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Supplementary Figure 8 (Relates to Figure 6) - SIK2 regulates a cell cycle network and a ER stress network related to cancer.

Supplementary Figure 2 (Relates to Figure 2) - SIK2 knock-down reduces proliferation and viability of LNCaP-Bic, C4-2 and DuCaP cells.

Supplementary Figure 1 (Relates to Figure 2) - Kinase-dead SIK2 and SIK2 knock-down reduce proliferation of LNCaP cells.

Supplementary Figure 7 (Relates to Figure 6) - Validation of microarray hits.

Supplementary Figure Legend

Supplementary Table S1. qRT-PCR primer sequences. When primer sequences were not from published studies, they were designed using Integrated DNA Technologies software.

Supplementary Figure 4 (Relates to Figure 4) - SIK2 localises at the centrosomes of SKOv3 and LNCaP cells.

Supplementary Figure 6 (Relates to Figure 5) - SIK2 regulates CREB1 activity via the phosphorylation of TORC2 and TORC3 and their sequestration in the cytoplasm.

Supplementary Figure 5. Validation of SIK2, TORC1, TORC2 and TORC3 antibodies by Western-Blot.

Supplementary Figure 3 (Relates to Figure 4) - SIK2 knock-down induces a cell cycle arrest in G1 phase and delays mitotic progression of LNCaP cells.

The growing use of neoadjuvant chemotherapy to treat advanced stage high-grade serous ovarian cancer (HGSOC) creates an opportunity to better understand chemotherapy-induced mutational and gene expression changes. Here we performed a cohort study including 34 patients with advanced stage IIIC or IV HGSOC to assess changes in the tumor genome and transcriptome in women receiving neoadjuvant chemotherapy. RNA sequencing and panel DNA sequencing of 596 cancer-related genes was performed on paired formalin-fixed paraffin-embedded specimens collected before and after chemotherapy, and differentially expressed genes (DEG) and copy-number variations (CNV) in pre- and post-chemotherapy samples were identified. Following tissue and sequencing quality control, the final patient cohort consisted of 32 paired DNA and 20 paired RNA samples. Genomic analysis of paired samples did not reveal any recurrent chemotherapy-induced mutations. Gene expression analyses found that most DEGs were upregulated by chemotherapy, primarily in the chemotherapy-resistant specimens. AP-1 transcription factor family genes (FOS, FOSB, FRA-1) were particularly upregulated in chemotherapy-resistant samples. CNV analysis identified recurrent 11q23.1 amplification, which encompasses SIK2. In vitro, combined treatment with AP-1 or SIK2 inhibitors with carboplatin or paclitaxel demonstrated synergistic effects. These data suggest that AP-1 activity and SIK2 copy-number amplification are induced by chemotherapy and may represent mechanisms by which chemotherapy resistance evolves in HGSOC. AP-1 and SIK2 are druggable targets with available small molecule inhibitors and represent potential targets to circumvent chemotherapy resistance. Significance: Genomic and transcriptomic analyses identify increased AP-1 activity and SIK2 copy-number amplifications in resistant ovarian cancer following neoadjuvant chemotherapy, uncovering synergistic effects of AP-1 and SIK2 inhibitors with chemotherapy.

Supplementary Figure 4

Table S1. Cell lines and culture media Table S2. GE Dharmacon siRNA catalogues Table S3. Primers Table S4. siRNA transfection conditions for custom screens Table S5. Antibodies for Western blot and immunohistochemistry Table S6. Paclitaxel sensitivity in 12 ovarian cancer cell lines. Table S7. Paclitaxel sensitivity of two siRNA combination in six ovarian cancer cell lines. Table S8. TMA patient information Figure S1. Effects of single kinase siRNA on microtubule stability. Figure S2. Knockdown efficiency was examined in SKOv3ip cells. Figure S3. Sequential treatment of siIKBKB followed by siSTK39 or siEDN2 followed by siTBK1 increases microtubule stability. Figure S4. Sequential treatment of siIKBKB followed by siSTK39 or siEDN2 followed by siTBK1 increases microtubule stability and increasing microtubule stability by depletion of IKBKB/STK39 or EDN2/TBK1 is dependent upon p38-mdiated phosphorylation MAP4. Figure S5. Increasing paclitaxel sensitivity by depletion of IKBKB and TK39 or EDN2 and TBK1 is dependent upon MAP4. Figure S6. Mice tolerate the siRNA and paclitaxel treatment.

