Journal: Cancer immunology research

Article Title: Targeting WEE1/AKT Restores p53-Dependent Natural Killer-Cell Activation to Induce Immune Checkpoint Blockade Responses in "Cold" Melanoma.

doi: 10.1158/2326-6066.CIR-21-0587

Figure Lengend Snippet: Figure 1. Modulating p53 pathway signaling in tumors to induce immunogenic cell death. B16F10 p53 knockdown (KD) and 1205 Lu p53 KD cells were created using shRNA. (A) 1205 Lu cells (control) and 1205 p53 KD cells were treated with doxorubicin and AKT/ WEE1 inhibitors for 24 hours. Cell lysates were probed for Western blot analysis with p53, and ERK2 served as a loading control. Representative of two independent experiments is shown. (B) Melanoma cells with wildtype p53 (1205 Lu and B16F10) and those lacking p53 (1205 Lu p53 KD and B16F10 p53 KD) were treated with the indicated concentrations (μM) of AKT (AZD5363) and WEE1 (MK1775) inhibitors for 72 hours, followed by estimation of cell viability using an MTS assay. Representative of two independent experiments is shown. (C) Nude mice bearing 1205 Lu (control) and 1205 Lu p53 KD subcutaneous tumors were treated with AZD5363 and MK1775 at 150 mg/kg and 50 mg/kg, respectively, daily through oral gavage. Tumor growth was measured over time. Mean + SEM is shown for 6 mice per group. Experiment was repeated three times. A representative is shown. (D) B16F10 (control) and B16F10 p53 KD were treated with AKT (6μM AZD5363) and WEE1 (3μM MK1775) inhibitors alone and in combination for 24 hrs. Calreticulin was measured by flow cytometry. DMSO-treated cells served as controls. Representative of two independent experiments is shown. (E) 1205 Lu cells (control) and 1205 p53 KD cells were

Article Snippet: NK cell ligands B16F10 and 1205 Lu (parental and p53 KD) cells were treated with p53 modulators, 6 μM AZD5363, 2 μM MK1775, 20 μM nutlin (Tocris, Minneapolis, Minnesota), 40 μM SJ172550 (AdooQ Bioscience, Irvine, California), and 0.05% DMSO and incubated for 24 hours.

Techniques: Knockdown, shRNA, Control, Western Blot, MTS Assay, Flow Cytometry

Journal: Cancer immunology research

Article Title: Targeting WEE1/AKT Restores p53-Dependent Natural Killer-Cell Activation to Induce Immune Checkpoint Blockade Responses in "Cold" Melanoma.

doi: 10.1158/2326-6066.CIR-21-0587

Figure Lengend Snippet: Figure 2. Increasing p53 pathway signaling in tumor cells induces NK cell ligands and enhances immunogenicity. 1205 Lu, 1205 Lu p53 KD, B16F10, and B16F10 p53 KD cells were treated with p53 modulators (6μM AZD5363+2μM MK1775, 20μM nutlin, or 40μM SJ172250) and incubated for 24 hours. Mean fluorescence intensities (MFI) of the indicated NK cell ligands in (A) 1205 Lu and 1205 Lu p53 KD or (B) B16F10 and B16F10 p53 KD cells following treatment were measured by flow cytometry. (C) 1205 Lu and 1205 Lu p53 KD cells were cultured with the indicated doses of AZD5363+MK1775 for 24 hours, washed, and co-cultured with NK-92 cells for 4 hours at a NK cell:tumor cell ratio of 8:1. Tumor cell viability was monitored by AnnexinV by flow cytometry. Representative of two independent experiments is shown for all panels. Statistical test: ANOVA + Dunnett’s post-hoc. Error bars represent mean + S.E.M.

Article Snippet: NK cell ligands B16F10 and 1205 Lu (parental and p53 KD) cells were treated with p53 modulators, 6 μM AZD5363, 2 μM MK1775, 20 μM nutlin (Tocris, Minneapolis, Minnesota), 40 μM SJ172550 (AdooQ Bioscience, Irvine, California), and 0.05% DMSO and incubated for 24 hours.

Techniques: Immunopeptidomics, Incubation, Fluorescence, Flow Cytometry, Cell Culture

Journal: Cancer immunology research

Article Title: Targeting WEE1/AKT Restores p53-Dependent Natural Killer-Cell Activation to Induce Immune Checkpoint Blockade Responses in "Cold" Melanoma.

doi: 10.1158/2326-6066.CIR-21-0587

Figure Lengend Snippet: Figure 3. AKT/WEE1 targeting enhances the effectiveness of anti-PD-1 treatment via NK-cell recruitment and activity in tumors. B16F10 cells were injected subcutaneously into male (n = 10) or female (n = 20) C57BL6 mice. Experiments were repeated 2 and 4 times respectively with 5 mice per group. After tumors reached 50 mm3, mice were treated with IgG (control), anti-PD-1 (200 μg twice a week), AZD5363 (150 mg/kg)+MK1775 (50 mg/kg), or triple combination daily. (A) Tumor growth was measured over time until tumor size in any group reached ethical limits. (B) Animal survival was also monitored every three days in a separate cohort until tumors reached ethic size limits in each group or stopped at day 40 in the AZD+MK group. Log Rank Test was performed. Experiment was repeated twice with 5 mice per group. At day 21, tumors from female mice were excised and phenotyped by FACS for (C) total NK

Article Snippet: NK cell ligands B16F10 and 1205 Lu (parental and p53 KD) cells were treated with p53 modulators, 6 μM AZD5363, 2 μM MK1775, 20 μM nutlin (Tocris, Minneapolis, Minnesota), 40 μM SJ172550 (AdooQ Bioscience, Irvine, California), and 0.05% DMSO and incubated for 24 hours.

