Rekha Rao

Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells

The polycomb repressive complex (PRC) 2 contains 3 core proteins, EZH2, SUZ12, and EED, in which the SET (suppressor of variegation-enhancer of zeste-trithorax) domain of EZH2 mediates the histone methyltransferase activity. This induces trimethylation of lysine 27 on histone H3, regulates the expression of HOX genes, and promotes proliferation and aggressiveness of neoplastic cells. In this study, we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) depletes EZH2 levels, and inhibits trimethylation of lysine 27 on histone H3 in the cultured human acute myeloid leukemia (AML) HL-60 and OCI-AML3 cells and in primary AML cells. DZNep treatment induced p16, p21, p27, and FBXO32 while depleting cyclin E and HOXA9 levels. Similar findings were observed after treatment with small interfering RNA to EZH2. In addition, DZNep treatment induced apoptosis in cultured and primary AML cells. Furthermore, compared with treatment with each agent alone, cotreatment with DZNep and the pan-histone deacetylase inhibitor panobinostat caused more depletion of EZH2, induced more apoptosis of AML, but not normal CD34(+) bone marrow progenitor cells, and significantly improved survival of nonobese diabetic/severe combined immunodeficiency mice with HL-60 leukemia. These findings indicate that the combination of DZNep and panobinostat is effective and relatively selective epigenetic therapy against AML cells.

Coordinated Silencing of MYC-Mediated miR-29 by HDAC3 and EZH2 as a Therapeutic Target of Histone Modification in Aggressive B-Cell Lymphomas

We investigated the transcriptional and epigenetic repression of miR-29 by MYC, HDAC3, and EZH2 in mantle cell lymphoma and other MYC-associated lymphomas. We demonstrate that miR-29 is repressed by MYC through a corepressor complex with HDAC3 and EZH2. MYC contributes to EZH2 upregulation via repression of the EZH2 targeting miR-26a, and EZH2 induces MYC via inhibition of the MYC targeting miR-494 to create positive feedback. Combined inhibition of HDAC3 and EZH2 cooperatively disrupted the MYC-EZH2-miR-29 axis, resulting in restoration of miR-29 expression, downregulation of miR-29-targeted genes, and lymphoma growth suppression in vitro and in vivo. These findings define a MYC-mediated miRNA repression mechanism, shed light on MYC lymphomagenesis mechanisms, and reveal promising therapeutic targets for aggressive B-cell malignancies.

HDAC6 inhibition enhances 17-AAG–mediated abrogation of hsp90 chaperone function in human leukemia cells

Histone deacetylase 6 (HDAC6) is a heat shock protein 90 (hsp90) deacetylase. Treatment with pan-HDAC inhibitors or depletion of HDAC6 by siRNA induces hyperacetylation and inhibits ATP binding and chaperone function of hsp90. Treatment with 17-allylamino-demothoxy geldanamycin (17-AAG) also inhibits ATP binding and chaperone function of hsp90, resulting in polyubiquitylation and proteasomal degradation of hsp90 client proteins. In this study, we determined the effect of hsp90 hyperacetylation on the anti-hsp90 and antileukemia activity of 17-AAG. Hyperacetylation of hsp90 increased its binding to 17-AAG, as well as enhanced 17-AAG-mediated attenuation of ATP and the cochaperone p23 binding to hsp90. Notably, treatment with 17-AAG alone also reduced HDAC6 binding to hsp90 and induced hyperacetylation of hsp90. This promoted the proteasomal degradation of HDAC6. Cotreatment with 17-AAG and siRNA to HDAC6 induced more inhibition of hsp90 chaperone function and depletion of BCR-ABL and c-Raf than treatment with either agent alone. In addition, cotreatment with 17-AAG and tubacin augmented the loss of survival of K562 cells and viability of primary acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) samples. These findings demonstrate that HDAC6 is an hsp90 client protein and hyperacetylation of hsp90 augments the anti-hsp90 and antileukemia effects of 17-AAG.

