Journal: Clinical Cancer Research

Article Title: Phase II Trial of HER-Vaxx, a B-cell Peptide-Based Vaccine, in HER2-Overexpressing Advanced Gastric Cancer Patients Under Platinum-Based Chemotherapy (HERIZON)

doi: 10.1158/1078-0432.CCR-24-0742

Figure Lengend Snippet: Detected levels of HER2-specific total IgG antibodies. The HER2-specific total IgG antibodies in patients treated with chemotherapy alone A, and 50 µg of HER-Vaxx plus chemotherapy B, measured by ELISA, are shown. The results are representative of at least two experiments.

Article Snippet: The level of intracellular HER2 phosphorylation in the lysates of the treated cells was evaluated by ELISA, using capture mouse antihuman HER2 (R&D Systems, USA; cat. No. MAB1129, RRID:AB_357477), phospho-HER2/ErbB2 (Tyr1221/1222; 6B12) rabbit mAb (Cell Signaling Technology, cat. No. 2243, RRID:AB_490899), and HRP-conjugated goat antirabbit IgG (Sigma-Aldrich; cat. No. 8275, RRID:AB_258382).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Clinical Cancer Research

Article Title: Phase II Trial of HER-Vaxx, a B-cell Peptide-Based Vaccine, in HER2-Overexpressing Advanced Gastric Cancer Patients Under Platinum-Based Chemotherapy (HERIZON)

doi: 10.1158/1078-0432.CCR-24-0742

Figure Lengend Snippet: Correlation between the induced HER2-specific antibody levels and antitumor effect and mediation of ADCC. The total IgG A, and IgG1 B, antibody levels in all subjects’ available sera after four vaccinations (week 12; ) and the observed antitumor effect (as fold change compared with baseline) in each respective patient are shown. The correlation of the induced HER2-specific IgG C, or IgG1 D, antibody levels in representative sera of low, high, and very high responders [after four vaccinations (week 12) with 50- or 100-µg vaccine dose], and ADCC expressed in percent of the positive control (trastuzumab) are presented. The levels of correlation and significance are indicated in the boxes. The target cells NCI-N87 were incubated with effector cells (i.e., PBMC) after treatment with 50 μg of the examined patients' isolated and purified total IgG antibodies. Trastuzumab as a positive control and serum samples from a healthy individual as a negative control were included in the assays. The results represent at least two experiments.

Article Snippet: The level of intracellular HER2 phosphorylation in the lysates of the treated cells was evaluated by ELISA, using capture mouse antihuman HER2 (R&D Systems, USA; cat. No. MAB1129, RRID:AB_357477), phospho-HER2/ErbB2 (Tyr1221/1222; 6B12) rabbit mAb (Cell Signaling Technology, cat. No. 2243, RRID:AB_490899), and HRP-conjugated goat antirabbit IgG (Sigma-Aldrich; cat. No. 8275, RRID:AB_258382).

Techniques: Positive Control, Incubation, Isolation, Purification, Negative Control

Journal: Scientific Reports

Article Title: Ganetespib targets multiple levels of the receptor tyrosine kinase signaling cascade and preferentially inhibits ErbB2-overexpressing breast cancer cells

doi: 10.1038/s41598-018-25284-0

Figure Lengend Snippet: Ganetespib inhibits cell proliferation in ErbB2 + breast cancer cells. ( a ) BT474 and SKBR3 cells were treated with ganetespib (0, 1, 5, 10, 50, or 100 nM) and for 5 days, followed by an MTT assay to assess cell viability. The average percentages of viable cells relative to the untreated control cells in each cell line are graphed. ( b ) For the clonogenic assay, BT474, and SKBR3 cells were treated with ganetespib (0, 1.25, 2.5, 5, or 10 nM) for 21 and 14 days, respectively. Then, colonies were stained with crystal violet and quantified. Representative images of the crystal violet-stained colonies are shown in the top panels with the corresponding graphs in the lower panels. All values are presented as the means ± S.E. (* P ≤ 0.05, ** P ≤ 0.01).

