Supporting Information

Pfeifer et al. 10.1073/pnas.1305656110 SI Materials and Methods we used the Benjamini and Hochberg method to calculate a false Cell Culture, Retroviral Constructs, and Transductions. dif- discovery rate (FDR) for every α-significance threshold. Gen- fuse large B-cell lymphoma (DLBCL) cell lines were cultured in erally, more were up-regulated (546 genes; P < 0.01 and RPMI (Invitrogen) with 10% (vol/vol) FCS (Sigma), except for FDR < 0.11) than down-regulated (279 genes; P < 0.01 and OCI-Ly1, OCI-Ly2, OCI-Ly4, OCI-Ly7, OCI-Ly10, OCI-Ly19, FDR < 0.11) by PTEN. Using tighter cutoffs, we identified 72 and TMD8, which were cultured in Iscove’s modified Dulbecco genes that were significantly down-regulated (P < 0.0025; FDR < medium supplemented with either 20% human plasma or 10% 0.06) and 82 genes that were up-regulated (P < 0.00025; FDR < 0.02) across all time points after PTEN induction (paired t test FCS. All cell lines were maintained at 37 °C with 5% CO2. For efficient retroviral transductions, cell lines were engineered based on two independent replicates) (Fig. 4B and Table S6). to express the murine ecotropic as previously described To obtain a better understanding of the expression changes, (1). Additionally, these cell lines were engineered to express the we performed an unbiased gene set enrichment analysis (GSEA) as bacterial tetracycline repressor, allowing doxycycline-inducible previously described using a previously curated shRNA or cDNA expression. shRNA-mediated RNA inter- signature database (5, 6). The most enriched gene signatures from > ference was performed as described (1). The targeting sequences this query (enrichment score 0.5) are reported in Table S7. fi of shRNAs 1 and 2 were CGATTCCTTCTAACAGAAATG Additionally, we performed gene expression pro ling of 34 and CCTATGAACTTGTTTCAAATG, respectively. As a negative primary DLBCL patient samples using Affymetrix GeneChip control, we used a previously described shRNA directed against Human 1.0 ST v2 microarrays. Samples from this cohort MSMO1 (2). PTEN (NM_000314.4), MYC (NM_002467.2), and a were later used as the basis for PTEN staining. These microar- constitutive active AKT cDNA (NM_005163.2) (3) were inserted rays were preprocessed with robust multiarray average (RMA) into either a modified version of the inducible pRetroSuper dual background subtraction and quantile normalization. Probes were vector or a noninducible pMSCV vector as previously aggregated by median polish on gene level based on the v14.1 of described (4). PTEN- and mutant PTEN-induced toxicity was the Gene CDF for Affymetrix GeneChip Human Exon assessed as previously described (4). Briefly, PTEN or mutant 1.0 ST v2 (7). fi PTEN cDNA was transduced using retroviruses. The expression TheDLBCLsampleswereclassied into activated B-cell-like vector coexpressed GFP, allowing us to monitor the proportion of (ABC) and GCB DLBCL as previously described (8). We used two + − GFP vs. GFP cells over time as a measure of toxicity of the co- reference samples from a previous cohort (9) to transfer the molec- expressed PTEN cDNA. Rescue experiments were performed as ular subtype prediction to Affymetrix GeneChip Human Exon arrays. previously described (2). Each experiment was completely repro- PCR Amplification and Sequencing. PCR amplification and sequenc- duced at least two to eight times for each cell line. ing were performed as previously described (10). The sequences PTEN Gene Expression Profiling. Gene expression profiling after reex- for primers applied to amplify are summarized in pression of PTEN cDNA was performed in the PTEN-deficient Table S4. germinal center B-cell-like (GCB) DLBCL cell line HT. HT cells Array CGH. Array comparative genomic hybridization (aCGH) in were transduced with PTEN cDNA and selected with puromycin, DLBCL cell lines was performed as previously described (11). and PTEN cDNA expression was induced with doxycycline; 6, 12, aCGH data from 10 DLBCL cell lines were obtained from GEO 18, and 24 h after PTEN induction, total RNA was isolated using through GEO accession no. GSE43272. The aCGH data from the NucleoSpin RNA II Kit (Macherey & Nagel) according to the remaining cell lines have been deposited in the GEO data- the manufacturer’s protocol. RNA was amplified and labeled base (accession no. GSE45495). with the TotalPrep RNA Amplification Kit (Illumina), and la- beled samples were hybridized on HumanHT-12 v4 Expression ’ Quantitative Genomic PCR. To determine the DNA copy number of BeadChips (Illumina) using the manufacturer s protocol. The the PTEN , we used a predesigned assay according to the gene expression data have been deposited in the Gene Expres- manufacturer’s protocol (Invitrogen). sion Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/; accession no. GSE45495). FISH. Primary DLBCL patient samples from cohorts 1 and 2 were PTEN-induced changes in gene expression were measured in investigated for the presence of MYC translocations using FISH two completely independent biologic replicates, and gene ex- as previously described (12, 13). pression changes in PTEN-transduced cells were compared with cells that were transduced with the empty vector alone. The in- Cell Viability Assay, Annexin-V Staining, and SNARF-1 Proliferation dependent measurements were preprocessed and normalized Assay. GCB DLBCL cells were incubated with DMSO or differ- in the following manner. Data were imported on raw bead level, ent concentrations of the pan-PI3K inhibitor Ly294002 (Cayman and subsequently, a bead-level spot filter was applied for each Chemicals). Cell viability was measured after incubation for 4 d microarray based on the fitted density mode for the background using the Cell Proliferation Kit II (Roche) according to the intensities. Afterward, bead intensities of all measured micro- manufacturer’s recommendations. arrays were quantile-normalized, and beads were grouped by Annexin-V PE (BD Pharmingen) staining was performed in the measured sequence to form bead sets. For genes having more GCB DLBCL cell lines BJAB, HT, and K422 2 d after PTEN than one measured sequence, additional merged bead sets were cDNA transduction according to the manufacturer’s protocol. created. Bead sets with more than 50% of their beads excluded Annexin-V staining of GFP-positive cells was measured by flow by the spot filter were also excluded. Additional analyses was cytometry. performed on gene level using median aggregation. SNARF-1 (carboxylic acid, acetate, and succinimidyl ester; Life Differentially expressed genes were identified in the following Technologies) staining was performed in the DLBCL cell lines manner. A one-tailed paired t test was used to calculate P values BJAB, HT, and K422 before PTEN cDNA transduction. SNARF-1 for every gene based on the eight microarray pairs. Additionally, dilutions were measured 2 and 6 d after PTEN transduction.

