scholarly journals Clinical Evaluation of Combined Epigenetic Therapies on the Induction of Fetal Hemoglobin in Patients with Hematologic Malignancies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 960-960
Author(s):  
Katharine Rose Press ◽  
Jeffrey Keefer ◽  
Steven D. Gore ◽  
Hetty E. Carraway ◽  
Sarah Sakoian ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Hdac Inhibitors ◽  
Fetal Hemoglobin ◽  
Fold Increase ◽  
Methyl Transferase ◽  
Disease Response ◽  
Deacetylase Inhibitor ◽  
Hbf Induction ◽  
Hbf Level

Fetal hemoglobin induction with hydroxyurea (HU) is a mainstay of therapy for β-hemoglobinopathies, especially sickle cell disease (SCD). A high level of fetal hemoglobin (HbF) has a direct relationship with acute clinical status in SCD patients including pain crises, acute chest syndrome, and death. However, not all patients benefit from HU, and more effective HbF induction strategies are needed. DNA methyl transferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors have been shown in vitro to induce HbF production through epigenetic modification of the β-globin gene cluster. Azacitidine (AZA) is a DNMT already used in some SCD patients resistant to HbF modulation with HU. Entinostat (MS-275) is an orally available histone deacetylase inhibitor with a long half-life and established antitumor activity in preclinical models. Recent studies suggest that drugs, which act with different molecular and epigenetic mechanisms, have synergistic effects on induction of fetal hemoglobin (Fard et al. IJHOSCR 2013). In this study, we evaluated the effects of a combination of AZA and MS-275 on HbF levels. This was preformed as a correlative study of a phase I clinical trial (J0443 trial) of these drugs in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). We sequentially measured the level of HbF the peripheral blood in 33 patients receiving different doses of AZA (range: 30mg/m2 to 50mg/m2 per day for 10 doses) and MS-275 (range: 2 to 8 mg/m2 orally on days 3 and 10). Patients completed a minimum of four 28-day cycles of combined therapy. HbF levels were measured in peripheral blood at baseline, at day 15 or 16 and day 29 or 30 of cycle 1, and after cycles 2, 4, and if applicable 6. Azacitidine dose positively correlated with HbF fold increase (mean of 1.1, 2.3, and 2.1 for doses of 30, 40, and 50 mg respectively, p=0.07) while MS-275 dose had a slightly negative correlation with HbF level (mean of 3.0, 1.8, and 1.3 for doses of 2, 4, and 6mg respectively, p=0.13). There was no correlation between baseline HbF and HbF fold increase after exposure to treatment (p=NS) and no correlation between baseline HbF levels and clinical disease response (p=0.19). Interestingly, we demonstrated a correlation between HbF fold increase and clinical disease response: median fold increase of 3.5 for patients achieving hematologic normalization (complete response, partial response, or trilineage hematological improvement) versus 1.4 in non-responders (p=0.006). The positive correlation between AZA dose and HbF increase is consistent with prior work showing that this drug induces HbF production. The correlation between clinical response and HbF induction could reflect a greater susceptibility to AZA potentially related to differing methylomes. Alternatively, it may also represent a known increase in HbF in the setting of stress erythropoiesis. The slight inverse correlation between MS-275 and HbF level was surprising, as other HDAC inhibitors are known to induce HbF in vitro. However, these results are in line with the methylation data found in the more recent randomized phase 2 trial of AZA +/- MS-275 (E1905 trial) that showed a potential pharmacodynamic antagonism of the combination (Prebet et al. J Clin Oncol. 2014). Overall, this work supports the use of AZA as a clinical inducer of HbF. It also shows the importance of trialing various combinations of HbF inducers, as not all drugs work synergistically and some may even be antagonistic in combination. Disclosures Off Label Use: Azacitidine (AZA) is a DNA methyl transferase (DNMT) inhibitor. Entinostat (MS-275) is an orally available histone deacetylase inhibitor. Both drugs were used in a phase I clinical trial (J0443 trial) of these drugs in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). . Keefer:MAST therapeutics: Employment. Gore:Celgene: Consultancy, Honoraria, Research Funding. Prebet:CELGENE: Research Funding.

