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

Zhou L, Zhang Y, Chen S, Kmieciak M, Leng Y, Lin H, Rizzo KA, Dumur CI, Ferreira-Gonzalez A, Dai Y, Grant S - Leukemia (2014)

Bottom Line: Similarly, Wee1 shRNA knockdown significantly sensitized cells to HDACIs.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology and Oncology, Department of Medicine, Virginia Commonwealth University and the Massey Cancer Center, Richmond, VA, USA.

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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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).
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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).

Mentions: Effects on cell cycle checkpoints were then examined, using the mitotic inhibitor Taxol as a control. Cell cycle analysis of U937 cells revealed that while HDACIs increased the G2/M sub-population (33), this event was enhanced by AZD1775 as early as 8 h after drug treatment (Fig 5a, top, and Supplemental Fig S3A). Notably, whereas both AZD1775 and HDACIs alone modestly increased the mitotic index (MI) at 8 h, reflected by positivity of S10 phosphorylated histone H3 (p-H3), combined exposure strikingly increased the p-H3 MI (e.g., 3.4- and 3.7- fold increases for AZD1775/Vorinostat or /SBHA, respectively; Fig 5a, bottom), consistent with premature mitotic entry at this early interval. However, while AZD1775 ± HDACIs clearly decreased S-phase cells, neither agent alone or in combination obviously affected DNA replication, manifested by a minimal increase in EdU-positive cells at 8 h (Supplemental Fig S3B–C). Interestingly, AZD1775/Vorinostat or /SBHA co-treatment for 16 h sharply arrested cells in early S-phase (Fig 5b, top, and Supplemental Fig S4A) and increased newly replicated DNA incorporating EdU (34) (Fig 5b, bottom, and Supplemental Fig S4B), accompanied by persistent increases in premature mitotic entry (e.g., increased p-H3 MI; Fig 5b, bottom, and Supplemental Fig S4C). Fluorescence microscopy demonstrated robust increases in both p-H3- and EdU-positive cells after 16-h co-exposure to AZD1775 and Vorinostat or SBHA (Fig 5c and Supplemental Fig S4D). Furthermore, confocal microscopy of AZD1775/HDACI-treated cells (16 h) revealed markedly aberrant mitosis characterized by multiple mitotic abnormalities e.g., anaphase bridging, mono- or multi-polar spindles, centrosome clustering, etc. in p-H3-positive cells (22, 35) (Fig 5d). Such findings indicate that AZD1775/HDACI co-treatment induces premature mitotic entry, aberrant mitosis, and accumulation of early S phase cells with increased de novo DNA replication, and suggest that Wee1 and HDAC inhibitors interact reciprocally to disrupt both G2/M and G1/S checkpoints.


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

Zhou L, Zhang Y, Chen S, Kmieciak M, Leng Y, Lin H, Rizzo KA, Dumur CI, Ferreira-Gonzalez A, Dai Y, Grant S - Leukemia (2014)

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).
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Related In: Results  -  Collection

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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).
Mentions: Effects on cell cycle checkpoints were then examined, using the mitotic inhibitor Taxol as a control. Cell cycle analysis of U937 cells revealed that while HDACIs increased the G2/M sub-population (33), this event was enhanced by AZD1775 as early as 8 h after drug treatment (Fig 5a, top, and Supplemental Fig S3A). Notably, whereas both AZD1775 and HDACIs alone modestly increased the mitotic index (MI) at 8 h, reflected by positivity of S10 phosphorylated histone H3 (p-H3), combined exposure strikingly increased the p-H3 MI (e.g., 3.4- and 3.7- fold increases for AZD1775/Vorinostat or /SBHA, respectively; Fig 5a, bottom), consistent with premature mitotic entry at this early interval. However, while AZD1775 ± HDACIs clearly decreased S-phase cells, neither agent alone or in combination obviously affected DNA replication, manifested by a minimal increase in EdU-positive cells at 8 h (Supplemental Fig S3B–C). Interestingly, AZD1775/Vorinostat or /SBHA co-treatment for 16 h sharply arrested cells in early S-phase (Fig 5b, top, and Supplemental Fig S4A) and increased newly replicated DNA incorporating EdU (34) (Fig 5b, bottom, and Supplemental Fig S4B), accompanied by persistent increases in premature mitotic entry (e.g., increased p-H3 MI; Fig 5b, bottom, and Supplemental Fig S4C). Fluorescence microscopy demonstrated robust increases in both p-H3- and EdU-positive cells after 16-h co-exposure to AZD1775 and Vorinostat or SBHA (Fig 5c and Supplemental Fig S4D). Furthermore, confocal microscopy of AZD1775/HDACI-treated cells (16 h) revealed markedly aberrant mitosis characterized by multiple mitotic abnormalities e.g., anaphase bridging, mono- or multi-polar spindles, centrosome clustering, etc. in p-H3-positive cells (22, 35) (Fig 5d). Such findings indicate that AZD1775/HDACI co-treatment induces premature mitotic entry, aberrant mitosis, and accumulation of early S phase cells with increased de novo DNA replication, and suggest that Wee1 and HDAC inhibitors interact reciprocally to disrupt both G2/M and G1/S checkpoints.

Bottom Line: Similarly, Wee1 shRNA knockdown significantly sensitized cells to HDACIs.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology and Oncology, Department of Medicine, Virginia Commonwealth University and the Massey Cancer Center, Richmond, VA, USA.

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.

Show MeSH
Related in: MedlinePlus