylated hsp70, ATG7, ATG5, Beclin1 as well as LC3-II. These in vivo findings suggest that as breast tumors grow they exhibit the stress-phenotype involving proteotoxic stress and autophagy. Our findings demonstrate the combination therapy with PS and autophagy inhibitor, which eliminates breast cancer cells with stress phenotype including autophagy, may be especially effective against established and enlarging breast cancers. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4860.

High pressure behavior of ZrGeO4 has been investigated using Raman and photoluminescence (PL) spectroscopies up to 25 GPa in a diamond anvil cell. Under the application of pressure, the GeO4 librational mode exhibits softening, suggesting dynamical instability of the scheelite structure. Qualitative changes are noted in the Raman spectrum above 12 GPa, suggesting a possible transition around this pressure. High pressure PL behavior of Eu3+-related crystal field transitions indicates a clear change in the site symmetry of Eu3+ around 12 GPa, strongly supporting structural transition to a lower symmetry phase at this pressure.

Among the hallmarks of cancer is the stress phenotype, which is collectively induced by hypoxia, deprivation of nutrients, acidosis and increased reactive oxygen species, and exhibited as metabolic and proteinmisfolding/denaturing (proteotoxic) stress. This results in the constitutive activation of the heat shock response with elevated levels of heat shock proteins (hsp), and induction of autophagy, for avoiding cell death. Autophagy is a conserved catabolic pathway in which cytoplasmic macromolecules and organelles are sequestered in autophagosomes, which fuse with lysosomes for protein degradation and amino acid recycling. Elevated levels of hsp70 and hsp90 promote proper protein folding and inhibit both the intrinsic and extrinsic pathways of apoptosis. In our present studies, we have determined in vitro that the stress induced by nutrient withdrawal or treatment with pan-HDAC inhibitor (HDI) panobinostat (PS) (Novartis Pharmaceuticals) resulted in hyperacetylation of hsp70, which induced autophagy in the cultured breast cancer MB-231 and MCF-7 cells. The initial steps of autophagy include vesicle nucleation, vesicle expansion to form the phagophore, edges of which fuse to create the autophagosome. This process is triggered by a lipid kinase (PI3KC3) signaling complex, consisting of Vps34, Vps15, and autophagy-associated genes (ATG) 6 and ATG14, which mediate vesicle nucleation. We determined that hyperacetylated hsp70 significantly enhanced the stability and activity of VPS34, which led to binding of VPS34 to Beclin1 (ATG6). Hyperacetylated hsp70 also recruited the PHD domain containing E3 ligase for sumoylation, KAP1, and induced the sumoylation of VPS34 by SUMO-1. This was documented by confocal immunostaining with fluorescence-conjugated anti-VPS34 and anti-SUMO-1 antibodies, as well as by biochemical determination of the in vitro and in vivo sumoylation of VPS34. PS treatment also induced LC3II accumulation and formation of authophagosomes, the latter determined morphologically by electron microscopy. PS-induced, KAP-1 dependent sumoylation of VPS34 and downstream autophagosome formation were abrogated by siRNA to hsp70. Also, VPS34 expression was attenuated and the processing of LC3 was inhibited in the cells from the hsp70 knockout as compared to the control mice. Importantly, we also determined that, as compared to treatment with each agent alone, co-treatment with PS and the inhibitor of autophagy 3- methl-adenosine (3-MA) or chloroquine significantly enhanced cell death of breast cancer MB-231 and SUM159T cells. Furthermore, we determined the in vivo role of hyperacetylated hsp70 in autophagy. In cohorts of mice, following the growth of the orthotopically-implanted MB-231 xenografts in the mammary fat pads of NOD/SCID mice to a size of 100, 200, 500 and 1000 cu mm, tumor cell-lysates were obtained. Western blot analyses demonstrated size-dependent increase in the intracellular levels of hyperacetylated hsp70, Beclin1 and LC3II. Collectively, these findings suggest that stress phenotype associated heat shock response and increase in acetylated hsp70 promotes autophagy. Our findings also demonstrate that in established breast cancers, autophagy can be selectively targeted by cotreatment with the pan-HDAC inhibitor and an inhibitor of autophagy to eliminate breast cancer cells. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B21.