Potent Induction of Human γ Globin Gene Expression by Largazole, a New Histone Deacetylase Inhibitor.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2022-2022
Author(s):  
Hua Cao ◽  
Rui Gao Fei ◽  
Albert A. Bowers ◽  
Thomas J. Greshock ◽  
Tenaya Newkirt ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Histone Deacetylase ◽  
Hdac Inhibitor ◽  
Globin Gene ◽  
Control Level ◽  
Hdac Inhibitors ◽  
Fetal Hemoglobin ◽  
Vitro Effect

Abstract Abstract 2022 Poster Board I-1044 Previous studies have demonstrated that Histone Deacetylase (HDAC) inhibitors such as butyrate and several short chain fatty acids, can induce fetal hemoglobin in humans and animal models; however induction of Hb F is achieved in relatively high concentrations of these compounds. We have previously investigated the induction of human γ globin gene activity by the prototypical HDAC inhibitor, FK228. The results demonstrated that FK228 is a more potent γ globin gene inducer compared to other HDAC inhibitors we have tested before (Am J Hematol. 12:981). In this study, we investigated the induction of human γ globin gene function of largazole and it's thiol analogue in vitro in cultures of normal human adult BFUe and in vivo in the mice carrying a human γ globin transgene. Largazole is a HDAC inhibitor which was recently isolated from a marine vyanobacterium by Luesch and co-workers. Structural features of largazole, a macrocyclic depsopeptide, closely resemble those of FK228, FR901375 and spiruchostatin. We have reported that largazole and numerous synthetic analogues are highly potent Class I histone deacetylase inhibitors (J Am Chem Soc. 130:11219, J Am Chem Soc. 2009 Feb 4). We used flow cytometry to measure the in vitro effect of largazole and it's derivatives on the frequency of HbF-positive erythroblasts in BFUe cultures from normal individuals; real-time quantitative PCR (RT-qPCR) and high performance liquid chromatography (HPLC) were used to measure the in vivo effects of largazole on human γ globin induction in γ transgenic mice carrying a human γ globin gene.. Our results show that largazole and it's thiol derivative are potent γ hemoglobin gene inducers. In the human BFUe cultures, largazole increased the levels of fetal hemoglobin positive cells from 21.9% (control level) to 62.8% at a concentration of 0.1μM; largazole thiol increased the levels of fetal hemoglobin positive cells to 62.0% at a concentration of 1μM. Transgenic mice carrying the human μLCR Aγ construct continue to express the human γ gene in the adult stage (Blood. 77:1326). Largazole was administered through IP injection at the dosages of 0.3mg/kg/day and 0.6mg/kg/day, 5 days per week, for 2 weeks to two cohorts of transgenic mice. Largazole at the dose of 0.3mg/kg/day increased the level of human γ mRNA at the end of injection by 160.7%; at a dose of 0.6mg/kg/day human γ mRNA increased by 174.7%. At the 0.6mg/kg/day dosage the level of fetal hemoglobin in the peripheral blood of the animals increased by 3.4 and 3.2 fold at day 21 and day 28, respectively. These results provide strong in vitro and in vivo evidence that Largazole and it's thiol analogue are potent HbF inducers acting at low concentrations, and thus provide promising alternatives to compounds currently considered for induction of Hb F in patients with sickle cell disease and thalassemia. Disclosures: No relevant conflicts of interest to declare.

Induction of Fetal Hemoglobin by Inactivation of HDAC1 or HDAC2 without Altering Cellular Proliferation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 354-354
Author(s):  
Erica B. Esrick ◽  
Jian Xu ◽  
Katherine Lin ◽  
Marie Ellen McConkey ◽  
Alyse Frisbee ◽  
...  
Keyword(s):  
Hdac Inhibitor ◽  
Cell Cycle Progression ◽  
Hdac Inhibitors ◽  
Fetal Hemoglobin ◽  
Fold Increase ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Hydroxyurea Treatment

