Fig 1.
ACY-957 and ACY-1035 selectively inhibits HDAC1 and 2.
(A) In vitro biochemical assay for HDAC1, HDAC2 or HDAC3 inhibition by ACY-957 (left panel) and ACY-1035 (right panel). Mean ± SD, n = 3. (B) MV-4-11, Kasumi-1 or HL-60 cells were treated with the indicated doses of compounds for 24 hours and immunoblotting performed for acetylated H2BK5, acetylated H3K56 and total H4. A dose-dependent induction of histone acetylation by ACY-957 or ACY-1035 was observed.
Fig 2.
HDAC1/2 inhibition suppresses AML cells in vitro and primary AML blasts ex vivo.
(A) MV-4-11, Kasumi-1, HL-60, MOLM-13 and NB-4 cells were treated with increasing doses of ACY-957 or ACY-1035. After 72 hours, cell viability was measured and response curves plotted using GraphPad Prism 6. Mean ± SD, n ≥ 3. (B) IC50 values from curves in ‘A’ were calculated and listed. ACY-957 and ACY-1035 reduced viability of AML cells with an average IC50 value of 1.5 μM for ACY-957 and 2.6 μM for ACY-1035, respectively. (C) MV-4-11 (upper panel) and HL-60 (lower panel) cells were treated with increasing doses of ACY-957 or ACY-1035 for 72 hours. Surface levels of CD11b were measured by flow cytometry and percentage of CD11b positive cells plotted. ACY-957 and ACY-1035 increased the percentage of CD11b positive cells in a dose-dependent manner. Mean ± SD, n = 3 independent experiments. (D) Cells were treated as above. Cell cycle was analyzed by flow cytometry Edu positive population corresponding to S phase cells were quantified and percentage of S phase cells plotted. ACY-957 and ACY-1035 reduced S phase cells in a dose-dependent manner. Mean ± SD, n = 3 independent experiments. (E) Cells were treated at the indicated doses of compounds for 96 hours. Annexin-V positive cells were analyzed by flow cytometry. ACY-957 and ACY-1035 induced apoptosis in a dose-dependent manner. Mean ± SD, n = 3 independent experiments. (F) Fresh primary AML patient samples were plated into 96-well plates containing ACY-957 at 8 doses and incubated at 37°C for 96 hours. Leukemic cells were identified by surface markers as described in the Materials and Methods and viable leukemic cells corresponding to annexin V negative populations quantified by flow cytometry. IC50 values were calculated using GraphPad Prism 6 and plotted. ACY-957 inhibited the proliferation of primary leukemic cells with a mean IC50 value of 1.1 μM. Bars represent mean ± SD, n = 38. (G) Frozen primary AML patient samples were cultured in methylcellulose-based medium containing cytokines and 6 concentrations of ACY-957 for 14 days as described in the Materials and Methods. Colonies were quantified and IC50 values plotted. ACY-957 inhibited colony growth at a mean IC50 value of 2.6 μM. Bars represent mean ± SD, n = 7. For all panels, statistical analysis was performed by two-tailed Student’s t-test. * indicates P < 0.05 when comparing compound-treated group to DMSO control.
Fig 3.
HDAC1/2 inhibition synergizes with azacitidine to inhibit transformed phenotypes of AML cell lines.
(A–B) MV-4-11 and MOLM-13 cells were treated with 7 doses of ACY-957 plus azacitidine (left panels) or 7 doses of ACY-1035 plus azacitidine (right panels) with the ratio of the drug doses staying at approximately IC50: IC50 as described in the Materials and Methods. Viability was measured and CI values calculated and reported as the average of 3 independent experiments. (C) MV-4-11 (upper panel) and HL-60 (lower panel) cells were treated with azacitidine, ACY-957 or ACY-1035 either as single agents or in combinations at the indicated doses for 72 hours. Surface levels of CD11b were analyzed as in Fig 2C. Combination treatment of ACY-957 plus azacitidine or ACY-1035 plus azacitidine further increased percentage of CD11b positive cells compared to each single agent. Mean ± SD, n = 3 independent experiments. (D) Cells were treated as in ‘C’ and percentage of S phase cells analyzed and plotted as in Fig 2D. Combination treatment further reduced the percentage of S phase cells compared to single agent treatments. Mean ± SD, n = 3 independent experiments. (E) Cells were treated as above for 96 hours and annexin V positive cells quantified as in Fig 2E. Combination treatment further increased apoptotic cells compared to single agent treatments. Mean ± SD, n = 3 independent experiments. For all panels, statistical analysis was performed by two-tailed Student’s t-test. * indicates P < 0.05 for all 3 comparisons: combination treatment versus DMSO; combination treatment versus azacitidine alone; combination treatment versus HDAC1/2 inhibitor alone.
Fig 4.
ACY-957 enhances the anti-leukemic activity of azacitidine in primary AML blasts ex vivo.
