A diversity of novel type-2 innate lymphoid cell subpopulations revealed during tumour expansion.

Type 2 innate lymphoid cells (ILC2s) perform vital functions in orchestrating humoral immune responses, facilitating tissue remodelling, and ensuring tissue homeostasis. Additionally, in a role that has garnered considerably less attention, ILC2s can also enhance Th1-related cytolytic T lymphocyte immune responses against tumours. Studies have thus far generally failed to address the mystery of how one ILC2 cell-type can participate in a multiplicity of functions. Here we utilized single cell RNA sequencing analysis to create the first comprehensive atlas of naïve and tumour-associated lung ILC2s and discover multiple unique subtypes of ILC2s equipped with developmental gene programs that become skewed during tumour expansion favouring inflammation, antigen processing, immunological memory and Th1-related anti-tumour CTL responses. The discovery of these new subtypes of ILC2s challenges current paradigms of ILC2 biology and provides an explanation for their diversity of function.

Establishment and validation of preclinical models of SMARCA4-inactivated and ARID1A/ARID1B co-inactivated dedifferentiated endometrial carcinoma.

OBJECTIVE: Dedifferentiated endometrial cancer (DDEC) is an uncommon and clinically highly aggressive subtype of endometrial cancer characterized by genomic inactivation of SWItch/Sucrose Non-Fermentable (SWI/SNF) complex protein. It responds poorly to conventional systemic treatment and its rapidly progressive clinical course limits the therapeutic windows to trial additional lines of therapies. This underscores a pressing need for biologically accurate preclinical tumor models to accelerate therapeutic development. METHODS: DDEC tumor from surgical samples were implanted into immunocompromised mice for patient-derived xenograft (PDX) and cell line development. The histologic, immunophenotypic, genetic and epigenetic features of the patient tumors and the established PDX models were characterized. The SMARCA4-deficienct DDEC model was evaluated for its sensitivity toward a KDM6A/B inhibitor (GSK-J4) that was previously reported to be effective therapy for other SMARCA4-deficient cancer types. RESULTS: All three DDEC models exhibited rapid growth in vitro and in vivo, with two PDX models showing spontaneous development of metastases in vivo. The PDX tumors maintained the same undifferentiated histology and immunophenotype, and exhibited identical genomic and methylation profiles as seen in the respective parental tumors, including a mismatch repair (MMR)-deficient DDEC with genomic inactivation of SMARCA4, and two MMR-deficient DDECs with genomic inactivation of both ARID1A and ARID1B. Although the SMARCA4-deficient cell line showed low micromolecular sensitivity to GSK-J4, no significant tumor growth inhibition was observed in the corresponding PDX model. CONCLUSIONS: These established patient tumor-derived models accurately depict DDEC and represent valuable preclinical tools to gain therapeutic insights into this aggressive tumor type.

A Bifunctional PARP-HDAC Inhibitor with Activity in Ewing Sarcoma.

PURPOSE: Histone deacetylase (HDAC) inhibition has been shown to induce pharmacologic "BRCAness" in cancer cells with proficient DNA repair activity. This provides a rationale for exploring combination treatments with HDAC and PARP inhibition in cancer types that are insensitive to single-agent PARP inhibitors (PARPi). Here, we report the concept and characterization of a novel bifunctional PARPi (kt-3283) with dual activity toward PARP1/2 and HDAC enzymes in Ewing sarcoma cells. EXPERIMENTAL DESIGN: Inhibition of PARP1/2 and HDAC was measured using PARP1/2, HDAC activity, and PAR formation assays. Cytotoxicity was assessed by IncuCyte live cell imaging, CellTiter-Glo, and spheroid assays. Cell-cycle profiles were determined using propidium iodide staining and flow cytometry. DNA damage was examined by γH2AX expression and comet assay. Inhibition of metastatic potential by kt-3283 was evaluated via ex vivo pulmonary metastasis assay (PuMA). RESULTS: Compared with FDA-approved PARP (olaparib) and HDAC (vorinostat) inhibitors, kt-3283 displayed enhanced cytotoxicity in Ewing sarcoma models. The kt-3283-induced cytotoxicity was associated with strong S and G2-M cell-cycle arrest in nanomolar concentration range and elevated DNA damage as assessed by γH2AX tracking and comet assays. In three-dimensional spheroid models of Ewing sarcoma, kt-3283 showed efficacy in lower concentrations than olaparib and vorinostat, and kt-3283 inhibited colonization of Ewing sarcoma cells in the ex vivo PuMA model. CONCLUSIONS: Our data demonstrate the preclinical justification for studying the benefit of dual PARP and HDAC inhibition in the treatment of Ewing sarcoma in a clinical trial and provides proof-of-concept for a bifunctional single-molecule therapeutic strategy.