Abstract Abstract 354 Histone deacetylase (HDAC) inhibitors are effective inducers of fetal hemoglobin, and prior studies have shown that selective inactivation of HDAC1 or HDAC2 is sufficient to induce fetal hemoglobin in vitro. In our current work, we demonstrate that HDAC1 and HDAC2 are attractive targets for clinical translation for two reasons: 1) Selective inhibition will decrease off-target effects that currently limit the use of hydroxyurea and pan-HDAC inhibitors, and 2) HDAC inhibitors induce fetal hemoglobin in various preclinical models, and they can be combined with hydroxyurea to achieve further fetal hemoglobin induction. To investigate off-target effects, we selectively inactivated HDAC1, HDAC2 or HDAC3 in human erythroid progenitor cells, and examined the effect of each knockdown on cellular cytotoxicity and cell cycle progression. Although knockdown of HDAC3 negatively influenced growth, selective knockdown of HDAC1 or HDAC2 had no effect on expansion of erythroid progenitors. In addition, knockdown of HDAC2 does not block cell cycle progression. These data support the possibility that an HDAC1- or HDAC2-specific inhibitor may offer a therapeutic advantage by reducing side effects, while maintaining robust HbF induction. Armed with this knockdown data, we are now investigating HDAC inhibitor compounds of various selectivity in in vitro and in vivo models. To perform optimal clinical trials, and ultimately to benefit the most sickle cell disease patients, it would be ideal to combine HDAC inhibitor treatment with hydroxyurea. A combination treatment approach may ameliorate some of the limitations of hydroxyurea use, such as the unpredictable effect on fetal hemoglobin levels, and the lack of benefit in beta thalassemia patients. First, we combined HDAC2 inactivation with hydroxyurea treatment in vitro. Human bone marrow-derived CD34+ cells were infected with lentiviruses containing an shRNA targeting either HDAC2 or a luciferase control gene. The cells were then treated on day 4 of erythroid differentiation with hydroxyurea (10–20 uM dose). Compared to the untreated luciferase control samples, we observed a 7–9-fold increase in gamma-globin expression in the untreated HDAC2-knockdown samples, a 2.5-fold increase in the hydroxyurea-treated luciferase control samples, and a trend toward an additive effect on gamma-globin induction in the cells where HDAC2 knockdown was combined with hydroxyurea treatment. To investigate the effects of HDAC inhibitors in vivo, we administered compounds to BCL11A conditional knockout transgenic mice (by erythroid-selective EpoR-GFP Cre) containing the human beta-globin locus. As reported previously, BCL11A inactivation powerfully de-repressed gamma-globin expression, and administration of an HDAC inhibitor, SAHA, led to a further elevation of gamma-globin mRNA. We now demonstrate that administration of another pan-HDAC inhibitor, panobinostat (LBH589), results in an additional 1.5- to 2.5-fold increase in gamma-globin mRNA relative to pre-treatment baseline. We are currently evaluating the combination of panobinostat and hydroxyurea in these mice to confirm that the compounds have an additive effect in vivo as well as in vitro. Taken together, these experiments indicate that inhibiting HDAC1 or HDAC2 is a promising therapeutic approach to increasing fetal hemoglobin levels in patients with beta-hemoglobinopathies, both alone and in combination with hydroxyurea. Disclosures: Bradner: Acetylon: .

scholarly journals Truncated Erythropoietin Receptors Confer an In Vivo Selective Advantage in Gene-Modified Erythroid Cells Expressing Fetal Hemoglobin Due to BCL11A Interference

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2063-2063
Author(s):  
Naoya Uchida ◽  
Claire Drysdale ◽  
Morgan Yapundich ◽  
Jackson Gamer ◽  
Tina Nassehi ◽  
...  
Keyword(s):  
Rhesus Macaques ◽  
Fetal Hemoglobin ◽  
Selective Advantage ◽  
Erythroid Cells ◽  
Post Transplant ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Hbf Induction