The growing use of neoadjuvant chemotherapy to treat advanced stage high-grade serous ovarian cancer (HGSOC) creates an opportunity to better understand chemotherapy-induced mutational and gene expression changes. Here we performed a cohort study including 34 patients with advanced stage IIIC or IV HGSOC to assess changes in the tumor genome and transcriptome in women receiving neoadjuvant chemotherapy. RNA sequencing and panel DNA sequencing of 596 cancer-related genes was performed on paired formalin-fixed paraffin-embedded specimens collected before and after chemotherapy, and differentially expressed genes (DEG) and copy-number variations (CNV) in pre- and post-chemotherapy samples were identified. Following tissue and sequencing quality control, the final patient cohort consisted of 32 paired DNA and 20 paired RNA samples. Genomic analysis of paired samples did not reveal any recurrent chemotherapy-induced mutations. Gene expression analyses found that most DEGs were upregulated by chemotherapy, primarily in the chemotherapy-resistant specimens. AP-1 transcription factor family genes (FOS, FOSB, FRA-1) were particularly upregulated in chemotherapy-resistant samples. CNV analysis identified recurrent 11q23.1 amplification, which encompasses SIK2. In vitro, combined treatment with AP-1 or SIK2 inhibitors with carboplatin or paclitaxel demonstrated synergistic effects. These data suggest that AP-1 activity and SIK2 copy-number amplification are induced by chemotherapy and may represent mechanisms by which chemotherapy resistance evolves in HGSOC. AP-1 and SIK2 are druggable targets with available small molecule inhibitors and represent potential targets to circumvent chemotherapy resistance.Significance:Genomic and transcriptomic analyses identify increased AP-1 activity and SIK2 copy-number amplifications in resistant ovarian cancer following neoadjuvant chemotherapy, uncovering synergistic effects of AP-1 and SIK2 inhibitors with chemotherapy.

Supplementary Tables

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The extracellular matrix protein TGFBI enhances the cytotoxic response of cancer cells to paclitaxel by affecting integrin signals that stabilize microtubules. Extending the implications of this knowledge, we tested the more general hypothesis that cancer cell signals which increase microtubule stability before exposure to paclitaxel may increase its ability to stabilize microtubules and thereby enhance its cytotoxicity. Toward this end, we carried out an siRNA screen to evaluate how genetic depletion affected microtubule stabilization, cell viability, and apoptosis. High content microscopic analysis was carried out in the absence or presence of paclitaxel. Kinase knockdowns that stabilized microtubules strongly enhanced the effects of paclitaxel treatment. Conversely, kinase knockdowns that enhanced paclitaxel-mediated cytotoxicity sensitized cells to microtubule stabilization by paclitaxel. The siRNA screen identified several genes that have not been linked previously to microtubule regulation or paclitaxel response. Gene shaving and Bayesian resampling used to classify these genes suggested three pathways of paclitaxel-induced cell death related to apoptosis and microtubule stability, apoptosis alone, or neither process. Our results offer a functional classification of the genetic basis for paclitaxel sensitivity and they support the hypothesis that stabilizing microtubules prior to therapy could enhance antitumor responses to paclitaxel treatment. Cancer Res; 71(17); 5806–17. ©2011 AACR.

