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Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy characterized by a paucity of tumor-proximal CD8+ T cells and resistance to immunotherapeutic interventions. Cancer-associated mechanisms that elicit CD8+ T-cell exclusion and resistance to immunotherapy are not well-known. Here, using a Kras- and p53-driven model of PDA, we describe a mechanism of action for the protumorigenic cytokine IL35 through STAT3 activation in CD8+ T cells. Distinct from its action on CD4+ T cells, IL35 signaling in gp130+CD8+ T cells activated the transcription factor STAT3, which antagonized intratumoral infiltration and effector function of CD8+ T cells via suppression of CXCR3, CCR5, and IFNγ expression. Inhibition of STAT3 signaling in tumor-educated CD8+ T cells improved PDA growth control upon adoptive transfer to tumor-bearing mice. We showed that activation of STAT3 in CD8+ T cells was driven by B cell– but not regulatory T cell–specific production of IL35. We also demonstrated that B cell–specific deletion of IL35 facilitated CD8+ T-cell activation independently of effector or regulatory CD4+ T cells and was sufficient to phenocopy therapeutic anti-IL35 blockade in overcoming resistance to anti–PD-1 immunotherapy. Finally, we identified a circulating IL35+ B-cell subset in patients with PDA and demonstrated that the presence of IL35+ cells predicted increased occurrence of phosphorylated (p)Stat3+CXCR3−CD8+ T cells in tumors and inversely correlated with a cytotoxic T-cell signature in patients. Together, these data identified B cell–mediated IL35/gp130/STAT3 signaling as an important direct link to CD8+ T-cell exclusion and immunotherapy resistance in PDA.

Polymerase theta (Pol θ, gene name Polq) is a widely conserved DNA polymerase that mediates a microhomology-mediated, error-prone, double strand break (DSB) repair pathway, referred to as Theta Mediated End Joining (TMEJ). Cells with homologous recombination deficiency are reliant on TMEJ for DSB repair. It is unknown whether deficiencies in other components of the DNA damage response (DDR) also result in Pol θ addiction. Here we use a CRISPR genetic screen to uncover 140 Polq synthetic lethal (PolqSL) genes, the majority of which were previously unknown. Functional analyses indicate that Pol θ/TMEJ addiction is associated with increased levels of replication-associated DSBs, regardless of the initial source of damage. We further demonstrate that approximately 30% of TCGA breast cancers have genetic alterations in PolqSL genes and exhibit genomic scars of Pol θ/TMEJ hyperactivity, thereby substantially expanding the subset of human cancers for which Pol θ inhibition represents a promising therapeutic strategy., Polymerase theta is a widely conserved DNA polymerase that mediates Theta Mediated End Joining. Here authors present a synthetic lethal CRISPR screen to identify DDR gene mutations that induce cellular addiction to Pol theta.

Summary Proliferative and invasive breast tumors evolve heterogeneously in individual patients, posing significant challenges in identifying new druggable targets for precision, effective therapy. Here we present a functional multi-omics method, interaction-Correlated Multi-omic Aberration Patterning (iC-MAP), which dissects intra-tumor heterogeneity and identifies in situ the oncogenic consequences of multi-omics aberrations that drive proliferative and invasive tumors. First, we perform chromatin activity-based chemoproteomics (ChaC) experiments on breast cancer (BC) patient tissues to identify genetic/transcriptomic alterations that manifest as oncogenically active proteins. ChaC employs a biotinylated small molecule probe that specifically binds to the oncogenically active histone methyltransferase G9a, enabling sorting/enrichment of a G9a-interacting protein complex that represents the predominant BC subtype in a tissue. Second, using patient transcriptomic/genomic data, we retrospectively identified some G9a interactor-encoding genes that showed individualized iC-MAP. Our iC-MAP findings represent both new diagnostic/prognostic markers to identify patient subsets with incurable metastatic disease and targets to create individualized therapeutic strategies., Graphical Abstract, Highlights • ChaC dissects tumor heterogeneity for identifying oncogenic-active proteins • An oncogenic-active G9a-interactome represents the invasive tumor in a tissue • iC-MAP identifies multi-omics aberrations that drive invasive tumors • Patient-specific iC-MAP of select interactor genes are of prognostic value, Biological Sciences; Cancer Systems Biology; Cancer