Article Snippet: Primary antibodies specific to cleaved PARP, cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, Bcl-2, ErbB2, phospho-ErbB2 (pErbB2), Akt, pAkt, Erk2, pErk1/2, mTOR, pmTOR, Cyclin B1, Src, pSrc, Bad, pBad, GSK3, and pGSK3 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: MTT Assay, Control, Clonogenic Assay, Staining

Journal: Scientific Reports

Article Title: Ganetespib targets multiple levels of the receptor tyrosine kinase signaling cascade and preferentially inhibits ErbB2-overexpressing breast cancer cells

doi: 10.1038/s41598-018-25284-0

Figure Lengend Snippet: Ganetespib induces G2/M cell cycle arrest in ErbB2 + breast cancer cells. ( a ) BT474 and SKBR3 cells were treated with ganetespib (0 or 250 nM) for 24 hours, PI-stained, and analyzed for cell cycle distribution using flow cytometry. ( b) Cropped Western blot images of the indicated cell cycle regulators are shown in ganetespib-treated (0–300 nM for 16 hours) BT474 and SKBR3 cells. All values are graphed as the means ± S.E. (* P ≤ 0.05; ** P ≤ 0.01 as compared to the corresponding untreated controls).

Article Snippet: Primary antibodies specific to cleaved PARP, cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, Bcl-2, ErbB2, phospho-ErbB2 (pErbB2), Akt, pAkt, Erk2, pErk1/2, mTOR, pmTOR, Cyclin B1, Src, pSrc, Bad, pBad, GSK3, and pGSK3 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Staining, Flow Cytometry, Western Blot

Journal: Scientific Reports

Article Title: Ganetespib targets multiple levels of the receptor tyrosine kinase signaling cascade and preferentially inhibits ErbB2-overexpressing breast cancer cells

doi: 10.1038/s41598-018-25284-0

Figure Lengend Snippet: Ganetespib induces apoptosis in ErbB2 + breast cancer cells. ( a ) BT474 and SKBR3 cells were treated with ganetespib (25–250 nM) for 48 hours. Then, cells were Annexin V/PI-stained and analyzed with flow cytometry. The percentages of cells in early (Annexin V + /PI − ) and late (Annexin V + /PI + ) stages of apoptosis are graphed in the panels to the right of the representative dot plots. ( b ) Cropped images of Western blots at the target molecular weights for the indicated apoptotic markers are shown in ganetespib-treated (0, 10, 30, 100, 300 nM for 16 hours) BT474 and SKBR3 cells. All values are graphed as the means ± S.E. (* P ≤ 0.05; ** P ≤ 0.01 as compared to the corresponding untreated controls).

Article Snippet: Primary antibodies specific to cleaved PARP, cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, Bcl-2, ErbB2, phospho-ErbB2 (pErbB2), Akt, pAkt, Erk2, pErk1/2, mTOR, pmTOR, Cyclin B1, Src, pSrc, Bad, pBad, GSK3, and pGSK3 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Staining, Flow Cytometry, Western Blot

Journal: Scientific Reports

Article Title: Ganetespib targets multiple levels of the receptor tyrosine kinase signaling cascade and preferentially inhibits ErbB2-overexpressing breast cancer cells

doi: 10.1038/s41598-018-25284-0

Figure Lengend Snippet: Ganetespib inhibits RTK signaling in ErbB2 + breast cancer cells. BT474 and SKBR3 cells were treated with ganetespib (0, 10, 30, 100, or 300 nM) for 16 hours, followed by Western blot analysis (cropped images) of the expression and activation/phosphorylation of the indicated markers involved in RTK signaling.

Article Snippet: Primary antibodies specific to cleaved PARP, cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, Bcl-2, ErbB2, phospho-ErbB2 (pErbB2), Akt, pAkt, Erk2, pErk1/2, mTOR, pmTOR, Cyclin B1, Src, pSrc, Bad, pBad, GSK3, and pGSK3 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Expressing, Activation Assay, Phospho-proteomics

Journal: Scientific Reports

Article Title: Ganetespib targets multiple levels of the receptor tyrosine kinase signaling cascade and preferentially inhibits ErbB2-overexpressing breast cancer cells

doi: 10.1038/s41598-018-25284-0

Figure Lengend Snippet: Ganetespib decreases the protein stability and half-life of ErbB2 in ErbB2 + breast cancer cells. ( a ) BT474 and SKBR3 cells were treated with CHX (100 μg/mL) +/− ganetespib (1 μM) for the indicated treatment durations. Then, ErbB2 protein expression was analyzed using Western blotting. Cropped images of ErbB2 and β-actin protein expression are shown. ( b ) ErbB2 protein band intensities were quantified using ImageJ software and normalized to corresponding β-actin protein band intensities. Relative ErbB2 protein expression after CHX +/− ganetespib treatments for each cell line is graphed.