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Background and Objective:Ovarian cancer (OC) is a deadly gynaecological cancer with limited successful treatment options; approximately 70–80% of patients relapse, even those who initially respond well to treatment. It has been recently suggested that relapse occurs due to dormancy, an inactive cellular state which can evade traditional therapeutics targeting highly proliferating cells through different mechanisms. One is immune evasion, which conceals tumour cells from the body’s natural defence system. The cells can modulate their immunogenicity and that of the host to overcome the opposing tumour-immune system operation. Therefore, developing immunotherapies, which function to arm the host immune system against the tumour, is vital to patient survival. Considering the successes of immunotherapies in other cancers, this review will outline various tumour immune evasion strategies within its complex microenvironment and examine current significant developments in immunotherapies to inflame the ovarian tumour and overcome the resistance such that no cell is left behind. Methods:A PubMed search prioritising all types of literature since 2010 was conducted using the keywords “ovarian cancer”, “epithelial ovarian cancer”, “immunotherapy”, “immune evasion”, and “relapse” in various combinations. Secondary searches and other citations were based off reference lists. Key Content and Findings:Numerous molecular and cellular modifications are utilised by OC cells to evade the immune system. Further, the tumour microenvironment creates a physical barrier to immune infiltration and an immunosuppressive environment. In response, many immunotherapies have been created to combat OC, including antibodies, vaccines, adoptive cell therapy (ACT), immunomodulators and immunogenic cell death (ICD) inducers. Conclusions:Most immunotherapies targeting OC are still in early stages and far from being used clinically. While combination therapy is suggested, it may also be beneficial to recruit various types of immune cells to the tumour. Awareness of immune evasion strategies is critical to treatment development and targeting relapse.

Purpose: Using RNA sequencing, we recently developed the 52-gene–based Oxford classifier of carcinoma of the ovary (Oxford Classic, OxC) for molecular stratification of serous ovarian cancers (SOCs) based on the molecular profiles of their cell of origin in the fallopian tube epithelium. Here, we developed a 52-gene NanoString panel for the OxC to test the robustness of the classifier. Experimental Design: We measured the expression of the 52 genes in an independent cohort of prospectively collected SOC samples (n = 150) from a homogenous cohort who were treated with maximal debulking surgery and chemotherapy. We performed data mining of published expression profiles of SOCs and validated the classifier results on tissue arrays comprising 137 SOCs. Results: We found evidence of profound nongenetic heterogeneity in SOCs. Approximately 20% of SOCs were classified as epithelial-to-mesenchymal transition–high (EMT-high) tumors, which were associated with poor survival. This was independent of established prognostic factors, such as tumor stage, tumor grade, and residual disease after surgery (HR, 3.3; P = 0.02). Mining expression data of 593 patients revealed a significant association between the EMT scores of tumors and the estimated fraction of alternatively activated macrophages (M2; P < 0.0001), suggesting a mechanistic link between immunosuppression and poor prognosis in EMT-high tumors. Conclusions: The OxC-defined EMT-high SOCs carry particularly poor prognosis independent of established clinical parameters. These tumors are associated with high frequency of immunosuppressive macrophages, suggesting a potential therapeutic target to improve clinical outcome.

Key Points The Zinc finger protein, Zbtb18, is downregulated during PC differentiation. Enhanced expression of Zbtb18 leads to impaired PC development. Zbtb18 directly binds and inhibits expression of PI3K subunits., The PI3K pathway plays a key role in B cell activation and is important for the differentiation of Ab producing plasma cells (PCs). Although much is known about the molecular mechanisms that modulate PI3K signaling in B cells, the transcriptional regulation of PI3K expression is poorly understood. In this study, we identify the zinc finger protein Zbtb18 as a transcriptional repressor that directly binds enhancer/promoter regions of genes encoding class I PI3K regulatory subunits, subsequently limiting their expression, dampening PI3K signaling and suppressing PC responses. Following activation, dividing B cells progressively downregulated Zbtb18, allowing gradual amplification of PI3K signals and enhanced development of PCs. Human Zbtb18 displayed similar expression patterns and function in human B cells, acting to inhibit development of PCs. Furthermore, a number of Zbtb18 mutants identified in cancer patients showed loss of suppressor activity, which was also accompanied by impaired regulation of PI3K genes. Taken together, our study identifies Zbtb18 as a repressor of PC differentiation and reveals its previously unappreciated function as a transcription modulator of the PI3K signaling pathway.