Functional mapping of androgen receptor enhancer activity.

BACKGROUND: Androgen receptor (AR) is critical to the initiation, growth, and progression of prostate cancer. Once activated, the AR binds to cis-regulatory enhancer elements on DNA that drive gene expression. Yet, there are 10-100× more binding sites than differentially expressed genes. It is unclear how or if these excess binding sites impact gene transcription. RESULTS: To characterize the regulatory logic of AR-mediated transcription, we generated a locus-specific map of enhancer activity by functionally testing all common clinical AR binding sites with Self-Transcribing Active Regulatory Regions sequencing (STARRseq). Only 7% of AR binding sites displayed androgen-dependent enhancer activity. Instead, the vast majority of AR binding sites were either inactive or constitutively active enhancers. These annotations strongly correlated with enhancer-associated features of both in vitro cell lines and clinical prostate cancer samples. Evaluating the effect of each enhancer class on transcription, we found that AR-regulated enhancers frequently interact with promoters and form central chromosomal loops that are required for transcription. Somatic mutations of these critical AR-regulated enhancers often impact enhancer activity. CONCLUSIONS: Using a functional map of AR enhancer activity, we demonstrated that AR-regulated enhancers act as a regulatory hub that increases interactions with other AR binding sites and gene promoters.

Multiomics Characterization of Low-Grade Serous Ovarian Carcinoma Identifies Potential Biomarkers of MEK Inhibitor Sensitivity and Therapeutic Vulnerability.

Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates in patients with metastatic disease. There is a pressing need to develop effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use multiomics integration of whole-exome sequencing, RNA sequencing, and mass spectrometry-based proteomics on 14 LGSOC cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Comparison of LGSOC cell line data with LGSOC tumor data enabled predictive biomarker identification of MEK inhibitor (MEKi) efficacy, with KRAS mutations found exclusively in MEKi-sensitive cell lines and NRAS mutations found mostly in MEKi-resistant cell lines. Distinct patterns of Catalogue of Somatic Mutations in Cancer mutational signatures were identified in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes were more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. These LGSOC cell lines were representative models of the molecular aberrations found in LGSOC tumors. For prediction of in vitro MEKi efficacy, proteomic data provided better discrimination than gene expression data. Condensin, minichromosome maintenance, and replication factor C protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. This study suggests that CDKN2A/B or MTAP deficiency may be exploited using synthetically lethal treatment strategies, highlighting the importance of using proteomic data as a tool for molecular drug prediction. Multiomics approaches are crucial to improving our understanding of the molecular underpinnings of LGSOC and applying this information to develop new therapies. SIGNIFICANCE: These findings highlight the utility of global multiomics to characterize LGSOC cell lines as research models, to determine biomarkers of MEKi resistance, and to identify potential novel therapeutic targets.

Well-Differentiated Papillary Mesothelioma of the Peritoneum Is Genetically Distinct from Malignant Mesothelioma.

Well-differentiated papillary mesothelioma (WDPM) is an uncommon mesothelial proliferation that is most commonly encountered as an incidental finding in the peritoneal cavity. There is controversy in the literature about whether WDPM is a neoplasm or a reactive process and, if neoplastic, whether it is a variant or precursor of epithelial malignant mesothelioma or is a different entity. Using whole exome sequencing of five WDPMs of the peritoneum, we have identified distinct mutations in EHD1, ATM, FBXO10, SH2D2A, CDH5, MAGED1, and TP73 shared by WDPM cases but not reported in malignant mesotheliomas. Furthermore, we show that WDPM is strongly enriched with C > A transversion substitution mutations, a pattern that is also not found in malignant mesotheliomas. The WDPMs lacked the alterations involving BAP1, SETD2, NF2, CDKN2A/B, LASTS1/2, PBRM1, and SMARCC1 that are frequently found in malignant mesotheliomas. We conclude that WDPMs are neoplasms that are genetically distinct from malignant mesotheliomas and, based on observed mutations, do not appear to be precursors of malignant mesotheliomas.