Article Snippet: Primary antibodies specific to cleaved PARP, cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, Bcl-2, ErbB2, phospho-ErbB2 (pErbB2), Akt, pAkt, Erk2, pErk1/2, mTOR, pmTOR, Cyclin B1, Src, pSrc, Bad, pBad, GSK3, and pGSK3 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Western Blot, Software

Journal: Scientific Reports

Article Title: Ganetespib targets multiple levels of the receptor tyrosine kinase signaling cascade and preferentially inhibits ErbB2-overexpressing breast cancer cells

doi: 10.1038/s41598-018-25284-0

Figure Lengend Snippet: Ganetespib preferentially inhibits ErbB2 + cancer cells. ( a ) MDA-MB-435/Control, MDA-MB-435/ErbB2, MCF7/Control, and MCF7/ErbB2 cells were treated with ganetespib (0, 1, 5, or 10 nM) and for 5 days, followed by an MTT assay to assess cell viability. The average percentages of viable cells relative to the untreated control cells in each cell line are graphed. For the clonogenic assay, these cells were treated with ganetespib (0 or 2.5 nM) for 14 days. Then, colonies were stained with crystal violet and quantified. Representative images of the crystal violet-stained colonies are shown in ( b ) with the corresponding graphs in ( c ). All values are presented as the means ± S.E. (* P ≤ 0.05; ** P ≤ 0.01 as indicated; ## P ≤ 0.01 as compared to the ganetespib-treated parental Control cells).

Article Snippet: Primary antibodies specific to cleaved PARP, cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, Bcl-2, ErbB2, phospho-ErbB2 (pErbB2), Akt, pAkt, Erk2, pErk1/2, mTOR, pmTOR, Cyclin B1, Src, pSrc, Bad, pBad, GSK3, and pGSK3 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Control, MTT Assay, Clonogenic Assay, Staining

Journal: Scientific Reports

Article Title: Ganetespib targets multiple levels of the receptor tyrosine kinase signaling cascade and preferentially inhibits ErbB2-overexpressing breast cancer cells

doi: 10.1038/s41598-018-25284-0

Figure Lengend Snippet: Ganetespib significantly inhibits ErbB2-overexpressing mammary tumors in vivo in a syngeneic transplantation mouse model. 10-week-old MMTV-ErbB2 mice (N = 6 mice per group) were subcutaneously injected with 78617 cells for syngeneic tumor growth. Once tumors were palpable 8 days after the initial tumor cell inoculations, ganetespib (30 mg/kg) was administered via i.p. injections every other day until the 20 th day after tumor cell inoculations. Tumor volumes were measured every 2 days. All values are presented as the means ± S.E. (** P ≤ 0.01).

Article Snippet: Primary antibodies specific to cleaved PARP, cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, Bcl-2, ErbB2, phospho-ErbB2 (pErbB2), Akt, pAkt, Erk2, pErk1/2, mTOR, pmTOR, Cyclin B1, Src, pSrc, Bad, pBad, GSK3, and pGSK3 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: In Vivo, Transplantation Assay, Injection

