www.sciencesignaling.org/cgi/content/full/5/224/rs4/DC1

Supplementary Materials for

MicroSCALE Screening Reveals Genetic Modifiers of Therapeutic Response in Melanoma

Kris C. Wood,* David J. Konieczkowski, Cory M. Johannessen, Jesse S. Boehm, Pablo Tamayo, Olga B. Botvinnik, Jill P. Mesirov, William C. Hahn, David E. Root, Levi A.

Garraway, David M. Sabatini*

*To whom correspondence should be addressed. E-mail: kcwood@alum.mit.edu (K.C.W.); sabatini@wi.mit.edu (D.M.S.)

Published 15 May 2012, Sci. Signal. 5, rs4 (2012)

DOI: 10.1126/scisignal.2002612 This PDF file includes:

Fig. S1. Strategy for restricting cell adhesion to printed features. Fig. S2. Requirement for purification and concentration of lentiviruses before microarray printing. Fig. S3. High-throughput lentivirus concentration using polyelectrolyte complexation. Fig. S4. Puromycin selection of infected cells on MicroSCALE features. Fig. S5. Quantification of cell number on MicroSCALE features using a DNA microarray scanner. Fig. S6. Printing and infections with ORF-expressing lentiviruses containing inserts of varying sizes. Fig. S7. Modifier screening pipeline. Fig. S8. Percentage of the total validated hits identified in a screen with six replicate arrays as a function of the number of replicate (drug-treated) microarrays used. Fig. S9. Sensitivity of A375 and MDA-MB-453 cells to targeted inhibitors. Fig. S10. Confirmation of hits identified in primary screens. Fig. S11. GI50 concentrations for B-RAFV600–mutant melanoma cell lines treated with MAPK inhibitors. Fig. S12. Effect of IKBKB overexpression on sensitivity to inhibitors of the MAPK pathway and the mTOR and PI3K pathway. Fig. S13. Comparison of resistance to PLX4720 conferred by overexpression of IKBKB, exogenous TNFα, or overexpression of C-RAF. Fig. S14. Phosphorylation of RelA and ERK in response to NF-κB pathway manipulations and PLX4720 treatment in SkMel28 cells.

Fig. S15. Effects of TNFα on PLX4720-induced apoptosis and cell cycle arrest in Colo679 cells. Table S1. Examples of cell lines compatible with MicroSCALE screening. Table S2. Examples of cell lines incompatible with MicroSCALE screening. Table S3. Constructs present on Kinome ORF MicroSCALEs. Table S5. Materials requirements and throughput estimates for kinome-scale drug modifier screens in MicroSCALE and 384-well formats.

Other Supplementary Material for this manuscript includes the following: (available at www.sciencesignaling.org/cgi/content/full/5/224/rs4/DC1)

Table S4 (Microsoft Excel format). Results of a PLX4720 modifier screen in A375 cells.

A549 786-0

(A) (B) Strategy: Print lentiviruses along with a cell adhesive

material onto an adhesion resistant substrate

Blue = lentiviruses + cell adhesive material (gelatin) Red = cell resistant substrate (polyacrylamide-coated glass)

(C)

Result: Cell adhesion and infection are restricted only to printed regions of the microarray surface

A549 786-0

Arrays printed on glass

Arrays printed with gelatin on polyacrylamide-coated glass

Fig S1. Strategy for restricting cell adhesion to printed features. (A) GFP-expressing lentiviruses printed on aminosilane-coated glass slides following lentivirus concentration. Arrays were seeded with the indicated cell lines. GFP is visualized. Note that cells adhere uniformly to the entire surface of aminosilane-coated slides, but only those cells that were infected with GFP are visible. Scale bar, 1 mm. (B) Schematic depicting the strategy for localizing cell adhesion, spreading, and infection. (C) Cell adhesion and infection on features printed on polyacrylamide hydrogel-coated glass slides and seeded with the indicated cell lines. Features were printed in two stages, first with gelatin and second with lentiviruses (GFP is visualized; note that cells adhere only to the printed features). Scale bar, 1 mm.

