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. 2015 Apr;29(4):807-18.
doi: 10.1038/leu.2014.296. Epub 2014 Oct 6.

A regimen combining the Wee1 inhibitor AZD1775 with HDAC inhibitors targets human acute myeloid leukemia cells harboring various genetic mutations

L Zhou  1 , Y Zhang  2 , S Chen  1 , M Kmieciak  1 , Y Leng  3 , H Lin  1 , K A Rizzo  4 , C I Dumur  4 , A Ferreira-Gonzalez  4 , Y Dai  1 , S Grant  5
Affiliations

A regimen combining the Wee1 inhibitor AZD1775 with HDAC inhibitors targets human acute myeloid leukemia cells harboring various genetic mutations

L Zhou et al. Leukemia. 2015 Apr.

Abstract

AZD1775 targets the cell cycle checkpoint kinase Wee1 and potentiates genotoxic agent cytotoxicity through p53-dependent or -independent mechanisms. Here, we report that AZD1775 interacted synergistically with histone deacetylase inhibitors (HDACIs, for example, Vorinostat), which interrupt the DNA damage response, to kill p53-wild type (wt) or -deficient as well as FLT3-ITD leukemia cells in association with pronounced Wee1 inhibition and diminished cdc2/Cdk1 Y15 phosphorylation. Similarly, Wee1 shRNA knockdown significantly sensitized cells to HDACIs. Although AZD1775 induced Chk1 activation, reflected by markedly increased Chk1 S296/S317/S345 phosphorylation leading to inhibitory T14 phosphorylation of cdc2/Cdk1, these compensatory responses were sharply abrogated by HDACIs. This was accompanied by premature mitotic entry, multiple mitotic abnormalities and accumulation of early S-phase cells displaying increased newly replicated DNA, culminating in robust DNA damage and apoptosis. The regimen was active against patient-derived acute myelogenous leukemia (AML) cells harboring either wt or mutant p53 and various next-generation sequencing-defined mutations. Primitive CD34(+)/CD123(+)/CD38(-) populations enriched for leukemia-initiating progenitors, but not normal CD34(+) hematopoietic cells, were highly susceptible to this regimen. Finally, combining AZD1775 with Vorinostat in AML murine xenografts significantly reduced tumor burden and prolonged animal survival. A strategy combining Wee1 with HDACI inhibition warrants further investigation in AML with poor prognostic genetic aberrations.

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Conflict of interest statement