Inactivating somatic mutation of LKB1 is found in approximately 30% of NSCLC and is often coincident with activating K-RAS mutation. In a mutant K-RAS-driven model of mouse lung cancer, strong cooperation and aggressive phenotype was seen with hemizygous inactivation of LKB1. Loss of LKB1 results in activation of mTOR pathway while somatic activating mutation in K-RAS results in activation of PI3K/AKT and RAS/RAF/ERK. Therefore therapeutic stratagies which target these pathways may have significant affect on the outcome of these cancers. NVP-BEZ235 is a dual PI3K and mTOR inhibitor and panobinostat (PS) (Novartis Pharmaceuticals Inc) is a pan-histone deacetylase (HDAC) inhibitor, which has been shown to induce proteasomal degradation and depletion of the levels of hsp90 client proteins, including AKT and c-RAF. Here, we determined the effects of BEZ235 and/or PS or AUY922 (an hsp90 inhibitor) on NSCLC A549 and H460 cells with LKB1 and K-RAS mutations (A549 and H460). Treatment with BEZ235 (200 to 1000 nM), PS (25 to 50 nM) or AUY922 (25 to 100 nM) dose-dependently increased % of cells in G2/M and decreased % of cells in S phase of cell cycle, as well as induced apoptosis of the NSCLC cells. Co-treatment with AUY922 or PS increased BEZ235 mediated depletion of p-AKT, p-4EBP1 and p-p70S6K levels, as well as synergistically induced apoptosis of A549 and H460 cells (combination indices < 1.0). Combined therapy with BEZ235 (25 mg/Kg) and PS (10 mg/Kg) or AUY922 (20 mg/Kg) resulted in significantly more tumor growth delay than treatment with each of the agents alone of A549 cell xenografts in nude mice (p < 05). We also determined the activity of BEZ235 and/or PS in the NSCLC H368 cells with mutant K-RAS and either wild-type (H358 cells) or stable knock down of LKB1 expression (H358/LKB1-KD cells), achieved by shRNA against LKB1, mimicking the presence of mutant LKB1. As compared to each agent alone, co-treatment with BEZ235 and PS exhibited a higher level of antitumor synergy against H358/LKB1-KD versus H358 cells. These pre-clinical in vitro and in vivo studies demonstrate that combined treatment with the dual PI3K/mTOR inhibitor BEZ235 and PS or AUY922 could potentially be a promising targeted therapy for NSCLC that harbors K-ras and LKB1 mutations. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C196.

