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Figure S1: Hit validation of siRNA screen for determinants of WEE1 inhibitor sensitivity. Figure S2: WEE1 inhibition by MK-1775 induces replication stress without DSBs and is dependent on CDK1/2 activity. Figure S3: Dual inhibition of WEE1 and MYT1 by PD0166285 induces premature mitosis. Figure S4: Increased sensitivity to WEE1 inhibition in FANCM- or BRIP1-depleted cells is caused by replication stress and unscheduled mitosis. Figure S5: Nucleoside supplementation defers the onset of replication stress and unscheduled mitosis triggered by WEE1 inhibition in cells with FA defects. Figure S6: Co-depletion of MRE11, but not MUS81 or SLX4, partially rescued the replication stress and unscheduled mitosis induced by WEE1 inhibition in FANCM- and BRIP1-depleted cells.

Supplier and catalogue number of compounds included in high-throughput drug screen

Lagging chromosomes in E-cadherin defective cell exposed to foretinib leading to failed cytokinesis

PDF file - 139K

Additional compounds included in the isogenic high-throughput drug screen

Supplementary Figure 1 ARID1A mutations in the panel of OCCC tumour cell line models with corresponding protein expression. Supplementary Figure 2 Un-cropped western blots from main and supplementary figures. Supplementary Figure 3 ARID1A selective effects from the high throughput drug screen. Supplementary Figure 4 Inhibitors of the PI3K/mTOR signalling pathway in the panel of OCCC cell lines and ARID1A selectivity. Supplementary Figure 5 Dasatinib is a synthetic lethal drug in ARID1A mutant OCCC tumour cell line models - see also Figure 2. Supplementary Figure 6 Dasatinib sensitivity in ARID1A mutant isogenic HCT116 colorectal tumour cell line model. Supplementary Figure 7 Dasatinib siRNA screen results. Supplementary Figure 8 Apoptosis assay in four OCCC cell line models, see also Figure 4. Supplementary Figure 9 Dasatinib sensitivity in ARID1A mutant OCCC is dependent upon G1/S checkpoint effectors, see also Figure 5. Supplementary Figure 10 Determining the optimal method of dasatinib delivery in vivo.

The cell adhesion glycoprotein E-cadherin (CDH1) is commonly inactivated in breast tumors. Precision medicine approaches that exploit this characteristic are not available. Using perturbation screens in breast tumor cells with CRISPR/Cas9-engineered CDH1 mutations, we identified synthetic lethality between E-cadherin deficiency and inhibition of the tyrosine kinase ROS1. Data from large-scale genetic screens in molecularly diverse breast tumor cell lines established that the E-cadherin/ROS1 synthetic lethality was not only robust in the face of considerable molecular heterogeneity but was also elicited with clinical ROS1 inhibitors, including foretinib and crizotinib. ROS1 inhibitors induced mitotic abnormalities and multinucleation in E-cadherin–defective cells, phenotypes associated with a defect in cytokinesis and aberrant p120 catenin phosphorylation and localization. In vivo, ROS1 inhibitors produced profound antitumor effects in multiple models of E-cadherin–defective breast cancer. These data therefore provide the preclinical rationale for assessing ROS1 inhibitors, such as the licensed drug crizotinib, in appropriately stratified patients.Significance: E-cadherin defects are common in breast cancer but are currently not targeted with a precision medicine approach. Our preclinical data indicate that licensed ROS1 inhibitors, including crizotinib, should be repurposed to target E-cadherin–defective breast cancers, thus providing the rationale for the assessment of these agents in molecularly stratified phase II clinical trials. Cancer Discov; 8(4); 498–515. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371

siRNA library gene list for dasatinib resistance screen, see also figure 5

PDF file - 32K, Paediatric GBM tissue microarray samples.

