Journal: Cancer Discovery

Article Title: Sevabertinib, a Reversible HER2 Inhibitor with Activity in Lung Cancer

doi: 10.1158/2159-8290.CD-25-0605

Figure Lengend Snippet: Sevabertinib shows potent antiproliferative activity in isogenic Ba/F3 cells ectopically expressing HER2 exon 20 insertions or HER2 missense mutations. A, HER2 exon 20 insertions and single-nucleotide HER2 missense mutations in the extracellular domain, transmembrane domain (TM), and kinase domain of HER2 protein. B, Dose–response curves of sevabertinib in isogenic Ba/F3 cell lines ectopically expressing HER2 exon 20 insertions. C, IC 50 values of sevabertinib and the EGFR/HER2 TKIs zongertinib, tucatinib, lapatinib, and neratinib in isogenic Ba/F3 cell lines ectopically expressing HER2 missense mutations. D, Dose–response curves of sevabertinib in isogenic Ba/F3 cell lines ectopically expressing HER2 missense mutations. E, IC 50 values of sevabertinib and the EGFR/HER2 TKIs zongertinib, tucatinib, lapatinib, and neratinib in isogenic Ba/F3 cell lines ectopically expressing HER2 missense mutations. Dose–response curves of sevabertinib in isogenic Ba/F3 cell lines ectopically expressing HER2 exon 20 insertions (A775insYVMA, P780insGSP) or point mutation (S310F) coupled with ( F ) HER2 C805S mutation or ( G ) HER2 T798I or T798M mutations, predicted to cause TKI resistance. Ba/F3 cells transduced with an empty vector and maintained in the presence of IL3 were included as a control.

Article Snippet: EGFR/HER2 TKIs tucatinib, lapatinib, neratinib, and zongertinib were purchased from Selleck Chemicals, and STX-721 from MedChemExpress.

Techniques: Activity Assay, Expressing, Mutagenesis, Transduction, Plasmid Preparation, Control

Journal: Cancer Research Communications

Article Title: Increased Microtubule Growth Triggered by Microvesicle-mediated Paracrine Signaling is Required for Melanoma Cancer Cell Invasion

doi: 10.1158/2767-9764.CRC-22-0010

Figure Lengend Snippet: MVs comprising HER2 mediate enhanced MT growth. A, Identification of HER2/ERBB2 as a constituent of MVs upon induction of supernumerary centrosomes. Isolated MVs derived from noninvasive SK-Mel-173 cells with or without overexpression of PLK4 were subjected to MS analysis. The volcano plot depicts HER2/ERBB2 and HER3/ERBB3 as well as several marker proteins for MVs. B, Detection of HER2 in whole-cell lysates and in MVs derived from noninvasive SK-Mel-173 cells with or without PLK4 overexpression. Representative Western blots detecting HER2, PLK4, GAPDH (loading control for MVs) and α-tubulin (loading control for whole-cell lysates) are shown. C, Representative Western blots showing mild overexpression of HER2 in noninvasive SK-Mel-173 cells. β-actin was detected as a loading control. D, Measurements of MT growth rates in noninvasive melanoma cells after mild overexpression of HER2 in the absence or presence of the irreversible HER2 kinase inhibitor canertinib. Scatter dot plots show average MT growth rates (20 MTs/cell, mean ± SD, n = 30, t test). E, Measurements of MT growth rates in the indicated invasive melanoma cells after treatment with DMSO (control) or the HER2 inhibitor trastuzumab. The scatter dot plots show average MT growth rates (20 MTs/cell, mean ± SD, n = 30, t test). F, 3D outgrowth of spheroids derived from invasive melanoma cells and treated with DMSO (control) or with the HER2 inhibitor canertinib. Left: Representative example images. Scale bar, 25 μm. Right: Quantification of the 3D outgrowth area of spheroids derived from the indicated invasive melanoma cells in the absence or presence of canertinib. The bar graphs show mean values ± SD ( n = 19–27 spheroids, t test). G, Measurements of MT growth rates in noninvasive melanoma cells (SK-Mel-173) after treatment with MVs derived from noninvasive (SK-Mel-173) or invasive cells (SK-Mel-147 or SK-Mel-103) that were transiently treated with reversible (trastuzumab) or irreversible (canertinib) HER2 inhibitor. The scatter dot plots show average MT growth rates (20 MTs/cell, mean ± SD, n = 30, t test).