During the in vivo growth of breast cancers, hypoxia, deprivation of nutrients, acidosis and increased reactive oxygen species collectively induce the metabolic and protein-misfolding/denaturing stress. This increases the levels of heat shock proteins (hsp) and induces autophagy to avoid cell death. Autophagy is a conserved catabolic pathway in which cytoplasmic macromolecules and organelles are sequestered in autophagosomes, which fuse with lysosomes for protein degradation and amino acid recycling. Elevated levels of hsp70 and hsp90 promote protein re-folding and prevent protein aggregation. Increase in hsp70 levels are also known to inhibit both intrinsic and extrinsic pathway of apoptosis. In our present studies, we have determined that the in vitro stress induced by nutrient withdrawal or treatment with pan-HDAC inhibitor (HDI) panobinostat (PS) (Novartis Pharmaceuticals) results in hyperacetylation of hsp70, which induces autophagy of the cultured breast cancer MB-231 and MCF-7 cells. The initial steps of autophagy include vesicle nucleation, vesicle expansion to form the phagophore, edges of which fuse to form the autophagosome. This process is triggered by a lipid kinase (PI3KC3) signaling complex, consisting of Vps34, Vps15, and autophagy associated genes (ATG) 6 and ATG14, that mediates vesicle nucleation. We have determined that hyperacetylated hsp70 significantly enhances the stability and activity of VPS34, which leads to binding of VPS34 to Beclin1 (ATG6). Hyperacetylated hsp70 also recruits the PHD domain containing E3 ligase for sumoylation, KAP1, to induce the sumoylation of VPS34 by SUMO-1. This was documented by confocal immunostaining with fluorescence-conjugated anti-VPS34 and anti-SUMO-1 antibodies, as well as by biochemical determination of in vitro and in vivo sumoylation of VPS34. PS treatment also induced LC3II accumulation and formation of authophagosomes, the latter determined morphologically by electron microscopy. PS-induced, KAP-1 dependent sumoylation of VPS34 and downstream autophagosome formation were abrogated by siRNA to hsp70. Also, VPS34 expression was attenuated and the processing of LC3 was inhibited in cells from the hsp70 knockout as compared to the control mice. Importantly, we also determined that, as compared to treatment with each agent alone, co-treatment with PS and the inhibitor of autophagy 3-methl-adenosine (3-MA) or chloroquine significantly enhanced cell death of breast cancer MB-231 and SUM159T cells. Furthermore, the in vivo role of hyperacetylated hsp70 in autophagy induced during growth of the orthotopically implanted MB-231 xenografts in the mammary fat pads of NOD/SCID mice was also established. In cohorts of mice, following growth to a size of 100, 200, 500 and 1000 cu mm, tumor cell-lysates demonstrated tumor size-dependent increase in the intracellular levels of hyperacetylated hsp70, Beclin1 and LC3 II. Collectively, these novel findings suggest that, stress-induced heat shock response and increase in acetylated hsp70 promotes autophagy. Our findings also demonstrate that in established breast cancers autophagy can be selectively targeted by co-treatment with a pan-HDAC inhibitor and an inhibitor of autophagy to eliminate breast cancer cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3124.

Abstract 2401 Poster Board II-378 MicroRNA (miR) alterations are highly involved in the pathogenesis of leukemia. However, the role of miRs in de novo or acquired resistance of cancer cells to therapeutic agents has not been fully elucidated. Recently, we reported the isolation and characterization of HL-60/LR cells, derived from human acute myeloid leukemia HL-60 cells, that are resistant to pan-histone deacetylase (HDAC) inhibitors (HDIs), including vorinostat and panobinostat (Blood. 2008; 112: 2896). To explore the role of miRs in acquiring resistance to HDIs, we performed a miR microarray analysis of the parental HL-60 and HL-60/LR cells. Compared to HL-60 cells, expression of thirteen microRNAs were discovered to be significantly increased (> 4-fold) and fourteen miRs were markedly down-regulated (> 4-fold) in HL-60/LR cells. Alterations in the expression of three of the most promising upregulated (miR-21, miR-126 and miR-146a) and down-regulated (miR-223, miR-148a and miR-342) miRs were confirmed by Q-PCR in HL-60/LR cells. The expression of miR-223, miR-148a and miR-342 was also significant lower in the relatively HDI-resistant K562 cells as compared to HDI-sensitive U937 and HL-60 cells. Conversely, miR-126 and miR-146a expressions were higher in K562 cells compared to U937 and HL-60 cells. Short term (24 hours) treatment with panobinostat (10 to 50 nM) did not alter the expression of miR-223 or miR-148a expression in HL-60 cells. As compared to treatment with either agent alone, co-treatment with the histone methyl transferase EZH2 antagonist 3-deazaneplanocin (DZnep, 1.0 uM) and DNA methyl transferase inhibitor decitabine (2.0 uM) induced miR-223 and miR-148a levels and mediated apoptosis of HL-60/LR cells, suggesting that an epigenetic silencing mechanism(s) may be involved in the down-modulation of miR-223 and miR-148a in HL-60/LR cells. To determine whether the alterations in the miR levels were mechanistically involved in conferring resistance to HDIs, we engineered through retroviral transduction stable ectopic expressions of miR-223, miR-148a and miR-342 into HL-60/LR cells and miR-21 and miR-146a into HL-60 cells. Ectopic expression of miR-223 and miR-148a significantly increased the sensitivity of HL-60/LR cells to panobinostat and vorinostat. In contrast, re-expression of miR-342 had an insignificant effect on HDI sensitivity. Increased expression of miR-21 and miR-146a did not confer resistance to the HDIs in HL-60 and U937 cells. Next, by Western analyses, we compared the expression levels of several of the predicted target proteins of miR-223 and miR-148a, (as predicted by the computer programs TargetScan and picTAR), in HL-60 versus HL-60/LR cells, as well as in the HL-60/LR cells with stable ectopic expression of miR-223 and miR-148a. Several candidate proteins including GRP94, Ribosomal protein S6 kinase MSK1, MEF2C and DNMT1 showed higher level of expression in HL-60/LR versus HL-60 and were down-regulated in miR-223 or miR-148a transduced HL-60/LR cells, suggesting that these proteins may confer resistance against HDI. Parenthetically, miR-223 was shown to be a myeloid-specific miR which negatively regulates progenitor proliferation and granulocyte differentiation and activation. miR-223 mutant mice have an expanded granulocytic compartment resulting from an increase in the number of granulocyte progenitors. In summary, our observations indicate that high miR-223 and miR-148a levels may be predictive biomarkers for sensitivity to HDIs in human AML cells. Additionally, induction of miR-223 and miR-148a by EZH2 antagonist may increase sensitivity and overcome resistance to HDIs in human AML cells. Targeting the levels and/or activity of the miR-223 and miR-148a target proteins may also be an effective strategy in enhancing the activity of HDI based combination therapy against AML. Disclosures: Atajada: Novartis: Employment. Bhalla:Merck: Honoraria; Novartis: Honoraria, Research Funding.

