Supplementary Figures 1-13 Supplementary Figure 1: Simple mutations and copy number alterations in NOTCH1, NOTCH2 and NOTCH3. Supplementary Figure 2: Simple mutations and copy number alterations in NOTCH4. Supplementary Figure 3: Complex alterations identified by TopNotch Supplementary Figure 4: Intra-NOTCH1 deletion in TCGA-BH-A1FC. Supplementary Figure 5: BRD4-NOTCH3 fusion and PEST domain deletion in TCGA-AN-A0AR. Supplementary Figure 6: Three PEST domain alterations identified in the TCGA breast cancer dataset. Supplementary Figure 7: Notch altered tumors often exhibit increased pathway activity. Supplementary Figure 8: Notch aberrations in pre-clinical GSI sensitive models. Supplementary Figure 9: NOTCH1 aberrations in the HBCx-14 breast cancer PDX model. Supplementary Figure 10: Tumor growth inhibition plots of PF-03084014 in Notch wt models Supplementary Figure 11: Comparison of NICD1 between Notch wt and mutant models. Supplementary Figure 12: Quantitative RT-PCR of HES and HEY target genes in Notch wt and mutant xenograft models Supplementary Figure 13:Quantitative RT-PCR of Notch target genes in Notch wt and mutant xenograft models
ARTICLE ABSTRACT
Purpose: To identify and characterize novel, activating mutations in Notch receptors in breast cancer and to determine response to the gamma secretase inhibitor (GSI) PF-03084014.Experimental Design: We used several computational approaches, including novel algorithms, to analyze next-generation sequencing data and related omic datasets from The Cancer Genome Atlas (TCGA) breast cancer cohort. Patient-derived xenograft (PDX) models were sequenced, and Notch-mutant models were treated with PF-03084014. Gene-expression and functional analyses were performed to study the mechanism of activation through mutation and inhibition by PF-03084014.Results: We identified mutations within and upstream of the PEST domains of NOTCH1, NOTCH2, and NOTCH3 in the TCGA dataset. Mutations occurred via several genetic mechanisms and compromised the function of the PEST domain, a negative regulatory domain commonly mutated in other cancers. Focal amplifications of NOTCH2 and NOTCH3 were also observed, as were heterodimerization or extracellular domain mutations at lower incidence. Mutations and amplifications often activated the Notch pathway as evidenced by increased expression of canonical Notch target genes, and functional mutations were significantly enriched in the triple-negative breast cancer subtype (TNBC). PDX models were also identified that harbored PEST domain mutations, and these models were highly sensitive to PF-03084014.Conclusions: This work suggests that Notch-altered breast cancer constitutes a bona fide oncogenic driver segment with the most common alteration being PEST domain mutations present in multiple Notch receptors. Importantly, functional studies suggest that this newly identified class can be targeted with Notch inhibitors, including GSIs. Clin Cancer Res; 21(6); 1487–96. ©2015 AACR.
