Genome-wide functional screen identifies a compendium of genes affecting sensitivity to tamoxifen.

Therapies that target estrogen signaling have made a very considerable contribution to reducing mortality from breast cancer. However, resistance to tamoxifen remains a major clinical problem. Here we have used a genome-wide functional profiling approach to identify multiple genes that confer resistance or sensitivity to tamoxifen. Combining whole-genome shRNA screening with massively parallel sequencing, we have profiled the impact of more than 56,670 RNA interference reagents targeting 16,487 genes on the cellular response to tamoxifen. This screen, along with subsequent validation experiments, identifies a compendium of genes whose silencing causes tamoxifen resistance (including BAP1, CLPP, GPRC5D, NAE1, NF1, NIPBL, NSD1, RAD21, RARG, SMC3, and UBA3) and also a set of genes whose silencing causes sensitivity to this endocrine agent (C10orf72, C15orf55/NUT, EDF1, ING5, KRAS, NOC3L, PPP1R15B, RRAS2, TMPRSS2, and TPM4). Multiple individual genes, including NF1, a regulator of RAS signaling, also correlate with clinical outcome after tamoxifen treatment.

Functional screening reveals HORMAD1-driven gene dependencies associated with translesion synthesis and replication stress tolerance.

HORMAD1 expression is usually restricted to germline cells, but it becomes mis-expressed in epithelial cells in ~60% of triple-negative breast cancers (TNBCs), where it is associated with elevated genomic instability (1). HORMAD1 expression in TNBC is bimodal with HORMAD1-positive TNBC representing a biologically distinct disease group. Identification of HORMAD1-driven genetic dependencies may uncover novel therapies for this disease group. To study HORMAD1-driven genetic dependencies, we generated a SUM159 cell line model with doxycycline-inducible HORMAD1 that replicated genomic instability phenotypes seen in HORMAD1-positive TNBC (1). Using small interfering RNA screens, we identified candidate genes whose depletion selectively inhibited the cellular growth of HORMAD1-expressing cells. We validated five genes (ATR, BRIP1, POLH, TDP1 and XRCC1), depletion of which led to reduced cellular growth or clonogenic survival in cells expressing HORMAD1. In addition to the translesion synthesis (TLS) polymerase POLH, we identified a HORMAD1-driven dependency upon additional TLS polymerases, namely POLK, REV1, REV3L and REV7. Our data confirms that out-of-context somatic expression of HORMAD1 can lead to genomic instability and reveals that HORMAD1 expression induces dependencies upon replication stress tolerance pathways, such as translesion synthesis. Our data also suggest that HORMAD1 expression could be a patient selection biomarker for agents targeting replication stress.

Synthetic lethal targeting of PTEN-deficient cancer cells using selective disruption of polynucleotide kinase/phosphatase.

A recent screen of 6,961 siRNAs to discover possible synthetic lethal partners of the DNA repair protein polynucleotide kinase/phosphatase (PNKP) led to the identification of the potent tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Here, we have confirmed the PNKP/PTEN synthetic lethal partnership in a variety of different cell lines including the PC3 prostate cancer cell line, which is naturally deficient in PTEN. We provide evidence that codepletion of PTEN and PNKP induces apoptosis. In HCT116 colon cancer cells, the loss of PTEN is accompanied by an increased background level of DNA double-strand breaks, which accumulate in the presence of an inhibitor of PNKP DNA 3'-phosphatase activity. Complementation of PC3 cells with several well-characterized mutated PTEN cDNAs indicated that the critical function of PTEN required to prevent toxicity induced by an inhibitor of PNKP is most likely associated with its cytoplasmic lipid phosphatase activity. Finally, we show that modest inhibition of PNKP in a PTEN knockout background enhances cellular radiosensitivity, suggesting that such a "synthetic sickness" approach involving the combination of PNKP inhibition with radiotherapy may be applicable to PTEN-deficient tumors.

NLK is a novel therapeutic target for PTEN deficient tumour cells.

PTEN (Phosphatase and tensin homolog) is a tumour suppressor gene commonly defective in human cancer, and is thus a potentially important therapeutic target. Targeting tumour suppressor loss-of-function is possible by exploiting the genetic concept of synthetic lethality (SL). By combining the use of isogenic models of PTEN deficiency with high-throughput RNA interference (RNAi) screening, we have identified Nemo-Like Kinase (NLK) inhibition as being synthetically lethal with PTEN deficiency. This SL is likely mediated by the transcription factor FOXO1 (Forkhead box O1), an NLK substrate, as the selectivity of NLK gene silencing for PTEN deficient cells can be reversed by FOXO1 knockdown. In addition, we provide evidence that PTEN defective cells targeted by NLK gene depletion undergo senescence, suggesting that NLK function is critical for the continued proliferation of PTEN deficient cells. Taken together, these data provide new insight into the potential of targeting of NLK to treat a range of tumourigenic conditions characterised by PTEN deficiency.