ARID1A mutation status in OCCC panel determined by whole exome sequencing

Results from the high throughput drug screen in the non-isogenic OCCC cell line panel, see also Figure 1

Supplementary Tables

Compounds used in the high throughput drug screen in the panel of OCCC cell lines

New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G1–S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1A-mutant OCCC. Mol Cancer Ther; 15(7); 1472–84. ©2016 AACR.

Pearson's correlation and Z prime values for isogenic cell lines demonstrating robust reproducibility and dynamic range from the high-throughput drug screen, see also Figure 1

This file contains the legends for Supplementary Figures S1-S6.

WEE1 kinase regulates CDK1 and CDK2 activity to facilitate DNA replication during S-phase and to prevent unscheduled entry into mitosis. WEE1 inhibitors synergize with DNA-damaging agents that arrest cells in S-phase by triggering direct mitotic entry without completing DNA synthesis, resulting in catastrophic chromosome fragmentation and apoptosis. Here, we investigated how WEE1 inhibition could be best exploited for cancer therapy by performing a functional genetic screen to identify novel determinants of sensitivity to WEE1 inhibition. Inhibition of kinases that regulate CDK activity, CHK1 and MYT1, synergized with WEE1 inhibition through both increased replication stress and forced mitotic entry of S-phase cells. Loss of multiple components of the Fanconi anemia (FA) and homologous recombination (HR) pathways, in particular DNA helicases, sensitized to WEE1 inhibition. Silencing of FA/HR genes resulted in excessive replication stress and nucleotide depletion following WEE1 inhibition, which ultimately led to increased unscheduled mitotic entry. Our results suggest that cancers with defects in FA and HR pathways may be targeted by WEE1 inhibition, providing a basis for a novel synthetic lethal strategy for cancers harboring FA/HR defects. Mol Cancer Ther; 14(4); 865–76. ©2015 AACR.

This file contains the Supplementary Materials and Methods.

XLSX file - 33K, Core enrichment of genes between differential expression analysis in primary tumours and ChIP-Seq in paediatric GBM cells.

Supplementary Figures

PDF file - 12611K, Datasets used for gene expression profiling analysis.

PDF file - 495K, Collated supplementary figures. S1 - Expression of histone H3 methylation marks in paediatric glioma and normal cell lines. S2 - Further characterisation of KNS42 cells. S3 - H3K36me3 binding in H3F3A wild-type SF188 paediatric GBM cells. S4 -Spatio-temporal gene expression of H3F3A mutation-specific signatures in the human brain S5-S12 - Validation of differential H3K36me3 binding and expression of developmental transcription factors and genes associated with stem cell maintenance and pluripotency S13 - MYCN expression in G34V isogenic cells

XLSX file - 1310K, Differentially bound ChIP-Seq regions and genes in paediatric GBM cells.

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Press Conference from Functional Viability Profiles of Breast Cancer

Pearson's correlation and Z prime values for OCCC panel demonstrating robust reproducibility and dynamic range from the high-throughput drug screen

Supplementary Figure legends and methods

Figure legends for Supplementary figures and supplementary methods

MS Stats output from dasatinib bead proteomics, see also Figure 3

Supplementary Figure from PBRM1 Deficiency Confers Synthetic Lethality to DNA Repair Inhibitors in Cancer

PDF file - 150K, Supplementary Figure 1: BMN 673 induces DNA damage at lower doses and with higher intensity than olaparib. Supplementary Figure 2: BMN 673 is active in vivo against xenograft models with PTEN deficiency. Supplementary Figure 3: Balb/c nude mice carrying sc MDA-MB-468 xenograft tumors were treated with BMN673 at 0.33 mg/kg, QD or BMN673 at 0.165 mg/kg, BID for 28 consecutive days (Days 15-42 post tumor implantation) and the animals were terminated at Day 64 post tumor implantation.