(A) Fresh bone marrow samples from AML patients (n = 30) were treated with 3 doses of ACY-957 plus 3 doses of azacitidine. Single agent treatment at 8 doses per drug was performed as well. CI values for 8 combinations were calculated and shown in the squares. The matrix shows one representative bone marrow sample. (B) The median of the 8 CI values was calculated for each sample and plotted. Twenty-two out of 30 samples had a CI value below 1, indicating synergism in majority of the samples tested. (C) Frozen bone marrow samples from AML patients (n = 5) were treated with the indicated doses of ACY-957, azacitidine, ACY-957 plus azacitidine or DMSO and cultured in methylcellulose-based medium for 14 days as in Fig 2G. Colony numbers were quantified and percentage of colonies in DMSO control was plotted. Combination treatment with ACY-957 plus azacitidine significantly reduced colony growth compared to each single agent in 4 out of 5 samples tested. Mean ± SD, n = 3. For all panels, statistical analysis was performed by two-tailed Student’s t-test. * indicates P < 0.05 for all 3 comparisons: combination treatment versus DMSO; combination treatment versus azacitidine alone; combination treatment versus ACY-957 alone.
Fig 5.
ACY-957 enhances the anti-leukemic activity of azacitidine in MOLM-13 xenograft model.
(A-B) Female Crl:NU(NCr)-Foxn1nu mice implanted with MOLM-13 cells (n = 10/group) were treated with vehicle, ACY-957 (125 mg/kg, po, daily), azacitidine (5.5 mg/kg, iv, bi-weekly), or the combination of ACY-957 plus azacitidine for 28 days. (A) Tumor volume was monitored and TTE for each mouse was calculated and plotted as described in the Materials and Methods. Combination treatment of ACY-957 with azacitidine significantly increased the survival of MOLM-13 model over azacitidine alone (P < 0.0001) as well as ACY-957 alone (P = 0.005). (B) Body weight was measured on the indicated days and percentage of body weight plotted. Mean ± SD, n = 10. Statistical analysis was performed by Log-rank (Mantel-Cox) test.
Fig 6.
Gene expression profiling identifies differentially regulated genes.
(A–B) MV-4-11 cells were treated with vehicle DMSO, azacitidine at 1 μM, ACY-1035 at 1 μM and the combination for 48 hours. Total RNA was prepared and analyzed by Affymetrix GeneChip PrimeView™ Human Gene Expression Array. (A) Genes that are up-regulated (red) or down-regulated (blue) by single or combo treatment relative to vehicle. (B) Genes exhibiting additive or synergistic response in the combination treatment relative to each single agent treatment were clustered into 8 categories and represented in a heatmap: A, genes unaffected by single agent treatments and up-regulated in the combination; B, genes unaffected by single agent treatments and down-regulated in the combination; C, genes up-regulated by azacitidine, unaffected by ACY-1035 and up-regulated in the combination more than azacitidine alone; D, genes down-regulated by azacitidine, unaffected by ACY-1035 and down-regulated by the combination more than azacitidine alone; E, genes unaffected by azacitidine, up-regulated by ACY-1035 and up-regulated by the combination more than ACY-1035 alone; F, genes unaffected by azacitidine, down-regulated by ACY-1035 and down-regulated by the combination more than ACY-1035 alone; G, genes up-regulated by azacitidine, up-regulated by ACY-1035 and up-regulated by the combination more than either single agent alone; H, genes down-regulated by azacitidine, down-regulated by ACY-1035 and down-regulated by the combination more than either single agent alone. (C) MV-4-11 and HL-60 cells were treated for 48 hours with the indicated doses of ACY-957, ACY-1035, azacitidine or the combinations. qPCR analysis was performed for the following genes from category G: CDKN1A, CDKN1C, GATA2 and HES1. ACTB was used as the normalizing control. Combinations of ACY-957 plus azacitidine or ACY-1035 plus azacitidine further increased the expression of these genes compared to single agent treatments. Mean ± SD, n = 3. Data shows one representative of 3 independent experiments. (D–E) MV-4-11 cells were transduced with GATA2 or GFP overexpression vectors and stable cell lines derived. (D) Overexpression of GATA2 was confirmed by qPCR. Mean ± SD, n = 3. (E) Stable cells were subjected to proliferation assay in 96-well plates for 6 days. Cell viability was determined at the indicated time points and viable cell numbers plotted. GATA2 overexpressing cells grew much slower than GFP expressing cells. Mean ± SD, n = 3. Data shows one representative of 3 independent experiments. (F) GSEA was performed on the array data using the ‘C3 motif TFT: transcription factor genes’ gene set database. Genes containing GATA binding sites within their promoter regions were enriched in the combination of ACY-1035 and azacitidine compared to azacitidine alone. Significant enrichment is illustrated by the positive running enrichment score (ES) marked by the green line, normalized enrichment score (NES) = 1.6, and false discovery rate (FDR) = 0.03.