human kinase domain focused crispr knockout ko library  (Addgene inc)

Journal: Blood Neoplasia

Article Title: Targeting HASPIN kinase disrupts SR protein–mediated RNA splicing and synergizes with BCL-2 inhibitor venetoclax in AML

doi: 10.1016/j.bneo.2025.100107

Figure Lengend Snippet: A kinase domain–targeted CRISPR screen identifies HASPIN as a novel dependency in AML cells. (A) Schematic of human kinase domain–targeted CRISPR screen in 2 t(8;21) AML cell lines, Kasumi-1 and SKNO-1. (B) Gene rank plots (left) and volcano plots (right) depicting significant kinase hits in the kinase domain–targeted CRISPR screens. Top candidates determined by CRISPR score as calculated by MAGeCK robust ranking aggregation (RRA) (left) and a log 2 (fold change) ≤−1.0 and false discovery rate (FDR) ≤0.05 significance cutoff (right). White diamonds indicate top 10 kinase hits in each plot. (C) Bubble plot of preranked gene set enrichment analysis results performed on top kinases identified by CRISPR screen in Kasumi-1 and SKNO-1 AML cell lines. Fill color indicates normalized enrichment score (NES). Size indicates significance by –log 10 (FDR). Facets indicate Molecular Signatures Database (MSigDB) gene set collection. (D) Density plot of all individual sgRNA log 2 (fold change) values in the kinase domain–targeted library. For selected genes, log 2 (fold change) values of corresponding sgRNAs depicted for Kasumi-1 and SKNO-1 cell lines relative to all other library sgRNAs (red, blue, and gray, respectively). (E) Competitive proliferation assay of Kasumi-1 or SKNO-1 cells expressing nontargeting negative control, RPA3-targeting positive control, or 1 of 2 HASPIN-targeting sgRNAs derived from CRISPR screen. Relative changes in cell proliferation rate measured by percentage of GFP-positive cells relative to nontargeting control on each day. Data are mean ± standard deviation (SD) of 4 independent experiments. CGP, chemical, genetic perturbation; CP, canonical pathway.

Article Snippet: CRISPR screen was performed using the human kinase domain–focused CRISPR knockout (KO) library (Addgene 117725; a gift from Christopher Vakoc) and 2 t(8;21) AML cell lines.

Techniques: CRISPR, Proliferation Assay, Expressing, Negative Control, Positive Control, Derivative Assay, Control, Standard Deviation

Journal: Blood Neoplasia

Article Title: Targeting HASPIN kinase disrupts SR protein–mediated RNA splicing and synergizes with BCL-2 inhibitor venetoclax in AML

doi: 10.1016/j.bneo.2025.100107

Figure Lengend Snippet: HASPIN is a clinically relevant, general leukemia dependency. (A) Bar plot depicting mean log 2 (fold change) of HASPIN targeting sgRNA genome-wide CRISPR screen performed in several leukemia cell lines as reported by Wang et al. Screen data were retrieved from BIOGRID ORCS. Dotted line indicates author-specified significance cutoff. (B) Competitive proliferation assay of THP-1 or OCI-AML3 cells expressing nontargeting negative control, RPA3-targeting positive control, or 1 of 2 HASPIN-targeting sgRNAs derived from CRISPR screen. Relative changes in cell proliferation rate measured by percentage of GFP-positive cells relative to nontargeting control on each day. Data are mean ± SD of 4 independent experiments per cell line. (C) Box plots depicting median HASPIN mRNA expression in the TCGA-LAML patient cohort separated by AML subtype. MLL (KMT2A) or RUNX1-RUNX1T1 t(8;21) translocation cohorts are highlighted in green and orange, respectively. Individuals with KMT2A structural variants are indicated with purple diamonds. (D) Box plots depicting median HASPIN mRNA expression in the BEAT-AML (2022) patient cohort separated by AML subtype. MLL (KMT2A) or RUNX1-RUNX1T1 t(8;21) translocation cohorts are highlighted in green and orange, respectively. Individuals with KMT2A structural variants are indicated with purple diamonds. (E) Kaplan-Meier survival curve depicting comparison of overall survival of patients with TCGA-LAML belonging to the top quartile (red) and bottom quartile (blue) of HASPIN expression. Plot and data derived from GEPIA2. (F) Forest plot of hazard ratios from multivariate Cox proportional hazard analysis of overall survival of patients with TCGA LAML incorporating HASPIN expression level and significant clinical and genetic factors. High and low HASPIN -expressing patients belong to the top and bottom expression quartiles, respectively. Clinical variables include the following: patient sex (Sex), age at first diagnosis (Diagnosis_Age), genetic risk group (Risk_Group), FLT3 mutation status (FLT3_Status), NPM1 mutation status (NPM1_Status), DNMT3A mutation status (DNMT3A_Status), TP53 mutation status (TP53_Status), and NRAS mutation status (NRAS_Status). Clinical metadata and mutation calls derived from the Genomic Data Commons TCGA LAML project patient information. N.D., not defined; NOS, not otherwise specified; NP, not profiled.

Article Snippet: CRISPR screen was performed using the human kinase domain–focused CRISPR knockout (KO) library (Addgene 117725; a gift from Christopher Vakoc) and 2 t(8;21) AML cell lines.

Techniques: Genome Wide, CRISPR, Proliferation Assay, Expressing, Negative Control, Positive Control, Derivative Assay, Control, Translocation Assay, Comparison, Biomarker Discovery, Mutagenesis

Journal: Blood Neoplasia

Article Title: Targeting HASPIN kinase disrupts SR protein–mediated RNA splicing and synergizes with BCL-2 inhibitor venetoclax in AML

doi: 10.1016/j.bneo.2025.100107

Figure Lengend Snippet: HASPIN inhibitor CHR-6494 effectively targets AML and synergizes with BCL-2 inhibition. (A) Dose-response curves (left) and IC comparison (right) of Kasumi-1 and healthy CD34 + hematopoietic progenitor cells treated with CHR-6494. IC values determined by nonlinear regression. Data on curve are mean ± SD of technical triplicates. Representative curves of 3 independent experiments revealed. Data on bar plot are mean ± SD of 3 independent experiments. Significance determined by unpaired 2-tailed Student t test. ∗∗∗∗ P < .0001. (B) Bar plots comparing CHR-6494 IC values in leukemia cell lines. IC values determined by dose-response curve with nonlinear regression for each cell line. Data are mean ± SD of 3 independent experiments. Dotted line indicates CHR-6494 IC value of healthy CD34 + hematopoietic progenitor cells determined in panel A. (C) Bar plots depicting normalized HASPIN sgRNA counts in a genome-wide CRISPR screen in MOLM-13 cells treated with either DMSO or VEN for 8 or 16 days as performed by Chen et al. Screen data were retrieved from BIOGRID ORCS. Counts were normalized to initial time point (d0). One data point was removed from DMSO (d16) as a significant outlier. Data are mean ± SD. Significance determined by 1-way ANOVA with Holm-Sidak multiple comparison correction. ∗ P < .05; ∗∗ P < .01. (D) Dose-response matrix (left) and corresponding zero interaction potency (ZIP) drug synergy contour plot (right) of Kasumi-1 cells treated with CHR-6494 and VEN combination for 48 hours. Each cell represents drug combined at indicated concentrations. Treatment response is percent inhibition; higher values indicate lower cell viability. Synergy scores represent ZIP synergy calculations of inhibition effects exceeding values expected between 2 noninteracting agents. Mean synergy scores and significance reported at top of respective contour plot. Representative plots of 3 independent experiments revealed. (E) Dose-response matrix (left) and corresponding ZIP drug synergy contour plot (right) of THP-1 cells treated with CHR-6494 and VEN combination for 48 hours. Each cell represents drug combined at indicated concentrations. Treatment response is percent inhibition; higher values indicate lower cell viability. Synergy scores represent ZIP synergy calculations of inhibition effects exceeding values expected between 2 noninteracting agents. Mean synergy scores and significance reported at top of respective contour plot. Representative plots of 3 independent experiments revealed. (F) Dose-response matrix (left) and corresponding ZIP drug synergy contour plot (right) of OCI-AML3 cells treated with CHR-6494 and VEN combination for 48 hours. Each cell represents drug combined at indicated concentrations. Treatment response is percent inhibition; higher values indicate lower cell viability. Synergy scores represent ZIP synergy calculations of inhibition effects exceeding values expected between 2 noninteracting agents. Mean synergy scores and significance reported at top of respective contour plot. Representative plots of 3 independent experiments revealed. DMSO, dimethyl sulfoxide.

Article Snippet: CRISPR screen was performed using the human kinase domain–focused CRISPR knockout (KO) library (Addgene 117725; a gift from Christopher Vakoc) and 2 t(8;21) AML cell lines.

Techniques: Inhibition, Comparison, Genome Wide, CRISPR

Journal: Nature Communications

Article Title: An all-in-one pipeline for the in vitro discovery and in vivo testing of Plasmodium falciparum malaria transmission blocking drugs

doi: 10.1038/s41467-025-62014-3

Figure Lengend Snippet: Results from the primary screening of small- to medium-sized compound libraries. A – C Effect of compounds of the Epigenetics Screening Library (Cayman Chemical) ( A ), human kinase inhibitors (SelleckChem, Enzo Life Sciences) ( B ) or compounds of the Prestwick Chemical Library ( C ) on NF54/iGP1_RE9H ulg8 stage V (day 12) gametocyte viability (1 µM concentration). Each assay plate included eight treated (50 µM MB; blue dots) and untreated (0.1% DMSO; orange dots) samples each as positive and negative controls, respectively. Values on the y-axis represent RLUs normalized to the mean signal emitted from the negative controls, obtained from n = 1 experiment for each library. Compounds with >50% inhibitory activity (blue shaded areas) are highlighted by numbers (1, SGI-1027; 2, SU4312; 3, monensin; 4, alexidine dihydrocholride; 5, indoprofen; 6, equilin). D Dose-response curves of SGI-1027, monensin and alexidine dihydrochloride tested against NF54/iGP1_RE9H ulg8 stage V gametocytes (day 12). Values on the y-axis represent RLUs normalized to the mean signal emitted from cells exposed to the lowest drug concentration, obtained from n = 3 biological replicates (mean ± s.e.m.) E Dose-response curves of SGI-1027, monensin and alexidine dihydrochloride tested against NF54 wild type asexual blood stage parasite multiplication. Values on the y-axis represent [ 3 H]-hypoxanthine incorporation normalized to the mean signal emitted from eight untreated control samples per plate, obtained from n = 3 biological replicates (mean ± s.e.m.). IC 50 values and 95% confidence intervals (CI) are indicated below the graphs.

Article Snippet: A – C Effect of compounds of the Epigenetics Screening Library (Cayman Chemical) ( A ), human kinase inhibitors (SelleckChem, Enzo Life Sciences) ( B ) or compounds of the Prestwick Chemical Library ( C ) on NF54/iGP1_RE9H ulg8 stage V (day 12) gametocyte viability (1 μM concentration).

Techniques: Concentration Assay, Activity Assay, Control

Journal: Cancers

Article Title: CRISPR/Cas9 Screening Highlights PFKFB3 Gene as a Major Contributor to 5-Fluorouracil Resistance in Esophageal Cancer

doi: 10.3390/cancers17101637

Figure Lengend Snippet: Human protein kinase library screen identified the PFKFB3 gene involved in esophageal cancer chemoresistance ( A ) Schematic outline of 5-FU drug resistance screening. ( B ) Identification of good sgRNA using MAGeCK-VISPR analysis. ( C – E ) PFKFB3 knockout KYSE-70, KYSE-270, and KYSE-150 cell lines were generated using CRISPR/Cas9 technology, and Western blot analysis was employed to confirm the efficiency of the PFKFB3 gene knockout.

Article Snippet: The human protein kinase library, which was a gift from John Doench and David Root (RRID: Addgene_75312), and additional sgRNAs designed to target genes involved in protein kinase [ ] contained 6934 sgRNAs targeting 1053 human genes and 107 NTCs (non-targeting-controls).

Techniques: Knock-Out, Generated, CRISPR, Western Blot, Gene Knockout

Journal: Cell Reports

Article Title: AMPK regulates Bcl2-L-13-mediated mitophagy induction for cardioprotection