Cotreatment with panobinostat and JAK2 inhibitor TG101209 attenuates JAK2V617F levels and signaling and exerts synergistic cytotoxic effects against human myeloproliferative neoplastic cells

The mutant JAK2V617F tyrosine kinase (TK) is present in the majority of patients with BCR-ABL-negative myeloproliferative neoplasms (MPNs). JAK2V617F activates downstream signaling through the signal transducers and activators of transcription (STAT), RAS/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3 (PI3)/AKT pathways, conferring proliferative and survival advantages in the MPN hematopoietic progenitor cells (HPCs). Treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) is known to inhibit the chaperone function of heat shock protein 90, as well as induce growth arrest and apoptosis of transformed HPCs. Here, we demonstrate that PS treatment depletes the autophosphorylation, expression, and downstream signaling of JAK2V617F. Treatment with PS also disrupted the chaperone association of JAK2V617F with hsp90, promoting proteasomal degradation of JAK2V617F. PS also induced apoptosis of the cultured JAK2V617F-expressing human erythroleukemia HEL92.1.7 and Ba/F3-JAK2V617F cells. Treatment with the JAK2 TK inhibitor TG101209 attenuated JAK2V617F autophosphorylation and induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with PS and TG101209 further depleted JAK/STAT signaling and synergistically induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with TG101209 and PS exerted greater cytotoxicity against primary CD34(+) MPN cells than normal CD34(+) HPCs. These in vitro findings suggest combination therapy with HDAC and JAK2V617F inhibitors is of potential value for the treatment of JAK2V617F-positive MPN.

Hydroxamic Acid Analogue Histone Deacetylase Inhibitors Attenuate Estrogen Receptor-α Levels and Transcriptional Activity: A Result of Hyperacetylation and Inhibition of Chaperone Function of Heat Shock Protein 90

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

Histone deacetylase inhibitors deplete enhancer of zeste 2 and associated polycomb repressive complex 2 proteins in human acute leukemia cells

Human enhancer of zeste 2 (EZH2) protein belongs to the multiprotein polycomb repressive complex 2, which also includes suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). The polycomb repressive complex 2 complex possesses histone methyltransferase activity mediated by the Su(var)3-9, enhancer of zeste, and trithorax domain of EZH2, which methylates histone H3 on lysine (K)-27 (H3K27). In the present studies, we determined that treatment with the hydroxamate histone deacetylase inhibitor LBH589 or LAQ824 depleted the protein levels of EZH2, SUZ12, and EED in the cultured (K562, U937, and HL-60) and primary human acute leukemia cells. This was associated with decreased levels of trimethylated and dimethylated H3K27, with concomitant depletion of the homeobox domain containing HOXA9 and of MEIS1 transcription factors. Knockdown of EZH2 by EZH2 small interfering RNA also depleted SUZ12 and EED, inhibited histone methyltransferase activity, and reduced trimethylated and dimethylated H3K27 levels, with a concomitant loss of clonogenic survival of the cultured acute myelogenous leukemia (AML) cells. EZH2 small interfering RNA sensitized the AML cells to LBH589-mediated depletion of EZH2, SUZ12, and EED; loss of clonogenic survival; and LBH589-induced differentiation of the AML cells. These findings support the rationale to test anti-EZH2 treatment combined with hydroxamate histone deacetylase inhibitors as an antileukemia epigenetic therapy, especially against AML with coexpression of EZH2, HOXA9, and MEIS1 genes. [Mol Cancer Ther 2006;5(12):3096–104]

Auranofin Induces Lethal Oxidative and Endoplasmic Reticulum Stress and Exerts Potent Preclinical Activity against Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) exhibits high remission rates after initial chemoimmunotherapy, but with relapses with treatment, refractory disease is the most common outcome, especially in CLL with the deletion of chromosome 11q or 17p. In addressing the need of treatments for relapsed disease, we report the identification of an existing U.S. Food and Drug Administration-approved small-molecule drug to repurpose for CLL treatment. Auranofin (Ridaura) is approved for use in treating rheumatoid arthritis, but it exhibited preclinical efficacy in CLL cells. By inhibiting thioredoxin reductase activity and increasing intracellular reactive oxygen species levels, auranofin induced a lethal endoplasmic reticulum stress response in cultured and primary CLL cells. In addition, auranofin displayed synergistic lethality with heme oxygenase-1 and glutamate-cysteine ligase inhibitors against CLL cells. Auranofin overcame apoptosis resistance mediated by protective stromal cells, and it also killed primary CLL cells with deletion of chromosome 11q or 17p. In TCL-1 transgenic mice, an in vivo model of CLL, auranofin treatment markedly reduced tumor cell burden and improved mouse survival. Our results provide a rationale to reposition the approved drug auranofin for clinical evaluation in the therapy of CLL.