Abstract 2656 Poster Board II-632 NPM1 is a nucleolar phosphoprotein that forms oligomers and functions as a molecular chaperone for both proteins and nucleic acids. NPM1 normally shuttles between the nucleus and cytoplasm but is mutated and aberrantly localized to the cytoplasm in 35% of patients with AML, Mutant (m) NPM1 contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal (NES), which causes mNPM1 to remain in the cytoplasm. In AML, the presence of mNPM1 is often co-detected with internal tandem duplications of FLT-3, which confers a poor prognosis. The effects of the cytosolic mNPM1 on the biology and drug-sensitivity of AML are unclear. In the present studies we determined the expression and biologic effects of mNPM1 in cultured and primary AML cells. Utilizing RT-PCR, immunoblot analysis and immunoflourescence microscopy (with a polyclonal anti-mNPM1 antibody, we confirmed that in the cultured AML OCI-AML3 cells (hetrozygous for NPM1 mutation) mNPM1 protein was localized in the cytosol, while the un-mutated NPM1 was nuclear. In AML HL-60 cells NPM1 was un-mutated and entirely nuclear. As compared to control siRNA, treatment with siRNA to NPM1 for 72 hours significantly induced p21 expression, decreased % of S phase cells in the cell cycle, as well as induced morphologic differentiation of OCI-AML3 but not of HL-60 cells. This was associated with marked induction of CEBPα in OCI-AML3 cells. NPM1 siRNA also attenuated the expression of HOXA9 and MEIS1 (which are known to be leukemogenic), associated with a marked loss of clonogenic survival of OCI-AML3 cells. NPM1 siRNA mediated depletion of Meis1, which is known to transactivate FLT3 tyrosine kinase in AML progenitor cells, was associated with down regulation of FLT-3 expression in OCI-AML3 cells. Importantly, treatment with NPM1 siRNA significantly sensitized OCI-AML3 more than HL-60 cells to all-trans retinoic acid (ATRA, 0.25 to 2.0 uM) and Ara-C (0.5 to 5.0 uM). Treatment with leptomycin B (5 nM for 24 hours), which is an inhibitor of the NES receptor CRM1/exportin-1, shifted the localization of mNPM1 from the cytosol to the nucleus, abrogated the levels of Meis1 and HoxA9 and induced apoptosis of OCI-AML3 but not HL-60 cells. Recently, treatment with NSC348884, a small molecule inhibitor of NPM1 oligomerzation, was shown to induce apoptosis of colon and prostate cancer cells. Our studies determined that NSC348884 (3.0 uM) was highly active in disrupting the oligomerization of mNPM1 and induce apoptosis of 90 % of OCI-AML but only 20% of HL-60 cells. Additionally, treatment with 5.0 uM of NSC34884 induced apoptosis of approximately 75% of primary AML cells with mNPM1, without inducing apoptosis of normal CD34+ progenitor cells. Collectively, these findings suggest that strategies targeting mNPM1 levels or oligomerization, or reversing the aberrant cytosolic localization of mNPM1, would induce relatively selective differentiation and apoptosis, as well as sensitize AML cells with mNPM1 to Ara-C and ATRA. Disclosures: No relevant conflicts of interest to declare.