Epitopes derived from mutated cancer proteins elicit strong antitumor T-cell responses that correlate with clinical efficacy in a proportion of patients. However, it remains unclear whether the subcellular localization of mutated proteins influences the efficiency of T-cell priming. To address this question, we compared the immunogenicity of NY-ESO-1 and OVA localized either in the cytosol or in mitochondria. We showed that tumors expressing mitochondrial-localized NY-ESO-1 and OVA proteins elicit significantdly higher frequencies of antigen-specific CD8+ T cells in vivo. We also demonstrated that this stronger immune response is dependent on the mitochondrial location of the antigenic proteins, which contributes to their higher steady-state amount, compared with cytosolic localized proteins. Consistent with these findings, we showed that injection of mitochondria purified from B16 melanoma cells can protect mice from a challenge with B16 cells, but not with irrelevant tumors. Finally, we extended these findings to cancer patients by demonstrating the presence of T-cell responses specific for mutated mitochondrial-localized proteins. These findings highlight the utility of prioritizing epitopes derived from mitochondrial-localized mutated proteins as targets for cancer vaccination strategies.

In 2001, a group of European academic surgeons created the Enhanced Recovery After Surgery (ERAS) study group and established the first official ERAS protocol. One of the most significant challenges during ERAS implementation is variability of drugs used throughout the perioperative period. Pharmacogenomic testing (blood or saliva) results (obtained within approximately 48 hrs) provide guidelines on how to prescribe the optimal drug with the optimal dosage to each patient based on an individual's unique genetic profile. Pharmacogenomic testing of various methods of multimodal analgesia is an essential element of ERAS protocols spanning the entire perioperative period to ultimately optimize postoperative pain control. The key goal for anesthetic management in ERAS protocols is to facilitate rapid emergence by using the shortest acting agents available, thus accelerating recovery and reducing length of stay, hospital expenses, and postoperative complications. Postoperative nausea and vomiting (PONV) is an additional challenge that should be overcome to ensure an enhanced recovery and shorter length of stay with the use of antiemetics. Postoperative ileus (POI) can result in longer hospital stay with increasing susceptibility to associated morbidities along with an increase in associated hospitalization costs. Genetics-guided pharmacotherapy and its impact on clinical outcomes should be thoroughly studied for better understanding and managing drug administration in the settings of ERAS.

Similar to tumor-initiating cells (TICs), minimal residual disease (MRD) is capable of reinitiating tumors and causing recurrence. However, the molecular characteristics of solid tumor MRD cells and drivers of their survival have remained elusive. Here we performed dense multiregion transcriptomics analysis of paired biopsies from 17 ovarian cancer patients before and after chemotherapy. We reveal that while MRD cells share important molecular signatures with TICs, they are also characterized by an adipocyte-like gene expression signature and a portion of them had undergone epithelial-mesenchymal transition (EMT). In a cell culture MRD model, MRD-mimic cells showed the same phenotype and were dependent on fatty acid oxidation (FAO) for survival and resistance to cytotoxic agents. These findings identify EMT and FAO as attractive targets to eradicate MRD in ovarian cancer and make a compelling case for the further testing of FAO inhibitors in treating MRD.

Single-cell RNA sequencing (scRNA-Seq) datasets that are produced from clinical samples are often confounded by batch effects and inter-patient variability. Existing batch effect removal methods typically require strong assumptions on the composition of cell populations being near identical across patients. Here we present a novel meta-clustering workflow, CIDER, based on inter-group similarity measures. We demonstrate that CIDER outperforms other scRNA-Seq clustering methods and integration approaches in both simulated and real datasets. Moreover, we show that CIDER can be used to assess the biological correctness of integration in real datasets, while it does not require the existence of prior cellular annotations.