Article Snippet: Cells were treated either with 0.5 nmol/L Taxol (Sigma-Aldrich), 40 μmol/L trastuzumab (reversible HER2 inhibitor, kindly provided by Gerald Wulf) or 200 nmol/L canertinib (irreversible HER2 kinase inhibitor, Selleck Chemicals).

Techniques: Isolation, Derivative Assay, Over Expression, Marker, Western Blot, Control

Journal: Glia

Article Title: AXON CONTACT-DRIVEN SCHWANN CELL DEDIFFERENTIATION

doi: 10.1002/glia.23131

Figure Lengend Snippet: The axonal dedifferentiating activity is different from the mitogenic activity. A–D. Lack of a dedifferentiating activity in membranes enriched in neuregulin-1. cAMP-differentiated SCs (dSCs) were left untreated (control) or stimulated with an equal protein concentration (20 μg) of membranes isolated from DRG neurons (DRG membranes) or HEK293 overexpressing neuregulin-1 (NRG-1) Type I and III. The incorporation of [3H]-thymidine (C) and the activation of ErbB signaling (D, Western blots) in undifferentiated SCs was used to confirm the mitogenic activity of all membrane preparations. E–G. Effect of tyrosine kinase antagonists on proliferation and dedifferentiation. SCs and dSCs were stimulated with DRG membranes in the absence or presence of CPT-cAMP provided alone or together with the indicated pharmacological inhibitors. These inhibitors were chosen based on their demonstrated specificity for ErbB receptors (EGFR/ErbB2 inhibitor), Src (PP2) and tyrosine kinases in general (genistein). Note that inhibitors effectively blocked S-phase entry, as determined by the incorporation of BrDU in SCs (E and G) and dSCs (E–F), without preventing O1 loss in response to DRG membrane stimulation (E–F). The dose-dependent anti-mitogenic action of the inhibitors was determined by [3H]-thymidine incorporation assays in undifferentiated SCs. Inhibitors were used at 10 μM unless indicated in the figure.

Article Snippet: The tyrosine kinase inhibitors EGFR/ErbB2 (4557W), AG825 (tyrphostin), genistein and PP2 were from Calbiochem-Novabiochem Corp. (La Jolla, CA).

Techniques: Activity Assay, Protein Concentration, Isolation, Activation Assay, Western Blot

Journal: Cell metabolism

Article Title: Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

doi: 10.1016/j.cmet.2018.08.008

Figure Lengend Snippet: (A) Chromatograms of BT474 cellular metabolites extracted from control and lapatinib (1 μM) treated cells for 2 hours at 37°C. Representative of three independent experiments, all of which showed similar results. (B) Cr kinase enzyme activity assays from the fractionated lysates of BT474 cells treated with vehicle control or 1 μM lapatinib for 2 hours at 37°C. The corresponding immunoblotting (WB) results of the fractionated samples with antibodies against MtCK1, anti-phospho-tyrosine (pTyr), PDHK1 (mitochondria marker), and β-actin (cytosol marker) are also shown. (C) Top: Immunoblots of lysates from 293T cells expressing vector control (−) and HER2 WT. Bottom: qRT-PCR results of MtCK1 mRNA in 293T cells expressing vector control (−) or HER2 WT. (D) Immunoblots of anti-Flag immunoprecipitates from cells expressing Flag-MtCK1 WT and Y153F mutant. (E) Immunoblots of cells treated with 1 μM lapatinib for 2 hours at 37°C. MtCK1 total protein levels were normalized between the control and lapatinib-treated samples to compare the levels of phospho-Y153 MtCK1 between the samples. All results are representative experiments of three independent replicates and P values were determined by a two-tailed Student’s t test (**P<0.01).

Article Snippet: Human HER2 WT , Addgene , Plasmid # 16257.