Abstract 2651 Poster Board II-627 The oncoprotein PIM1 is a cytoplasmic serine/threonine kinase, which is overexpressed and implicated in the pathogenesis of hematologic malignancies. PIM1 activity promotes cell survival and proliferation, and is a downstream mediator of cytokine receptor signaling involving the JAK/STATs. PIM1 phosphorylates and inactivates Bad, which enhances the anti-apoptotic functions of Bcl-2. PIM1 also mediates the phosphorylation and inactivation of cell cycle inhibitors p21 and p27, thereby promoting progression through G1 and G2/M phases of the cell cycle. PIM1 phosphorylates serine 10 on histone H3, and has been shown to phosphorylate and stabilize Myc, thereby collaborating in Myc-induced transformation. Further, PIM1 is known to phosphorylate 4EBP1 on the same residues (threonine 37 and 46) as mTOR kinase. It can also phosphorylate eIF4B, thereby maintaining high levels of protein translation. PIM1 is known to be chaperoned as a heat shock protein 90 client protein. In the present studies, we demonstrate that treatment with the PIM kinase inhibitor SGI-1776 (SuperGen Inc.) dose-dependently (500 nM to 2.0 μM) depleted p-BAD (Ser112), p-p70S6K, p-4EBP1, p-Ser10 Histone H3 and c-Myc levels, while inducing p27 and p21 expression, in the cultured human CML-BC K562 and erythroleukemia HEL92.1.7 (with homozygous JAK2-V617F mutation). Treatment with SGI-1776 induced apoptosis of K562 and HEL92.1.7 (HEL) cells as well as Ba/F3-hEpoR-JAK2-V617F but not Ba/F3-hEpoR cells. Similar effects of SGI-1776 were observed in primary CML-BC and MPN (myeloproliferative neoplasm) cells. Treatment with the non-geldanamycin analogue hsp90 inhibitor AUY922 (Novartis Pharma) or with the pan-HDAC inhibitor panobinostat (PS) (Novartis Pharma) disrupted the chaperone association of PIM1 with hsp90 and increased binding to hsp70, thereby promoting the proteasomal degradation of PIM1 in K562 and HEL cells. Treatment with PS (20–100 nM) also depleted PIM1 mRNA as measured by qPCR. Co-treatment with SGI-1776 (0.5 to 2.0 μM) and clinically achievable concentrations of AUY922 (e.g., 25 nM) caused greater depletion of pBAD (Ser112), pp70S6K, p4EBP1, cyclin B1, c-Raf, c-Myc, and AKT and synergistically induced apoptosis of K562 and HEL92.1.7 cells (combination indices < 1.0). As compared to treatment with either agent alone, co-treatment with SGI-1776 and AUY922 or PS also exerted greater cytotoxicity against primary CML-BC and MF-MPN cells. Importantly, co-treatment with PS significantly enhanced SGI-1776-induced apoptosis of purified primary CD34+CD38-Lin- hematopoietic stem cells expressing JAK2-V617F derived from a patient with primary myelofibrosis. Taken together, these pre-clinical findings indicate that simultaneously depleting PIM kinase levels by AUY922 or PS and inhibiting PIM1 kinase activity with SGI-1776 induces synergistic apoptosis against cultured and primary CML-BC and MPN cells. Additionally, our findings suggest that co-treatment with PS and SGI-1776 may be an effective treatment strategy against hematopoietic stem cells expressing JAK2-V617F in patients with MF-MPN. Disclosures: Atajada: Novartis: Employment. Quadt:Novartis: Employment. Bhalla:Merck: Honoraria; Novartis: Honoraria, Research Funding.

