www.sciencesignaling.org/cgi/content/full/7/357/ra121/DC1

Supplementary Materials for

Systematic identification of signaling pathways with potential to confer anticancer drug resistance

Colin A. Martz, Kathleen A. Ottina, Katherine R. Singleton, Jeff S. Jasper,

Suzanne E. Wardell, Ashley Peraza-Penton, Grace R. Anderson, Peter S. Winter, Tim Wang, Holly M. Alley, Lawrence N. Kwong, Zachary A. Cooper, Michael Tetzlaff,

Pei-Ling Chen, Jeffrey C. Rathmell, Keith T. Flaherty, Jennifer A. Wargo, Donald P. McDonnell, David M. Sabatini,* Kris C. Wood*

*Corresponding author. E-mail: sabatini@wi.mit.edu (D.M.S.); kris.wood@duke.edu (K.C.W.)

Published 23 December 2014, Sci. Signal. 7, ra121 (2014) DOI: 10.1126/scisignal.aaa1877

This PDF file includes:

Fig. S1. Schematic of the screen. Fig. S2. Stable transfection of constructs. Fig. S3. Results of a screen in UACC-62 melanoma cells (BRAFV600E) for pathways conferring resistance to MEK1/2 inhibitor AZD6244. Fig. S4. Meta-analysis of screening results across 13 targeted therapies. Fig. S5. Resistance to etoposide mediated by stable expression of non-cleavable caspases in MCF-7 breast cancer cells. Fig. S6. Immunoblotting of BRAFV600 melanoma cells expressing pathway-activating constructs and treated with MAPK pathway inhibitors. Fig. S7. Resistance to targeted and cytotoxic drugs in breast cancer cells treated with a soluble Notch agonist. Fig. S8. Differentiation markers and signaling in MCF-7 breast cancer cells expressing Notch1. Fig. S9. Characterization of dedifferentiation-associated phenotypes in breast cancer cells with activated Notch1. Fig. S10. Analysis of Notch1 pathway members and EMT markers in a mouse model of tamoxifen-resistant breast cancer (TamR). Fig. S11. Analysis of Notch1 pathway members and EMT markers in human breast cancer patients. Fig. S12. Estrogen receptor–driven resistance to MAPK inhibitors. Fig. S13. Notch1 target gene expression in melanoma cells with activated Notch1. Fig. S14. Western blot analysis of differentiation markers and signaling in BRAF-mutant melanoma cells expressing Notch1.

Fig. S15. Characterization of dedifferentiation-associated phenotypes in melanoma cells with activated Notch1. Fig. S16. Notch1 hairpin validation. Fig. S17. Characterization of evolved MAPK inhibitor–resistant, BRAF-mutant melanoma cell lines. Fig. S18. Analysis of patient tumors in cohort 1. Fig. S19. Analysis of patient tumors in cohort 2. Fig. S20. Resistance pathway inhibitors sensitize intrinsically resistant melanoma cells to VX-11E independently of inhibitor effects on cell viability. Fig. S21. Vector maps for vectors used in this study. Legends for tables S1 to S7 Legend for date file S1

Other Supplementary Material for this manuscript includes the following: (available at www.sciencesignaling.org/cgi/content/full/7/357/ra121/DC1)

Table S1 (Microsoft Excel format). List of pathway-activating constructs and controls used in this study. Table S2 (Microsoft Excel format). List of all drugs, drug concentrations, and cell lines screened. Table S3 (Microsoft Excel format). Results of primary screens. Table S4 (Microsoft Excel format). Characterization of cell lines and clonal derivates with evolved resistance to MAPK inhibitors. Table S5 (Microsoft Excel format). Metastatic melanoma patient characteristics (cohorts 1 and 2). Table S6 (Microsoft Excel format). List of attB1/B2 primers used to barcode and amplify constructs by PCR. Table S7 (Microsoft Excel format). Sequences of additional primers used in this study. Data file S1 (Microsoft Word format). Nucleotide sequences.

Figure S1. Schematic of the screen. Schematic depicting design, construction, validation, and screening with the library of pathway-activating constructs used in this study.