A549

U2OS

HepG2

5 x 108 5 x 107

p24 staining

serum-free

10% serum

(A) (B) Virus titer (IFU/mL)

Fig. S2. Requirement for purification and concentration of lentiviruses before microarray printing. (A) Lentiviral p24 staining of array features printed using concentrated lentiviruses (5 × 108 IFU/mL) suspended in serum-free or 10% serum-containing print buffer prior to printing. Scale bar, 500 µM. (B) GFP expression on microarray spots printed on aminosilane-coated glass slides using lentiviruses at the indicated titers in serum-free print buffer and seeded with the indicated cell lines. Images were obtained 5 days after seeding. Scale bar, 500 µM. (A single microarray feature such as those shown above, printed with concentrated lentivirus at ~108 IFU/mL, typically yields an MOI of 5-10, depending on parameters such as seeding density and pin size.)

Fig. S3. High-throughput lentivirus concentration using polyelectrolyte complexation. (A) Schematic depicting the lentivirus concentration method. (B) Functional recovery of lentiviruses following polyelectrolyte complexation with centrifugation (1150g) for indicated times, aspiration to remove residual media, and resuspension in a smaller volume of printing buffer. (n=3 replicate experiments per condition).

Fig. S4. Puromycin selection of infected cells on MicroSCALE features. Polyacrylamide hydrogel-coated slides were printed with features containing gelatin and either GFP-expressing lentiviruses encoding the puromycin resistance gene (PAC) or no virus. Arrays were seeded with A549 cells and treated with puromycin (or vehicle) on day 1 after seeding. Images of individual features following 1, 3, and 4 days of puromycin selection (or vehicle treatment) are shown. Scale bar, 250 µM.

(A)

(B) Data generated by cell counting Data generated by measuring total staining

intensity (on the same microarray)

Fig. S5. Quantification of cell number on MicroSCALE features using a DNA microarray scanner. (A) Total intensity of the Syto82 DNA stain on individual spots versus the number of cells present on those spots. (B) Growth curves generated by counting the number of cells on individual MicroSCALE spots (as shown in Figure 2A) versus those generated by quantifying the total Syto82 DNA staining intensity on each spot using a DNA microarray scanner (n=4 replicate spots per condition).

Fig. S6. Printing and infections with ORF-expressing lentiviruses containing inserts of varying sizes. (A) Lentiviral ORFs representing the range of sizes found in the kinase collection were produced, concentrated, and printed as microarrays. Arrays were seeded with HBL100 cells and selected with blasticidin for 4 days. Blue: Hoeschst, Red: Phalloidin. Scale bar, 500 µM. (B) Relative number of infected cells per feature following 4 days of blasticidin selection. Relative cell numbers were determined using the background subtracted Hoechst staining intensity on each feature (n=4 replicate spots per condition)

Fig. S7. Modifier screening pipeline. Schematic outlining the MicroSCALE ORF-based functional screening approach used to uncover kinases that drive resistance to targeted therapies in B-RAFV600-mutant melanoma cells.

Fig. S8. Percentage of the total validated hits identified in a screen with six replicate arrays as a function of the number of replicate (drug-treated) microarrays used. Hits were defined as the 8 previously-identified wild-type ORFs and 5 positive control ORFs with activating MAPK pathway mutants (described in Figure 2 and the accompanying text). Hits were considered correctly identified if they scored in the top 10% (62) of all ORFs in the screen, which is consistent with the hit-calling procedure used throughout this study.

Fig. S9. Sensitivity of A375 and MDA-MB-453 cells to targeted inhibitors. A375 cells are BRAFV600E mutant melanoma and MDA-MB-453 cells are Her2 amplified, PIK3CA mutant breast cancer. Cells were seeded in 96-well plates, treated with drugs after 1 day, and assayed for viability after 4 days (n=3 replicate wells per condition).