The phase stability of YbVO4 under pressure has been investigated using synchrotron based angle dispersive x-ray diffraction and Raman spectroscopic techniques up to 34.5 and 26.5 GPa, respectively. The results indicate that the compound transforms from the ambient pressure zircon structure to the scheelite structure above 5.9 GPa with 11.8% volume discontinuity. The coexistence of the two phases is observed over a large pressure range. At 15.8 GPa, the (011) peak of the scheelite phase develops asymmetry, and the pattern at further high pressures could be fitted to a fergusonite-type monoclinic structure. On reducing the pressure, the fergusonite phase reverses back to the scheelite phase; the latter phase could be recovered as a metastable phase at ambient pressure. The refined structural parameters along with the equation of state are given for various phases of YbVO4. Changes in the vibrational properties across these transitions, particularly across the scheelite↔fergusonite transition, have been inv...

Lysine specific histone methylation and deacetylation and DNA hypermethylation are involved in the epigenetic silencing of tumor suppressor genes (TSG), e.g., p16 and JunB. The multi-protein complex PRC (polycomb repressive complex) 2 that contains the three core proteins EZH2, SUZ12 and EED, has intrinsic histone methyltransferase (HMTase) activity. This is mediated by the SET domain of EZH2, which induces tri-methylation (3Me) of lysine (K)-27 on histone H3, as well as promotes cell proliferation and aggressiveness of neoplastic cells. EZH2 is preferentially overexpressed in proliferating but not resting Mantle Cell Lymphoma (MCL) cells. In the present studies we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) dose-dependently (500 nM to 2.0 uM) depletes EZH2, SUZ12 and EED levels, as well as inhibits 3Me K27 on H3 while inducing K27 H3 acetylation. DZNep treatment also induces the levels of p21, p27, JunB and FBXO32, while depleting cyclin D1 and cyclin E levels in the cultured human MCL Jeko-1, MO2058 and Z138 cells and in primary patient-derived MCL cells. Treatment with DZNep induces PARP cleavage activity of the caspases and apoptosis in the cultured and primary MCL cells. DZNep promoted proteasomal degradation of EZH2 and SUZ12, since co-treatment with bortezpmib significantly restored EZH2 and SUZ12 levels in the MCL cells. We had previously reported that treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) (LBH589, Novartis Pharmaceutical Corp) depletes the levels of EZH2, SUZ12 and EED in cultured and primary AML cells (Mol Cancer Ther.2006; 5:3096). Within the PRC2 complex, EZH2 bound and recruited the DNA methyltransferases DNMT1, and treatment with PS also disrupted the interaction of EZH2 with DNMT1, attenuated DNMT1 levels and its binding to the EZH2-targeted gene promoters, e,g, JunB. Here, we also demonstrate that, PS treatment depletes DNMT1 levels and induces JunB levels in cultured MCL cells. As compared to treatment with either agent alone, co-treatment with DZNep and PS caused more depletion of EZH2 and SUZ12, but not of DNMT1, more induction of JunB, p21 and p27, as well as synergistically induced apoptosis of cultured MCL cells (combination indices < 1.0). Taken together, these findings indicate that DZNep and PS mediated targeting of EZH2 and the PRC2 complex is an effective epigenetic therapy of MCL, which also results in undermining several molecular determinants of MCL cell proliferation and survival. Additionally, combined epigenetic therapy with DZNep and PS exerts synergistic in vitro activity against human MCL cells, suggesting that this combination may be a promising novel treatment for MCL.