High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing.

RNA interference (RNAi) screening is a state-of-the-art technology that enables the dissection of biological processes and disease-related phenotypes. The commercial availability of genome-wide, short hairpin RNA (shRNA) libraries has fueled interest in this area but the generation and analysis of these complex data remain a challenge. Here, we describe complete experimental protocols and novel open source computational methodologies, shALIGN and shRNAseq, that allow RNAi screens to be rapidly deconvoluted using next generation sequencing. Our computational pipeline offers efficient screen analysis and the flexibility and scalability to quickly incorporate future developments in shRNA library technology.

Functional viability profiles of breast cancer.

UNLABELLED: The design of targeted therapeutic strategies for cancer has largely been driven by the identification of tumor-specific genetic changes. However, the large number of genetic alterations present in tumor cells means that it is difficult to discriminate between genes that are critical for maintaining the disease state and those that are merely coincidental. Even when critical genes can be identified, directly targeting these is often challenging, meaning that alternative strategies such as exploiting synthetic lethality may be beneficial. To address these issues, we have carried out a functional genetic screen in >30 commonly used models of breast cancer to identify genes critical to the growth of specific breast cancer subtypes. In particular, we describe potential new therapeutic targets for PTEN-mutated cancers and for estrogen receptor-positive breast cancers. We also show that large-scale functional profiling allows the classification of breast cancers into subgroups distinct from established subtypes. SIGNIFICANCE: Despite the wealth of molecular profiling data that describe breast tumors and breast tumor cell models, our understanding of the fundamental genetic dependencies in this disease is relatively poor. Using high-throughput RNA interference screening of a series of pharmacologically tractable genes, we have generated comprehensive functional viability profiles for a wide panel of commonly used breast tumor cell models. Analysis of these profiles identifies a series of novel genetic dependencies, including that of PTEN-null breast tumor cells upon mitotic checkpoint kinases, and provides a framework upon which additional dependencies and candidate therapeutic targets may be identified.

PTEN deficiency in endometrioid endometrial adenocarcinomas predicts sensitivity to PARP inhibitors.

PTEN (phosphatase and tensin homolog) loss of function is the most common genetic aberration in endometrioid endometrial carcinomas. In addition to its well-described role in cell signaling, PTEN is involved in the maintenance of genomic stability. Loss of PTEN function causes defects in repair of DNA double-strand breaks by homologous recombination and, therefore, sensitizes cells to inhibition of the poly(adenosine diphosphate ribose) polymerase (PARP). Here, we determined the PTEN status of eight endometrioid endometrial carcinoma cell lines and correlated it with in vitro sensitivity to the PARP inhibitor KU0058948. PTEN-deficient cells showed a significantly greater sensitivity to KU0058948 than the two endometrioid endometrial carcinoma cell lines with wild-type PTEN. The cell lines lacking PTEN expression were unable to elicit a homologous recombination damage response as assayed by RAD51 focus function (a marker of competent homologous recombination DNA repair) upon irradiation and treatment with PARP inhibitors. PTEN silencing in PTEN wild-type Hec-1b cells resulted in reduced RAD51 foci formation after DNA damage and increased sensitivity to PARP inhibition. PTEN reexpression in PTEN-null cell lines resulted in enhanced RAD51 foci formation and in relative resistance to KU0058948. Given that up to 80% of endometrioid endometrial cancers lack PTEN expression, our results suggest that PARP inhibitors may be therapeutically useful for a subset of endometrioid endometrial cancers.

Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors.

The tumour suppressor gene, phosphatase and tensin homolog (PTEN), is one of the most commonly mutated genes in human cancers. Recent evidence suggests that PTEN is important for the maintenance of genome stability. Here, we show that PTEN deficiency causes a homologous recombination (HR) defect in human tumour cells. The HR deficiency caused by PTEN deficiency, sensitizes tumour cells to potent inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), both in vitro and in vivo. PARP inhibitors are now showing considerable promise in the clinic, specifically in patients with mutations in either of the breast cancer susceptibility genes BRCA1 or BRCA2. The data we present here now suggests that the clinical assessment of PARP inhibitors should be extended beyond those with BRCA mutations to a larger group of patients with PTEN mutant tumours.