Techniques: Control, Activity Assay, Western Blot, Marker, Expressing, Plasmid Preparation, Quantitative RT-PCR, Mutagenesis, Two Tailed Test

Journal: Cell metabolism

Article Title: Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

doi: 10.1016/j.cmet.2018.08.008

Figure Lengend Snippet: (A) Top: Immunoblots of lysates from 293T cells co-expressing Flag-MtCK1 WT or Y153F along with vector control (−) or HER2 WT. Bottom: Densitometric quantification of four independent anti-Flag and anti-β-actin immunoblots based on the densitometric analysis showing a dose-dependent linear correlation of anti-Flag and anti-β-actin immunoblots of the lysates (Figure S2A). (B) Top: Immunoblots of SKBR3 cells stably expressing Flag-MtCK1 WT or Y153F that were treated with 200 μg/ml cycloheximide (CHX) for the indicated times. Bottom: Densitometric quantification of three independent anti-Flag and anti-β-actin immunoblots. (C) Left: MtCK1 enzyme activity in anti-Flag immunoprecipitates from 293T cells co-expressing Flag-MtCK1 WT, MtCK1 Y153F or MtCK1 E227L and HER2. Right: Immunoblots of the corresponding anti-Flag immunoprecipitates (IP) and whole-cell lysates (WCL) from 293T cells. (D) Immunoblots of 293T cells co-expressing either vector control (−) or HER2 and Flag-MtCK1 E227L. All results are representative experiments of three independent replicates. P values were determined by a two-tailed Student’s t test (*P<0.05, **P<0.01, ***p < 0.001).

Article Snippet: Human HER2 WT , Addgene , Plasmid # 16257.

Techniques: Western Blot, Expressing, Plasmid Preparation, Control, Stable Transfection, Activity Assay, Two Tailed Test

Journal: Cell metabolism

Article Title: Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

doi: 10.1016/j.cmet.2018.08.008

Figure Lengend Snippet: (A) Immunoblots of lysates (left) and qRT-PCR for MtCK1 mRNA (right) from “rescued” BT474 cells that stably express Flag-MtCK1 WT or Y153F with stable knockdown of endogenous MtCK1. (B) Immunoblots of “rescued” BT474 cells expressing Flag-MtCK1 WT and Y153F after treatment with MG132 for 6 h. (C) Immunoblots of anti-Flag immunoprecipitates and whole-cell lysates (WCL) from Flag-MtCK1 WT and Y153F “rescued” BT474 cells. (D) Immunoblots (WB) of anti-TRAP1 or anti-IgG immunoprecipitates and whole-cell lysates (WCL) from BT474 cells with or without lapatinib treatment (500 nM, 6 h). (E) Immunoblots (WB) of anti-Flag immunoprecipitates and WCL from 293T cells expressing Flag-MtCK1 WT expressing control vector (−) or HER2. (F) Immunoblots of lysates (left) and PCr levels (right) from 293T cells expressing vector control (−) or HER2 with or without stable knockdown of endogenous TRAP1. All results are representative experiments of three independent replicates. P values were determined by a two-tailed Student’s t test (*P<0.05).

Article Snippet: Human HER2 WT , Addgene , Plasmid # 16257.

Techniques: Western Blot, Quantitative RT-PCR, Stable Transfection, Knockdown, Expressing, Control, Plasmid Preparation, Two Tailed Test

Journal: Cell metabolism

Article Title: Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

doi: 10.1016/j.cmet.2018.08.008

Figure Lengend Snippet: (A) Immunoblots of an in vitro tyrosine kinase assay using recombinant ABL with purified recombinant MtCK1 WT protein. (B) Immunoblots of in vitro ABL kinase assay using recombinant ABL and MtCK1 WT or Y153F proteins. (C) Immunoblots of anti-Flag immunoprecipitates and WCL from 293T cells co-expressing Flag-MtCK1 WT and vector control (−), HER2 WT or ABL. (D) Immunoblots of BT474 cells treated with DMSO, or imatinib (20 βM), or lapatinib (500 nM) for 6 h. (E) Immunoblots of anti-Flag immunoprecipitates and WCL from BT474 cells treated with imatinib (10 βM) overnight. (F) Immunoblots of anti-TRAP1 or anti-IgG immunoprecipitates and WCL from BT474 cells with or without imatinib treatment (20 βM) for 6 h. (G) Immunoblots of anti-IgG control and anti-MtCK1 antibody immunoprecipitates and input lysate from BT474 cells. (H) Isolated mitochondria from BT474 cells were treated with 5 μg/mL proteinase K in the absence and presence of 1% Triton X-100 and immunoblotted for the indicated proteins. All immunoblots results are representative experiments of three independent replicates.