Although various methods have been developed for sequencing cytosine modifications, it is still challenging for specific and quantitative sequencing of individual modification at base-resolution. For example, to obtain both true 5-methylcytosine (5mC) and true 5-hydroxymethylcytosine (5hmC) information, the two major epigenetic modifications, it usually requires subtraction of two methods, which increases noise and requires high sequencing depth. Recently, we developed TET-assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of 5mC and 5hmC. Here we demonstrate that two sister methods, TAPSβ and chemical-assisted pyridine borane sequencing (CAPS), can be effectively used for subtraction-free and specific whole-genome sequencing of 5mC and 5hmC, respectively. We also demonstrate pyridine borane sequencing (PS) for whole-genome profiling of 5-formylcytosine and 5-carboxylcytosine, the further oxidized derivatives of 5mC and 5hmC. This work completes the set of versatile borane reduction chemistry-based methods as a comprehensive toolkit for direct and quantitative sequencing of all four cytosine epigenetic modifications., Specific and quantitative sequencing of cytosine modifications is challenging at base-resolution. Here the authors present TAPSβ and CAPS for subtraction-free whole genome sequencing of 5mC and 5hmC.

Cancer is a leading cause of death worldwide and, despite new targeted therapies and immunotherapies, many patients with advanced-stage- or high-risk cancers still die, owing to metastatic disease. Adoptive T-cell therapy, involving the autologous or allogeneic transplant of tumour-infiltrating lymphocytes or genetically modified T cells expressing novel T-cell receptors or chimeric antigen receptors, has shown promise in the treatment of cancer patients, leading to durable responses and, in some cases, cure. Technological advances in genomics, computational biology, immunology and cell manufacturing have brought the aspiration of individualised therapies for cancer patients closer to reality. This new era of cell-based individualised therapeutics challenges the traditional standards of therapeutic interventions and provides opportunities for a paradigm shift in our approach to cancer therapy. Invited speakers at a 2020 symposium discussed three areas—cancer genomics, cancer immunology and cell-therapy manufacturing—that are essential to the effective translation of T-cell therapies in the treatment of solid malignancies. Key advances have been made in understanding genetic intratumour heterogeneity, and strategies to accurately identify neoantigens, overcome T-cell exhaustion and circumvent tumour immunosuppression after cell-therapy infusion are being developed. Advances are being made in cell-manufacturing approaches that have the potential to establish cell-therapies as credible therapeutic options. T-cell therapies face many challenges but hold great promise for improving clinical outcomes for patients with solid tumours.

Bulk whole genome sequencing (WGS) enables the analysis of tumor evolution but, because of depth limitations, can only identify old mutational events. The discovery of current mutational processes for predicting the tumor's evolutionary trajectory requires dense sequencing of individual clones or single cells. Such studies, however, are inherently problematic because of the discovery of excessive false positive mutations when sequencing picogram quantities of DNA. Data pooling to increase the confidence in the discovered mutations, moves the discovery back in the past to a common ancestor. Here we report a robust whole genome sequencing and analysis pipeline (DigiPico/MutLX) that virtually eliminates all false positive results while retaining an excellent proportion of true positives. Using our method, we identified, for the first time, a hyper-mutation (kataegis) event in a group of ∼30 cancer cells from a recurrent ovarian carcinoma. This was unidentifiable from the bulk WGS data. Overall, we propose DigiPico/MutLX method as a powerful framework for the identification of clone-specific variants at an unprecedented accuracy.