BACKGROUND: High tumor mutation burden (TMB-H) has been proposed as a predictive biomarker for response to immune checkpoint blockade (ICB), largely due to potential for tumor mutations to generate immunogenic neoantigens. Despite recent pan-cancer approval of ICB treatment for any TMB-H tumor, as assessed by the targeted Foundation One CDx assay in 9 tumor types, the utility of this biomarker has not been fully demonstrated across all cancers. PATIENTS AND METHODS: Data from over 10,000 patient tumors included in The Cancer Genome Atlas were used to compare approaches to determine TMB and identify the correlation between predicted neoantigen load and CD8 T cells. Association of TMB with ICB treatment outcomes was analyzed by both objective response rates (ORRs, N=1551) and overall survival (OS, N=1936). RESULTS: In cancer types where CD8 T cell levels positively correlated with neoantigen load, such as melanoma, lung, and bladder cancers, TMB-H tumors exhibited a 39.8% ORR to ICB (95% CI 34.9–44.8), which was significantly higher than that observed in low TMB (TMB-L) tumors (odds ratio (OR) = 4.1, 95% CI 2.9–5.8, P < 2×10(−16)). In cancer types that showed no relationship between CD8 T cell levels and neoantigen load, such as breast cancer, prostate cancer, and glioma, TMB-H tumors failed to achieve a 20% ORR (ORR = 15.3%, 95% CI 9.2–23.4, P = 0.95), and also exhibited a significantly lower ORR relative to TMB-L tumors (OR = 0.46, 95% CI 0.24–0.88, P = 0.02). Bulk ORRs were not significantly different between the two categories of tumors (P = 0.10) for patient cohorts assessed. Equivalent results were obtained by analyzing OS and by treating TMB as a continuous variable. CONCLUSIONS: Our analysis failed to support application of TMB-H as a biomarker for treatment with ICB in all solid cancer types. Further tumor type specific studies are warranted.

MotivationQuantification estimates of gene expression from single-cell RNA-seq (scRNA-seq) data have inherent uncertainty due to reads that map to multiple genes. Many existing scRNA-seq quantification pipelines ignore multi-mapping reads and therefore underestimate expected read counts for many genes. alevin accounts for multi-mapping reads and allows for the generation of “inferential replicates”, which reflect quantification uncertainty. Previous methods have shown improved performance when incorporating these replicates into statistical analyses, but storage and use of these replicates increases computation time and memory requirements.ResultsWe demonstrate that storing only the mean and variance from a set of inferential replicates (“compression”) is sufficient to capture gene-level quantification uncertainty. Using these values, we generate “pseudo-inferential” replicates from a negative binomial distribution and propose a general procedure for incorporating these replicates into a proposed statistical testing framework. We show reduced false positives when applying this procedure to trajectory-based differential expression analyses. We additionally extend the Swish method to incorporate pseudo-inferential replicates and demonstrate improvements in computation time and memory consumption without any loss in performance. Lastly, we show that the removal of multi-mapping reads can result in significant underestimation of counts for functionally important genes in a real dataset.Availability and implementationmakeInfReps and splitSwish are implemented in the development branch of the R/Bioconductor fishpond package available at http://bioconductor.org/packages/devel/bioc/html/fishpond.html. Sample code to calculate the uncertainty-aware p-values can be found on GitHub at https://github.com/skvanburen/scUncertaintyPaperCode.Contactmichaelisaiahlove@gmail.com

Over 55,000 people in the United States are diagnosed with pancreatic ductal adenocarcinoma (PDAC) yearly, and fewer than 20% of these patients survive a year beyond diagnosis. Chemotherapies are considered or used in nearly every PDAC case, but there is limited understanding of the complex signaling responses underlying resistance to these common treatments. Here, we take an unbiased approach to study protein kinase network changes following chemotherapies in patient-derived xenograft (PDX) models of PDAC to facilitate design of rational drug combinations. Proteomics profiling following chemotherapy regimens reveals that activation of JNK-JUN signaling occurs after 5-fluorouracil plus leucovorin (5-FU + LEU) and FOLFOX (5-FU + LEU plus oxaliplatin [OX]), but not after OX alone or gemcitabine. Cell and tumor growth assays with the irreversible inhibitor JNK-IN-8 and genetic manipulations demonstrate that JNK and JUN each contribute to chemoresistance and cancer cell survival after FOLFOX. Active JNK1 and JUN are specifically implicated in these effects, and synergy with JNK-IN-8 is linked to FOLFOX-mediated JUN activation, cell cycle dysregulation, and DNA damage response. This study highlights the potential for JNK-IN-8 as a biological tool and potential combination therapy with FOLFOX in PDAC and reinforces the need to tailor treatment to functional characteristics of individual tumors.