Journal: Scientific Reports

Article Title: Ganetespib targets multiple levels of the receptor tyrosine kinase signaling cascade and preferentially inhibits ErbB2-overexpressing breast cancer cells

doi: 10.1038/s41598-018-25284-0

Figure Lengend Snippet: Ganetespib potentiates the anti-cancer effects of lapatinib in ErbB2 + breast cancer cells. ( a ) BT474 and SKBR3 cells were treated with ganetespib (0 or 5 nM) and lapatinib (0, 15.6, 31.25, 62.5, 125, 250, or 500 nM) for 5 days, followed by an MTT assay to assess cell viability. ( b ) BT474 and SKBR3 cells were treated with ganetespib alone (25 or 50 nM), lapatinib alone (100 or 125 nM), or ganetespib + lapatinib for 24 hours. Then, cells were Annexin V/PI-stained and analyzed with flow cytometry. The percentages of cells in early (Annexin V + /PI − ) and late (Annexin V + /PI + ) stages of apoptosis are graphed in the panels to the right of the representative dot plots. ( c ) BT474 cells were treated with ganetespib (50 nM) +/− lapatinib (250 nM) for 16 hours, and SKBR3 cells were treated ganetespib (50 nM) +/− lapatinib (500 nM) for 16 hours. Then, Western blot analysis was performed to detect the expression and activation/phosphorylation of the indicated markers. Cropped Western blot images are shown at the target molecular weights for the indicated markers. ( d ) BT474 and SKBR3 cells were treated with a series of increasing doses of ganetespib and lapatinib for 5 days. Then the viable fraction of each cell line (fraction affected; FA) was determined with an MTT assay. The Combination Index (CI) was calculated using CompuSyn software and graphed for each cell line. Dose combinations with a CI < 1.0 are highlighted in red. All values are presented as the means ± S.E. (* P ≤ 0.05; ** P ≤ 0.01 as compared to the corresponding untreated controls).

Article Snippet: Primary antibodies specific to cleaved PARP, cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, Bcl-2, ErbB2, phospho-ErbB2 (pErbB2), Akt, pAkt, Erk2, pErk1/2, mTOR, pmTOR, Cyclin B1, Src, pSrc, Bad, pBad, GSK3, and pGSK3 were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: MTT Assay, Staining, Flow Cytometry, Western Blot, Expressing, Activation Assay, Phospho-proteomics, Software

Journal: Genes & development

Article Title: Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas cancer.

doi: 10.1101/gad.351037.123

Figure Lengend Snippet: Figure 1. Cancer-associated fibroblasts promote resistance to KRAS∗extinction in a PDAC model. (A) Crystal violet staining of iKPC1 cell colony formation assay, with Kras∗on (on dox), MRTX1133, or Kras∗off (off dox). The presence of 50% conditional medium from pCAF1, pCAF2, or CAF1 significantly increased the capacity of cell growth 4 d after treatment. (B) Measurement of cell viability using Celltiter-Glo in the same settings as in A 4 d after treatment. (C) Measurement of colony diameter of iKPC1 in Matrigel culturing assay in the same settings as in A 7 d after treatment. (D) Luciferase image of mice injected with 1 × 106 iKPC1-luc cells alone, iKPC2-luc cells alone, or iKPC1-luc or iKPC2-luc cells together with 1 × 106 pCAF1 or CAF1 cells 7 d after injection. Coinjection of iKPC and CAFs sig- nificantly increased KRAS∗-independent growth. (E) Quantification of luciferase signal from D. (F) Histology (HE, trichome, GFP, and KRASG12D staining) of tissue mass from iKPC + CAF1 injection from D. (G) Measurement of colony diameter of iKPC1 in Matrigel cul- turing assay 7 d after treatment with the indicated condition and treatment. (DMSO) Dox on with DMSO, (MRTX) dox on with 100 nM MRTX1133, (Kras∗off) dox off with DMSO, (CM) the presence of 50% CAF-CM in culture, (CM-HI) the presence of 50% heat-inactivated CAF-CM in culture. (H) Summary of the strategy for isolating CAF-secreted protein (CAF-pro). (I) Experimental settings and Western blot analysis of iKPC1 cells at the indicated time points after adding CAF-pro. (J) RTK array analysis of two iKPC cell lines after 5 min of CAF- pro or heat-inactivated CAF-pro (HI-CAF-pro) treatment. (K) Quantification of J. (L) Confirming ERBB2, ERBB3, and AKT phosphorylation in four different iKPC cell lines. Data are represented as mean ± SD for B, C, and G, and mean only for E. For B, C, E, and G, Student’s t-test was performed to calculate statistical values.