BAP1 haploinsufficiency predicts a distinct immunogenic class of malignant peritoneal mesothelioma.

BACKGROUND: Malignant peritoneal mesothelioma (PeM) is a rare and fatal cancer that originates from the peritoneal lining of the abdomen. Standard treatment of PeM is limited to cytoreductive surgery and/or chemotherapy, and no effective targeted therapies for PeM exist. Some immune checkpoint inhibitor studies of mesothelioma have found positivity to be associated with a worse prognosis. METHODS: To search for novel therapeutic targets for PeM, we performed a comprehensive integrative multi-omics analysis of the genome, transcriptome, and proteome of 19 treatment-naïve PeM, and in particular, we examined BAP1 mutation and copy number status and its relationship to immune checkpoint inhibitor activation. RESULTS: We found that PeM could be divided into tumors with an inflammatory tumor microenvironment and those without and that this distinction correlated with haploinsufficiency of BAP1. To further investigate the role of BAP1, we used our recently developed cancer driver gene prioritization algorithm, HIT'nDRIVE, and observed that PeM with BAP1 haploinsufficiency form a distinct molecular subtype characterized by distinct gene expression patterns of chromatin remodeling, DNA repair pathways, and immune checkpoint receptor activation. We demonstrate that this subtype is correlated with an inflammatory tumor microenvironment and thus is a candidate for immune checkpoint blockade therapies. CONCLUSIONS: Our findings reveal BAP1 to be a potential, easily trackable prognostic and predictive biomarker for PeM immunotherapy that refines PeM disease classification. BAP1 stratification may improve drug response rates in ongoing phases I and II clinical trials exploring the use of immune checkpoint blockade therapies in PeM in which BAP1 status is not considered. This integrated molecular characterization provides a comprehensive foundation for improved management of a subset of PeM patients.

Alignment-free clustering of UMI tagged DNA molecules.

MOTIVATION: Next-Generation Sequencing has led to the availability of massive genomic datasets whose processing raises many challenges, including the handling of sequencing errors. This is especially pertinent in cancer genomics, e.g. for detecting low allele frequency variations from circulating tumor DNA. Barcode tagging of DNA molecules with unique molecular identifiers (UMI) attempts to mitigate sequencing errors; UMI tagged molecules are polymerase chain reaction (PCR) amplified, and the PCR copies of UMI tagged molecules are sequenced independently. However, the PCR and sequencing steps can generate errors in the sequenced reads that can be located in the barcode and/or the DNA sequence. Analyzing UMI tagged sequencing data requires an initial clustering step, with the aim of grouping reads sequenced from PCR duplicates of the same UMI tagged molecule into a single cluster, and the size of the current datasets requires this clustering process to be resource-efficient. RESULTS: We introduce Calib, a computational tool that clusters paired-end reads from UMI tagged sequencing experiments generated by substitution-error-dominant sequencing platforms such as Illumina. Calib clusters are defined as connected components of a graph whose edges are defined in terms of both barcode similarity and read sequence similarity. The graph is constructed efficiently using locality sensitive hashing and MinHashing techniques. Calib's default clustering parameters are optimized empirically, for different UMI and read lengths, using a simulation module that is packaged with Calib. Compared to other tools, Calib has the best accuracy on simulated data, while maintaining reasonable runtime and memory footprint. On a real dataset, Calib runs with far less resources than alignment-based methods, and its clusters reduce the number of tentative false positive in downstream variation calling. AVAILABILITY AND IMPLEMENTATION: Calib is implemented in C++ and its simulation module is implemented in Python. Calib is available at https://github.com/vpc-ccg/calib. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genomic Alterations in Cell-Free DNA and Enzalutamide Resistance in Castration-Resistant Prostate Cancer.