Epigenomics, the study of the human genome and its interactions with proteins and other cellular elements, has become of significant interest in recent years. Such interactions have been shown to regulate essential cellular functions and are associated with multiple complex diseases. Therefore, understanding how these interactions may change across conditions is central in biomedical research. Chromatin immunoprecipi-tation followed by massively-parallel sequencing (ChIP-seq) is one of several techniques to detect local changes in epigenomic activity (peaks). However, existing methods for differential peak calling are not optimized for the diversity in ChIP-seq signal profiles, are limited to the analysis of two conditions, or cannot classify specific patterns of differential change when multiple patterns exist. To address these limitations, we present a flexible and efficient method for the detection of differential epigenomic activity across multiple conditions. We utilize data from the ENCODE Consortium and show that the presented method, mixNBHMM, exhibits superior performance to current tools and it is among the fastest algorithms available. Our method also allows the classification of combinatorial patterns of differential epigenomic activity, and the characterization of chromatin regulatory states. Supplementary materials, including code to replicate the results, are available as an online supplement. The implemented R package is available for download at https://github.com/plbaldoni/mixNBHMM .

Purpose: Molecular subtyping for pancreatic cancer has made substantial progress in recent years, facilitating the optimization of existing therapeutic approaches to improve clinical outcomes in pancreatic cancer. With advances in treatment combinations and choices, it is becoming increasingly important to determine ways to place patients on the best therapies upfront. Although various molecular subtyping systems for pancreatic cancer have been proposed, consensus regarding proposed subtypes, as well as their relative clinical utility, remains largely unknown and presents a natural barrier to wider clinical adoption. Experimental Design: We assess three major subtype classification schemas in the context of results from two clinical trials and by meta-analysis of publicly available expression data to assess statistical criteria of subtype robustness and overall clinical relevance. We then developed a single-sample classifier (SSC) using penalized logistic regression based on the most robust and replicable schema. Results: We demonstrate that a tumor-intrinsic two-subtype schema is most robust, replicable, and clinically relevant. We developed Purity Independent Subtyping of Tumors (PurIST), a SSC with robust and highly replicable performance on a wide range of platforms and sample types. We show that PurIST subtypes have meaningful associations with patient prognosis and have significant implications for treatment response to FOLIFIRNOX. Conclusions: The flexibility and utility of PurIST on low-input samples such as tumor biopsies allows it to be used at the time of diagnosis to facilitate the choice of effective therapies for patients with pancreatic ductal adenocarcinoma and should be considered in the context of future clinical trials.

Summary: The Mre11-Rad50-Nbs1 complex is a DNA double-strand break sensor that mediates a tumor-suppressive DNA damage response (DDR) in cells undergoing oncogenic stress, yet the mechanisms underlying this effect are poorly understood. Using a genetically inducible primary mammary epithelial cell model, we demonstrate that Mre11 suppresses proliferation and DNA damage induced by diverse oncogenic drivers through a p53-independent mechanism. Breast tumorigenesis models engineered to express a hypomorphic Mre11 allele exhibit increased levels of oncogene-induced DNA damage, R-loop accumulation, and chromosomal instability with a characteristic copy number loss phenotype. Mre11 complex dysfunction is identified in a subset of human triple-negative breast cancers and is associated with increased sensitivity to DNA-damaging therapy and inhibitors of ataxia telangiectasia and Rad3 related (ATR) and poly (ADP-ribose) polymerase (PARP). Thus, deficiencies in the Mre11-dependent DDR drive proliferation and genome instability patterns in p53-deficient breast cancers and represent an opportunity for therapeutic exploitation. : The origins of genome instability in cancer remain poorly understood. Fagan-Solis et al. reveal a p53-independent genome integrity checkpoint pathway mediated by Mre11 that protects against genome instability in breast cancer. Mre11 dysfunction in breast cancer models induces a genomic loss signature and vulnerability to PARP and ATR inhibitors. Keywords: breast cancer, genome instability, chromosomal instability, DNA damage response, oncogenic stress, Mre11, R loops, genomic scar, replication stress

