cx 4945 sodium salt  (MedChemExpress)

Journal: Cancers

Article Title: Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia

doi: 10.3390/cancers13051127

Figure Lengend Snippet: CK2 overexpression in AML cells causes IKAROS phosphorylation which is reversed by CK2 inhibitor, CX-4945. ( A ) Baseline protein levels of CK2α, CK2α’, CK2β, IKAROS and BCL-XL in the myeloid leukemia cell panel [U937, THP-1, K562, and primary AML cells (labelled AML-1) were measured by western blot and compared to CD34+ Hematopoietic Stem Cells (HSC). Multiple IKAROS bands represent Ikaros isoforms 1 and 2. ( B ) qRT-PCR showing mRNA level of CK2α and BCL-XL in various AML cells compared to CD34+ HSC. **** ( p < 0.0001). ( C ) Radio-blot showing increased Phosphorylated IKAROS (p-IKAROS) in AML cells compared to HSC. ( D ) Radio-immunoblot showing dose-dependent decrease in Phospho-IKAROS level following CX-4945 treatment. U937 and THP-1 were treated with 10 µM and AML-1 was treated with 5μM (IC50–3.791 μM) CX-4945 for 48 h. IC50 values of CX-4945 treated cells are shown in . ( E ) Western blot showing decrease in amount of phosphorylated CK2 substrates with molecular masses of approximately 175, 120, 80, 70 and 56 kDa as indicated by arrows (left panel) and western blot showing phosphorylation extent of specific CK2 substrate, AKT1 at Ser129 (right panel).

Article Snippet: The CK2 inhibitor CX-4945 sodium salt was a gift from Senhwa Biosciences.

Techniques: Over Expression, Phospho-proteomics, Western Blot, Quantitative RT-PCR

Journal: Cancers

Article Title: Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia

doi: 10.3390/cancers13051127

Figure Lengend Snippet: CK2 inhibition suppresses BCL-XL expression in AML cells and xenograft model. ( A ) U937 and AML-1 were treated with 5 and 10 µM concentration of CX-4945 for 48 h before RNA and protein extraction. mRNA level of BCL-XL was measured by qRT-PCR. ( B ) U937 and K562 cells were treated with 5 and 10 µM of CX-4945 for 48 hours and protein was extracted. BCL-XL protein level was measured by western blot. ( C ) Downregulation of CK2α in U937 was achieved using shRNA. Validation of CK2α protein knockdown in U937-shCK2α cell line is shown in . qRT-PCR shows mRNA level of CK2α and BCL-XL in CK2α shRNA treated U937 cells. ( D ) Overexpression of CK2α was achieved by retroviral transduction of U937 cells. Validation of CK2α protein overexpression is shown in . qRT-PCR showed increased BCL-XL mRNA level in CK2α overexpressed U937 (left panel) and THP-1 cells (right panel). ( E ) WST assay showing increased cell viability in CK2α overexpressing U937 and THP-1 cells. U937 cells transduced with retroviral vector CK2α-gfp-luc were transplanted into NRG-S mice. 25,000 cells were injected via the tail vein. Bioluminescence imaging using IVIS 100 was obtained weekly following transplant. ( F ) Bioluminescence signal in xenograft mice engrafting U937-gfp-luc cell or control (U937-ctl-gfp-luc) shown at week 2 and 3 post transplantation. ( G ) Kaplan Maier plot showing survival probability in U937-CK2-gfp-luc cells vs control. P -value summaries are as follows: p > 0.05 (ns-not significant); p < 0.01 (**); p < 0.001 (***); p < 0.0001 (****).

Article Snippet: The CK2 inhibitor CX-4945 sodium salt was a gift from Senhwa Biosciences.

Techniques: Inhibition, Expressing, Concentration Assay, Protein Extraction, Quantitative RT-PCR, Western Blot, shRNA, Biomarker Discovery, Knockdown, Over Expression, Retroviral, Transduction, WST Assay, Plasmid Preparation, Injection, Imaging, Control, Transplantation Assay

Journal: Cancers

Article Title: Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia

doi: 10.3390/cancers13051127

Figure Lengend Snippet: CX-4945 treatment shows therapeutic efficacy in AML Patient Derived Xenograft (PDX). ( A ) Schema showing AML PDX generation and treatment. AML-1 PDX was developed by injecting NRG-S mice with one million cells per mouse via the tail vein. Treatment was started when 2–5% human CD45+ cells were detected in peripheral blood (PB). Mice received 100 mg/kg of CX-4945 via gavage twice daily for 21 days. ( B ) Percent AML (human CD45+, CD13+,CD33+) in bone marrow (BM) and spleen of treated and untreated AML-1 PDX mice by flow cytometry. ( C ) Kaplan Maier plot showing survival probability of treated and untreated AML-1 PDX mice. Cell line derived xenograft (CDX) mouse models was developed using luciferase, and green fluro-protein (gfp) labeled U937 cells (U937-gfp-luc) or THP-1 cells (THP1-gfp-luc) transplanted into immunocompromised NRG-S mice as described in methods. Mice were treated with vehicle or CX-4945 at a dose of 100 mg/kg twice daily via gavage for up to 7 days. ( D ) Engraftment was monitored using bioluminescence imaging shown as the mean of the total flux in photons/second of mice in each group. ( E ) Following treatment, mice were sacrificed, and bone marrow mononuclear cells were collected. Human CD45+ cells in bone marrow were measured using flow cytometry. Flow cytometry using conjugated BCL-XL antibody was used to quantify intracellular BCL-XL protein level in FACS enriched human CD45+cells in the bone marrow of treated and untreated mice. shows histogram of BCL-XL and CK2α protein level in vehicle and CX-4945 treated U937 and THP-1 CDX. ( F ) Mean fluorescence intensity(MFI) is graphed, showing decreased BCL-XL protein level. P -value summaries are as follows: p > 0.05 (ns-not significant); p ≤ 0.05 (*); p < 0.01 (**); p < 0.001 (***).

