Journal: Molecular cancer therapeutics

Article Title: Anti-Leukemia Effects of Notch-Mediated Inhibition of Oncogenic PLK1 in B-Cell Acute Lymphoblastic Leukemia

doi: 10.1158/1535-7163.MCT-18-0706

Figure Lengend Snippet: (A) Intracellular PLK1 expression in cell lines (top panels) and patient samples (bottom panels) representing the 3 B-ALL subsets; PH-ve, PH-like, and PH+, was assessed by flow cytometry and (B) median fluorescence intensity (MFI) was summarized. Pale and dark shades represent cell lines and patient samples, respectively, within each subset. PH-like cells had significantly higher PLK1 MFI when compared to both PH-ve and PH+ cells (p<0.01) using a one-way ANOVA with post-hoc Tukey’s test. (C) Intracellular PLK1 expression in the same cells was measured after 48 hours of co-culture with HS5 cells expressing DLL1. (D) Summary of the relative reduction of PLK1 MFI after co-culture with HS5-DLL1 compared to baseline. PLK1 MFI reduction was significantly greater in PH-like when compared to PH-ve cells (p<0.05) using a one-way ANOVA with post-hoc Tukey’s test. (E) T-ALL (CEM; a HES1-positive control) and B-ALL cell lines along with two representative Ph-like B-ALL patient samples (Pt.B1, Pt.B2) were assessed by Western blot for cleaved intra-cellular Notch 2 (ICN2), HES1(abcam ab), PLK1, and β-actin after co-culture on HS5 cells expressing either GFP or DLL1 for 48 hours. (F) Representative flow plots of PLK1 expression from the B-ALL cell line, SB, expressing intra-cellular Notch domains 1-4 (ICN1-4) or a GFP control 48 hours post induction. (G) T-ALL (CEM, SupT1, Molt4, Jurkat) and B-ALL (SB, JM1, Nalm6, 697) cells were transduced with GFP control or HES1. HES1(Origene ab) and PLK1 protein expression in transduced cells was determined by Western blot.

Article Snippet: Doxycycline-induced red fluorescent protein expression was confirmed by flow cytometry analysis. siRNA transfection Oligonucleotide small-interfering RNA (siRNA) targeting human PLK1 (sc36277) or CHFR (sc37567) and siFITC-nontargeting control scramble (sc36869) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Flow Cytometry, Fluorescence, Co-Culture Assay, Positive Control, Western Blot, Control, Transduction

Journal: Molecular cancer therapeutics

Article Title: Anti-Leukemia Effects of Notch-Mediated Inhibition of Oncogenic PLK1 in B-Cell Acute Lymphoblastic Leukemia

doi: 10.1158/1535-7163.MCT-18-0706

Figure Lengend Snippet: (A) Cells representing T-ALL (CEM) or B-ALL (SB, JM1, Nalm6 [N6]) and primary samples from patients with Ph-like B-ALL were treated with a vehicle control or PLK1 inhibitor, BI2536 (100 nM), for 1 week. Cells were counted by trypan blue daily and the counts normalized the seed number. (B, C) Intracellular p53 (B) and pMDM2 (ser260) (C) for all cells in (A) were determined by flow cytometry at 48 hours. (D) Similarly, cell lysates were collected at the same time point and probed for pMDM2(ser260), MDM2, p53, and β-actin. (E) NSG-SGM3 mice engrafted with cells from a patient with Ph-like B-ALL (Pt.B1) were treated, beginning 3 weeks after initial tail vein injection, with vehicle (control), PLK1 inhibitor BI2536 (15 mg/kg), or BI6727 (volasertib; 15 mg/kg) by gavage twice per week for 2 weeks (n=10 mice per group) and continuously monitored for 2 additional weeks. The leukemia burden was measured weekly in the peripheral blood by hCD45 staining. (F) Mice were euthanized 4 weeks after beginning treatment and the leukemia burden was measured in the bone marrow and spleen by hCD45 staining. (G) Intracellular pMDM2 and p53 levels in the bone marrow of the treated mice were quantified by flow-based analysis. (H) SB B-ALL were treated with various combinations of dexamethasome (DEX) or vincristine (Vi) and PLK1 inhibitor BI2536 (BI) for 3 days (n=3 per treatment group). Cell viability was determined by alamar blue. *p<0.05; **p<0.01.

Article Snippet: Doxycycline-induced red fluorescent protein expression was confirmed by flow cytometry analysis. siRNA transfection Oligonucleotide small-interfering RNA (siRNA) targeting human PLK1 (sc36277) or CHFR (sc37567) and siFITC-nontargeting control scramble (sc36869) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Control, Flow Cytometry, Injection, Staining

Journal: Molecular cancer therapeutics

Article Title: Anti-Leukemia Effects of Notch-Mediated Inhibition of Oncogenic PLK1 in B-Cell Acute Lymphoblastic Leukemia

doi: 10.1158/1535-7163.MCT-18-0706

Figure Lengend Snippet: (A) Primary cells from B-ALL (left) and T-ALL (right) patients (each n=3) were transduced with doxycycline- inducible PLK1 or scrambled shRNAs and cultured in low-dose doxycycline (2 μg/mL). Cell viability was determined on days 1 and 4 by trypan blue staining. (B) The PLK1-shRNA knockdown was confirmed both at the RNA and protein level by qRT-PCR and Western blot, respectively. PLK1 RNA was significantly reduced in both B-ALL patient samples (p<0.05, n=3) by two sample t-tests. (C) Representative flow cytometry data summarizing the differences in intracellular p53 expression from a T-ALL and 5 B-ALL patient samples transduced with the PLK1 or scrambled shRNA after 48 hours.

Article Snippet: Doxycycline-induced red fluorescent protein expression was confirmed by flow cytometry analysis. siRNA transfection Oligonucleotide small-interfering RNA (siRNA) targeting human PLK1 (sc36277) or CHFR (sc37567) and siFITC-nontargeting control scramble (sc36869) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transduction, Cell Culture, Staining, shRNA, Knockdown, Quantitative RT-PCR, Western Blot, Flow Cytometry, Expressing

Journal: Molecular cancer therapeutics

Article Title: Anti-Leukemia Effects of Notch-Mediated Inhibition of Oncogenic PLK1 in B-Cell Acute Lymphoblastic Leukemia

doi: 10.1158/1535-7163.MCT-18-0706

Figure Lengend Snippet: (A) Ph-like B-ALL patient samples (Pt.B1, Pt.B2) and B-ALL cell line, SB, were co-cultured on HS5 cells expressing either the GFP-control or DLL1. MDM2 (phospho and total), p53, PLK1, HES1, and β-actin protein expression were quantified by Western blot. (B) Intracellular p53 was measured in primary B-ALL patient cells (Pt.B1) treated with soluble DLL1, γ-secretase (Notch) inhibitor DAPT (200 nM), or both for 48 h by flow cytometry. (C) Similarly, HES1 was quantified by qRT-PCR in the same primary B-ALL cells treated with DLL1, DAPT, or both, relative to untreated controls. Only the DLL1 treated cells had a significant increase in HES1 RNA expression (p<0.05) by one-way ANOVA with Dunnett’s post-hoc test. (D) A representative Ph-like B-ALL patient sample was cultured on either control (Fc) or DLL1 (DLL1Fc) plate-bound ligand for 48 hours. Cell lysates were immunoprecipitated with IgG control, MDM2, or p53 and the membrane was probed with pMDM2 and p53. (E,F) Similarly, intracellular pMDM2 and p53 was measured by flow cytometry for 2 B-ALL patient samples after 48 hours of culture on control (Fc) or DLL1 (DLL1Fc) plate-bound ligand. Intracellular p53 expression was significantly increased in both patient samples (p<0.05 and p<0.01) cultured on DLL1Fc when compared to their corresponding control cultures.

Article Snippet: Doxycycline-induced red fluorescent protein expression was confirmed by flow cytometry analysis. siRNA transfection Oligonucleotide small-interfering RNA (siRNA) targeting human PLK1 (sc36277) or CHFR (sc37567) and siFITC-nontargeting control scramble (sc36869) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Cell Culture, Expressing, Control, Western Blot, Flow Cytometry, Quantitative RT-PCR, RNA Expression, Immunoprecipitation, Membrane

Journal: Molecular cancer therapeutics

Article Title: Anti-Leukemia Effects of Notch-Mediated Inhibition of Oncogenic PLK1 in B-Cell Acute Lymphoblastic Leukemia

doi: 10.1158/1535-7163.MCT-18-0706

Figure Lengend Snippet: (A) PLK1 mRNA expression was determined by qRT-PCR after co-culture with HS5-DLL1 or HS5-GFP for 48 hours (n=3). (B) B-ALL cell lines were treated with MG132 4 hours prior to various time points (12, 24, or 48 h) of co-culture with HS5-GFP or HS5-DLL1. Cell lysates were subjected to immunoprecipitation with ubiquitin and probed for PLK1. (C) Similary, after 48 hours of co-culture cells were treated with MG132 for 4 hours and harvested. Cell lysates were subjected to immunoprecipitation with PLK1 and blots were probed with PLK1 and ubiquitin. (D) The same panel of B-ALL cells was transduced with retrovirus-mediated HES1 for 48 hours to induce ectopic expression of the gene. The cells were treated with MG132 and the cell lysates subjected to immunoprecipitation with ubiquitin and probed for PLK1. (E) Primary B-ALL cells (Pt. B1) were either transduced with HES1 retrovirus, transfected with CHFR siRNA (siCHFR), or both for 24 hours; these cells and untreated controls were probed for CHFR and PLK1 expression. (F) Cell lysates from B-ALL cell lines co-culture with HS5-GFP or HS5-DLL1 were immunoprecipitated with CHFR and probed for PLK1. (G) T-ALL cells (CEM) and B-ALL cells (SB, JM1, Nalm6) were either co-cultured on control or DLL1-expressing HS5 cells and subjected to CHFR depletion via siRNA for 4 days. Viability was determined by trypan blue. (H) Cell lysates from (E) were immunoprecipitated with p53, and blots were probed with pMDM2(ser260) and p53. (I) B-ALL cells were co-cultured on control or DLL1-expressing HS5 cells for 48 hours, and cell lysates were immunoprecipitated with CHFR and resolved on 4-15% non-denaturing gradient gels; the membrane was probed for polyADP ribosylation (PAR). (J) Cells from patients with Ph-like B-ALL (Pt.B1, Pt.B2) were co-cultured on control or DLL1-expressing HS5 cells and treated for 48 hours with the PARP inhibitor, 3ABA. Viability was determined by trypan blue. *p<0.05; **p<0.01.

Article Snippet: Doxycycline-induced red fluorescent protein expression was confirmed by flow cytometry analysis. siRNA transfection Oligonucleotide small-interfering RNA (siRNA) targeting human PLK1 (sc36277) or CHFR (sc37567) and siFITC-nontargeting control scramble (sc36869) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Quantitative RT-PCR, Co-Culture Assay, Immunoprecipitation, Ubiquitin Proteomics, Transduction, Transfection, Cell Culture, Control, Membrane