Screening of an inhibitor library targeting kinases and epigenetic regulators identified several molecules having antiproliferative synergy with extraterminal domain (BET) bromodomain (BD) inhibitors (JQ1, OTX015) in triple-negative breast cancer (TNBC). GSK2801, an inhibitor of BAZ2A/B BDs, of the imitation switch chromatin remodeling complexes, and BRD9, of the SWI/SNF complex, demonstrated synergy independent of BRD4 control of P-TEFb–mediated pause-release of RNA polymerase II. GSK2801 or RNAi knockdown of BAZ2A/B with JQ1 selectively displaced BRD2 at promoters/enhancers of ETS-regulated genes. Additional displacement of BRD2 from rDNA in the nucleolus coincided with decreased 45S rRNA, revealing a function of BRD2 in regulating RNA polymerase I transcription. In 2D cultures, enhanced displacement of BRD2 from chromatin by combination drug treatment induced senescence. In spheroid cultures, combination treatment induced cleaved caspase-3 and cleaved PARP characteristic of apoptosis in tumor cells. Thus, GSK2801 blocks BRD2-driven transcription in combination with BET inhibitor and induces apoptosis of TNBC. Implications: Synergistic inhibition of BDs encoded in BAZ2A/B, BRD9, and BET proteins induces apoptosis of TNBC by a combinatorial suppression of ribosomal DNA transcription and ETS-regulated genes.

Objective A novel approach to regulate obesity-associated adipose inflammation may be through metabolic reprogramming of macrophages (MΦs). Broadly speaking, MΦs dependent on glucose are pro-inflammatory, classically activated MΦs (CAM), which contribute to adipose inflammation and insulin resistance. In contrast, MΦs that primarily metabolize fatty acids are alternatively activated MΦs (AAM) and maintain tissue insulin sensitivity. In actuality, there is much flexibility and overlap in the CAM-AAM spectrum in vivo dependent upon various stimuli in the microenvironment. We hypothesized that specific lipid trafficking proteins, e.g. fatty acid transport protein 1 (FATP1), would direct MΦ fatty acid transport and metabolism to limit inflammation and contribute to the maintenance of adipose tissue homeostasis. Methods Bone marrow derived MΦs (BMDMs) from Fatp1−/− and Fatp1+/+ mice were used to investigate FATP1-dependent substrate metabolism, bioenergetics, metabolomics, and inflammatory responses. We also generated C57BL/6J chimeric mice by bone marrow transplant specifically lacking hematopoetic FATP1 (Fatp1B−/−) and controls Fatp1B+/+. Mice were challenged by high fat diet (HFD) or low fat diet (LFD) and analyses including MRI, glucose and insulin tolerance tests, flow cytometric, histologic, and protein quantification assays were conducted. Finally, an FATP1-overexpressing RAW 264.7 MΦ cell line (FATP1-OE) and empty vector control (FATP1-EV) were developed as a gain of function model to test effects on substrate metabolism, bioenergetics, metabolomics, and inflammatory responses. Results Fatp1 is downregulated with pro-inflammatory stimulation of MΦs. Fatp1−/− BMDMs and FATP1-OE RAW 264.7 MΦs demonstrated that FATP1 reciprocally controled metabolic flexibility, i.e. lipid and glucose metabolism, which was associated with inflammatory response. Supporting our previous work demonstrating the positive relationship between glucose metabolism and inflammation, loss of FATP1 enhanced glucose metabolism and exaggerated the pro-inflammatory CAM phenotype. Fatp1B−/− chimeras fed a HFD gained more epididymal white adipose mass, which was inflamed and oxidatively stressed, compared to HFD-fed Fatp1B+/+ controls. Adipose tissue macrophages displayed a CAM-like phenotype in the absence of Fatp1. Conversely, functional overexpression of FATP1 decreased many aspects of glucose metabolism and diminished CAM-stimulated inflammation in vitro. FATP1 displayed acyl-CoA synthetase activity for long chain fatty acids in MΦs and modulated lipid mediator metabolism in MΦs. Conclusion Our findings provide evidence that FATP1 is a novel regulator of MΦ activation through control of substrate metabolism. Absence of FATP1 exacerbated pro-inflammatory activation in vitro and increased local and systemic components of the metabolic syndrome in HFD-fed Fatp1B−/− mice. In contrast, gain of FATP1 activity in MΦs suggested that Fatp1-mediated activation of fatty acids, substrate switch to glucose, oxidative stress, and lipid mediator synthesis are potential mechanisms. We demonstrate for the first time that FATP1 provides a unique mechanism by which the inflammatory tone of adipose and systemic metabolism may be regulated., Graphical abstract, Highlights • FATP1-mediated activation of fatty acids is a novel approach to limit inflammation. • Fatp1 deficiency primed macrophages for pro-inflammatory activation. • Lack of Fatp1 led to greater HFD-induced adipose inflammation. • Fatp1−/− adipose tissue macrophages were classically activated.