Pfeifer et al. www.pnas.org/cgi/content/short/1305656110 1of13 SNARF staining was assessed by flow cytometry according to the transferred to polyvinylidene difluoride membranes (Millipore). manufacturer’s protocol. All antibodies used in this study were obtained from Cell Signal- ing, except for PTEN (Santa Cruz), MYC (Epitomics), p-MYC Western Blotting. Western blotting was performed as previously (Abcam), , and α-tubulin (Sigma). described (2). In brief, whole-cell lysates were harvested Western blotting for p-AKT was performed in DLBCL cell from DLBCL cell lines or primary patient samples in Phospho- lines and 16 primary GCB DLBCL samples from cohort 1. In safe lysis buffer (EMD Millipore), and protein was quantified addition, four GCB DLBCL samples with known PTEN sta- using the BCA assay (Thermo Scientific). Subsequently, lysates tus, for which frozen material was available, were analyzed for were separated by SDS/PAGE on 12% polyacryalmide gels and p-AKT levels.

1. Ngo VN, et al. (2006) A loss-of-function RNA interference screen for molecular targets 8. Wright G, et al. (2003) A gene expression-based method to diagnose clinically distinct in cancer. Nature 441(7089):106–110. subgroups of diffuse large B cell lymphoma. Proc Natl Acad Sci USA 100(17): 2. Wenzel SS, et al. (2013) MCL1 is deregulated in subgroups of diffuse large B-cell 9991–9996. lymphoma. Leukemia 27(6):1381–1390. 9. Hummel M, et al. (2006) A biologic definition of Burkitt’s lymphoma from 3. Kharas MG, et al. (2010) Constitutively active AKT depletes hematopoietic stem cells transcriptional and genomic profiling. N Engl J Med 354(23):2419–2430. and induces leukemia in mice. Blood 115(7):1406–1415. 10. Lenz G, et al. (2008) Oncogenic CARD11 mutations in human diffuse large B cell 4. Hailfinger S, et al. (2011) Malt1-dependent RelB cleavage promotes canonical NF- lymphoma. Science 319(5870):1676–1679. kappaB activation in lymphocytes and lymphoma cell lines. Proc Natl Acad Sci USA 108 11. Lenz G, et al. (2008) Molecular subtypes of diffuse large B-cell lymphoma arise by (35):14596–14601. distinct genetic pathways. Proc Natl Acad Sci USA 105(36):13520–13525. 5. Shaffer AL, et al. (2006) A library of gene expression signatures to illuminate normal 12. Obermann EC, Csato M, Dirnhofer S, Tzankov A (2009) Aberrations of the MYC gene and pathological lymphoid biology. Immunol Rev 210:67–85. in unselected cases of diffuse large B-cell lymphoma are rare and unpredictable by 6. Subramanian A, et al. (2005) Gene set enrichment analysis: A knowledge-based morphological or immunohistochemical assessment. J Clin Pathol 62(8):754–756. approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 13. Kwanhian W, et al. (2012) MicroRNA-142 is mutated in about 20% of diffuse large B- 102(43):15545–15550. cell lymphoma. Cancer Med 1(2):141–155. 7. Dai M, et al. (2005) Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data. Nucleic Acids Res 33(20):e175.

A OCI-Ly1 cells K422 cells 3 3

2 PTEN 2 PTEN locus locus 1 1 Log2 Log2 (ratios) 0 (ratios) 0

−1 −1

−2 −2

−3 −3 85 86 87 88 89 90 91 92 93 94 80 82 84 86 88 90 92 94 96 98 position on Base pair position on 10 in megabases chromosome 10 in megabases

B 4.5

4

3.5

3 PTEN GCB DLBCL DNA 2.5 copy ABC DLBCL number 2 1.5

1

0.5

0 HBL-1 TMD8 OCI- BJAB DB HT K422 OCI- OCI- OCI- OCI- OCI- RL SUD Ly10 Ly1 Ly2 Ly4 Ly7 Ly19 HL-10

C Exon 5 Exon 6 Exon 7 Exon 8 Exon 9

HBL-1OCI-Ly1NTC HBL-1OCI-Ly1NTC HBL-1OCI-Ly1NTC HBL-1OCI-Ly1NTC HBL-1OCI-Ly1NTC 700 bp 500 bp 300 bp 200 bp

Fig. S1. PTEN is deregulated in GCB DLBCL. (A) OCI-Ly1 and K422 cells are characterized by heterozygous deletions of the PTEN locus. (B) Analysis of PTEN DNA copy number using quantitative PCR. Each bar represents a DLBCL cell line. (C) OCI-Ly1 cells are characterized by an internal PTEN deletion affecting exons 6–9. NTC, nontemplate control.

