Journal: Cancer discovery

Article Title: Discovery of biomarkers predictive of GSI response in triple negative breast cancer and adenoid cystic carcinoma

doi: 10.1158/2159-8290.CD-13-0830

Figure Lengend Snippet: (A) Summary of overlapping direct Notch1 target genes in the T-ALL cell line CUTLL1, the mantle cell lymphoma cell line REC-1, and the triple negative breast cancer cell line HCC1599. (B) Chromatin landscapes near HES4 in CUTLL1 cells. A gamma-secretase inhibitor-sensitive RBPJ/NOTCH1 binding site is present in the HES4 promoter. (C) NOTCH1/RBPJ complexes associate with the HES4 promoter site in REC-1 cells. Local ChIP for NOTCH1 and RBPJ was performed under steady state conditions (DMSO), in cells treated with the GSI compound E (1µM) for 72hr (the Notch-off state), and in cells treated for 72hr with GSI followed by 4hr of recovery following GSI washout (w4h). (D) HES4 expression in HCC1599 cells is Notch dependent. RT-PCR with HES4 specific primers was performed under steady state conditions (DMSO), in cells treated with the GSI compound E (1µM) for 72hr, and in cells treated for 72hr with GSI followed by 4hr of recovery following GSI washout (w4h).

Article Snippet: Xenograft models From 1–8×10 6 HCC1599, HCC1187, MB157, or MDA-MD-231 triple negative breast cancer cells were inoculated subcutaneously into the left flank of 4–6 week-old immunodeficient (nu/nu or NOD-SCID) female mice (Charles River Laboratories).

Techniques: Binding Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer discovery

Article Title: Discovery of biomarkers predictive of GSI response in triple negative breast cancer and adenoid cystic carcinoma

doi: 10.1158/2159-8290.CD-13-0830

Figure Lengend Snippet: (A) Western blot analysis on lysates prepared from cell lines with or without NOTCH1 gene rearrangement following treatment with MRK-003 or vehicle (DMSO) for 24hr. Primary antibodies used were specific for activated NOTCH1 (N1-ICD), pRb, MYC, p21, p-ERK, p-PRAS40 and β-actin (loading control). (B) Cell cycle analysis performed following treatment of NOTCH1-rearranged MB157 cells and HCC1143 cells with wild type NOTCH1 alleles with 1µM MRK-003 for 72hr. (C) HCC1599 cells harboring a NOTCH1 rearrangement and HCC1187 cells harboring a NOTCH2 rearrangement were treated with increasing concentrations of MRK-003 for 24hr. Western blots were stained with antibodies specific for N1-ICD, cleaved PARP (c-PARP), p21, and total AKT (loading control). (D) Induction of senescence. β-galactosidase staining was performed after once weekly treatment of MB157 cells with 1uM MRK-003 or DMSO (vehicle) for four weeks. Over 95% of cells treated with MRK-003 were positive for β-galactosidase, versus 1% of the cells exposed to DMSO.

Article Snippet: Xenograft models From 1–8×10 6 HCC1599, HCC1187, MB157, or MDA-MD-231 triple negative breast cancer cells were inoculated subcutaneously into the left flank of 4–6 week-old immunodeficient (nu/nu or NOD-SCID) female mice (Charles River Laboratories).

Techniques: Western Blot, Control, Cell Cycle Assay, Staining

Journal: Cancer discovery

Article Title: Discovery of biomarkers predictive of GSI response in triple negative breast cancer and adenoid cystic carcinoma

doi: 10.1158/2159-8290.CD-13-0830

Figure Lengend Snippet: Summary of anti-tumor activity of MRK-003 alone or in combination with Paclitaxel in TNBC xenograft models.

Article Snippet: Xenograft models From 1–8×10 6 HCC1599, HCC1187, MB157, or MDA-MD-231 triple negative breast cancer cells were inoculated subcutaneously into the left flank of 4–6 week-old immunodeficient (nu/nu or NOD-SCID) female mice (Charles River Laboratories).

Techniques: Activity Assay

Journal: Cancer discovery

Article Title: Discovery of biomarkers predictive of GSI response in triple negative breast cancer and adenoid cystic carcinoma

doi: 10.1158/2159-8290.CD-13-0830

Figure Lengend Snippet: (A, B) Xenograft models of HCC1599 and HCC1187 cells were treated with 150mg/kg or 300mg/kg MRK-003 once a week, vehicle control, or 15mg/kg paclitaxel as indicated. A summary of tumor growth inhibition (TGI) is presented in Table 1. MRK-003 treatment at both doses (150mg/kg and 300mg/kg) resulted in significant tumor growth inhibition (p<0.001). (C) qRT-PCR analysis of tumor tissues from HCC1599 xenografts treated with 150mg/kg, 300mg/kg MRK-003 or vehicle control showing effects of Notch inhibition on a 9 gene signature 6hr after dosing. HES and HEY family members were significantly down-regulated together with MYC, NRARP and SHQ1, whereas DTX1 (which does not score as a target gene in breast cancer cells) was up-regulated. (D) Immunohistochemistry of formalin fixed paraffin embedded tumor sections from HCC1599 xenografts treated with 300mg/kg MRK-003 or vehicle control with a N1-ICD specific antibody, showing decreased nuclear levels in the MRK-003 treated mice. Tissue was harvested 6hr after dosing. (E, F) MB-157 and HBCx-14 xenograft models, treated with 150mg/kg or 300mg/kg MRK-003 by oral gavage once a week, vehicle control (methylcellulose) or 15mg/kg paclitaxel, alone or in combination with 300mg/kg MRK-003. The MB-157 model was treated for 70 days, while the HBCx-14 model was treated for 28 days. MRK-003 alone or in combination with Paclitaxel resulted in significant tumor growth inhibition (p<0.001) in the MB-157 model. In HBCx-14 model, only combination therapy with MRK-003 and paclitaxel was effective (p<0.001).

Article Snippet: Xenograft models From 1–8×10 6 HCC1599, HCC1187, MB157, or MDA-MD-231 triple negative breast cancer cells were inoculated subcutaneously into the left flank of 4–6 week-old immunodeficient (nu/nu or NOD-SCID) female mice (Charles River Laboratories).

Techniques: Control, Inhibition, Quantitative RT-PCR, Immunohistochemistry, Formalin-fixed Paraffin-Embedded