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique to detect genomic regions containing protein-DNA interaction, such as transcription factor binding sites or regions containing histone modifications. One goal of the analysis of ChIP-seq experiments is to identify genomic loci enriched for sequencing reads pertaining to DNA bound to the factor of interest. The accurate identification of such regions aids in the understanding of epigenomic marks and gene regulatory mechanisms. Given the reduction of massively parallel sequencing costs, methods to detect consensus regions of enrichment across multiple samples are of interest. Here, we present a statistical model to detect broad consensus regions of enrichment from ChIP-seq technical or biological replicates through a class of Zero-Inflated Mixed Effects Hidden Markov Models. We show that the proposed model outperforms existing methods for consensus peak calling in common epigenomic marks by accounting for the excess zeros and sample-specific biases. We apply our method to data from the Encyclopedia of DNA Elements (ENCODE) and Roadmap Epigenomics projects and also from an extensive simulation study.

Cisplatin is a major cancer chemotherapeutic drug. It kills cancer cells by damaging their DNA, mainly in the form of Pt-d(GpG) diadducts. However, it also has serious side effects, including nephrotoxicity and hepatotoxicity that limit its usefulness. Chronotherapy is taking circadian time into account during therapy to improve the therapeutic index, by improving efficacy and/or limiting toxicity. To this end, we tested the impact of clock time on excision repair of cisplatin-induced DNA damage at single-nucleotide resolution across the genome in mouse kidney and liver. We found that genome repair is controlled by two circadian programs. Repair of the transcribed strand (TS) of active, circadian-controlled genes is dictated by each gene's phase of transcription, which falls across the circadian cycle with prominent peaks at dawn and dusk. In contrast, repair of the nontranscribed strand (NTS) of all genes, repair of intergenic DNA, and global repair overall peaks at Zeitgeber time ZT08, as basal repair capacity, which is controlled by the circadian clock, peaks at this circadian time. Consequently, the TS and NTS of many genes are repaired out of phase. As most cancers are thought to have defective circadian rhythms, these results suggest that future research on timed dosage of cisplatin could potentially reduce damage to healthy tissue and improve its therapeutic index.