Article Snippet: The CK2 inhibitor CX-4945 sodium salt was a gift from Senhwa Biosciences.

Techniques: Drug discovery, Derivative Assay, Flow Cytometry, Luciferase, Labeling, Imaging, Fluorescence

Journal: Cancers

Article Title: Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia

doi: 10.3390/cancers13051127

Figure Lengend Snippet: CK2 inhibitor increases IKAROS DNA-binding to BCL-XL . Chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) and analysis of genome-wide occupancy of IKAROS was performed on U937 following the CX-4945 treatment at 10 µM concentration for 72 h. A change in IKAROS binding to promoter regions of the BCL-XL gene was analyzed following the CX-4945 treatment. ( A ) A chIP-seq signal map for IKAROS binding to the BCL-XL/BCL- 2L1 promoter region in U937-untreated labeled as WT (wild-type) (top panel) and CX-4945 treated U937 (bottom panel). Y-axis represents the log-2-fold change enrichment of IKAROS binding (** p < 0.01). ( B ) U937 and primary AML cells (AML-1) cells were treated with 10 and 5 μM of CX-4945, respectively (based on the IC50 value) for 48 and 72 h. IKAROS binding to the BCL-XL promoter region was confirmed using the qChIP assay in WT and CX-4945 treated cells. Binding at 72 h was not significantly increased compared to the 48 h treatment (not shown in the graph). Results are the mean +/– SD of three independent experiments. P-value summaries are as follows: p > 0.05 (ns-non significant); p < 0.001 (***); p < 0.0001 (****).

Article Snippet: The CK2 inhibitor CX-4945 sodium salt was a gift from Senhwa Biosciences.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Next-Generation Sequencing, ChIP-sequencing, Genome Wide, Concentration Assay, Labeling

Journal: Cancers

Article Title: Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia

doi: 10.3390/cancers13051127

Figure Lengend Snippet: CK2 and IKAROS regulate sensitivity towards daunorubicin. CK2 overexpressing U937 ( A , B ) and THP-1 ( C , D ) were treated with 10 nM or 50 nM of daunorubicin for 48 hand stained with 7-AAD and Annexin V for flow cytometry to determine apoptosis. Flow plots ( A , C ) and percent apoptotic cells (early + late apoptosis) are shown in ( B , D ). Flow plots showing representative results from three replicates. The percentage of cells in the right upper and lower quadrant of each flow chart represents the percentage of late and early apoptotic cells, respectively. Q1-dead, Q2-late apoptosis, Q3-early apoptosis, Q4-live. ( E ) Cytotoxicity and drug response measured by MTT assay after treating CK2 overexpressing U937 and THP-1 cells and respective controls with various concentrations of daunorubicin for 48 h. p > 0.05 (ns); p < 0.05 (*); p < 0.001 (***); p < 0.0001 (****).

Article Snippet: The CK2 inhibitor CX-4945 sodium salt was a gift from Senhwa Biosciences.

Techniques: Staining, Flow Cytometry, MTT Assay

Journal: Cancers

Article Title: Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia

doi: 10.3390/cancers13051127

Figure Lengend Snippet: CK2 inhibition potentiates daunorubicin-induced apoptosis in AML cells. Cells were treated with 10 µM of CX-4945 or a combination of 10 µM CX-4945 with 10 nM of daunorubicin for up to 48 h. Cells were stained with 7-AAD and Annexin V for flow cytometry to assess apoptosis. ( A ) Flow plots showing representative results from three replicate experiments. The percentage of cells in the right upper and lower quadrant of each flow chart represents the percentage of late and early apoptotic cells, respectively, in U937 (top row), THP-1 (middle row), and AML-1 cells (bottom row) Q1: Dead, Q2: Late apoptosis, Q3: Early apoptosis, Q4: Live. Graphed in ( B ) are the mean +/−SD of triplicates from two independent experiments showing the percent of apoptosis cells following the drug treatment, as indicated above. ( C ) CK2α silencing in U937 cells achieved using ShCK2α and sorted for GFP after 24 h. Sorted cells were treated with 10 nM of DNR for 24 h before staining for Annexin V and 7AAD to assess apoptosis. The graph shows the combined percent apoptotic cells (early + late) in each group with and without the DNR treatment. ( D ) Cytotoxic drug response measured by the MTT assay after treating CK2α ShRNA treated U937 cells and the respective controls with various daunorubicin concentrations for 24 h. ( E ) Cells were pretreated as above for 48 h and were plated in a Methocult medium. Colonies were counted under an inverted light microscope. Colonies that contained around 50 cells or more were counted for analysis. Graphed in E is the number of colonies after 14 days as the mean of three replicates +/− SD of two independent experiments. ( F ) The qRT-PCR showing decreases in the mRNA level in U937 cells treated with daunorubicin alone (10 nM) or a combination of CX-4945 and daunorubicin. p -value summaries are as follows: p > 0.05 (ns); p < 0.01 (**); p < 0.001 (***); p < 0.0001 (****).