Pfeifer et al. www.pnas.org/cgi/content/short/1305656110 2of13 A Wildtype OCI-Ly7 Y68H SUDHL-10 Wildtype OCI-Ly7 D162H SUDHL-10

B Wildtype OCI-Ly7 N276S, T277A RL

C Wildtype OCI-Ly7 L320* OCI-Ly4 T321 Frame shift OCI-Ly4

D Wildtype OCI-Ly7 Splice acceptor mutation Ex 3 K422

E F ΔY177 patient_028 tumor DNA Germ-line DNA patient_028 G209FS patient_019 tumor DNA Germ-line DNA patient_019

G

Fig. S2. PTEN is deregulated by somatic PTEN mutations in GCB DLBCL. (A) SUDHL-10 cells are characterized by two different PTEN missense mutations. Arrows indicate mutations compared with WT sequence detected in OCI-Ly7 cells. (B) In RL cells, two different PTEN missense mutations are detectable. Arrows indicate mutations compared with WT sequence detected in OCI-Ly7 cells. (C) OCI-Ly4 cells are characterized by a nonsense and a frame shift PTEN mutation. Arrows indicate mutations compared with WT sequence detected in OCI-Ly7 cells (depicted are cloned and sequenced PCR products). (D)K422cellsharboraspliceacceptormu- tation. Arrows indicate the mutation compared with WT sequence detected in OCI-Ly7 cells. (E) Three base pair deletion detected in the GCB DLBCL patient sample 028. Arrows indicate the position of the first nucleotide of the deletion compared with WT sequence detected in the patients’ germ-line DNA. (F) Frame shift mutation detected in the ABC DLBCL patient sample 019. Arrows indicate aberration compared with WT sequence detected in the patients’ germ-line DNA. (G)GCB DLBCL patient sample 028, which is characterized by a three base pair PTEN deletion, does not express detectable PTEN protein levels by immunohistochemistry.

Pfeifer et al. www.pnas.org/cgi/content/short/1305656110 3of13 A OCI- B BJAB HT Ly19 BJAB HT

PTEN cDNA ---++ + PTEN cDNA -+-+ PTEN PTEN Caspase 9 FLAG Cleaved caspase 9 Tubulin Caspase 3 Cleaved caspase 3 Tubulin

C RL OCI-Ly19 140 140 120 120 Wildtype 100 100 Live Y68H transduced 80 80 D162H cells (% of 60 60 Y68H/D162H d2 fraction) Δ 40 40 Y177 N276S/T277A 20 20 0 0 d2 d4 d6 d8 d10 d12 d2 d4 d6 d8 d10 d12 Time after transduction Time after transduction

PTEN C124S D 200

PTEN-positive PTEN-deficient 150 Live Cell DLBCL Cell DLBCL transduced line subtype line subtype HBL-1 ABC RL GCB cells (% of 100 OCI-Ly19 GCB SUDHL-10 GCB d2 fraction) OCI-Ly4 GCB OCI-Ly1 GCB 50 K422 GCB HT GCB BJAB GCB 0 d2 d4 d6 d8 d10 d12 Time after transduction

Fig. S3. PTEN-deficient GCB DLBCL cell lines are addicted to PI3K/AKT signaling. (A) PTEN-deficient (BJAB and HT) and PTEN-positive (OCI-Ly19) DLBCL cell lines express similar levels of exogenous PTEN after retroviral transduction. (B) Reexpression of PTEN induces apoptosis in BJAB but not in HT measured by an increase in cleaved caspases 3 and 9 by Western blotting. (C) PTEN mutants induce reduced or no toxicity in the PTEN-deficient GCB DLBCL cell line RL compared with PTEN WT and no toxicity in the PTEN-positive cell line OCI-Ly19. (D) Phosphatase-inactive PTEN mutant C124S does not induce toxicity in DLBCL cell lines.

Pfeifer et al. www.pnas.org/cgi/content/short/1305656110 4of13 A BJAB HT K422 B BJAB HT Ly294002 (5 µM): - + -++ - MYC cDNA: -++ -

MYC MYC

Tubulin Actin

C Cell line: BJAB HT

shRNA: Control MYC MYC Control MYC MYC #1 #2 #1 #2

MYC

Tubulin D MYC shRNA #2 Control shRNA 120 150

Cell PTEN 120 line expression Live 90 transduced OCI-Ly19 + 90 OCI-Ly7 + cells (% of RL - d2 fraction) 60 SUDHL-10 - 60 OCI-Ly4 - OCI-Ly1 - 30 K422 - 30 HT - BJAB -

0 0 d2 d4 d6 d8 d10 d12 d2 d4 d6 d8 d10 d12 Time after transduction Time after transduction E BJAB HT K422 120 120 120

100 100 100 Live transduced 80 80 80 cells (% of d2 fraction) 60 60 60 40 40 40

20 20 20

0 0 0 d2 d4 d6 d8 d10 d12 d2 d4 d6 d8 d10 d12 d2 d4 d6 d8 d10 d12 Time after shRNA transduction Time after shRNA transduction Time after shRNA transduction

MYC shRNA #1 MYC shRNA #2

MYC shRNA #1 + MYC cDNA MYC shRNA #2 + MYC cDNA

F ABC DLBCL GCB DLBCL cell lines cell lines

TMD8 OCI-Ly10 HBL-1 OCI-Ly1 OCI-Ly2 OCI-Ly4 OCI-Ly7 OCI-Ly19 BJAB HT SUDHL-10K422 DB RL

MYC

Tubulin

Fig. S4. PTEN loss up-regulates MYC in GCB DLBCL. (A) PI3K/AKT inhibition using the pan-PI3K inhibitor Ly294002 down-regulates MYC protein expression in PTEN-deficient GCB DLBCL cell lines BJAB, HT, and K422. (B) Exogeneous MYC protein expression in BJAB and HT cells to perform the MYC rescue experiment (Fig. 4F). (C) MYC shRNA 1 and 2 significantly down-regulate MYC protein measured by Western blotting. (D) shRNA-mediated MYC knockdown (MYC shRNA 2) is toxic to PTEN-positive and -deficient GCB DLBCL cell lines. A negative control shRNA does not induce toxicity. (E)ExpressionofanMYC cDNA rescues BJAB, HT, and K422 cells transduced with MYC shRNAs 1 and 2 (targeting the MYC 3′UTR) from toxicity. Representative results from two independent replicates are shown. (F) MYC protein expression in ABC and GCB DLBCL cell lines determined by Western blotting.