Figure S2. Stable transfection of constructs. Relative abundances of all screened constructs in (A) BT-20 cells and (B) MDA-MB-453 cells immediately after infection (t=0) and after 4 weeks of culture (t=f), indicating that all constructs are present and stably persist in pooled cell populations. (continued next page)

Figure S2. (continued) (C) Relative abundances of all screened constructs in T47D cells immediately after infection (t=0) and after 4 weeks of culture (t=f), indicating that all constructs are present and stably persist in pooled cell populations.

Figure S3. Results of a screen in UACC-62 melanoma cells (BRAFV600E) for pathways conferring resistance to MEK1/2 inhibitor AZD-6244. The fold enrichment for each construct, which represents the fractional representation of that construct in the presence of drug normalized to the same quantity in the absence of drug, is shown. Shaded region represents no enrichment. Hit pathways are indicated by arrows.

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Figure S4. Meta-analysis of screening results across 13 targeted therapies. (A) Percentage of screens in which at least one construct for each indicated pathway scored. (B) Percentage of screens in which the indicated number of resistance pathways were identified for a given drug and cell line. Resistance pathways were considered hits when at least one construct activating that pathway yielded an Enrichment Score of >1.5 and scored in at least 2 of 3 drug concentrations screened.

Figure S5. Resistance to etoposide mediated by stable expression of non-cleavable caspases in MCF-7 breast cancer cells. Cells expressing indicated constructs were incubated for four

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Figure S6. Immunoblotting of BRAFV600 melanoma cells expressing pathway-activating constructs and treated with MAPK pathway inhibitors. A375 melanoma cells were treated with a RAF inhibitor (RAFi, P), MEK inhibitor (MEKi, A), or ERK inhibitor (ERKi, V). Blots are representative of two experiments.

Figure S7. Resistance to targeted and cytotoxic drugs in breast cancer cells treated with a soluble Notch agonist. GI50 values for MCF-7 cells treated with the indicated drugs and incubated with either vehicle or the indicated concentrations of a Notch1-activating ligand, the DSL peptide. Data are means ± S.D. from three experiments. ** p < 0.05; *** p < 0.01.

Figure S8. Differentiation markers and signaling in MCF7 breast cancer cells expressing Notch1. (A) Western blotting for mesenchymal differentiation markers in cells expressing luciferase (Luc) or Notch1 ICD (N1ICD). (B) Western blotting for phosphorylated and total AKT in cells expressing indicated cDNAs and treated with vehicle (-) or PI3K–mTOR inhibitor BEZ-235 (+). Blots are representative of three experiments.

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Figure S9. Characterization of dedifferentiation-associated phenotypes in breast cancer cells with activated Notch1. Migration (A), mammosphere formation (B), and apoptosis resistance (C) in T47D cells stably expressing Notch1 ICD, the EMT transcription factor Slug, or luciferase. Images in (A) are representative of three experiments and data in (B and C) are means ± S.D. from three experiments. *p < 0.1, **p < 0.01.

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Figure S10. Analysis of Notch1 pathway members and EMT markers in a mouse model of tamoxifen-resistant breast cancer (TamR). (A) Expression of Notch1 pathway genes in TamR versus parental MCF-7 tumors by qRT-PCR. (continued next page)

Figure S10. (continued) (B) qRT-PCR analysis of the Notch1 pathway in TamR cells. Pathway map was modified using PathVisio.

Figure S11. Analysis of Notch1 pathway members and EMT markers in human breast cancer patients. (A) Metaplot showing the Log2 DMFS hazard ratios for Notch1 pathway members in tumors from patients with luminal B breast cancer (Figure 3, E and F). Indicators are colored according to the expression of the Notch1 high group described in Figure 3E. (B) Correlation plot of Notch1, Notch1 pathway, Notch1 target genes, and dedifferentiation markers in luminal B breast tumors. (continued next page)

Figure S11. (continued) (C) Gene Set Enrichment Analysis (GSEA) results based on the signature of Notch1 coexpressed genes in luminal B breast tumors. The top 20, as ranked by the Nominal Enrichment Score (NES), are plotted as a bar graph and colored according to gene-set size.