Article Snippet: The CK2 inhibitor CX-4945 sodium salt was a gift from Senhwa Biosciences.

Techniques: Inhibition, Staining, Flow Cytometry, MTT Assay, shRNA, Light Microscopy, Quantitative RT-PCR

Journal: Cancers

Article Title: Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia

doi: 10.3390/cancers13051127

Figure Lengend Snippet: Model illustration of the regulation of apoptosis in AML by CK2 and IKAROS via repression of BCL-XL.

Article Snippet: The CK2 inhibitor CX-4945 sodium salt was a gift from Senhwa Biosciences.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Transcriptional Regulation of PIK3CD and PIKFYVE in T-Cell Acute Lymphoblastic Leukemia by IKAROS and Protein Kinase CK2

doi: 10.3390/ijms22020819

Figure Lengend Snippet: CK2 is overexpressed in T-ALL compared to normal mononuclear cells . ( A ) Schematic showing PIK3CD and PIKFYVE genes in the PI3K pathway. ( B ) Baseline protein levels of CK2α, pCK2, and IKAROS in the T cell leukemia cell panel (CCRF-CEM, MOLT4, and primary T-ALL cells (labeled T-ALL#1)) were measured by Western blot and compared to peripheral blood mononuclear cells (MNCs). The protein level is graphed relative to vinculin as a loading control. ( C ) Radio-immunoblot showing phospho-IKAROS in a leukemia cell panel (CCRF-CEM, MOLT4, and primary T-ALL cells (labeled ALL#1-5)) compared to MNCs. ( D ) Radio-immunoblot showing a decrease in the phospho-IKAROS level following CX-4945 treatment. MOLT4 cells were treated with 10 μM of CX-4945 for 24 h. EV, endosomal vesicle; PIK3CD, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta; and PIKFYVE, phosphoinositide kinase, FYVE-type zinc finger containing; PI3K, phosphoinositide 3-kinase; P, phospho; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PDK1, phosphoinositide-dependent kinase; PI3P, phosphatidylinositol-3-phosphate; PI(3,5)P2, phosphotydile inositol 3,5-biphosphate.

Article Snippet: CK2 inhibitor CX-4945 sodium salt was purchased from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Labeling, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Transcriptional Regulation of PIK3CD and PIKFYVE in T-Cell Acute Lymphoblastic Leukemia by IKAROS and Protein Kinase CK2

doi: 10.3390/ijms22020819

Figure Lengend Snippet: CK2 inhibition restores IKAROS DNA binding and repression of PIK3CD and PIKFYVE . MOLT4, CEM, and T-ALL#1 cells were treated with 10 μM of CX-4945 for 24 h. IKAROS binding to the ( A ) PIK3CD and ( B ) PIKFYVE promoter region was confirmed using qChIP assay in vehicle- and CX-4945-treated cells. Results are mean ± SD of triplicates representative of one of three independent experiments. Molecular inhibition of CK2α in CEM and MOLT4 cells was achieved using shRNA. Two of four shRNAs showed a significant and similar decrease in CK2α. qRT-PCR shows the mRNA level of ( C ) CK2α and ( D ) PIK3CD and PIKFYVE in CK2-silenced MOLT4 (left panel) and CEM (right panel) cells. CEM and MOLT4 cells were treated with 10 and 20 μM of CX-4945 for 48 h, and T-ALL#1 primary leukemia cells were treated with 10 μM of CX-4945 for 12 h. mRNA and protein were extracted. ( E ) The mRNA level of PIK3CD and PIKFYVE was measured in CX-4945-treated CEM, MOLT4, and T-ALL#1 cells. ( F ) AKT and phosphorylated-AKT (p-AKT) protein levels were measured by Western blot. The protein level is expressed relative to vinculin. The p -value summaries are as follows: p ≤ 0.05 (*); p < 0.01 (**).

Article Snippet: CK2 inhibitor CX-4945 sodium salt was purchased from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Inhibition, Binding Assay, shRNA, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Transcriptional Regulation of PIK3CD and PIKFYVE in T-Cell Acute Lymphoblastic Leukemia by IKAROS and Protein Kinase CK2

doi: 10.3390/ijms22020819

Figure Lengend Snippet: Model illustration of regulation of PI3K pathway genes PIK3CD and PIKFYVE in T-ALL by CK2 and IKAROS.

Article Snippet: CK2 inhibitor CX-4945 sodium salt was purchased from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: