Journal: Cell reports

Article Title: ABL kinases regulate translation in HER2+ cells through Y-box-binding protein 1 to facilitate colonization of the brain

doi: 10.1016/j.celrep.2022.111268

Figure Lengend Snippet: (A–C) Nude mice were injected intracranially with HCC1954-LCC1 cells on day 0. On day 10, mice were divided between treatment groups (vehicle [n = 9] or GNF5 [n = 10]). Tumors were monitored weekly by bioluminescent imaging (BLI) until experimental endpoint (day 55). (B) Representative BLI images on day 33. (C) Overall survival curve of mice. (D and E) Cell viability of HCC1954-LCC1 (D) or SUM190-BrM (E) cells treated with DMSO, 10 μM GNF5, or 10 μM ABL001 as measured by CellTiter-Glo. (F) Immunoblots of ABL1 and ABL2 in HCC1954-LCC1 cells virally transduced with Cas9 and gRNAs targeting ABL1 and ABL2 (ABL1/2 knockout [KO] #1). Control cells are virally transduced with empty gRNA backbones (control). (G) Cell viability of HCC1954-LCC1 ABL1/2 KO #1 and control cells as measured by CellTiter-Glo. (H–J) Nude mice were injected intracranially with HCC1954-LCC1 control (n = 15) or ABL1/2 KO #1 (n = 15) cells. (H) Representative BLI images of mice on day 21. (I and J) Brain flux over time (I) and overall survival curve (J) of mice. (K–M) FVB mice were injected intracranially with ErbB2-BrM2 cells on day 0. On day 7, mice were divided between treatment groups (vehicle or ABL001) (n = 13/group). Tumors were monitored weekly by BLI until experiment endpoint (day 21). (L) Representative BLI images of mice on day 21 are shown. (M) Brain flux over time in mice was measured by BLI. Statistical analysis was performed using a repeated measures ANOVA (n = 3) or log rank (Mantel-Cox) test for survival studies. *p < 0.05, ***p < 0.001, ****p < 0.0001. Data are mean ± SEM. p/s, photons/second.

Article Snippet: ABL2 (6D5) , Abnova , Cat# H00000027-M03; RRID: AB_565433.

Techniques: Injection, Imaging, Western Blot, Transduction, Knock-Out, Control

Journal: Cell reports

Article Title: ABL kinases regulate translation in HER2+ cells through Y-box-binding protein 1 to facilitate colonization of the brain

doi: 10.1016/j.celrep.2022.111268

Figure Lengend Snippet: (A) Immunoblot and quantification of HER2 in HCC1954-LCC1 cells treated with DMSO, 10 μM GNF5, or 10 μM DPH for 48 h. (B) Immunoblot and quantification of HER2 in HCC1954-LCC1 virally transduced with shRNAs targeting ABL1 and ABL2 (shAA) or a scramble control (shScr). (C) Immunoblot and quantification of HER2 in ErbB2-BrM2 cells treated with DMSO, 10 μM GNF5, or 10 μM ABL001 for 48 h. (D) HER2 cell-surface levels were measured by FACS analysis of mean fluorescence intensity. ErbB2-BrM2 cells were treated with DMSO or 10 μM GNF5 for 48 h. For immunoblots, p-CrkL serves as a marker of ABL kinase activity, and HER2 protein levels were normalized to GAPDH. Statistical analysis was performed using Student’s t test and one-way ANOVA with Bonferroni’s post hoc testing (n = 3). *p < 0.05, ***p < 0.001, ****p < 0.0001. Data are mean ± SEM.

Article Snippet: ABL2 (6D5) , Abnova , Cat# H00000027-M03; RRID: AB_565433.

Techniques: Western Blot, Transduction, Control, Fluorescence, Marker, Activity Assay

Journal: Cell reports

Article Title: ABL kinases regulate translation in HER2+ cells through Y-box-binding protein 1 to facilitate colonization of the brain

doi: 10.1016/j.celrep.2022.111268

Figure Lengend Snippet:

Article Snippet: ABL2 (6D5) , Abnova , Cat# H00000027-M03; RRID: AB_565433.

Techniques: Western Blot, Recombinant, Labeling, Silver Staining, Mass Spectrometry, Immunoprecipitation, Caspase-Glo Assay, Binding Assay, shRNA, Control, Software

Journal: bioRxiv

Article Title: ABL Kinases Modulate EZH2 Phosphorylation and Signaling in Metastatic Triple Negative Breast Cancer

doi: 10.1101/2025.02.18.638898

Figure Lengend Snippet: (A.) ABL kinases were knocked down using short hairpin RNAs in bone-metastatic triple-negative breast cancer cells (1833), RNA was extracted from knockdown cells followed analysis via by RNA-sequencing. Gene set enrichment analysis (GSEA) was retrospectively conducted on the generated dataset assessing changes in genes involved in epigenetic regulation of gene expression. (B.) GSEA was also performed to evaluate how genes altered by PRC2 knockdown are impacted in ABL knockdown (AA) cells. (C.)Schema of PRC2 complex. (D.) Genes significantly enriched in the retrospective RNA-seq analysis were confirmed via RT-qPCR in 1833 cells. Gene expression was normalized to a housekeeping gene (18s) before being calculated relative to SCR transduced cells. Significantly enriched genes are marked as indicated. (E.,F.) shRNAs targeting either ABL1 and ABL2 Kinase (AA) or a scramble control (SCR) were transduced into MDA-MB-231 and SUM159 TNBC cell lines to knock down both ABL Kinases. Cells lysates were analyzed by immunoblotting for changes in H3K27me3 levels. Actin was used as a loading control. All data represented in this figure represent n=3 independent experiments. Significance between SCR and shAA groups for gene expression data was determined using Welch’s T-test *,p<0.05, ns=not significant.

Article Snippet: EV (pN1-eGFP) and ABL2 eGFP (pN1-ABL2-eGFP WT) constructs were previously generated and described previously( , ). pcDNA3.1-3’3xFlag was a gift from Quanfu Ma (Addgene plasmid # 208616; http://n2t.net/addgene:208616 ; RRID:Addgene_208616); pcDNA3.1_3xFlagEzh2 WT was a gift from Thomas Cech (Addgene plasmid # 173717; http://n2t.net/addgene:173717 ; RRID:Addgene_173717)( ) pCMVHA hEZH2 was a gift from Kristian Helin (Addgene plasmid # 24230; http://n2t.net/addgene:24230 ; RRID:Addgene_24230)( ). pGFP FAK and pGFP FAK Y397F were a gift from Kenneth Yamada (Addgene plasmid # 50515; http://n2t.net/addgene:50515 ; RRID:Addgene_50515)( ).

Techniques: Knockdown, RNA Sequencing, Generated, Gene Expression, Quantitative RT-PCR, Control, Western Blot

Journal: bioRxiv

Article Title: ABL Kinases Modulate EZH2 Phosphorylation and Signaling in Metastatic Triple Negative Breast Cancer

doi: 10.1101/2025.02.18.638898

Figure Lengend Snippet: ( A. ) MDA-MB-231 cells were treated with either DMSO (control), 10uM ABL001, 20uM GNF5, or 5uM GMB475 for 48 hours, cells were then assessed by immunoblotting for changes in H3K27me3 levels. Histone H3 was used as a loading control. (B.) HEK 293T cells were transiently transduced with either empty vector (EV), ABL1 eGFP, or ABL2 eGFP wildtype. Cell lysates were then assessed by immunoblotting for changes in H3K27me3 levels. Actin was used as a loading control. (C.) HEK 293T cells were transiently transfected with plasmids expressing EV, ABL2 eGFP, or ABL2eGFP and HA EZH2. ABL2 was pulled down from prepared cell lysates using the GFPtrap nanobody system. Actin was used as a loading control in the Input samples. (D.) HEK 293T cells were transiently transfected with plasmids expressing EV, ABL1 eGFP, or ABL1eGFP and HA EZH2. ABL1 was pulled down from prepared cell lysates using the GFPtrap nanobody system. Actin was used as a loading control in the Input samples. (E.) SUM159 TNBC cells were treated with 10uM GNF5 for 24 hours afterwhich endogenous EZH2 interactions were assessed via immunoprecipitation (IP) from whole cell lysate (WCL). Actin was used as a loading control in the WCL. (F.) HEK293T cells were transiently transfected with ABL2 eGFP, afterwhich ectopic ABL2 was pulled down using the GFP-Trap Nanobody system. GFP Trap pulldown lysates were probed for PRC2 complex members in tandem with WCL were analyzed by immunoblotting with indicated antibodies. Actin was used as a loading control. All data represented in this figure represent n=3 independent experiments.

Article Snippet: EV (pN1-eGFP) and ABL2 eGFP (pN1-ABL2-eGFP WT) constructs were previously generated and described previously( , ). pcDNA3.1-3’3xFlag was a gift from Quanfu Ma (Addgene plasmid # 208616; http://n2t.net/addgene:208616 ; RRID:Addgene_208616); pcDNA3.1_3xFlagEzh2 WT was a gift from Thomas Cech (Addgene plasmid # 173717; http://n2t.net/addgene:173717 ; RRID:Addgene_173717)( ) pCMVHA hEZH2 was a gift from Kristian Helin (Addgene plasmid # 24230; http://n2t.net/addgene:24230 ; RRID:Addgene_24230)( ). pGFP FAK and pGFP FAK Y397F were a gift from Kenneth Yamada (Addgene plasmid # 50515; http://n2t.net/addgene:50515 ; RRID:Addgene_50515)( ).

Techniques: Control, Western Blot, Transduction, Plasmid Preparation, Transfection, Expressing, Immunoprecipitation

Journal: bioRxiv

Article Title: ABL Kinases Modulate EZH2 Phosphorylation and Signaling in Metastatic Triple Negative Breast Cancer

doi: 10.1101/2025.02.18.638898

Figure Lengend Snippet: ( A. ) EZH2 and SUZ12 tyrosine phosphorylation is unchanged upon ABL inhibition. SUM159 cells were treated with either DMSO (vehicle control) or ABL001 for 24 hours, after which cells were lysed and an antibody against IgG (control),EZH2,or SUZ12 was added to cell lysates. Tyrosine Phosphorylation was detected using 4G10 pan phosphotyrosine antibody. Actin was used as a loading control in the WCL. (B.) EZH2 phosphorylation mark abundance was profiled from data generated from TNBC clinical samples hosted on the Proteomic Data Commons.( C.) SUM159 cells were treated with ABL kinase inhibitor ABL001 for 24 hours with ABL001. Lysates were prepared from these cells and cells were probed for endogenous levels of phosphorylated forms of EZH2. (D.) Bone metastatic TNBC cells were lentivirally transduced with either a nontargeting shRNA (SCR) or and shRNA directed against both ABL1 and ABL2 (AA). P-EZH2 (T487) levels were assessed via immunoblot. Actin was used as a loading control. (E.) Linear protein structure of EZH2 with T487 indicated. (F.)HEK293T cells were transiently transfected with constructs expressing either pcDNA3.1_3xFlagEzh2: WT, T487A (phosphodeficient mutant), or T487D (phosphomimetic). Cell lysates were prepared and ectopically expressed EZH2 was pulled down using the fab-trap nanobody system recognizing 3xFlag. Interactions with endogenous proteins were assessed as indicated. Vinculin was used as a loading control for the input. (G.) Bone metastatic TNBC cells were treated with 10uM ABL001 for 72 hours afterwhich EZH2 was immunoprecipitated from the whole cell lysate (WCL) and probed for endogenous interactors via immunoblot. Vinculin was used as a loading control. All data represent n=3 independent experiments, with the exception of (B.).

Article Snippet: EV (pN1-eGFP) and ABL2 eGFP (pN1-ABL2-eGFP WT) constructs were previously generated and described previously( , ). pcDNA3.1-3’3xFlag was a gift from Quanfu Ma (Addgene plasmid # 208616; http://n2t.net/addgene:208616 ; RRID:Addgene_208616); pcDNA3.1_3xFlagEzh2 WT was a gift from Thomas Cech (Addgene plasmid # 173717; http://n2t.net/addgene:173717 ; RRID:Addgene_173717)( ) pCMVHA hEZH2 was a gift from Kristian Helin (Addgene plasmid # 24230; http://n2t.net/addgene:24230 ; RRID:Addgene_24230)( ). pGFP FAK and pGFP FAK Y397F were a gift from Kenneth Yamada (Addgene plasmid # 50515; http://n2t.net/addgene:50515 ; RRID:Addgene_50515)( ).

Techniques: Inhibition, Control, Generated, Transduction, shRNA, Western Blot, Transfection, Construct, Expressing, Mutagenesis, Immunoprecipitation