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, requires high-level gene marking and robust therapeutic globin expression in erythroid cells (>20% of γ- or β-globin production) for widespread successful clinical application. We previously demonstrated that lentiviral transduction of a truncated human erythropoietin receptor (thEpoR) gene allows for erythropoietin-dependent selective proliferation of gene-modified human erythroid cells during in vitro differentiation (ASH 2017). In this study, we sought to evaluate whether thEpoR can enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous non-human primate transplantation models. To investigate this hypothesis, we designed lentiviral vectors encoding both thEpoR and BCL11A-targeting micro RNA-adapted short hairpin RNA (shmiBCL11A), driven off an erythroid specific ankyrin 1 (ANK1) promoter. Both selective proliferation and high-level fetal hemoglobin (HbF) induction were observed in in vitro erythroid differentiation cultures using transduced human CD34+ cells. Healthy donor CD34+ cells were transduced with shmiBCL11A vector, thEpoR-shmiBCL11A vector, and GFP vector (control). Transduced cells were transplanted into immunodeficient NBSGW mice. Five months post-transplant, xenograft bone marrow cells were evaluated for human cell engraftment (human CD45+) and vector copy number (VCN) in both human CD34+ progenitor cells and glycophorin A+ (GPA+) erythroid cells. HbF production was also measured in GPA+ erythroid cells by reverse phase HPLC. We observed efficient transduction in transduced CD34+ cells in vitro (VCN 2.1-5.1) and similar human cell engraftment among all groups (84-89%). The VCN with thEpoR-shmiBCL11A transduction was 3-fold higher in human erythroid cells when compared to CD34+ cells (p<0.01), but not with shmiBCL11A or GFP vectors. HbF levels were significantly elevated in thEpoR-shmiBCL11A vector (43±6%, p<0.01) when compared to no transduction control (1±0%), but not for either shmiBCL11A vector (3±1%) or GFP vector (1±0%). These data demonstrate selective proliferation of gene-modified erythroid cells, as well as enhanced HbF induction with thEpoR-shmiBCL11A transduction. We then performed autologous rhesus CD34+ cell transplantation using either shmiBCL11A vector (142562 and RA0706, n=2, compared to a GPA promoter-derived shmiBCL11A vector) or thEpoR-shmiBCL11A vector (ZL50 and ZM24, n=2, compared to a Venus-encoding vector). Transduced CD34+ cells were transplanted into autologous rhesus macaques following 2x5Gy total body irradiation. Efficient transduction was observed in CD34+ cells in vitro among all 4 macaques (VCN 3.8-8.7) using a high-density culture protocol (Uchida N, Mol Ther Methods Clin Dev. 2019). In shmiBCL11A transduction animals, engraftment of gene-modified cells (VCN 0.2-1.0) and robust HbF induction (14-16%) were observed 1 month post-transplant. However, VCN and HbF levels were reduced down to VCN ~0.1 and HbF ~0.4% in both animals 6 months post-transplant. In contrast, a thEpoR-shmiBCL11A transduction animal (ZL50) resulted in engraftment of gene-modified cells (VCN 0.8-1.0) and robust HbF induction (~18%) 1 month post-transplant, with both gene marking and HbF levels remaining high at VCN 0.6-0.7 and HbF ~15% 4 months post-transplant. These data suggest that shmiBCL11A transduction results in transient HbF induction in gene-modified erythroid cells, while thEpoR-based selective advantage allows for sustained HbF induction with shmiBCL11A. In summary, we developed erythroid-specific thEpoR-shmiBCL11A expressing vectors, enhancing HbF induction in gene-modified erythroid cells in xenograft mice and rhesus macaques. While further in vivo studies are desirable, the use of thEpoR appears to provide a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals Design, synthesis and evaluation of belinostat analogs as histone deacetylase inhibitors

2019 ◽  
Vol 11 (21) ◽  
pp. 2765-2778
Author(s):  
Jie-Huan Zhang ◽  
Madhusoodanan Mottamal ◽  
Hai-Shan Jin ◽  
Shanchun Guo ◽  
Yan Gu ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Active Compound ◽  
Histone Deacetylase Inhibitors ◽  
Hdac Inhibitors ◽  
Antitumor Therapy ◽  
Design Synthesis ◽  
Inhibitory Activities ◽  
Antiproliferative Activities

Aim: Histone deacetylase (HDAC) is an attractive target for antitumor therapy. Therefore, the development of novel HDAC inhibitors is warranted. Materials & methods: A series of HDAC inhibitors based on N-hydroxycinnamamide fragment was designed as the clinically used belinostat analog using amide as the connecting unit. All target compounds were evaluated for their in vitro HDAC inhibitory activities and some selected compounds were tested for their antiproliferative activities. Conclusion: Among them, compound 7e showed an IC50 value of 11.5 nM in inhibiting the HDAC in a pan-HDAC assay, being the most active compound of the series.

Effects of FK228, a novel histone deacetylase inhibitor, on human lymphoma U-937 cells in vitro and in vivo

2002 ◽  
Vol 64 (7) ◽  
pp. 1079-1090 ◽  
Author(s):  
Yuka Sasakawa ◽  
Yoshinori Naoe ◽  
Takeshi Inoue ◽  
Tatsuya Sasakawa ◽  
Masahiko Matsuo ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitor ◽  
Deacetylase Inhibitor ◽  
Human Lymphoma

scholarly journals Histone deacetylase inhibitor MPT0B291 suppresses Glioma Growth in vitro and in vivo partially through acetylation of p53

2020 ◽  
Vol 16 (16) ◽  
pp. 3184-3199
Author(s):  
Batsaikhan Buyandelger ◽  
Eli E Bar ◽  
Kuo-Sheng Hung ◽  
Ruei-Ming Chen ◽  
Yung-Hsiao Chiang ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitor ◽  
Deacetylase Inhibitor ◽  
Glioma Growth
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