Techniques: Activity Assay, Injection, Control

Journal: Cancer immunology research

Article Title: Targeting WEE1/AKT Restores p53-Dependent Natural Killer-Cell Activation to Induce Immune Checkpoint Blockade Responses in "Cold" Melanoma.

doi: 10.1158/2326-6066.CIR-21-0587

Figure Lengend Snippet: Figure 4. AKT+WEE1+anti-PD-1 therapeutic response requires P53, NK cells, and CD8+ T cells. B16F10 or B16F10 p53 KD tumors where engrafted s.c. into C57BL/6 mice. NK cells and CD8+ T cells were depleted in mice by treating mice with anti-NK1.1 and anti-CD8a, respectively, on days −7, −3, −1, 1, 3, 7, 14, and 21, with tumors being inoculated on day 0. After tumors reached 50 mm3 (Day 9), mice were treated with anti-PD-1 twice a week, or AZD5363 (150 mg/kg)+MK1775 (50 mg/kg), or triple combination daily. (A) Tumor size was monitored once every three days. P53KD experiment: N=15. Experiments were repeated 3 times with N=5 for each group. NK cell depletion experiment and control

Article Snippet: NK cell ligands B16F10 and 1205 Lu (parental and p53 KD) cells were treated with p53 modulators, 6 μM AZD5363, 2 μM MK1775, 20 μM nutlin (Tocris, Minneapolis, Minnesota), 40 μM SJ172550 (AdooQ Bioscience, Irvine, California), and 0.05% DMSO and incubated for 24 hours.

Techniques: Clinical Proteomics, Control

Journal: Breast Cancer Research : BCR

Article Title: Estrogen promotes estrogen receptor negative BRCA1-deficient tumor initiation and progression

doi: 10.1186/s13058-018-0996-9

Figure Lengend Snippet: Pharmaceutical inhibition of Akt suppresses epithelial-mesenchymal transition and cell proliferation preventing Brca1- deficient tumor progression. a p18 −/− ;Brca1 MGKO tumor cells were treated with dimethyl sulfoxide (DMSO) or 5 nM E2 in the presence or absence of different dosage of AZD5363 for 2 h, the expression of p-mTor, p-4E-bp1, p-Gsk3β, p-Fra1 and vimentin (Vim) were analyzed by western blot. b p18 −/− ; Brca1 MGKO tumor cells were treated with DMSO or 5 nM E2 in the presence or absence of different dosage of AZD5363, and the numbers of viable cells were determined on day 1, day 3, and day 5. * p < 0.05 between E2-treated and E2 + AZD5363-treated groups at the time points (Student t test). Data are represented as mean ± SD ( n = 4). c p18 −/− ;Brca1 MGKO tumor cells were treated with 5 nM E2 with or without 1 μM AZD5363 for 24 h, and bromodeoxyuridine (Brdu) incorporation was then determined by fluorescence-activated cell sorting (FACS). d - g We transplanted 1 × 10 6 p18 −/− ; Brca1 MGKO tumor cells into the mammary fat pads of NSG mice along with E2 pellet under the skin, and tumors were allowed to reach ~ 250 mm 3 in size. Mice were then treated with AZD5363 at 150 mg/kg body weight or vehicle daily by oral gavage. The tumor size was determined and plotted ( d ). Data in d are represented as mean ± SD of four tumors in each group. * p < 0.05 between two groups at each time point (Student t test). p18 −/− ; Brca1 MGKO tumors treated with AZD5363 or vehicle for 7 days ( d ) were analyzed by western blot ( e ), immunohistochemical staining ( f ), and immunofluorescent staining ( g ). Samples in e were derived from eight different tumors developed in eight individual mice. The percentages of Ki67 + and/or Ck14 + cells ( g ) were quantitated in four randomly selected fields for each section of a tumor, and the results represent the mean ± SD of three individual tumors per group

Article Snippet: For treatment of estrogen, AZD5363, and 4OHT, tumor cells were cultured in 10% charcoal-stripped FBS in the presence of E2, AZD5363 (ApexBio Technology), 4OHT (Sigma), or dimethyl sulfoxide (DMSO) for the indicated times and then collected for further analysis.

Techniques: Inhibition, Expressing, Western Blot, BrdU Incorporation Assay, Fluorescence, FACS, Immunohistochemical staining, Staining, Derivative Assay