Pfeifer et al. www.pnas.org/cgi/content/short/1305656110 5of13 Table S1. PTEN expression in subtypes of DLBCL determined by immunohistochemistry using a cutoff level of <10% of positive lymphoma cells Molecular subtype and patient cohort PTEN positive PTEN negative n

GCB DLBCL cohort 1 9 9 18 Non-GCB DLBCL cohort 1 12 4 16 GCB DLBCL cohort 2 29 42 71 Non-GCB DLBCL cohort 2 88 55 143 GCB DLBCL combined 38 51 89 Non-GCB DLBCL combined* 100 59 159

*P = 0.003.

Table S2. Summary of PTEN genomic analyses in DLBCL cell lines PTEN mutation PTEN protein p-AKT Cell line DLBCL subtype status PTEN DNA copy number PTEN inactivation pattern expression status

HBL-1 ABC WT 2 No aberration detectable ++ OCI-Ly10 ABC WT 2 No aberration detectable ++ TMD8 ABC WT 2 No aberration detectable ++ DB GCB WT 1 Monoallelic inactivation + − OCI-Ly2 GCB WT 2 No aberration detectable + − OCI-Ly7 GCB WT 2 No aberration detectable + − OCI-Ly19 GCB WT 2 No aberration detectable + − OCI-Ly1 GCB ΔExons 6-9 1 exons 1-5, 0 exons 6-9 Biallelic inactivation − + OCI-Ly4 GCB L320*, T321FS 4 Monoallelic inactivation − + BJAB GCB WT 3 No aberration detectable − + HT GCB WT 2 No aberration detectable − + RL GCB N276S, T277A 2 Monoallelic inactivation −− SUDHL-10 GCB Y68H, D162H 1 Biallelic inactivation − + K422 GCB SA Exon3 1 Biallelic inactivation − +

Pfeifer et al. www.pnas.org/cgi/content/short/1305656110 6of13 Table S3. Summary of PTEN genomic analyses in primary DLBCL patient samples of cohort 1 DLBCL PTEN mutation PTEN DNA copy PTEN inactivation PTEN protein p-AKT MYC translocation Patient ID subtype status number pattern expression status status

DLBCL_017 ABC WT 2 No aberration detectable + n.a. Negative DLBCL_019 ABC G209FS 2 Monoallelic inactivation + n.a. Negative DLBCL_041 ABC WT 1 Monoallelic inactivation − n.a. Negative DLBCL_050 ABC WT 2 No aberration detectable + n.a. Negative DLBCL_069 ABC WT 3 No aberration detectable + n.a. Negative DLBCL_073 ABC WT 2 No aberration detectable + n.a. Negative DLBCL_092 ABC WT 2 No aberration detectable + n.a. Negative DLBCL_111 ABC WT 2 No aberration detectable + n.a. Negative DLBCL_016 GCB WT 3 No aberration detectable + − Negative DLBCL_021 GCB WT 3 No aberration detectable − + Negative DLBCL_025 GCB WT 2 No aberration detectable + n.a. Negative DLBCL_028 GCB ΔY177 1 Biallelic inactivation − + Negative DLBCL_034 GCB WT 2 No aberration detectable + − Negative DLBCL_037 GCB WT 2 No aberration detectable + n.a. Negative DLBCL_054 GCB WT 1 Monoallelic inactivation + − Negative DLBCL_058 GCB WT 2 No aberration detectable + − Positive DLBCL_059 GCB WT 2 No aberration detectable ++Negative DLBCL_061 GCB WT 2 No aberration detectable − + Negative DLBCL_071 GCB WT 1 Monoallelic inactivation − + Negative DLBCL_076 GCB WT 2 No aberration detectable − + Negative DLBCL_079 GCB WT 2 No aberration detectable − + Negative DLBCL_085 GCB WT 2 No aberration detectable − + Positive DLBCL_090 GCB WT 3 No aberration detectable ++n.a. DLBCL_104 GCB WT 2 No aberration detectable − + Negative DLBCL_114 GCB WT 2 No aberration detectable − + n.a. DLBCL_099 GCB WT 3 No aberration detectable + − Negative DLBCL_006 Unclassified WT 2 No aberration detectable + n.a. Negative DLBCL_013 Unclassified WT 1 Monoallelic inactivation + n.a. Negative DLBCL_040 Unclassified WT 2 No aberration detectable + n.a. Positive DLBCL_042 Unclassified WT 1 Monoallelic inactivation + n.a. Negative DLBCL_057 Unclassified WT 2 No aberration detectable − n.a. n.a. DLBCL_063 Unclassified WT 1 Monoallelic inactivation − n.a. n.a. DLBCL_081 Unclassified WT 2 No aberration detectable + n.a. Negative DLBCL_107 Unclassified WT 2 No aberration detectable + n.a. Negative

n.a., not available.