RAFi (22) MEK1/2i (21)

(B)

(3) (2)

(19)

BEZ-235 (27)

Torin1 (19)

(C)

(A)

ACVR1C AK3

BCL-XL BIRC2 FRK

GALK2 MAPKAPK3

NEK5 NME5

PIK3CG PIP5K3 PRKAB2 PRKACB PRKACG PRKAR1A

PTK2B RPS6KA5

TK1 TRAF2

EPHB4 MAP2K5 MKNK2 PCTK1

PDXK PRKAR2A

SNX16 TP53RK

AXL ERBB2 C334S ERBB2 V777L

FGR FRK HCK

HRAS V12 IKBKB

KRAS V12 LCK

MEK DD MEK1 P124L MEK1 Q56P

PIM1 PRKCE PRKCH PRKCQ TANK TRAF2

MOS RAF1 TBK1

MAP3K14 TTK

Fig. S10. Confirmation of hits identified in primary screens. (A) Approximately 30-40 ORFs scoring in each primary screen were validated in secondary assays in 96-well plate format. Confirmed hits are those yielding viability scores (viability in the presence of drug / viability in the presence of vehicle) greater than MEK1, a negative control. (B) Venn diagram representing the overlap in ORFs scoring in RAF inhibitor and MEK1/2 inhibitor secondary assays. (C) Venn diagram representing the overlap in ORFs scoring in Torin1 and BEZ-235 secondary assays. Names of hit genes in each category are provided.

(A) (B)

(C)

Fig. S11. GI50 concentrations for B-RAFV600–mutant melanoma cell lines treated with MAPK inhibitors. (A) Cells exposed to PLX4720. (B) Cells exposed to AZD-6244. (C) Cells exposed to Vertex 11e.

Fig. S12. Effect of IKBKB overexpression on sensitivity to inhibitors of the MAPK pathway and the mTOR and PI3K pathway. MEK1 overexpression serves as a negative control. Measurements were taken at a single concentration of each drug (GDC-0879 = 1µM, AZD-6244 = 250 nM, Torin-1 = 200 nM, and BEZ-235 = 200 nM; n=3 replicates per condition).

Fig. S13. Comparison of resistance to PLX4720 conferred by overexpression of IKBKB, exogenous TNFα, or overexpression of C-RAF. MEK1 overexpression serves as a neutral control. (A) Fold-change in PLX4720 GI50 following TNFα addition, IKBKB overexpression, or C-RAF overexpression in A375 and UACC62 cell lines (n=3 replicate GI50 curves per condition). (B) Effect of wild-type MEK1 overexpression on PLX4750 GI50 relative to parental cell lines (n=3 replicate GI50 curves per condition).

Fig. S14. Phosphorylation of RelA and ERK in response to NF-κB pathway manipulations and PLX4720 treatment in SkMel28 cells. Three replicate experiments were performed, and representative blots are shown.

Fig. S15. Effects of TNFα on PLX4720-induced apoptosis and cell cycle arrest in Colo679 cells. Three replicate experiments were performed and representative plots are shown.

Table S1. Examples of cell lines compatible with MicroSCALE screening. Cell lines were deemed compatible with MicroSCALE screening if they exhibited good attachment, growth, infection, and drug selection on microarrays.

A549 293T SW480

HeLa Panc1 HCT116

786-0 SW620 HCC827

HepG2 HBL100 A375

U2OS MCF7 DDLS8817

DU145 PC3 LPS141

90-8T MDA-MB-231 SkBr3

U87 MDA-MB-453 UACC812

p53-/- MEF Various human mammary epithelial cell

derivatives

ZR-75-1

Table S2. Examples of cell lines incompatible with MicroSCALE screening.

LS174T Low cell adhesion

T47D Low cell adhesion

BT474 Low cell adhesion

LNCAP Low cell adhesion

HT29 Low infection

Cell line� Reason for failure�

Table S3. Constructs present on Kinome ORF MicroSCALEs. Kinome ORF MicroSCALEs contain 597 previously published human kinase ORFs (32) plus several additional constructs, including mutants of Ras, RAF, and MEK that are relevant to melanoma progression and drug resistance and an assortment of mutant receptor tyrosine kinases and protein kinases and related signaling molecules associated with apoptosis and survival.