Supplementary Table from PBRM1 Deficiency Confers Synthetic Lethality to DNA Repair Inhibitors in Cancer

PDF file - 133K, Supplementary Table 1. Inhibition of PARP1 enzymatic activity by chiral isomers of BMN 673. Supplementary Table 2. List of genes that are targeted by the siRNA library. Supplementary Table 3: Drug effect (DE) scores for significant siRNA sensitization genes from siRNA PARPi drug sensitization screens. Supplementary Table 4: siRNA drug sensitization profiles for all PARP inhibitors tested. Supplementary Table 5. Chiral selectivity of anti-tumor activities and PARP inhibition potency. Supplementary Table 6. Metabolic stability of BMN 673 in human, dog and rat liver microsomes.

Purpose: PARP1/2 inhibitors are a class of anticancer agents that target tumor-specific defects in DNA repair. Here, we describe BMN 673, a novel, highly potent PARP1/2 inhibitor with favorable metabolic stability, oral bioavailability, and pharmacokinetic properties.Experimental Design: Potency and selectivity of BMN 673 was determined by biochemical assays. Anticancer activity either as a single-agent or in combination with other antitumor agents was evaluated both in vitro and in xenograft cancer models.Results: BMN 673 is a potent PARP1/2 inhibitor (PARP1 IC50 = 0.57 nmol/L), but it does not inhibit other enzymes that we have tested. BMN 673 exhibits selective antitumor cytotoxicity and elicits DNA repair biomarkers at much lower concentrations than earlier generation PARP1/2 inhibitors (such as olaparib, rucaparib, and veliparib). In vitro, BMN 673 selectively targeted tumor cells with BRCA1, BRCA2, or PTEN gene defects with 20- to more than 200-fold greater potency than existing PARP1/2 inhibitors. BMN 673 is readily orally bioavailable, with more than 40% absolute oral bioavailability in rats when dosed in carboxylmethyl cellulose. Oral administration of BMN 673 elicited remarkable antitumor activity in vivo; xenografted tumors that carry defects in DNA repair due to BRCA mutations or PTEN deficiency were profoundly sensitive to oral BMN 673 treatment at well-tolerated doses in mice. Synergistic or additive antitumor effects were also found when BMN 673 was combined with temozolomide, SN38, or platinum drugs.Conclusion: BMN 673 is currently in early-phase clinical development and represents a promising PARP1/2 inhibitor with potentially advantageous features in its drug class. Clin Cancer Res; 19(18); 5003–15. ©2013 AACR.

Supplementary data from PBRM1 Deficiency Confers Synthetic Lethality to DNA Repair Inhibitors in Cancer

Inactivation of Polybromo 1 (PBRM1), a specific subunit of the PBAF chromatin remodeling complex, occurs frequently in cancer, including 40% of clear cell renal cell carcinomas (ccRCC). To identify novel therapeutic approaches to targeting PBRM1-defective cancers, we used a series of orthogonal functional genomic screens that identified PARP and ATR inhibitors as being synthetic lethal with PBRM1 deficiency. The PBRM1/PARP inhibitor synthetic lethality was recapitulated using several clinical PARP inhibitors in a series of in vitro model systems and in vivo in a xenograft model of ccRCC. In the absence of exogenous DNA damage, PBRM1-defective cells exhibited elevated levels of replication stress, micronuclei, and R-loops. PARP inhibitor exposure exacerbated these phenotypes. Quantitative mass spectrometry revealed that multiple R-loop processing factors were downregulated in PBRM1-defective tumor cells. Exogenous expression of the R-loop resolution enzyme RNase H1 reversed the sensitivity of PBRM1-deficient cells to PARP inhibitors, suggesting that excessive levels of R-loops could be a cause of this synthetic lethality. PARP and ATR inhibitors also induced cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) innate immune signaling in PBRM1-defective tumor cells. Overall, these findings provide the preclinical basis for using PARP inhibitors in PBRM1-defective cancers.Significance:This study demonstrates that PARP and ATR inhibitors are synthetic lethal with the loss of PBRM1, a PBAF-specific subunit, thus providing the rationale for assessing these inhibitors in patients with PBRM1-defective cancer.