Table S4. Sequences of primers used for amplification of PTEN exons Exon Primer name Sequence

1 PTEN_1_f 5′-TTTCCATCCTGCAGAAGAAG-3′ 1 PTEN_1_r 5′-CATTTTCGCATCCGTCTACT-3 2 PTEN_2_f 5′-CTCCAGCTATAGTGGGGAAAACTTTC-3′ 2 PTEN_2_r 5′-CCCCTGAAGTCCATTAGGTACGG-3′ 3 PTEN_3_f 5′-ATATTCTCTGAAAAGCTCTGG-3′ 3 PTEN_3_r 5′-TTAATCGGTTTAGGAATACAA-3′ 4 PTEN_4_f 5′-TTGAAAAAGGTGATCGTTGG-3′ 4 PTEN_4_r 5′-ATTGTTATGACAGTAAGATACAGTCTATCG-3′ 5 PTEN_5_f 5′-GACCTATGCTACCAGTCCGTA-3′ 5 PTEN_5_r 5′-CACCTCAATAAAACTGAAGGAAAAA-3′ 6 PTEN_6_f 5′-AATGTATATATGTTCTTAAATGGCTACGA-3′ 6 PTEN_6_r 5′-GCTTCAGAAATATAGTCTCCTGCAT-3′ 7 PTEN_7_f 5′-CAGATACAGAATCCATATTTCGTG-3′ 7 PTEN_7_r 5′-AAGCAAAACACCTGCAGATCTAATA-3′ 8 PTEN_8_f 5′-AAATGCAACAGATAACTCAGATTG-3′ 8 PTEN_8_r 5′-AGCAAGTTCTTCATCAGCTGTACT-3′ 9 PTEN_9_f 5′-GTTTAAGATGAGTCATATTTG-3′ 9 PTEN_9_r 5′-TGGTGTTTTATCCCTCTTGAT-3′

Pfeifer et al. www.pnas.org/cgi/content/short/1305656110 7of13 Table S5. Patient samples analyzed by Western blotting for p-AKT Sample no. in PTEN protein status by Western blot Patient ID immunohistochemistry DLBCL subtype

1 DLBCL_8862 0 GCB 2 DLBCL_10483 0 GCB 3 DLBCL_5332 0 GCB 4 DLBCL_085 0 GCB 5 DLBCL_104 0 GCB 6 DLBCL_028 0 GCB 7 DLBCL_061 0 GCB 8 DLBCL_071 0 GCB 9 DLBCL_076 0 GCB 10 DLBCL_114 0 GCB 11 DLBCL_021 0 GCB 12 DLBCL_079 0 GCB 13 DLBCL_18143 1 GCB 14 DLBCL_090 1 GCB 15 DLBCL_054 1 GCB 16 DLBCL_058 1 GCB 17 DLBCL_059 1 GCB 18 DLBCL_016 1 GCB 19 DLBCL_034 1 GCB 20 DLBCL_099 1 GCB

Pfeifer et al. www.pnas.org/cgi/content/short/1305656110 8of13 Table S6. Up- and down-regulated genes after PTEN induction Gene Signature name symbol Probe set name Gene ID Gene description

Down-regulated genes AATF ILMN_1703743 26574 Apoptosis antagonizing factor Down-regulated genes ACTL8 ILMN_1704078 81569 Actin-like 8 Down-regulated genes ACTN4 ILMN_1725534 81 Actinin, α4 Down-regulated genes APOL3 ILMN_1756862 80833 Apolipoprotein L, 3 Down-regulated genes BRMS1 ILMN_2398432 25855 Breast cancer metastasis suppressor 1 Down-regulated genes CACTIN 1) ILMN_2350421, 2) ILMN_2262462 58509 Cactin, spliceosome C complex subunit Down-regulated genes CASP1 1) ILMN_2326509, 2) ILMN_2326512 834 Caspase 1, apoptosis-related cysteine peptidase Down-regulated genes CD48 ILMN_2061043 962 CD48 molecule Down-regulated genes CDC42P2 ILMN_3282321 643336 Cell division cycle 42 2 Down-regulated genes CDK18 ILMN_2284222 5129 Cyclin-dependent kinase 18 Down-regulated genes CEACAM1 1) ILMN_2371724, 2) ILMN_1716815, 3) ILMN_1664330 634 Carcinoembryonic antigen- related cell adhesion molecule 1 (biliary glycoprotein) Down-regulated genes CENPN ILMN_1720526 55839 Centromere protein N Down-regulated genes CHN1 1) ILMN_1679638, 2) ILMN_1678493 1123 Chimerin (chimaerin) 1 Down-regulated genes COL5A1 ILMN_1706505 1289 Collagen, type V, α1 Down-regulated genes CORO1C ILMN_1745954 23603 Coronin, actin binding protein, 1C Down-regulated genes DHX37 1) ILMN_2192683, 2) ILMN_1805742 57647 DEAH (Asp-Glu-Ala-His) box polypeptide 37 Down-regulated genes EPSTI1 ILMN_2388547 94240 Epithelial stromal interaction 1 (breast) Down-regulated genes FAM136BP ILMN_3241756 387071 Family with sequence similarity 136, member B, pseudogene Down-regulated genes FAM153C ILMN_1765002 653316 Family with sequence similarity 153, member C, pseudogene Down-regulated genes FAM203B ILMN_3243302 728071 Family with sequence similarity 203, member B Down-regulated genes FAM207BP ILMN_3306019 729535 Family with sequence similarity 207, member B, pseudogene Down-regulated genes FAM53B 1) ILMN_2053490, 2) ILMN_1704571 9679 Family with sequence similarity 53, member B Down-regulated genes FOXD4 ILMN_2173524 2298 Forkhead box D4 Down-regulated genes FYN ILMN_1781207 2534 FYN oncogene related to SRC, FGR, YES Down-regulated genes GCFC1 1) ILMN_1682896, 2) ILMN_2331197, 3) ILMN_1712936 94104 GC-rich sequence DNA-binding factor 1 Down-regulated genes GEMIN4 ILMN_1770206 50628 Gem (nuclear organelle) associated protein 4 Down-regulated genes GPR97 ILMN_1765941 222487 -coupled receptor 97 Down-regulated genes GUSBP3 ILMN_1763628 653188 Glucuronidase, β-pseudogene 3 Down-regulated genes HLA-DRA 1) ILMN_2157441, 2) ILMN_1689655 3122 Major histocompatibility complex, class II, DR alpha Down-regulated genes HOXC13 ILMN_1759676 3229 C13 Down-regulated genes HTR3A ILMN_2371079 3359 5-Hydroxytryptamine (serotonin) receptor 3A, ionotropic Down-regulated genes IFI44 ILMN_1760062 10561 IFN-induced protein 44 Down-regulated genes KRT8P44 ILMN_3182942 100129958 Keratin 8 pseudogene 44 Down-regulated genes LAPTM5 ILMN_1772359 7805 Lysosomal protein transmembrane 5 Down-regulated genes LOC723805 1) ILMN_3293173, 2) ILMN_3205404 723805 IL-like Down-regulated genes LOC90499 ILMN_3223843 90499 Uncharacterized LOC90499 Down-regulated genes MCM7 1) ILMN_1663195, 2) ILMN_1704702 4176 Minichromosome maintenance complex component 7 Down-regulated genes METTL12 ILMN_3239525 751071 Methyltransferase like 12