Figure S12. Estrogen receptor–driven resistance to MAPK inhibitors. VX-11E GI50 values for BRAF-mutant UACC-62 melanoma cells expressing luciferase, ERα Y537S, or treated with estradiol. O/E, overexpression. Data are means ± S.D. from three experiments.

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Figure S13. Notch1 target gene expression in melanoma cells with activated Notch1. Expression of canonical Notch1 target genes in UACC-62 melanoma cells expressing Notch1 ICD or luciferase measured by qRT-PCR. Data are means ± S.D. from three experiments. ** p< 0.05; *** p < 0.01.

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Figure S14. Western blot analysis of differentiation markers and signaling in BRAF-mutant melanoma cells expressing Notch1. (A) Expression of melanocyte differentiation markers in UACC-62 cells expressing luciferase (Luc) or Notch1 ICD. (B) ERK phosphorylation in cells expressing the indicated cDNAs and treated with PLX4720 (RAF inhibitor; Colo679 cells). Blots are representative of two experiments in each.

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Figure S15. Characterization of dedifferentiation-associated phenotypes in melanoma cells with activated Notch1. (A) Migration, (B) melanosphere formation, and (C) apoptosis resistance in UACC-62 cells stably expressing Notch1 ICD, Slug, or luciferase. Images in (A) are representative of three experiments, and data in (B and C) are means ± S.D. from three experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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Figure S16. Notch1 hairpin validation. Notch1 knockdown by the indicated shRNAs in melanoma cell lines assessed by immunoblotting. Image is representative of two experiments.

Figure S17. Characterization of evolved MAPK inhibitor–resistant, BRAF-mutant melanoma cell lines. (A) Apoptosis induction by VX-11E in Notch1-dependent, and PLX- or AZD-resistant derivatives (+, 1 μM; ++, 3 μM). Data are means ± SD from three experiments. * p < 0.05, ** p < 0.01. (B) Western blot of phosphorylated (P) and total (T) ERK in parental cell lines and Notch1-dependent, MAPK inhibitor (MAPKi)-resistant derivatives. S, cells derived using the slow or step-wise method; F, cells derived using the fast or constant-dose method (Methods). Blots are representative of two experiments. (C) Notch1-dependent and independent clonal derivatives of cell lines in Fig. 4C. (D) Representative clonogenic assays that are used to calculate S Score and dependence. S Score is defined in table S4; S Scores significantly less than 1.0 indicate Notch1-dependent resistance.

Figure S18. Analysis of patient tumors in cohort 1. Expression of Notch1-driven reporter genes (A) and genes encoding Notch1 ligands (B) in relapsed patient tumors (coded 3a, 3b, 8a, or 12) from cohort 1 with evidence of Notch1 activation (normalized to pretreatment expression). (C) Candidate resistance genes queried from tumor cDNA in all tumors from cohort 1. (D) Patient 10 from cohort 1 harbored evidence of coincident Notch1 activation and a second putative resistance mechanism, IGF-1R overexpression (O/E) (34). Rx, treatment.

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Figure S19. Analysis of patient tumors in cohort 2. (A) Expression of HEY2 in relapsed patient tumors (coded 3 and 24) from cohort 2 with evidence of Notch1 activation (normalized to pretreatment transcript abundance). Data are means ± SD from three technical replicate measurements; **p<0.05. (B) Candidate resistance genes queried from tumor cDNA in all tumors from cohort 2.

A B Candidate resistance genes assayed

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Figure S20. Resistance pathway inhibitors sensitize intrinsically resistant melanoma cells to VX-11E independently of inhibitor effects on cell viability. Blue bars (left axis): VX-11E GI50 values in the presence of pathway inhibitors normalized to GI50 in the absence of inhibitors. Sensitizing drug interactions are indicated by GI50 ratios less than 1.0. GI50 ratios represent the ratio of mean GI50 values in the “+” and “-“ inhibitor conditions, each calculated from three experiments. Red boxes (right axis): Cell viability in the presence of pathway inhibitors normalized to viability in the absence of inhibitors. Data are means ± SD from three experiments.

