Journal: HemaSphere

Article Title: The Novel Oral BET-CBP/p300 Dual Inhibitor NEO2734 Is Highly Effective in Eradicating Acute Myeloid Leukemia Blasts and Stem/Progenitor Cells

doi: 10.1097/HS9.0000000000000610

Figure Lengend Snippet: NEO2734 and NEO1132 reduce AML cell growth by inducing apoptosis, and efficiently downregulate c-Myc and Bcl2 expression. For all in vitro experiments, AML cell lines were incubated with increasing concentrations of CPI-637, NEO1132, or NEO2734 for 96 h, unless stated otherwise. Data are plotted as mean ± SEM and P values were calculated using a 2-way ANOVA with post-hoc Tuckey’s multiple comparison test. (A), Cell viability of AML cell lines was measured using an MTT assay, quantified as relative absorbance and normalized against untreated controls. Graphs are representative of 3 independent experiments (in triplicate). IC50 values and statistics are shown in Supplemental Digital Content, Table S1, http://links.lww.com/HS/A176 . (B and C), Immunoblot analysis of c-Myc and Bcl2 expression in AML cell lines treated with NEO2734. (D–E), Percentage of (D) Annexin-V + and (E) 7AAD + cells in AML cell lines after treatment with increasing concentrations of indicated drugs. Untreated control samples were set to 0%. (F), Immunoblot analysis of caspase 3, cleaved caspase 3, and p21 in THP1 cells treated with NEO2734 for 48 h. AML = acute myeloid leukemia; IC50 = the concentration whereby a drug reduces cell survival by half; MTT = 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.

Article Snippet: For in vitro and ex vivo experiments, NEO1132 (Neomed Institute), NEO2734 (Neomed Institute), and CPI-637 (MedChem Express) were dissolved at a concentration of 10 mM in DMSO and preserved at −20°C for future use.

Techniques: Expressing, In Vitro, Incubation, MTT Assay, Western Blot, Concentration Assay

Journal: HemaSphere

Article Title: The Novel Oral BET-CBP/p300 Dual Inhibitor NEO2734 Is Highly Effective in Eradicating Acute Myeloid Leukemia Blasts and Stem/Progenitor Cells

doi: 10.1097/HS9.0000000000000610

Figure Lengend Snippet: NEO2734 and NEO1132 induce a G1-phase cell cycle arrest, and efficiently inhibit tumor growth of MV4;11 cells in a tumor xenograft mouse model. (A), Cell cycle analysis of AML cell lines incubated with 100 and 500 nM of CPI-637, NEO1132, or NEO2734 for 24 h (THP1 and MV4;11) or 48 h (HL60 and KG1). The percentage of cells in G1, S, and G2 phase was measured using flow cytometry in 3 independent experiments and plotted as mean ± SEM. P values were calculated using a 2-way ANOVA with post-hoc Tuckey’s multiple comparison test for statistical comparison of the G1 phase. (B), Schematic overview of the experiment (left). 3 × 10 6 MV4;11 cells were subcutaneously injected on the right side of the lower flanks of SCID (CB17) mice. 3 wk postinjection, mice were treated once daily (ED) for 11 consecutive days during treatment period 1 (day 1-11) or for 11 times EOD during treatment period 1 (day 1-11) and treatment period 2 (day 19-27) with 10 mg/kg NEO1132, NEO2734, or control (40% PEG400 in distilled water), administered by oral gavage at 5 mL/kg. Tumor volume from 6 mice each group was monitored until study termination (right). P values were calculated using a 2-way repeated measure ANOVA with post-hoc Tukey’s multiple comparison test for comparison of 3 or more groups, * vs control group, + vs NEO1132 EOD, # vs NEO2734 EOD. For comparison of 2 groups, P values were calculated using a paired Student’s t test, ‡NEO1132 vs NEO2734. AML = acute myeloid leukemia; ED = every day; EOD = every other day.

Article Snippet: For in vitro and ex vivo experiments, NEO1132 (Neomed Institute), NEO2734 (Neomed Institute), and CPI-637 (MedChem Express) were dissolved at a concentration of 10 mM in DMSO and preserved at −20°C for future use.

Techniques: Cell Cycle Assay, Incubation, Flow Cytometry, Injection

Journal: HemaSphere

Article Title: The Novel Oral BET-CBP/p300 Dual Inhibitor NEO2734 Is Highly Effective in Eradicating Acute Myeloid Leukemia Blasts and Stem/Progenitor Cells