Pfeifer et al. www.pnas.org/cgi/content/short/1305656110 9of13 Table S6. Cont. Gene Signature name symbol Probe set name Gene ID Gene description

Down-regulated genes MGC27345 ILMN_1660412 157247 Uncharacterized protein MGC27345 Down-regulated genes MT1IP ILMN_2136089 644314 Metallothionein 1I, pseudogene Down-regulated genes MT2A ILMN_1686664 4502 Metallothionein 2A Down-regulated genes MYBPC2 ILMN_1799743 4606 Myosin binding protein C, fast type Down-regulated genes NQO1 ILMN_1720282 1728 NAD(P)H dehydrogenase, quinone 1 Down-regulated genes NXPH4 1) ILMN_2237211, 2) ILMN_1696333, 3) ILMN_1695893 11247 Neurexophilin 4 Down-regulated genes OR2T11 ILMN_1760065 127077 Olfactory receptor, family 2, subfamily T, member 11 Down-regulated genes OSBPL9 ILMN_2313856 114883 Oxysterol binding protein-like 9 Down-regulated genes PAG1 1) ILMN_2055156, 2) ILMN_1736806, 3) ILMN_1673640 55824 Phosphoprotein associated with glycosphingolipid microdomains 1 Down-regulated genes PCDHGC3 ILMN_1675428 5098 Protocadherin-γ subfamily C, 3 Down-regulated genes PIM3 1) ILMN_1789781, 2) ILMN_1707748 415116 Pim-3 oncogene Down-regulated genes PLD6 1) ILMN_3240586, 2) ILMN_1731518 201164 Phospholipase D family, member 6 Down-regulated genes POLR2J3 ILMN_1661516 548644 Polymerase (RNA) II (DNA directed) polypeptide J3 Down-regulated genes POLR3A ILMN_1681837 11128 Polymerase (RNA) III (DNA directed) polypeptide A, 155kDa Down-regulated genes PTAFR ILMN_1746836 5724 Platelet-activating factor receptor Down-regulated genes RGS16 ILMN_1808226 6004 Regulator of G protein signaling 16 Down-regulated genes RGS8 ILMN_1808215 85397 Regulator of G protein signaling 8 Down-regulated genes RNF112 ILMN_1744676 7732 Ring finger protein 112 Down-regulated genes S100A4 1) ILMN_1684306, 2) ILMN_1688780 6275 S100 calcium binding protein A4 Down-regulated genes SCARB1 ILMN_2183409 949 Scavenger receptor class B, member 1 Down-regulated genes SLC25A3 ILMN_1720703 5250 Solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 3 Down-regulated genes SNORD12C ILMN_3249286 26765 Small nucleolar RNA, C/D box 12C Down-regulated genes SNRPD3 ILMN_1794599 6634 Small nuclear ribonucleoprotein D3 polypeptide 18kDa Down-regulated genes TAGLN2 ILMN_1691892 8407 Transgelin 2 Down-regulated genes TMEM202 ILMN_2245523 338949 Transmembrane protein 202 Down-regulated genes TMEM38A ILMN_1765584 79041 Transmembrane protein 38A Down-regulated genes TNF ILMN_1728106 7124 Tumor necrosis factor Down-regulated genes TPM3P9 ILMN_1761801 147804 Tropomyosin 3 pseudogene 9 Down-regulated genes TSR1 ILMN_2092232 55720 TSR1, 20S rRNA accumulation, homolog () Down-regulated genes UNC45A ILMN_1709860 55898 Unc-45 homolog A () Down-regulated genes ZNF3 ILMN_2390739 7551 Zinc finger protein 3 Down-regulated genes ZNF624 ILMN_1670122 57547 Zinc finger protein 624 Down-regulated genes ZNF655 1) ILMN_2396292, 2) ILMN_1728528 79027 Zinc finger protein 655 Down-regulated genes ZRANB2 ILMN_1662383 9406 Zinc finger, RAN-binding domain containing 2 Up-regulated genes ABTB1 1) ILMN_2367165, 2) ILMN_1802096 80325 Ankyrin repeat and BTB (POZ) domain containing 1

Pfeifer et al. www.pnas.org/cgi/content/short/1305656110 10 of 13 Table S6. Cont. Gene Signature name symbol Probe set name Gene ID Gene description