doi: 10.1016/j.celrep.2024.115001

Figure Lengend Snippet: AMPKα2 is the kinase responsible for Bcl2-L-13 phosphorylation at Ser272 (A) Schematic of the responsible kinase screening workflow. (B) In vitro kinase assay in the third screening. Bacterially synthesized HA-Bcl2-L-13 was mixed with purified candidate proteins and ATP. After incubation at 37°C for 30 min, the reaction mix was subjected to western blotting using an anti-phospho-Bcl2-L-13 (Ser272) antibody. (C) The effect of AMPKα2 knockdown in CCCP-induced Bcl2-L-13 phosphorylation. HEK293A cells stably expressing HA-Bcl2-L-13 were transfected with control siRNA or siAMPKα2 for 72 h. Then, the cells were treated with DMSO or 15 μM CCCP for the indicated times, and cell lysates were subjected to western blot analysis. Densitometric analysis of phospho-Bcl2-L-13 (Ser272) is shown in the bar graph. The value for the group with control siRNA (siCtrl) transfection and 15 min of DMSO treatment in each experiment was set to 1 ( n = 3). (D) Upregulation of AMPKα2 activity by CCCP treatment. To analyze AMPKα2-specific activity, HEK293A cells stably expressing HA-Bcl2-L-13 were transfected with siAMPKα1 for 72 h and then treated with DMSO or 15 μM CCCP. The value for the group with 15-min DMSO treatment in each experiment was set to 1 ( n = 4). (E and F) HEK293A cells were transfected with control siRNA (siCtrl) or siAMPKα2 for 72 h, followed by transfection with an empty vector or HA-Bcl2-L-13. Forty-four hours after transfection, cells were treated with 100 nM bafilomycin A1 for 4 h and immunostained with anti-LC3B and anti-ATP synthase antibodies. Images in the box at higher magnification are shown on the right. White arrows indicate the puncta recognized as colocalized by the software. The number of LC3B dots colocalized with ATP synthase dots per cell is shown in (F). At least 20 cells were counted for each group ( n = 3). Scale bar: 10 μm. (G) Upregulation of AMPKα2 phosphorylation 5 days after TAC operation. To analyze AMPKα2-specific phosphorylation, lysates from the left ventricle were subjected to immunoprecipitation with an anti-AMPKα2 antibody followed by immunoblotting with an anti-phospho-AMPKα (Thr172) antibody. Densitometric analysis of phospho-AMPKα (Thr172) is shown in the right bar graph. The value for the WT sham group in each experiment was set to 1 ( n = 3). (H) Interaction between Bcl2-L-13 and AMPKα2 5 days after TAC. Lysates from the left ventricle were subjected to immunoprecipitation with an anti-AMPKα2 antibody. Co-precipitated Bcl2-L-13 was detected by immunoblotting. Densitometric analysis of Bcl2-L-13 is shown in the graph below. The value for the WT sham group in each experiment was set to 1 ( n = 3). Results are shown as mean with 95% CI. Statistical analysis by unpaired, two-tailed t tests in (C), (D), (F), and (H) and one-way ANOVA followed by Tukey-Kramer’s post hoc test in (G). All pairwise comparisons were performed. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ns, not significant. See also Figures S6 and .

Article Snippet: SilencerTM Human Kinase siRNA Library , ThermoFisher Scientific , Cat# A30079.

Techniques: Phospho-proteomics, In Vitro, Kinase Assay, Synthesized, Purification, Incubation, Western Blot, Knockdown, Stable Transfection, Expressing, Transfection, Control, Activity Assay, Plasmid Preparation, Software, Immunoprecipitation, Two Tailed Test

Journal: Cell Reports

Article Title: AMPK regulates Bcl2-L-13-mediated mitophagy induction for cardioprotection

doi: 10.1016/j.celrep.2024.115001

Figure Lengend Snippet:

Article Snippet: SilencerTM Human Kinase siRNA Library , ThermoFisher Scientific , Cat# A30079.

Techniques: Transduction, Virus, Recombinant, Western Blot, Protease Inhibitor, Immunoprecipitation, Purification, ATP Assay, In Situ, Kinase Assay, Real-time Polymerase Chain Reaction, Knock-Out, Knock-In, Negative Control, Software

Journal: Cell Reports

Article Title: AMPK regulates Bcl2-L-13-mediated mitophagy induction for cardioprotection

doi: 10.1016/j.celrep.2024.115001

Figure Lengend Snippet:

Article Snippet: For the primary screen, the Silencer Human Kinase siRNA Library (three siRNAs per gene) targeting 708 genes (ThermoFisher Scientific, A30079) was used.

Techniques: Transduction, Virus, Recombinant, Western Blot, Protease Inhibitor, Immunoprecipitation, Purification, ATP Assay, In Situ, Kinase Assay, Real-time Polymerase Chain Reaction, Knock-Out, Knock-In, Negative Control, Software