Targeting levels or oligomerization of nucleophosmin 1 induces differentiation and loss of survival of human AML cells with mutant NPM1

Nucleophosmin 1 (NPM1) is an oligomeric, nucleolar phosphoprotein that functions as a molecular chaperone for both proteins and nucleic acids. 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, which causes NPM1c+ to be localized in the cytoplasm. Here, we determined the effects of targeting NPM1 in cultured and primary AML cells. Treatment with siRNA to NPM1 induced p53 and p21, decreased the percentage 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 unmutated NPM1. Notably, knockdown of NPM1 by shRNA abolished lethal AML phenotype induced by OCI-AML3 cells in NOD/SCID mice. Knockdown of NPM1 also sensitized OCI-AML3 to all-trans retinoic acid (ATRA) and cytarabine. Inhibition of NPM1 oligomerization by NSC348884 induced apoptosis and sensitized OCI-AML3 and primary AML cells expressing NPM1c+ to ATRA. This effect was significantly less in AML cells coexpressing FLT3-ITD, or in AML or normal CD34+ progenitor cells expressing wild-type NPM1. Thus, attenuating levels or oligomerization of NPM1 selectively induces apoptosis and sensitizes NPM1c+ expressing AML cells to treatment with ATRA and cytarabine.

Combination of Pan-Histone Deacetylase Inhibitor and Autophagy Inhibitor Exerts Superior Efficacy against Triple-Negative Human Breast Cancer Cells

Histone deacetylase (HDAC) inhibitors (HDI) induce endoplasmic reticulum (ER) stress and apoptosis, while promoting autophagy, which promotes cancer cell survival when apoptosis is compromised. Here, we determined the in vitro and in vivo activity of the combination of the pan-HDI panobinostat and the autophagy inhibitor chloroquine against human estrogen/progesterone receptor and HER2 (triple)-negative breast cancer (TNBC) cells. Treatment of MB-231 and SUM159PT cells with panobinostat disrupted the hsp90/histone deacetylase 6/HSF1/p97 complex, resulting in the upregulation of hsp. This was accompanied by the induction of enhanced autophagic flux as evidenced by increased expression of LC3B-II and the degradation of the autophagic substrate p62. Treatment with panobinostat also induced the accumulation and colocalization of p62 with LC3B-II in cytosolic foci as evidenced by immunofluorescent confocal microscopy. Inhibition of panobinostat-induced autophagic flux by chloroquine markedly induced the accumulation of polyubiquitylated proteins and p62, caused synergistic cell death of MB-231 and SUM159PT cells, and inhibited mammosphere formation in MB-231 cells, compared with treatment with each agent alone. Finally, in mouse mammary fat pad xenografts of MB-231 cells, a tumor size–dependent induction of heat shock response, ER stress and autophagy were observed. Cotreatment with panobinostat and chloroquine resulted in reduced tumor burden and increased the survival of MB-231 breast cancer xenografts. Collectively, our findings show that cotreatment with an autophagy inhibitor and pan-HDI, for example, chloroquine and panobinostat results in accumulation of toxic polyubiquitylated proteins, exerts superior inhibitory effects on TNBC cell growth, and increases the survival of TNBC xenografts. Mol Cancer Ther; 11(4); 973–83. ©2012 AACR.

Molecular and biologic characterization and drug sensitivity of pan-histone deacetylase inhibitor–resistant acute myeloid leukemia cells

Hydroxamic acid analog pan-histone deacetylase (HDAC) inhibitors (HA-HDIs) have shown preclinical and clinical activity against human acute leukemia. Here we describe HA-HDI-resistant human acute myeloid leukemia (AML) HL-60 (HL-60/LR) cells that are resistant to LAQ824, vorinostat, LBH589, and sodium butyrate. HL-60/LR cells show increased expression of HDACs 1, 2, and 4 but lack HDAC6 expression, with concomitant hyperacetylation of heat shock protein 90 (hsp90). Treatment with HA-HDI failed to further augment hsp90 acetylation, or increase the levels of p21 or reactive oxygen species (ROSs), in HL-60/LR versus HL-60 cells. Although cross-resistant to antileukemia agents (eg, cytarabine, etoposide, and TRAIL), HL-60/LR cells are collaterally sensitive to the hsp90 inhibitor 17-AAG. Treatment with 17-AAG did not induce hsp70 or deplete the hsp90 client proteins AKT and c-Raf. HL-60/LR versus HL-60 cells display a higher growth fraction and shorter doubling time, along with a shorter interval to generation of leukemia and survival in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Thus, resistance of AML cells to HA-HDIs is associated with loss of HDAC6, hyperacetylation of hsp90, aggressive leukemia phenotype, and collateral sensitivity to 17-AAG. These findings suggest that an hsp90 inhibitor-based antileukemia therapy may override de novo or acquired resistance of AML cells to HA-HDIs.