IMPORTANCE: The molecular landscape underpinning response to the androgen receptor (AR) antagonist enzalutamide in patients with metastatic castration-resistant prostate cancer (mCRPC) is undefined. Consequently, there is an urgent need for practical biomarkers to guide therapy selection and elucidate resistance. Although tissue biopsies are impractical to perform routinely in the majority of patients with mCRPC, the analysis of plasma cell-free DNA (cfDNA) has recently emerged as a minimally invasive method to explore tumor characteristics. OBJECTIVE: To reveal genomic characteristics from cfDNA associated with clinical outcomes during enzalutamide treatment. DESIGN, SETTING, AND PARTICIPANTS: Plasma samples were obtained from August 4, 2013, to July 31, 2015, at a single academic institution (British Columbia Cancer Agency) from 65 patients with mCRPC. We collected temporal plasma samples (at baseline, 12 weeks, end of treatment) for circulating cfDNA and performed array comparative genomic hybridization copy number profiling and deep AR gene sequencing. Samples collected at end of treatment were also subjected to targeted sequencing of 19 prostate cancer-associated genes. EXPOSURE: Enzalutamide, 160 mg, daily orally. MAIN OUTCOMES AND MEASURES: Prostate-specific antigen response rate (decline ≥50% from baseline confirmed ≥3 weeks later). Radiographic (as per Prostate Cancer Working Group 2 Criteria) and/or clinical progression (defined as worsening disease-related symptoms necessitating a change in anticancer therapy and/or deterioration in Eastern Cooperative Group performance status ≥2 levels). RESULTS: The 65 patients had a median (interquartile range) age of 74 (68-79) years. Prostate-specific antigen response rate to enzalutamide treatment was 38% (25 of 65), while median clinical/radiographic progression-free survival was 3.5 (95% CI, 2.1-5.0) months. Cell-free DNA was isolated from 122 of 125 plasma samples, and targeted sequencing was successful in 119 of 122. AR mutations and/or copy number alterations were robustly detected in 48% (31 of 65) and 60% (18 of 30) of baseline and progression samples, respectively. Detection of AR amplification, heavily mutated AR (≥2 mutations), and RB1 loss were associated with worse progression-free survival, with hazard ratios of 2.92 (95% CI, 1.59-5.37), 3.94 (95% CI, 1.46-10.64), and 4.46 (95% CI, 2.28-8.74), respectively. AR mutations exhibited clonal selection during treatment, including an increase in glucocorticoid-sensitive AR L702H and promiscuous AR T878A in patients with prior abiraterone treatment. At the time of progression, cfDNA sequencing revealed mutations or copy number changes in all patients tested, including clinically actionable alterations in DNA damage repair genes and PI3K pathway genes, and a high frequency (4 of 14) of activating CTNNB1 mutations. CONCLUSIONS AND RELEVANCE: Clinically informative genomic profiling of cfDNA was feasible in nearly all patients with mCRPC and can provide important insights into enzalutamide response and resistance.

Androgen Receptor Gene Aberrations in Circulating Cell-Free DNA: Biomarkers of Therapeutic Resistance in Castration-Resistant Prostate Cancer.

PURPOSE: Although novel agents targeting the androgen-androgen receptor (AR) axis have altered the treatment paradigm of metastatic castration-resistant prostate cancer (mCRPC), development of therapeutic resistance is inevitable. In this study, we examined whether AR gene aberrations detectable in circulating cell-free DNA (cfDNA) are associated with resistance to abiraterone acetate and enzalutamide in mCRPC patients. EXPERIMENTAL DESIGN: Plasma was collected from 62 mCRPC patients ceasing abiraterone acetate (n = 29), enzalutamide (n = 19), or other agents (n = 14) due to disease progression. DNA was extracted and subjected to array comparative genomic hybridization (aCGH) for chromosome copy number analysis, and Roche 454 targeted next-generation sequencing of exon 8 in the AR. RESULTS: On aCGH, AR amplification was significantly more common in patients progressing on enzalutamide than on abiraterone or other agents (53% vs. 17% vs. 21%, P = 0.02, χ(2)). Missense AR exon 8 mutations were detected in 11 of 62 patients (18%), including the first reported case of an F876L mutation in an enzalutamide-resistant patient and H874Y and T877A mutations in 7 abiraterone-resistant patients. In patients switched onto enzalutamide after cfDNA collection (n = 39), an AR gene aberration (copy number increase and/or an exon 8 mutation) in pretreatment cfDNA was associated with adverse outcomes, including lower rates of PSA decline ≥ 30% (P = 0.013, χ(2)) and shorter time to radiographic/clinical progression (P = 0.010, Cox proportional hazards regression). CONCLUSIONS: AR gene aberrations in cfDNA are associated with resistance to enzalutamide and abiraterone in mCRPC. Our data illustrate that genomic analysis of cfDNA is a minimally invasive method for interrogating mechanisms of therapeutic resistance in mCRPC.