MTB/TICNX vs. TICNX Cell lines with A TICNX MTB/TICNX B N1 gene fusions 15 2.0 vs. wild-type ** * *** 1.5 10 1.0 237072 748 5 0.5 Relative expression 0 0.0 NICD1 Hey1 Hey2 C D 2.5 TICNX *** 2.0 P < 0.0001 MTB/TICNX P < 0.0001 2.0 trend 1.5 1.5 *** 1.0 1.0 0.5 0.5 Estimated Notch activity 0 Estimated Notch activity 0 48h + doxycycline 96h + doxycycline ControlNOTCH1 L1601P NICD1 Testing data Training data E F 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 Estimated Notch activity P = 0.0087 Estimated Notch activity P < 0.0001 0 0 Wild-type Constitutively active DMSO GSI NOTCH1 status Supplemental Figure 1 Generation and validation of a Notch pathway signature. (A) qRT-PCR for human NICD1 transgene and mouse Hey1 and Hey2 in TICNX and MTB/TICNX mice induced with doxycycline for 4d; n = 4. (B) Venn diagram showing overlap between differentially expressed genes in MTB/TICNX versus TICNX mice (GSE51628) and in human breast cancer cell lines with and without NOTCH1 gene fusions (GSE36133, ref. 31). (C) Estimation of Notch pathway activity in TICNX and MTB/TICNX mammary glands induced with doxycycline for 48h or 96h (GSE51628); n = 4. (D) Estimation of Notch pathway activity in MCF-10A cells transduced with increasingly potent NOTCH1 constructs (GSE20285, ref. 33); n = 4. (E) Estimation of Notch pathway activity in human T-ALL cell lines grouped by Notch1 activation status, wild-type (n = 4) or constitutively active (n =10) (GSE36133, refs. 31, 36, 89). The outlier in the wild-type group corresponds to TALL-1, a cell line that is known to be GSI sensitive (89) and exhibits activated Notch3 signaling (90). (F) Estimation of Notch pathway activity human T-ALL cell lines with activating NOTCH1 mutations treated with vehicle or GSI for 24h (GSE5716, ref. 36); n = 10. Data in A, C and D are shown as the mean ± s.d. Error bars in E represent mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 calculated by two-tailed Student’s t tests in A, C, E and F, one-way ANOVA in D. A B C 0.4 0.20 0.15 P = 2.6e−43 P = 3.8e−21 P = 2.8e−11 0.3 0.15 0.10 0.2 0.10 0.05 0.1 0.05 Relative Notch activity 0 0 0 Normal LumA LumBHER2 Basal G2G1 G3 ER+ ER− Intrinsic molecular subtype Tumor grade ER status D E Estimated Notch pathway activity versus RFS Estimated Notch pathway activity versus RFS adjusted for intrinsic molecular subtype adjusted for tumor grade P = 0.00042 P = 0.0051 F Overall p-value: 0.0051 Estimated Notch pathway activity versus RFS adjustedj for ER status Supplemental Figure 2 Notch pathway activity is independently associated with decreased relapse-free survival in women with breast cancer. (A-C) Association between estimated Notch pathway activity and established prognostic factors in breast cancer, and (D-F) forest plots of association between estimated Notch pathway activity and recurrence-free survival (RFS) in women with breast cancer after adjusting for correlated prognostic factors using multivariate Cox proportional hazards regression: (A and D) intrinsic molecular subtype, (B and E) increasing tumor grade (G1 – G3) and (C and F) estrogen receptor (ER) status. In A-C, P values were calculated by the two-tailed Student’s t test or one-way P = 0.0052 ANOVA. In D-F, the overall hazard ratios and associated confidence intervals are shown for the fixed-effects model. ABEstimated Notch pathway activity versus RFS Estimated Notch pathway activity versus RFS within basal-like tumors within high grade (G3) tumors P = 0.00084 P = 0.00025 C Estimated Notch pathway activity versus RFS within ER− tumors Supplemental Figure 3 Notch pathway activity is associated with decreased relapse-free survival within high-risk subgroups. (A-C) Forest plots of association between estimated Notch pathway activity and recurrence-free survival (RFS) within: (A) basal-like tumors, (B) high grade (G3) tumors, and (C) estrogen receptor (ER) negative tumors. The overall hazard ratios and associated confidence intervals are shown for the fixed-effects model. P = 0.012 A B 2.0 P = 0.015 NS 4 Vehicle GSI *** Ptrend = 0.006 LAP LAP + GSI 1.5 3 *** *** 1.0 2 *** *** NS NS 0.5 *** 1 Relative expression Estimated Notch activity 0 Vehicle 0.1 μM 1 μM 0 Hey1 Hey2 Dll1 Lapatinib Supplemental Figure 4 Notch signaling is activated in HER2+ SKBR3 human breast cancer cells following HER2 targeted therapy. (A) Estimation of Notch pathway activity in SKBR3 cells treated with vehicle or lapatinib for 24h (GSE38376, ref. 38). (B) qRT-PCR analyses of Notch signaling components in SKBR3 cells 48h after treatment with lapatinib alone