doi: 10.1097/HS9.0000000000000610

Figure Lengend Snippet: NEO2734 and NEO1132 eliminate primary AML blasts by inducing apoptosis. Primary AML samples were incubated for 4 d with increasing concentrations of CPI-637, NEO1132, or NEO2734. Percentages of AML cell populations were measured using flow cytometry, quantified relative to flow count beads, normalized against untreated controls and plotted as mean ± SEM. P values were calculated using a repeated measure 1- or 2-way ANOVA with post-hoc Tukey’s multiple comparison test. Patient characteristics and IC50 values are shown in Supplemental Digital Content, Table S2, http://links.lww.com/HS/A176 . (A and B), In 6 primary AML samples, cell viability was quantified using an MTT assay, (A) measured as relative absorbance (in triplicate) and normalized against untreated controls. (B), IC50 values in nM were calculated. (C), Viable CD45 dim CD33 + (top, blue) and CD45 dim CD34 + (bottom, red) cells in AML6 after treatment with 500 nM of indicated drugs. (D), Percentage of viable CD45 dim CD33 + and (E) CD45 dim CD34 + cells in 5 primary AML samples. (F), Annexin-V + (top) and 7AAD + (bottom) cells in AML5 after treatment with 1000 nM of indicated drugs. (G), Percentage of annexin-V + and (H) 7AAD + cells in 3 primary AML samples. Untreated control samples were set to 0%. AML = acute myeloid leukemia; IC50 = the concentration whereby a drug reduces cell viability by half.

Article Snippet: For in vitro and ex vivo experiments, NEO1132 (Neomed Institute), NEO2734 (Neomed Institute), and CPI-637 (MedChem Express) were dissolved at a concentration of 10 mM in DMSO and preserved at −20°C for future use.

Techniques: Incubation, Flow Cytometry, MTT Assay, Concentration Assay

Journal: HemaSphere

Article Title: The Novel Oral BET-CBP/p300 Dual Inhibitor NEO2734 Is Highly Effective in Eradicating Acute Myeloid Leukemia Blasts and Stem/Progenitor Cells

doi: 10.1097/HS9.0000000000000610

Figure Lengend Snippet: NEO2734 significantly impairs leukemic stem/progenitor cell survival. Primary AML samples were incubated with increasing concentrations of CPI-637, NEO1132, or NEO2734. Data were normalized against untreated controls and depicted as mean ± SD, unless stated otherwise. P values were calculated using a repeated measure 2-way ANOVA with post-hoc Tukey’s multiple comparison test. Patient characteristics are shown in Supplemental Digital Content, Table S2, http://links.lww.com/HS/A176 . (A–D), After 4 d of treatment, CD45 dim CD33 + LAIP + blasts and CD45 dim CD34 + CD38 − LAIP + cells in AML2 (LAIP = CD56) and AML5 (LAIP = CD7) were measured using flow cytometry and quantified relative to flow count beads. (A), Flow cytometric analysis after treatment with 1000 nM of indicated drugs and (B) percentages of CD45 dim CD33 + LAIP + cells. (C), Flow cytometry analysis after treatment with 1000 nM of indicated drugs and (D) percentages of CD45 dim CD34 + CD38 - LAIP + cells. (E), CFU progenitor assays (in duplicate) of primary AML samples. After treatment of the primary cells for 1 wk, cells were incubated in MethoCult without erythropoietin for 8-12 d, and colonies were quantified using a microscope. Data were normalized against untreated controls and plotted as mean ± SEM. AML = acute myeloid leukemia; CFU = colony-forming unit; LAIP = leukemia-associated immunophenotypic.

Article Snippet: For in vitro and ex vivo experiments, NEO1132 (Neomed Institute), NEO2734 (Neomed Institute), and CPI-637 (MedChem Express) were dissolved at a concentration of 10 mM in DMSO and preserved at −20°C for future use.

Techniques: Incubation, Flow Cytometry, Microscopy

Journal: HemaSphere

Article Title: The Novel Oral BET-CBP/p300 Dual Inhibitor NEO2734 Is Highly Effective in Eradicating Acute Myeloid Leukemia Blasts and Stem/Progenitor Cells

doi: 10.1097/HS9.0000000000000610

Figure Lengend Snippet: NEO2734 reduces leukemic engraftment and adds to chemotherapy treatment in a PDX AML mouse model. After IV injection of T-cell–depleted primary AML cells, NSG mice were IV treated at 10 mL/kg with combination chemotherapy (50 mg/kg cytarabine and 1.5 mg/kg doxorubicin) or control (PBS), and/or 10 mg/kg NEO1132, NEO2734, or control (40% PEG400 in distilled water) administered by oral gavage at 10 mL/kg. At study termination, bone marrows were collected and analyzed using flow cytometry. P values were calculated using a 1-way ANOVA with post-hoc Tukey’s multiple comparison. Patient characteristics are shown in Supplemental Digital Content, Table S2, http://links.lww.com/HS/A176 . (A), Schematic overview of the experiment (left). After injection of 1.5 × 10 6 AML8 cells, NSG mice were treated with NEO1132, NEO2734, or control for 11 times (week 11-12). At week 15, the bone marrows of the mice were analyzed for the presence of myeloid hCD45 + CD33 + LAIP + leukemia cells (LAIP = CD7, middle) and immature hCD45 + CD34 + cells (right). (B), Schematic overview of the experiment (top left). After injection of 2.4 × 10 6 AML9 cells, NSG mice were treated with combination chemotherapy for 2 consecutive days in week 8 and subsequently 10 times with NEO1132 or NEO2734 in week 8-11. At week 15, the bone marrows of the mice were analyzed for the presence of human CD45 + cells (top right), myeloid leukemia hCD45 + CD33 + LAIP + cells (LAIP = CD15 − /HLA-DR − , bottom left), and immature hCD45 + CD34 + cells (bottom right). (C), Schematic overview of the experiment (top). After injection of 1.6 × 10 6 AML10 cells, NSG mice were treated with combination chemotherapy for 2 consecutive days in week 6 and subsequently 10 times with NEO1132 or NEO2734 in week 6-9. At week 12, the bone marrows of the mice were analyzed for the presence of human CD45 + cells (left) and myeloid leukemia hCD45 + CD33 + LAIP + cells (LAIP = CD7, right). AML = acute myeloid leukemia; IV = intravenously; LAIP = leukemia-associated immunophenotypic; NSG = NOD/SCID/IL2r gamma; PBS = phosphate buffered saline; PDX = patient-derived xenograft; PEG400 = 40% polyethylene glycol.