GENE GENE ID DESCRIPTION KINASE CLASS

BCL2L1 598 BCL2L1 BCL2-like 1 (BCL-XL) BCL-2 protein family

BIRC2 329 BIRC2 baculoviral IAP repeat containing 2 TRAF-binding protein

BRAFV600E 673 v-raf murine sarcoma viral oncogene homolog B1 protein kinase (S/TK)

CABC1 56997 chaperone, ABC1 activity of bc1 complex like (S. pombe) protein kinase

CHUK 1147 conserved helix-loop-helix ubiquitous kinase protein kinase (S/TK)

eGFP N/A enhanced Green Fluorescent Protein control

EGFR A289V 1956 epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) protein kinase (RTK)

EPHA3 K761N 2042 EPH receptor A3 protein kinase (RTK)

ERBB2 A775_G776insYVMA 2064

v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) protein kinase (RTK)

ERBB2 C334S 2064 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) protein kinase (RTK)

ERBB2 V777L 2064 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) protein kinase (RTK)

ERBB4 H618P 2066 v-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian) protein kinase (RTK)

FGFR3 K650E 2261 fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism) protein kinase (RTK)

HRASV12 3265 v-Ha-ras Harvey rat sarcoma viral oncogene homolog (G12V mutant) Ras GTPase

IKBKB 3551 inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta protein kinase (S/TK)

IKBKG 8517 inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma protein kinase (S/TK)

KRASV12 3845 v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (G12V mutant) Ras GTPase

MEKP124L 5604 mitogen-activated protein kinase kinase 1 (P124L mutant) protein kinase (S/TK)

MEKQ56P 5604 mitogen-activated protein kinase kinase 1 (Q56P mutant) protein kinase (S/TK)

RETG691S 5979 ret proto-oncogene (multiple endocrine neoplasia and medullary thyroid carcinoma 1, Hirschsprung disease) protein kinase (RTK)

RIPK5 25778 receptor interacting protein kinase 5 protein kinase (S/TK)

TANK 10010 TRAF family member-associated NFKB activator TRAF-binding protein

TRAF2 7186 TNF receptor-associated factor 2 TNF-R binding protein

Table S5. Materials requirements and throughput estimates for kinome-scale drug modifier screens in MicroSCALE and 384-well formats. Calculations are based on the assumption of quadruplicate measurements per ORF per condition (drug/vehicle) for 384-well plate screens and sextuplicate measurements per ORF per condition for MicroSCALE screens, both of which are considered standard and yield approximately equivalent data in the drug modifier assays used in this manuscript. Based on current screening protocols and the cost recovery-based pricing structure used in the Broad Institute’s RNAi Platform as a basis for comparison, we conservatively estimate that the maximum throughput of MicroSCALE screens are approximately 25-50 screens / week / scientist and the maximum throughput of analogous 384 well-based screens are 5-10 screens / week / scientist (provided that appropriate fluid handling robotics are available for the latter). The costs of MicroSCALE screens are approximately 15- to 30-fold lower than 384-well plate screens depending on parameters such as the number of replicates used and the choice of detection reagent(s) ($100-200 per screen in MicroSCALE format versus $2900-3600 per screen in 384-well format as of spring 2012).

MicroSCALE 384-well plate

Cells required / screen 0.2-0.4 × 106 2.5-5.0 × 106

Media required / screen (mL) 50-100 750-1000

Drug required / screen

(mL, at screening concentration)

20-40 250-500

Detection reagent required / screen (mL)

(for example, viability stain or antibody)

< 5 50-125

Number of arrays or plates required / screen 4 (arrays) 12 (plates)

Legend to Table S4 provided as an Excel file

Table S4. Results of a PLX4720 modifier screen in A375 cells. PLX4720 was used at 1 µM. Average viability scores of individual ORFs are shown (each normalized to vehicle only treatment). ORFs yielding low infection have been removed. These results are graphically shown in Figure 2E. For details, refer to the Materials and Methods. Bold: Top hits from (32).