CONFLICT OF INTEREST

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. AZD1775 inhibits Wee1, an event enhanced by HDACIs
Human leukemia U937 (a), MV4-11 (b), MOLM-13 (c), and OCI-AML-3 (d) cells were exposed to indicated concentrations of AZD1775 +/− Vorinostat (Vor) or SBHA for 24 h, after which total and phosphorylated Wee1 (S642) and cdc2/Cdk1 (Y15) were monitored by Western blot analysis. For both MOLM-13 and OCI-AML-3, Vorinostat = 1.5 μM and SBHA = 20 μM. Blots for pWee1 (S642) and total Wee1 were quantified using ImageJ software (available online). Values indicate ratio of phosphorylated vs total Wee1.
Figure 2
Figure 2. AZD1775 interacts synergistically with HDACIs to induce apoptosis in p53-deficient leukemia cells
(a–b) U937 (p53-null) and MV4-11 (p53mut, FLT-ITD) cells were exposed to AZD1775 (nM) +/− 1.5μM Vorinostat or 15 μM SBHA for 24 h, after which the percentage of Annexin V+/PI− (early apoptosis) and Annexin V+/PI+ (late apoptosis) was determined by flow cytometry. (c) Alternatively, Western blot analysis was performed to detect cleavage of caspase-9 and PARP. CF = cleaved fragment. (d) After 24 h-exposure to AZD1775 (U937, 500 nM; MV4-11, 250 nM) +/− Vorinostat (U937, 1.5 μM; MV4-11, 0.5 μM), a soft-agar assay was performed to assess colony formation capacity (** P < 0.01). (e) Cells were exposed (24 h) to varying concentrations of AZD1775 and Vorinostat alone and in combination at a fixed ratio. At the end of this period, the percentage of Annexin V+ cells was determined for each condition, and Median Dose-Effect analysis was then employed to characterize the nature of the interaction between these agents. Combination Index (C.I.) values less than 1.0 denote a synergistic interaction. The results are representative for three separate experiments.
Figure 3
Figure 3. AZD1775 significantly potentiates HDACI lethality in leukemia cells expressing wild type p53
(a–b) OCI-AML-3 (p53wt) and MOLM-13 (p53wt, FLT3-ITD) cells were exposed to the indicated concentrations of AZD1775 +/− 1.5 μM Vorinostat or 20 μM SBHA for 24 h, after which apoptosis was monitored by Annexin V/PI staining and flow cytometry (*P < 0.05, **P < 0.01). (c) Median Dose-Effect analysis was performed to determine C.I. values (< 1.0 denote synergism) in OCI-AML-3 and MOLM-13 cells. The results are representative for three separate experiments. (d) After OCI-AML-3 and MOLM-13 cells were treated with 250 nM AZD1775 +/− 1.5 μM Vorinostat or 20 μM SBHA for 24 h, Western blot analysis was performed to monitor cleavage of caspase 9 and PARP.
Figure 4
Figure 4. Exposure to AZD1775 results in Chk1 activation and cdc2/Cdk1 T14 phosphorylation, events blocked by HDACIs
(a–c) U937 (a), MV-4-11 (b), and MOLM-13 (c) cells were treated (24 h) with the indicated concentrations of AZD1775 +/− Vorinostat or SBHA, after which Western blot analysis was performed to examine phosphorylation (S296, S317, S345) and protein levels of Chk1. (d–f) Alternatively, the mitotic entry marker histone H3 S10 phosphorylation, T14 phosphorylation of cdc2/Cdk1, and total protein levels were monitored in parallel.
Figure 5
Figure 5. Co-treatment with AZD1775 and HDACIs triggers premature mitotic entry and increases newly replicated DNA in early S phase
(a) U937 cells were treated with 250 nM AZD1775 +/− 1.5 μM Vorinostat or 15 μM SBHA for 8 h, after which cells were stained with AlexaFluor 488-conjugated anti-phospho-histone H3 (S10) and PI to determine cell cycle distribution (top, values indicate percentage of cells in G2/M) and the percentage of p-H3-positive cells (bottom, values indicate fold-increases in p-H3-positive cells vs untreated controls) by flow cytometry. (b) After 16 h-treatment, cells were pulse labeled with EdU for 30 min, followed by staining for cell cycle distribution (top, values indicate percentage of cells in early S) or double staining for p-H3 and EdU (bottom, values indicate fold-increases in p-H3-positive cells and EdU-positive cells vs untreated controls, respectively). (c–d) Alternatively, cytospin slides were prepared, followed by triple staining for p-H3 (red), EdU (green), and α-tubulin (red) together with DAPI (blue) nuclear counterstaining. Representative images of p-H3/EdU (top) and α-tubulin/DAPI (bottom) in the same field were shown for each condition. Arrow head, anaphase bridge; arrow, monopolar or multiple spindle; triangle, centrosome clustering and mitotic slippage).
Figure 6
Figure 6. Knock-down of Wee1 sensitizes leukemia cells to HDACI lethality in association with premature mitotic entry and increased DNA damage
(a) U937 cells were stably transfected with constructs encoding shRNA targeting human Wee1 (shWee1) or scrambled sequence as negative control (shNC). Two clones (designated D3 and E3) of shWee1 cells displayed marked down-regulation of Wee1, compared to shNC cells (inset). Cells were then exposed to 1.5 μM Vorinostat or 20 μM for 24 h, after which the percentage of Annexin V+/PI− and Annexin V+/PI+ were determined by flow cytometry (*P < 0.05 and *P < 0.01 vs shNC cells). (b–c) Alternatively, Western blot analysis was performed to monitor levels of Wee1 and total and Y15 phosphorylated cdc2/Cdk1 (B), or phosphorylated histone H3 (S10), γH2A.X, and PARP cleavage (c).
Figure 7
Figure 7. The AZD1775/Vorinostat regimen is active against primary AML cells, including the primitive CD34+/CD38−/CD123+ population, but displays minimal toxicity towards normal CD34+ cells
(a) Primary blasts from a patient with AML (top) and normal CB CD34+ cells (bottom) were exposed to 250 nM AZD1775 +/− 1.5 μM Vorinostat for 24 h, after which cells were stained with Annexin-V/PI and analyzed by flow-cytometry. Values indicate the percentage of Annexin V+/PI− and Annexin V+/PI+ cells in a total of 10,000 cells analyzed for each condition. (b) Parallel experiments were carried out with six AML primary samples. Median response was analyzed based on the percentage of cell death measured by DiOC6/7AAD double staining and flow cytometry. Squares highlight the weakly-responding sample expressing only the NPM1 mutation (patient #6; see panel c). P values were determined by One-way ANOVA with Tukey-Kramer Multiple Comparisons Test. (c) In five of these six AML patient samples, NGS was conducted to define genetic abnormalities using the Cancer Hotspot Panel. Fq. = frequency of the mutations. (d) Bone marrow mononuclear cells from a primary AML sample were stained with CD132-APC, CD38-PerCP, CD34-PE, and Annexin-V-FITC, after which the percentage of apoptosis was determined in the primitive CD34+/CD38−/CD123+ population. (e) Western blot analysis was performed to monitor γH2A.X levels and PARP degradation in one representative primary responding sample.
Figure 8
Figure 8. The combination of AZD1775 and Vorinostat suppresses tumor growth in a murine xenograft model and prolongs animal survival
NOD/SCID-gamma (NSG) mice were subcutaneously inoculated in the right rear flank with 5×106 U937 cells stably expressing luciferase. Treatment was initiated after luciferase activity was detected (5 days after injection of tumor cells). AZD1775 was freshly reconstituted with 0.5% methylcellulose and administrated at a dose of 50 mg/kg by oral gavage (p.o.) bid three days a week. Vorinostat in DMSO was diluted in PEG/H2O (1:1) and administrated at a dose of 100 mg/kg via intraperitoneal (i.p.) injection daily five days a week. Control animals were administered equal volumes of vehicle. (a) Tumor size was measured visually every other day. Representative median tumor volume for day 21 was shown. (b) Tumor growth was monitored every other day after i.p. injection with 150 mg/kg luciferin using the IVIS 200 imaging system. d = day. (c) Representative images of mice (day 21) and tumors removed from mice on the indicated day post-tumor cell injection. 1 = vehicle, 2 = 100 mg/kg Vorinostat, 3 = 50 mg/kg AZD1775, 4 = AZD1775 + Vorinostat. (d) Kaplan-Meier analysis performed to analyze survival of animals. Inset, median survival. (e) Tumor tissues were homogenized and subjected to Western blot analysis.
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