Up-regulated genes ADA ILMN_1803686 100 Adenosine deaminase Up-regulated genes AFF3 ILMN_1775235 3899 AF4/FMR2 family, member 3 Up-regulated genes BACH2 1) ILMN_2058468, 2) ILMN_1670695 60468 BTB and CNC 1, basic 2 Up-regulated genes C15orf52 ILMN_1775330 388115 Chromosome 15 ORF 52 Up-regulated genes C17orf58 1) ILMN_2398926, 2) ILMN_1700515, 3) ILMN_1712985 284018 ORF 58 Up-regulated genes C7orf41 ILMN_1672605 222166 ORF 41 Up-regulated genes CAB39L 1) ILMN_1783598, 2) ILMN_1660815 81617 Calcium binding protein 39-like Up-regulated genes CARD10 ILMN_1743714 29775 Caspase recruitment domain family, member 10 Up-regulated genes CBLN3 ILMN_2053829 643866 Cerebellin 3 precursor Up-regulated genes CCDC41 ILMN_1799113 51134 Coiled-coil domain containing 41 Up-regulated genes CCNG2 1) ILMN_1747244, 2) ILMN_2228732 901 Cyclin G2 Up-regulated genes CD40 1) ILMN_1779257, 2) ILMN_2367818 958 CD40 molecule, TNF receptor superfamily member 5 Up-regulated genes CDKN1B 1) ILMN_2196347, 2) ILMN_1722811 1027 Cyclin-dependent kinase inhibitor 1B (p27, Kip1) Up-regulated genes CDKN2C ILMN_1656415 1031 Cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) Up-regulated genes CORO2A ILMN_1813746 7464 Coronin, actin binding protein, 2A Up-regulated genes CTDSP2 ILMN_1692962 10106 CTD (carboxyl-terminal domain, RNA polymerase II, polypeptide A) small phosphatase 2 Up-regulated genes FAM53A ILMN_1658452 152877 Family with sequence similarity 53, member A Up-regulated genes FBXO32 ILMN_1703955 114907 F-box protein 32 Up-regulated genes FCRL2 ILMN_1791329 79368 Fc receptor-like 2 Up-regulated genes FCRL3 ILMN_1691693 115352 Fc receptor-like 3 Up-regulated genes FZD7 ILMN_1804351 8324 Frizzled family receptor 7 Up-regulated genes GCET2 ILMN_1667449 257144 Germinal center expressed transcript 2 Up-regulated genes HBP1 1) ILMN_1685415, 2) ILMN_2160764 26959 HMG-box transcription factor 1 Up-regulated genes HCG27 ILMN_1746436 253018 HLA complex group 27 (nonprotein coding) Up-regulated genes HES6 ILMN_1694268 55502 Hairy and enhancer of split 6(Drosophila) Up-regulated genes HIST1H2BD ILMN_1651496 3017 Histone cluster 1, H2bd Up-regulated genes HLA-DOB ILMN_1700428 3112 MHC, class II, DO-β Up-regulated genes HOMER2 ILMN_1671486 9455 Homer homolog 2 (Drosophila) Up-regulated genes HPCAL1 ILMN_1764850 3241 Hippocalcin-like 1 Up-regulated genes ID3 ILMN_1732296 3399 Inhibitor of DNA binding 3, dominant negative helix–loop–helix protein Up-regulated genes IFI16 ILMN_1710937 3428 IFN, γ-inducible protein 16 Up-regulated genes IL4R ILMN_1652185 3566 IL-4 receptor Up-regulated genes IRAK2 ILMN_1745964 3656 IL-1 receptor-associated kinase 2 Up-regulated genes ISG20 ILMN_1659913 3669 IFN-stimulated exonuclease gene 20kDa Up-regulated genes KIAA0125 1) ILMN_1707491, 2) ILMN_3187535 9834 KIAA0125 Up-regulated genes KLHL24 ILMN_1678671 54800 Kelch-like 24 (Drosophila) Up-regulated genes KREMEN2 ILMN_2382290 79412 Kringle containing transmembrane protein 2 Up-regulated genes LAMP3 1) ILMN_2170813, 2) ILMN_2170814 27074 Lysosomal-associated membrane protein 3 Up-regulated genes LHFPL2 ILMN_1747744 10184 Lipoma HMGIC fusion partner-like 2

Pfeifer et al. www.pnas.org/cgi/content/short/1305656110 11 of 13 Table S6. Cont. Gene Signature name symbol Probe set name Gene ID Gene description

Up-regulated genes LINC00324 ILMN_1681252 284029 Long intergenic nonprotein coding RNA 324 Up-regulated genes LMTK3 ILMN_1668194 114783 Lemur tyrosine kinase 3 Up-regulated genes LOC339352 ILMN_3289745 339352 Cytosolic thiouridylase subunit 1 homolog (Schizosaccharomyces pombe) pseudogene Up-regulated genes LOC730101 1) ILMN_3305735, 2) ILMN_3229424 730101 Uncharacterized LOC730101 Up-regulated genes LTB 1) ILMN_2376205, 2) ILMN_2376204 4050 Lymphotoxin-β (TNF superfamily, member 3) Up-regulated genes MAP1LC3A ILMN_1776188 84557 Microtubule-associated protein 1 light chain 3α Up-regulated genes METTL7A ILMN_1656285 25840 Methyltransferase-like 7A Up-regulated genes MIAT ILMN_1864900 440823 Myocardial infarction associated transcript (nonprotein coding) Up-regulated genes MID1IP1 1) ILMN_2165473, 2) ILMN_1668960 58526 MID1 interacting protein 1 Up-regulated genes MST1 ILMN_1707464 4485 Macrophage stimulating 1 (hepatocyte growth factor-like) Up-regulated genes MST1P9 ILMN_2099259 11223 Macrophage stimulating 1 (hepatocyte growth factor-like) pseudogene 9 Up-regulated genes MYL2 1) ILMN_1688417, 2) ILMN_2113807 4633 Myosin, light chain 2, regulatory, cardiac, slow Up-regulated genes N4BP2L1 1) ILMN_2344650, 2) ILMN_1799487 90634 NEDD4 binding protein 2-like 1 Up-regulated genes NINJ1 ILMN_1815086 4814 Minjurin 1 Up-regulated genes NLRP7 1) ILMN_1658632, 2) ILMN_1798063, 3) ILMN_1652366 199713 NLR family, pyrin domain containing 7 Up-regulated genes OAS1 1) ILMN_1672606, 2) ILMN_2410826, 3) ILMN_1675640 4938 2’-5′-oligoadenylate synthetase 1, 40/46kDa Up-regulated genes OPRL1 ILMN_2400922 4987 Opiate receptor-like 1 Up-regulated genes PHYH ILMN_1773073 5264 Phytanoyl-CoA 2-hydroxylase Up-regulated genes PIM2 ILMN_1748283 11040 Pim-2 oncogene Up-regulated genes PLTP ILMN_1773389 5360 Phospholipid transfer protein Up-regulated genes PNPLA8 1) ILMN_1680223, 2) ILMN_2180582 50640 Patatin-like phospholipase domain containing 8 Up-regulated genes PRR15L ILMN_1748970 79170 Proline rich 15-like Up-regulated genes RAB26 ILMN_1790317 25837 RAB26, member RAS oncogene family Up-regulated genes RASAL1 ILMN_1793517 8437 RAS protein activator like 1 (GAP1 like) Up-regulated genes RGL4 ILMN_1663422 266747 Ral guanine nucleotide dissociation stimulator-like 4 Up-regulated genes RHBDD1 1) ILMN_1681543, 2) ILMN_2209766 84236 Rhomboid domain containing 1 Up-regulated genes SEC14L1 1) ILMN_1732575, 2) ILMN_2391912, 3) ILMN_2285802 6397 SEC14-like 1 (S. cerevisiae) Up-regulated genes SLC25A29 1) ILMN_2350801, 2) ILMN_1697544 123096 Solute carrier family 25 (mitochondrial carnitine/ acylcarnitine carrier), member 29 Up-regulated genes SLC44A2 ILMN_1771987 57153 Solute carrier family 44, member 2 Up-regulated genes SLC44A3 ILMN_1658498 126969 Solute carrier family 44, member 3 Up-regulated genes SLC6A16 ILMN_1723287 28968 Solute carrier family 6, member 16 Up-regulated genes SNTA1 ILMN_1753241 6640 Syntrophin, α1 Up-regulated genes SPOCK2 ILMN_1656287 9806 Sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 2 Up-regulated genes STMN3 ILMN_3244117 50861 Stathmin-like 3 Up-regulated genes SUSD3 ILMN_1785570 203328 Sushi domain containing 3