Supplementary Figure Legend. Legends for Supplementary Figures S1-S6.

PDF file 32K, Supplementary Table S1 Antibody reagent list used in study

PDF file 58K, Supplementary Table S4. DNA repair involvement of PARP inhibitor sensitisation genes found in the screen. PMID numbers refer to pubmed entries

Small-molecule inhibitors of PARP1/2, such as olaparib, have been proposed to serve as a synthetic lethal therapy for cancers that harbor BRCA1 or BRCA2 mutations. Indeed, in clinical trials, PARP1/2 inhibitors elicit sustained antitumor responses in patients with germline BRCA gene mutations. In hypothesizing that additional genetic determinants might direct use of these drugs, we conducted a genome-wide synthetic lethal screen for candidate olaparib sensitivity genes. In support of this hypothesis, the set of identified genes included known determinants of olaparib sensitivity, such as BRCA1, RAD51, and Fanconi's anemia susceptibility genes. In addition, the set included genes implicated in established networks of DNA repair, DNA cohesion, and chromatin remodeling, none of which were known previously to confer sensitivity to PARP1/2 inhibition. Notably, integration of the list of candidate sensitivity genes with data from tumor DNA sequencing studies identified CDK12 deficiency as a clinically relevant biomarker of PARP1/2 inhibitor sensitivity. In models of high-grade serous ovarian cancer (HGS-OVCa), CDK12 attenuation was sufficient to confer sensitivity to PARP1/2 inhibition, suppression of DNA repair via homologous recombination, and reduced expression of BRCA1. As one of only nine genes known to be significantly mutated in HGS-OVCa, CDK12 has properties that should confirm interest in its use as a biomarker, particularly in ongoing clinical trials of PARP1/2 inhibitors and other agents that trigger replication fork arrest. Cancer Res; 74(1); 287–97. ©2013 AACR.

PDF file 470K, Supplementary Table S3 shRNA constructs with Olaparib Drug Effect Z scores >1.96

Supplementary Figures S1-S6. S1: Sensitivity to IGF�1R inhibitor AZ12253801 is influenced by levels of DVL3 protein but not IGF�1R or INSR S2: Effects of HUNK depletion on IGF signalling S3: Sensitivity to IGF�1R inhibition is enhanced by proximal but not distal WNT pathway inhibition S4: Investigating effects of DVL3 on IGF�induced ERK activation in prostate and breast cancer cells S5: ERK activation induced by DVL inhibition is independent of EGFR and IRS�1, and involves complex formation with adaptor proteins Grb2, DAB2 and SOS S6: DVL3 immunohistochemistry

PDF file 3292K, Supplementary Figure S1. Genome wide shRNA screen information. Supplementary Figure S2. Validation of ATAD5 as a genetic determinant of olaparib response. Supplementary Figure S3. A working model of PARP1/2 inhibitor-induced DNA repair. Supplementary Figure S4. Inhibition of CDK12 sensitises serous ovarian cancer cells to olaparib and cisplatin. Supplementary Figure S5. CDK12 silencing and the impact on expression of DNA repair proteins. Supplementary Figure S6. Targeting of CCNK sensitises serous ovarian cancer cells to olaparib and cisplatin. Supplementary Figure S7. Animal body weights from in vivo study

Supplementary Methods and References. Description of additional methods and procedures used in the study. Also includes supplementary references.

Supplementary Tables S1-S8. S1: Antibodies used for western blotting, immunoprecipitation and immunofluorescence S2: siRNA libraries and individual siRNAs used in this study S3: Sequences of qPCR primers S4: Z' values in primary siRNA screens S5: Candidate resistance mediators tested in second round screens S6: Validation of screen hits S7: DVL3 expression and clinical parameters in breast and prostate cancer S8:DVL3 IPS on tumor tissue from 22 patients recruited to trials of IGF-1R antibodies