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Figure S21. Vector maps for vectors used in this study. The Gateway-compatible donor vector pDONR223 and destination (expression) vector pcw107 are shown (C-terminal V5 tag not shown).

Table S1. List of pathway-activating constructs and controls used in this study. Thirty-six unique pathway-activating constructs, three ORF controls. Constructs containing a C-terminal V5 tag are indicated with a “+”, whereas those lacking the tag are indicated with a “-”. In some cases, both the V5-tagged and untagged versions of a construct were produced (indicated with “+/-” designation). Table S1 is provided as an Excel file. Table S2. List of all drugs, drug concentrations, and cell lines screened. Totals: 13 drugs, 12 cell lines, 110 screens. Table S2 is provided as an Excel file. Table S3. Results of primary screens. Fold enrichment (drug/vehicle treatment) for each pathway activating construct in each primary screen. Primary screens (columns) are annotated as Cell line_Drug_Drug dose. Table S3 is provided as an Excel file. Table S4. Characterization of cell lines and clonal derivates with evolved resistance to MAPK inhibitors. Six BRAF-mutant melanoma cell lines were evolved to resistance to PLX4720 (PLX), AZD6244 (AZD), or VX-11E (VX) by continuous selection using the slow (S) or fast (F) methods (refer to Methods). Pathway reactivation was assessed by immunoblotting for phospho-ERK and total-ERK in each cell line in the presence of treatment with the indicated MAPK inhibitor. Evolved resistance indicates the GI50 value for the indicated drug in the indicated cell line normalized to the same value in the parental cell line from which it arose. Notch1-dependent sensitization indicates the GI50 value for the indicated drug in the indicated cell line with concurrent Notch1 knockdown normalized to the GI50 of the same drug in the parental cell line. In the Summary column, Complete indicates complete resensitization of resistant derivatives via Notch1 knockdown (to GI50 levels at or below that of the parental cell lines); Partial indicates resensitization to intermediate levels between the parental and resistant derivative lines. S Score reflects the ratio of the area of cell growth in Notch1-knockdown wells normalized to GFP-knockdown wells in the presence of treatment with the indicated drug normalized to the same quantity in the absence of drug treatment (clonogenic growth assay; refer to Methods). S Score of less than 1.0 indicates sensitization by Notch1-knockdown. White boxes under the columns "Notch1-dependent Sensitization" and "S Score" indicate cell lines or clones with evidence of Notch1-dependent resistance while pink boxes indicate cell lines or clones whose resistance is Notch1-independent. Table S4 is provided as an Excel file. Table S5. Metastatic melanoma patient characteristics (cohorts 1 and 2). For each patient, the patient sample identifier, BRAF mutation, drug treatment (Rx), RECIST (Response Evaluation Criteria In Solid Tumors) response category, response (percentage change in size of

target lesions), progression-free survival interval, putative resistance mechanism, and putative Notch1 activation status are provided (34). Table S5 is provided as an Excel file. Table S6. List of attB1/B2 primers used to barcode and amplify constructs by PCR. Gateway recombination sequences are indicated in red text, construct barcodes in black text, spacer sequences in green text, and construct-specific 5' and 3' complementary sequences in blue text. F and R designations in primer names signify "forward" and "reverse". Table S6 is provided as an Excel file. Table S7. Sequences of additional primers used in this study. For primers, blue text indicates P5 and P7 sequences, red text indicates complementary sequences, and green text indicates index barcode sequences (one example is shown). For reads, black text indicates the 4 nucleotide construct barcode sequence, green text indicates the 17 nucleotide spacer sequence, and red text indicates the 6 nucleotide index barcode sequence. Table S7 is provided as an Excel file. Data File S1: Nucleotide sequences. Full nucleotide sequences for all pathway-activating and control ORFs used in this study. Red = Tag sequence; Black = Coding sequence. The data file is provided as an Excel file.