The STK32 kinases are a small subfamily of three uncharacterised serine/threonine kinases from the AGC kinase family whose functional role is so far unknown. Here, we analyse the consensus peptide sequence for STK32A phosphorylation, showing that STK32A is directed towards acidic substrate sequences and exhibits dual-specificity for serine/threonine and tyrosine residues. A crystal structure of STK32A reveals an overall structure typical of the AGC protein kinase family but with significant and unique features including an altered binding mode of the hydrophobic motif to the N-terminal lobe of the kinase domain, and a novel alpha-helix in between the turn motif and the hydrophobic motif. The crystal structure combined with phylogenetic analysis reveals the evolutionary conservation of the acidic substrate preference.In vitrobinding assays demonstrated that the STK32 kinases bind significant numbers of clinically used kinase inhibitors.

The inter-differentiation between cell states promotes cancer cell survival under stress and fosters non-genetic heterogeneity (NGH). NGH is, therefore, a surrogate of tumor resilience but its quantification is confounded by genetic heterogeneity. Here we show that NGH in serous ovarian cancer (SOC) can be accurately measured when informed by the molecular signatures of the normal fallopian tube epithelium (FTE) cells, the cells of origin of SOC. Surveying the transcriptomes of ∼6,000 FTE cells, predominantly from non-ovarian cancer patients, identified 6 FTE subtypes. We used subtype signatures to deconvolute SOC expression data and found substantial intra-tumor NGH. Importantly, NGH-based stratification of ∼1,700 tumors robustly correlated with survival. Our findings lay the foundation for accurate prognostic and therapeutic stratification of SOC. Using single-cell RNA sequencing, Hu et al. identify six subtypes of fallopian tube epithelium (FTE) cells in normal human fallopian tube tissues. The FTE cellular subtypes reveal intra-tumoral heterogeneity in serous ovarian cancer (SOC) and define SOC subtypes that correlate with patient prognosis.

// Charlotte Taylor 1, 2 , David Mannion 1, 2 , Fabrizio Miranda 1, 2 , Mohammad Karaminejadranjbar 1, 2 , Sandra Herrero-Gonzalez 1, 2 , Karin Hellner 1, 2 , Yiyan Zheng 1, 2 , Geoffrey Bartholomeusz 3 , Robert C. Bast Jr 3 , Ahmed Ashour Ahmed 1, 2 1 Ovarian Cancer Cell Laboratory, Weatherall Institute of Molecular Medicine, University of Oxford, Headington, Oxford, OX3 9DS, UK 2 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Women’s Centre, John Radcliffe Hospital, Oxford, OX3 9DU, UK 3 Department of Experimental Therapeutics, M.D. Anderson Cancer Center, University of Texas, Houston, TX 77030, USA Correspondence to: Ahmed Ashour Ahmed, email: ahmed.ahmed@obs-gyn.ox.ac.uk Keywords: ovarian cancer, paclitaxel, mitotic arrest, PFKFB4, PFKFB3 Received: July 26, 2016 Accepted: December 15, 2016 Published: January 31, 2017 ABSTRACT Taxanes represent some of the most commonly used chemotherapeutic agents for ovarian cancer treatment. However, they are only effective in approximately 40% of patients. Novel therapeutic strategies are required to potentiate their effect and improve patient outcome. A hallmark of many cancers is the constitutive activation of the PI3K/AKT pathway, which drives cell survival and metabolism. We discovered a striking decrease in AKT activity coupled with a significant reduction in glucose 6-phosphate and ATP levels during mitotic arrest in the majority of ovarian cancer cell lines tested, indicating a potential metabolic vulnerability. A high-content siRNA screen to detect novel metabolic targets in mitotically arrested ovarian cancer cells identified the glycolytic enzyme PFKFB4. PFKFB4 depletion increased caspase 3/7 activity, and levels of reactive oxygen species only in mitotically arrested cells, and significantly enhanced mitotic cell death after paclitaxel treatment. Depletion of PFKFB3 demonstrated a similar phenotype. The observation that some ovarian cancer cells lose AKT activity during mitotic arrest and become vulnerable to metabolic targeting is a new concept in cancer therapy. Thus, combining mitotic-targeted therapies with glycolytic inhibitors may act to potentiate the effects of antimitotics in ovarian cancer through mitosis-specific cell death.