Background CALGB (Alliance)/SWOG 80405 was a randomized phase III trial that found no difference in overall survival (OS) in first-line mCRC Patients (pts) treated with bevacizumab (Bev) or cetuximab (Cet) combined with the same chemotherapy. By applying NGS to tumour DNA collected from pts in the trial, we aimed to discover novel mutated genes associated with differential response to therapy with the biologics and prognosis. Methods Primary tumour DNA from 520 pts was profiled for somatic gene mutations. Mutations in 426 genes were determined by NGS (FoundationOne). OS was the primary endpoint, both as a time-to-event and a categorical ( Results In Bev-treated pts, tumours with mutated IGF1 (6%) conferred worse OS than WT tumours (HR 2.8 [1.5-5.2], p = 0.0009), and tumours with mutated KDR (11%) conferred worse OS than WT tumours (OR 4.4 [1.6-12.3], p = 0.004). In Cet-treated pts, tumours with mutated FANCD2 (6%) conferred worse OS than WT tumours (HR 2.7 [1.4-5.3], p = 0.004), and tumours with mutated APC (74%) conferred better OS than WT tumours (OR 0.3 [0.1-0.6], p = 0.0009). RNF43-mutated tumours (12%) and SMARCA4-mutated tumours (6%) conferred worse OS in Cet-treated pts than Bev-treated pts (interaction p-value 0.002 and 0.0009, respectively). In all patients in the study, FANCI-mutated tumours (4%) conferred worse OS than WT tumours (HR 2.0 [1.2-3.3], p = 0.005; OR 5.0 [1.9-14.8], p = 0.002). The full set of NGS results will be presented, including the mutational map of tumour location. Conclusions To our knowledge, this is the largest and most comprehensive NGS analysis of somatic DNA mutations in tumours from mCRC pts treated with standard of care therapy. It provides novel gene candidates affecting response to Bev and Cet, each combined with chemotherapy, as well as mutated genes affecting the prognosis of patients. If validated in other phase III studies, these results may be used to guide treatment decisions in pts with mCRC. Clinical trial identification NCT00265850. Legal entity responsible for the study The authors. Funding U10CA180821, U10CA180882, U10CA180888 and U10CA180830 (SWOG); Genentech, Eli Lilly & Co., Pfizer, Sanofi. Disclosure X. Qu: Shareholder / Stockholder / Stock options, Full / Part-time employment: Genentech. M. Bertagnolli: Research grant / Funding (self): BMS; Research grant / Funding (self): Eli Lilly; Research grant / Funding (self): Roche; Research grant / Funding (self): Sanofi; Research grant / Funding (self): Pfizer; Research grant / Funding (self): Genentech. A. Venook: Research grant / Funding (self), travel: Genentech; Research grant / Funding (self): BMS; Travel / Accommodation / Expenses: Roche. O. Kabbarah: Full / Part-time employment: Genentech; Shareholder / Stockholder / Stock options: Roche. H.J. Lenz: Honoraria (self): Roche; Advisory / Consultancy: Roche; Travel / Accommodation / Expenses: Roche. All other authors have declared no conflicts of interest.

We have previously demonstrated that interleukin-17A (IL-17) producing T helper 17 cells are significantly elevated in blood and bone marrow (BM) in multiple myeloma (MM) and IL-17A promotes MM cell growth via the expression of IL-17 receptor. In this study, we evaluated anti-human IL-17A human monoclonal antibody (mAb), AIN457 in MM. We observe significant inhibition of MM cell growth by AIN457 both in the presence and the absence of BM stromal cells (BMSCs). Although IL-17A induces IL-6 production, AIN457 significantly downregulated IL-6 production and MM cell adhesion in MM-BMSC co-culture. AIN457 also significantly inhibited osteoclast cell differentiation. More importantly, in the SCIDhu model of human myeloma administration of AIN457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth and reduced bone damage compared with isotype control mice. To understand the mechanism of action of anti-IL-17A mAb, we report, here, that MM cells express IL-17A. We also observed that IL-17A knockdown inhibited MM cell growth and their ability to induce IL-6 production in co-cultures with BMSC. These pre-clinical observations suggest efficacy of AIN457 in myeloma and provide the rationale for its clinical evaluation for anti-myeloma effects and for improvement of bone disease.

Sequencing techniques have been widely used to assess gene expression (i.e., RNA-seq) or the presence of epigenetic features (e.g., DNase-seq to identify open chromatin regions). In contrast to traditional microarray platforms, sequencing data are typically summarized in the form of discrete counts, and they are able to delineate allele-specific signals, which are not available from microarrays. The presence of epigenetic features are often associated with gene expression, both of which have been shown to be affected by DNA polymorphisms. However, joint models with the flexibility to assess interactions between gene expression, epigenetic features and DNA polymorphisms are currently lacking. In this paper, we develop a statistical model to assess the associations between gene expression and epigenetic features using sequencing data, while explicitly modeling the effects of DNA polymorphisms in either an allele-specific or nonallele-specific manner. We show that in doing so we provide the flexibility to detect associations between gene expression and epigenetic features, as well as conditional associations given DNA polymorphisms. We evaluate the performance of our method using simulations and apply our method to study the association between gene expression and the presence of DNase I Hypersensitive sites (DHSs) in HapMap individuals. Our model can be generalized to exploring the relationships between DNA polymorphisms and any two types of sequencing experiments, a useful feature as the variety of sequencing experiments continue to expand.