and in combination with GSI. Data are shown as the mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA followed by the post test for linear trend or Bonferroni’s multiple comparison test; n = 3. NS not statistically significant. A 4h treatment 48h treatment B 4h treatment 48h treatment doxycycline doxycycline doxycycline doxycycline AKTi AKTi AKTi AKTi MEKi+AKTi MEKi+AKTi DMSO MEKi DMSO MEKi DMSO MEKi MEKi+AKTi DMSO MEKi MEKi+AKTi − − − − pErk1/2 HER2/neu pAkt -Tubulin -Tubulin C 48h treatment DMSO -dox MEKi ERKi Supplemental Figure 5 Effects AKT, MEK and ERK inhibition in HER2/neu-dependent primary tumor NICD cells derived from MTB/TAN mice. (A and B) Western blot analysis of (A) pErk1/2 (B) pAkt and HER2/neu, 4h or 48h after doxycycline withdrawal or treatment with MEKi and/or AKTi. (C) Western blot alysis of NICD 48h after doxycycline withdrawal or treatment with MEKi or ERKi. β-tubulin and GAPDH Gapdh are shown as loading controls. Western blot results are representative of 3 different experiments. A pk1 B pk1 100 Hes1 10000 Nrarp 1000 10 100 10 1 1 Relative Hes1 expression 0.1 Relative Nrarp expression 0.1 DMSO GSI DMSO GSI DMSO GSI DMSO GSI +dox −dox +dox −dox C D 1000 MigR1 * 4 0 hours NICD1 24 hours Ptrend = 0.0007 48 hours 100 *** 3 10 2 Ptrend = NS 1 1 Relative expression normalized to MigR1 0.1 Relative Hey1 expression 0 Hey1 Hey2 MigR1 dnMAML Supplemental Figure 6 Experimental manipulation of Notch signaling in primary MTB/TAN tumor cells. (A) qRT-PCR analysis of Hes1 expression in primary MTB/TAN tumor cells transduced with control (pk1Empty) or Hes1 expression constructs. (B) qRT-PCR analysis of Nrarp expression in primary MTB/TAN tumor cells transduced with control (pk1Empty) or Nrarp expression constructs. (C) qRT-PCR for Hey1 and Hey2 expression in primary MTB/TAN tumor cells transduced with control (MigR1) or NICD1 expression constructs. (D) qRT-PCR for Hey1 at 0, 24h and 48h after doxycycline withdrawal in primary MTB/TAN tumor cells transduced with control (MigR1) or dnMAML expression constructs. β-tubulin is shown as a loading control. Data are shown as the mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA followed by Bonferroni multiple comparison test or the post test for linear trend; n = 3. A B 100 15 AdGFP ) 3 AdCre 75 10 50 5 25 Tumor volume (mm Tumor P = NS P = NS 0 to regression (days) Time 0 0 5 10 15 20 25 AdGFP AdCre Days post injection C D 100 Rbpjflox/flox + AdGFP Rbpjflox/flox + AdCre AdGFP AdCre 75 RbpJ 50 -Tub 25 P = NS 0 Recurrence-free survival (%) 020 40 60 80 100 Days post doxycycline withdrawal E F 1.5 flox/flox ) 100 Rbpj + AdGFP 3 Rbpjflox/flox + AdCre 1.0 75 50 0.5 25 Tumor volume (mm Tumor Relative RbpJ protein P = 0.008 p = ns P = NS 0 0 Rbpjflox/flox Rbpjflox/flox 07525 50 +AdGFP +AdCre Days post injection Supplemental Figure 7 Transduction with AdCre effectively excises floxed alleles without altering tumor progression. (A) Representative tumor growth rates for primary MTB/TAN tumor cells transduced with AdGFP (n = 16) or AdCre (n = 18). (B) Time to regression after doxycycline withdrawal for orthotopic tumors generated from primary MTB/TAN tumor cells transduced with AdGFP (n = 8) or AdCre (n = 15). (C) Kaplan-Meier survival curves showing indistinguishable latencies for recurrence-free survival in mice harboring fully regressed orthotopic tumors generated from primary MTB/TAN tumor cells transduced with AdGFP (n = 6) or AdCre (n = 14). (D) Western blot analysis of Rbpj and β-tubulin (control) in primary orthotopic tumors generated from MTB/TAN/Rbpjflox/flox tumor cells infected with AdGFP or AdCre. (E) Quantification of western blot in D showing Rbpj expression normalized to β-tubulin. (F) Tumor growth rates for primary orthotopic tumors generated from an MTB/TAN/Rbpjflox/flox tumor that was digested to yield a single cell suspension and infected with AdGFP (n = 30) or AdCre (n = 26). Error bars in A, B and D-F represent mean ± s.e.m. P values calculated by two-tailed Student’s t test in A, B, E and F, and by the Mantel-Haenszel method in C. NS not statistically significant. A Primary Recurrent AdGFP AdCre B Primary tumors Recurrent tumors AdGFPAdCre AdGFP AdCre Ladder Wild-type H2O Ladder Rbpj Δ 400bp Gapdh 590bp Supplemental Figure 8 Tumor cells with Rbpj deletion are selected against during tumor recurrence. (A) PCR analysis of Gapdh loading control and Rbpjflox/flox excision (Rbpj Δ) in primary and recurrent tumors generated from orthotopic injections of MTB/TAN/Rbpjflox/flox tumor cells infected with AdGFP or AdCre.