Pfeifer et al. www.pnas.org/cgi/content/short/1305656110 12 of 13 Table S6. Cont. Gene Signature name symbol Probe set name Gene ID Gene description

Up-regulated genes TCL1A ILMN_1788841 8115 T-cell leukemia/lymphoma 1A Up-regulated genes TMEM156 ILMN_2095660 80008 Transmembrane protein 156 Up-regulated genes TRAM2 ILMN_1788783 9697 Translocation-associated membrane protein 2 Up-regulated genes TXNIP ILMN_1697448 10628 Thioredoxin interacting protein Up-regulated genes XPNPEP2 ILMN_1743357 7512 X-prolyl aminopeptidase (aminopeptidase P) 2, membrane-bound Up-regulated genes YPEL3 ILMN_1791147 83719 Yippee-like 3 (Drosophila) Up-regulated genes ZNF627 1) ILMN_1708787, 2) ILMN_2197519 199692 Zinc finger protein 627

Table S7. Gene expression signatures that are enriched with down- or up-regulated genes after PTEN induction Enrichment P value lower Signature name Category Defined genes score than (GSEA) FDR (GSEA)

Down-regulated signatures Myc_overexpression_1.5x_up Signaling pathway 88 0.632 0.0010 0.0010 HIF1α_2x_down Transcription factor target 41 0.595 0.0013 0.0088 Tcell_cytokine_induced_IL2_IL7_IL15only Signaling pathway 24 0.563 0.0259 0.0470 Myc_overexpression_2x_up Signaling pathway 36 0.560 0.0020 0.0183 Notch_T-ALL_up_Palomero Signaling pathway 47 0.546 0.0021 0.0147 HIF1α_1.5x_down Transcription factor target 215 0.545 0.0013 0.0012 Myc_RNAi_OCILy3 Transcription factor target 54 0.539 0.0012 0.0084 Notch_T-ALL_up_Sharma Signaling pathway 37 0.536 0.0109 0.0360 Leucine_starve_down Cellular process 178 0.535 0.0010 0.0010 Myeloid_Node1536 Cellular differentiation 19 0.507 0.1021 1.0000 Tcell_cytokine_induced_prolif Signaling pathway 27 0.504 0.0457 0.0637 Glutamine_starve_down Cellular process 313 0.501 0.0010 0.0010 Up-regulated signatures Blood_Module-3.5_Undetermined Cellular differentiation 13 −0.626 0.0168 0.0474 p53_up_Xray Transcription factor target 17 −0.620 0.0229 0.2123 Blood_Module-1.3_B_cells Cellular differentiation 55 −0.591 0.0011 0.0023 GC_B_cell_BLhigh_DLBCLlow Cellular differentiation 36 −0.583 0.0012 0.0211 BCL6_repressed Transcription factor target 19 −0.554 0.0438 0.1555 Myeloma_PR_subgroup_down Cancer differential 50 −0.553 0.0010 0.0411 CNSonly_Node1460 Cellular differentiation 18 −0.542 0.0602 0.1571 B_cell_up_anergy Cellular process 17 −0.542 0.0728 0.1464 Thymic_DP_Tcell_gt_Thymic_progenitor_Tcell Cellular differentiation 65 −0.541 0.0012 0.0234 NFkB_ChIPCHIP_Young_5factors Transcription factor target 15 −0.525 0.0881 0.1238 GC_T_helper_up_Chtanova_and_Kim Cellular differentiation 19 −0.523 0.0986 0.1881 Pan_B_U133plus Cellular differentiation 86 −0.514 0.0012 0.0217 CLL_unmutated_gt_CLL_mutated Cancer differential 25 −0.509 0.0507 0.2735 Muscle_Node1645 Cellular differentiation 24 −0.503 0.0503 0.1265 Regulatory_Tcell_FOXP3+_4x_gt_CD4+Tcell Cellular differentiation 11 −0.501 0.2168 0.2046

Pfeifer et al. www.pnas.org/cgi/content/short/1305656110 13 of 13