Article Snippet: Human HER2 WT , Addgene , Plasmid # 16257.

Techniques: Western Blot, In Vitro, Tyrosine Kinase Assay, Recombinant, Purification, Kinase Assay, Expressing, Plasmid Preparation, Control, Isolation

Journal: Cell metabolism

Article Title: Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

doi: 10.1016/j.cmet.2018.08.008

Figure Lengend Snippet: (A) Immunoblots of three normal breast tissue lysates (#1-#3), six HER2+ patient-derived xenograft (PDX) tumor lysates (#4-#9), five TNBC PDX tumor lysates (#10-#14). (B) Tumor volumes in NSG mice (N=5) with PDX #4 cells with or without MtCK1 knockdown. Each dot represents tumor volume in an individual mouse with error bars ± standard deviation. Immunoblots of lysates from the corresponding cells are also shown. (C) Structures of Cr and Cyclo-Cr. (D) HPLC analysis of metabolites extracted from SKBR3-R cells treated with either water or Cyclo-Cr (1 mM) overnight. The experiment shown is representative of three independent experiments, all of which showed similar results. (E) Clonogenic assay results of breast cancer cell lines treated with Cyclo-Cr. (F) Cell proliferation assay results of SKBR3-R treated with Cyclo-Cr and/or PCr. (G) Cell proliferation assay results of normal primary mammary epithelial cells up to 6 mM of CCr. Error bars in E-G ± standard deviation of 3 independent measurements. P values were determined by a two-tailed Student’s t test. ***P<0.001.

Article Snippet: Human HER2 WT , Addgene , Plasmid # 16257.

Techniques: Western Blot, Derivative Assay, Knockdown, Standard Deviation, Clonogenic Assay, Proliferation Assay, Two Tailed Test

Journal: Cell metabolism

Article Title: Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

doi: 10.1016/j.cmet.2018.08.008

Figure Lengend Snippet: (A) Tumor growth in nude mice with BT474-R tumors treated with 0.3% Cyclo-Cr in drinking water versus regular drinking water. The Cyclo-Cr and PCr concentrations in the harvested BT474-R tumors determined by HPLC analysis are also shown below. N=8 in each group. (B) Tumor growth of HER2+ PDX #4 in NSG mice treated with 0.3% Cyclo-Cr in drinking water versus regular drinking water. The Cyclo-Cr and PCr concentrations in harvested PDX #4 tumors determined by HPLC analysis are also shown below. N=6 in each group. (C) Left: Tumor growth in NSG mice with trastuzumab-resistant HER2+ PDX #4 under various drug combinations (a total of 6 groups; control, Cyclo-Cr alone, lapatinib alone, Cyclo-Cr plus PCr, Cyclo-Cr plus lapatinib, and Cyclo-Cr plus lapatinib plus PCr). Cyclo-Cr was administered in drinking water at 0.3%, lapatinib was administrated orally at 50 mg/kg/day and PCr was administered at 400 mg/kg/day intraperitoneally. Right: Bar graphs represent tumor volume in mice with various treatments at day 20. N=9 in each group. P values were determined by a two-tailed Student’s t test. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: Human HER2 WT , Addgene , Plasmid # 16257.

Techniques: Control, Two Tailed Test

Journal: Cell metabolism

Article Title: Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

doi: 10.1016/j.cmet.2018.08.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human HER2 WT , Addgene , Plasmid # 16257.

Techniques: Derivative Assay, Recombinant, FLAG-tag, Plasmid Preparation, shRNA, Software, Cloning, Mutagenesis, SYBR Green Assay, CyQUANT Assay, Proliferation Assay, Cell Viability Assay, Protease Inhibitor