Lysine specific histone methylation and deacetylation and DNA hypermethylation are involved in the epigenetic silencing of tumor suppressor genes (TSG), e.g., p15 and p16. The multi-protein complex PRC (polycomb repressive complex) 2 that contains the three core proteins EZH2, SUZ12 and EED, has intrinsic histone methyltransferase (HMTase) activity. This is mediated by the SET domain of EZH2, which induces tri-methylation (3Me) of lysine (K)-27 on histone H3, regulates the expression of HOX genes and promotes cell proliferation and aggressiveness of neoplastic cells. In the present studies we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) dose-dependently (200 nM to 2.0 uM) depletes EZH2, SUZ12 and EED levels, inhibits 3Me K27 on H3 while inducing K27 H3 acetylation in the cultured human AML HL60 and OCI-AML3 cells and in primary, patient-derived AML blasts. DZNep treatment also induced the levels of p16, p21, p27 and FBXO32 while depleting cyclin E and HOXA9 levels. Treatment with DZNep induced PARP cleavage activity of the caspases and apoptosis in the cultured and primary AML cells. DZNep promoted proteasomal degradation of EZH2 and SUZ12, since co-treatment with bortezomib significantly restored EZH2 and SUZ12 levels in the AML cells. We had previously reported that treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) (LBH589, Novartis Pharmaceutical Corp) also depletes the levels of EZH2, SUZ12 and EED in cultured and primary AML cells (Mol Cancer Ther.2006; 5:3096). Within the PRC2 complex, EZH2 bound and recruited the DNA methyltransferases DNMT1, and treatment with PS also disrupted the interaction of EZH2 with DNMT1, attenuated DNMT1 levels and its binding to the EZH2-targeted gene promoters, e.g., p16 and JunB. Here, we also demonstrate that, as compared to treatment with either agent alone, co-treatment with DZNep and PS caused more depletion of EZH2, SUZ12 and EED, more induction of p16, p21 and p27, as well as synergistically induced apoptosis of AML cells (combination indices < 1.0). Additionally, DZNep induced apoptosis of HL-60/LR cells that are resistant to HDACs including PS, as well as sensitized HL-60/LR cells to PS. Taken together, these findings indicate that targeting EZH2 and the PRC2 complex is an effective epigenetic therapy of AML that also overcomes resistance to HDAC inhibitors. Additionally, combined epigenetic therapy with DZNep and PS exerts synergistic in vitro activity against human AML cells, suggesting that this combination may be a promising novel treatment for AML.