Article Snippet: For in vitro and ex vivo experiments, NEO1132 (Neomed Institute), NEO2734 (Neomed Institute), and CPI-637 (MedChem Express) were dissolved at a concentration of 10 mM in DMSO and preserved at −20°C for future use.

Techniques: IV Injection, Flow Cytometry, Injection, Derivative Assay

Journal: HemaSphere

Article Title: The Novel Oral BET-CBP/p300 Dual Inhibitor NEO2734 Is Highly Effective in Eradicating Acute Myeloid Leukemia Blasts and Stem/Progenitor Cells

doi: 10.1097/HS9.0000000000000610

Figure Lengend Snippet: NEO2734 shows higher activity in primary AML cells than in normal hematopoietic bone marrow cells, providing a therapeutic window. For all ex vivo experiments, NBM cells from healthy donors were incubated for 4 d with increasing concentrations of CPI-637, NEO1132, or NEO2734, and P values were calculated using a 1 or 2-way ANOVA with post-hoc Tukey’s multiple comparison test unless stated otherwise. NBM sample characteristics and IC50 values are shown in Supplemental Digital Content, Table S2, http://links.lww.com/HS/A176 . (A and B), Using an MTT assay, cell viability (in triplicate) was quantified in 2 NBM samples, (A) measured as relative absorbance and normalized against untreated controls, and (B) IC50 values (in nM) were calculated. Data are plotted as mean ± SD and P values were calculated using a repeated measure 1-way ANOVA with Dunnett’s multiple comparison test. For each drug, the mean IC50 ± SEM in primary AML and NBM samples (right table) was compared and P values were determined using a Student’s t test. (C–E), Percentage of (C) viable CD45 dim CD33 + , (D) CD3 + T-cells, and (E) CD19 + B-cells in 2 NBM samples, measured using flow cytometry, quantified against untreated controls and plotted as mean ± SD. (F), CFU progenitor assays (in duplicate) of 2 NBM samples. After treatment of the NBM cells for 1 wk, cells were incubated in MethoCult without erythropoietin for 8-12 d, and colonies were quantified using a microscope. Data were normalized against untreated controls and depicted as mean ± SEM. P values were calculated using a 2-way ANOVA with post-hoc Tukey’s multiple comparison test. (G–L), 3 × 10 6 MV4;11 cells were subcutaneously injected on the right side of the lower flanks of SCID (CB17) mice. 3 wk postinjection, mice were treated once daily (ED) for 11 consecutive days during treatment period 1 (day 1-11) or for 11 times EOD during treatment period 1 (day 1-11) and treatment period 2 (day 19-27) with 10 mg/kg NEO1132, NEO2734, or control (40% PEG400 in distilled water), administered by oral gavage at 5 mL/kg. Schematic overview of the experiment is shown in Figure B, left. Data are depicted as mean ± SEM, derived from 6 mice each group. (G), Body weight (in g) of the mice was monitored until study termination. The numbers of (H) WBC, (I) granulocytes, (J) RBC, (K) monocytes, and (L) lymphocytes were measured after treatment period 1 at day 12. AML = acute myeloid leukemia; CFU = colony-forming unit; ED = every day; EOD = every other day; IC50 = the concentration whereby a drug reduces cell viability by half; MTT = 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; NBM = normal bone marrow; PEG400 = 40% polyethylene glycol; RBC = red blood cells; SCID = severe combined immunodeficiency; WBC = white blood cells.

Article Snippet: For in vitro and ex vivo experiments, NEO1132 (Neomed Institute), NEO2734 (Neomed Institute), and CPI-637 (MedChem Express) were dissolved at a concentration of 10 mM in DMSO and preserved at −20°C for future use.

Techniques: Activity Assay, Ex Vivo, Incubation, MTT Assay, Flow Cytometry, Microscopy, Injection, Derivative Assay, Concentration Assay

Journal: Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy

Article Title: Targeting Myc-driven stress addiction in colorectal cancer

doi: 10.1016/j.drup.2023.100963

Figure Lengend Snippet: Selected Myc targeting agents in development.

Article Snippet: A novel dual BET and histone acetyltransferases (HAT) inhibitor NEO2734, reduces MYC mRNA and the growth of CRC cells and xenografts by inducing PUMA- and DR5-dependent apoptosis ( Kuang et al., 2022 ).

Techniques: Activation Assay