Article Snippet: Plasmid construction and gene knockdown, knockout, and overexpression LentiORFs were purchased from Origene for mouse Erbb2 (NM_001003817, MR227307), mouse Erbb3 (NM_010153, MR227559), mouse Nrg1 (NM_178591, MR226911L3), and human NRG1 (NM_013956, RC220134).

Techniques: Staining, Colony Assay, Luciferase, Injection, Western Blot, Phospho-proteomics

Journal: Genes & development

Article Title: Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas cancer.

doi: 10.1101/gad.351037.123

Figure Lengend Snippet: Figure 2. Paracrine Nrg1–Erbb2/3 signaling activation confers KRAS∗resistance. (A) Western blot of NRG1 protein levels in various cell lines. (B) Multiplexed RNAscope immunofluorescence staining of iKPC tumor with NRG1-specific RNA probe (white), GFP (green), PDPN (red), and DAPI (blue). (C) qPCR results of Nrg1 on flow cytometry-sorted GFP+ PDAC cells or PDPN+ CAFs from iKPC GEM tu- mors. Two independent tumors were pooled together for dissociation. (D) Measurement of colony diameter of iKPC1 in 3D Matrigel cul- turing assay with the indicated treatment for 7 d. (on) Kras∗on with dox, (off) Kras∗off without dox, (MRTX) 100 nM MRTX1133 treatment, (CAF) the presence of CAF1-pro in culture, (NRG1) the presence of 10 ng/mL rmNRG1 in culture. Data are represented as mean ± SEM; Student’s t-test. (E) Western blot of the indicated iKPC1 knockout clones Kras∗off for 48 h and serum-starved for 2 h prior to 5-min stimulation of the indicated treatments (CAF1-pro or 10 ng/mL rmNRG1). (F) Western blot of the indicated iKPC1 ERBB3 knock- out or rescued clones K Kras∗off for 48 h and serum-starved for 2 h prior to 5-min stimulation of the indicated treatments (CAF1-pro or 10 ng/mL rmNRG1). (G) Western blot of iKPC1 cells with 48 h of K Kras∗off and 2 h of serum starvation prior to 5-min stimulation of the indicated treatments (CAF1-pro cells were preincubated with 100 μg/mL mouse IgG or the indicated concentration of NRG1 neutraliza- tion antibody YW538.24.71 for 1 h at 37°C). (H) Western blot of AsPC1, DMSO, or 100 nM MRTX1133 treatment for 24 h and serum star- vation for 2 h with the presence of 10 μg/mL control human IgG or 10 μg/mL pertuzumab prior to 5-min stimulation of the indicated treatments (CAF-pro, 10 ng/mL rmNRG1, or CAF-sgNrg1-pro). (I) Cell viability assay with AsPC1 with the indicated treatment for 4 d. (MRTX) Treated with 100 nM MRTX1133 (with pCAF1-pro, CAF1-pro, or 10 ng/mL rmNRG1), (mIgG + hIgG) treated with 10 μg/ mL control mouse IgG and 10 μg/mL control human IgG, (anti-NRG1) the presence of 10 μg/mL YW538.24.71, (pertuzumab) the presence of 10 μg/mL pertuzumab. Data are represented as mean ± SD; two-way ANOVA. (J) Luciferase assay of iKPC1-luc cells coinjected with pCAF3 or CAF1. One day after cell implantation, luciferase signal was measured to acquire the initial value of luciferase activity, and IgG or YW538.24.71 was given at a dose of 25 mg/kg. Luciferase signal was acquired again at 7 d after implantation. (K) Quantification of luciferase signal from J. Paired multiple t-test at day 7.

Article Snippet: Plasmid construction and gene knockdown, knockout, and overexpression LentiORFs were purchased from Origene for mouse Erbb2 (NM_001003817, MR227307), mouse Erbb3 (NM_010153, MR227559), mouse Nrg1 (NM_178591, MR226911L3), and human NRG1 (NM_013956, RC220134).

Techniques: Activation Assay, Western Blot, RNAscope, Immunofluorescence, Staining, Flow Cytometry, Knock-Out, Clone Assay, Concentration Assay, Control, Viability Assay, Luciferase, Activity Assay