Lysine specific histone methylation and deacetylation and DNA hypermethylation are involved in the epigenetic silencing of tumor suppressor genes (TSG), e.g., p15 and p16. DNA methyltransferase (DNMT) inhibitors 5-azacytidine and 5-aza-2’-deoxycytidine demethylate the CpG dinucleotide islands in or near gene promoters, leading to derepression of TSGs in AML. SGI-110 (S110) (Cancer Res.2007; 67:6400) and SGI-1036 (SuperGen, Inc.) are novel, DNMT inhibitors, which also deplete DNMT1 levels. SGI-110 is a dinucleotide containing 5-aza-2’-deoxycytidine and SGI-1036 is a non-nucleoside heterocycle. The multi-protein complex PRC (polycomb repressive complex) 2 that contains the three core proteins EZH2, SUZ12 and EED, has intrinsic histone methyltransferase (HMTase) activity. This is mediated by the SET domain of EZH2, which induces trimethylation of histone H3 on lysine (K)-27. We recently reported that treatment with the pan-HDAC inhibitor panobinostat (LBH589, Novartis Pharmaceutical Corp) acetylates and inhibits the ATP binding and chaperone function of hsp90, as well as depletes the levels of EZH2, Suz12 and EED in cultured and primary AML cells (Mol Cancer Ther.2006; 5:3096). Within the PRC2 complex, EZH2 was shown to interact with and modulate the DNA methyltransferases DNMT1, DNMT3a and DNMT3b, which affects their binding to the EZH2-targeted gene promoters. In the present studies we determined the effects of SGI-110 or SGI-1036 and LBH589 on the PRC2 proteins EZH2 and SUZ12, and DNMT1, in the cultured (HL-60, OCI-AML3 and K562) and primary AML cells. Treatment with SGI-110 (0.5 to 2.0 μM) or SGI-1036 (0.5 and 1.0 μM) for 24 hours depleted protein levels of DNMT1 and EZH2 in the cultured and primary AML cells. SGI-110 and SGI-1036 promoted proteasomal degradation of DNMT1 and EZH2 since co-treatment with bortezomib significantly restored DNMT1 and EZH2 levels in the AML cells. Following treatment with SGI-110 or SGI-1036, bisulfite modification and methylation specific PCR demonstrated increase in unmethylated promoter DNA of p15 and JunB. This was associated with induction of the mRNA and protein levels of p15 and JunB, as well as caused inhibition of cell cycle progression (% of cells increased in G1 and increased in S phase) and colony growth in the soft agar. Treatment with 1.0 μM of SGI-110 or SGI-1036 also induced PARP cleavage activity of caspases and induced morphologic evidence of apoptosis in the AML cells. Co-treatment with 10 to 50 nM panobinostat enhanced SGI-110 or SGI-1036 mediated depletion of DNMT1 and EZH2, with more de-repression of the p15 and JunB and significant increase in apoptosis of AML cells. Collectively, these findings indicate that, SGI-110 and SGI-1036 deplete DNMT1 and EZH2 levels, as well as exert potent anti-AML activity. Additionally, combined epigenetic therapy consisting of SGI-110 or SGI-1036 in combination with panobinostat may represent a promising novel treatment of AML.

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.

Structural stability of HfW2O8 is investigated at high pressure using Raman spectroscopy. Irreversible amorphization is found to occur when pressurized to above 3 GPa under hydrostatic conditions. The Raman spectrum of the pressure-amorphized sample closely resembles that of amorphous WO3. On the other hand, spectrum of the recovered sample subjected to uniaxial compression of about 5 GPa showed bands characteristic of crystalline HfO2 besides the parent cubic phase and amorphous phase. Ex situ x-ray diffraction measurements on the pressure-cycled samples also indicated the presence of monoclinic HfO2. These results suggest that the observed pressure-induced amorphization (PIA) under hydrostatic compression is indeed due to hindered decomposition, which is facilitated under uniaxial compression. Thus, the behavior of HfW2O8 appears to be different from that of ZrW2O8, which exhibited only PIA and not pressure-induced decomposition at ambient temperature.

14542 Background: HDAC7 belongs to the class IIA histone deacetylases (HDACs) and is overexpressed in thymocytes, where it transcriptionally represses the proapoptotic nuclear orphan receptor Nur77...

14541 Background: Bone marrow stroma-derived factor-1 (SDF-1, CXCL12) binds and activates the chemokine receptor CXCR4, which regulates the trafficking and mobilization of normal and leukemic hemat...

14569 Background: Aurora A and B are essential for normal mitosis. Aurora A (AA) is amplified and/or co-overexpressed with Aurora B (AB) in over 50% of breast cancers, including the basaloid variet...

11054 Background: MK-0457 (M) is a pan-Aurora kinase inhibitor that also inhibits Bcr-Abl, including mutant Bcr-AblT315I. Aurora kinases are chaperoned by hsp90. Vorinostat (V) is a pan-histone dea...

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.

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.