Background Triple-negative breast cancer (TNBC), lacking expression of hormone and human epidermal growth factor receptor 2 receptors, is an aggressive subtype that frequently metastasizes to the brain and has no FDA-approved systemic therapies. Previous literature demonstrates mitogen-activated protein kinase kinase (MEK) pathway activation in TNBC brain metastases. Thus, we aimed to discover rational combinatorial therapies with MEK inhibition, hypothesizing that co-inhibition using clinically available brain-penetrant inhibitors would improve survival in preclinical models of TNBC brain metastases. Methods Using human-derived TNBC cell lines, synthetic lethal small interfering RNA kinase screens were evaluated with brain-penetrant inhibitors against MEK1/2 (selumetinib, AZD6244) or phosphatidylinositol-3 kinase (PI3K; buparlisib, BKM120). Mice bearing intracranial TNBC tumors (SUM149, MDA-MB-231Br, MDA-MB-468, or MDA-MB-436) were treated with MEK, PI3K, or platelet derived growth factor receptor (PDGFR; pazopanib) inhibitors alone or in combination. Tumors were analyzed by western blot and multiplexed kinase inhibitor beads/mass spectrometry to assess treatment effects. Results Screens identified MEK+PI3K and MEK+PDGFR inhibitors as tractable, rational combinations. Dual treatment of selumetinib with buparlisib or pazopanib was synergistic in TNBC cells in vitro. Both combinations improved survival in intracranial SUM149 and MDA-MB-231Br, but not MDA-MB-468 or MDA-MB-436. Treatments decreased mitogen-activated protein kinase (MAPK) and PI3K (Akt) signaling in sensitive (SUM149 and 231Br) but not resistant models (MDA-MB-468). Exploratory analysis of kinome reprogramming in SUM149 intracranial tumors after MEK ± PI3K inhibition demonstrates extensive kinome changes with treatment, especially in MAPK pathway members. Conclusions Results demonstrate that rational combinations of the clinically available inhibitors selumetinib with buparlisib or pazopanib may prove to be promising therapeutic strategies for the treatment of some TNBC brain metastases. Additionally, effective combination treatments cause widespread alterations in kinase pathways, including targetable potential resistance drivers.

Targeting the dysregulated BRAF–MEK–ERK pathway in cancer has increasingly emerged in clinical trial design. Despite clinical responses in specific cancers using inhibitors targeting BRAF and MEK, resistance develops often involving nongenomic adaptive bypass mechanisms. Inhibition of MEK1/2 by trametinib in patients with triple-negative breast cancer (TNBC) induced dramatic transcriptional responses, including upregulation of receptor tyrosine kinases (RTK) comparing tumor samples before and after one week of treatment. In preclinical models, MEK inhibition induced genome-wide enhancer formation involving the seeding of BRD4, MED1, H3K27 acetylation, and p300 that drives transcriptional adaptation. Inhibition of the P-TEFb–associated proteins BRD4 and CBP/p300 arrested enhancer seeding and RTK upregulation. BRD4 bromodomain inhibitors overcame trametinib resistance, producing sustained growth inhibition in cells, xenografts, and syngeneic mouse TNBC models. Pharmacologic targeting of P-TEFb members in conjunction with MEK inhibition by trametinib is an effective strategy to durably inhibit epigenomic remodeling required for adaptive resistance. Significance: Widespread transcriptional adaptation to pharmacologic MEK inhibition was observed in TNBC patient tumors. In preclinical models, MEK inhibition induces dramatic genome-wide modulation of chromatin, in the form of de novo enhancer formation and enhancer remodeling. Pharmacologic targeting of P-TEFb complex members at enhancers is an effective strategy to durably inhibit such adaptation. Cancer Discov; 7(3); 302–21. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 235

Human cytomegalovirus (HCMV) is a significant cause of disease in immune-compromised adults and immune naïve newborns. No vaccine exists to prevent HCMV infection, and current antiviral therapies have toxic side effects that limit the duration and intensity of their use. There is thus an urgent need for new strategies to treat HCMV infection. Repurposing existing drugs as antivirals is an attractive approach to limit the time and cost of new antiviral drug development. Virus-induced changes in infected cells are often driven by changes in cellular kinase activity, which led us to hypothesize that defining the complement of kinases (the kinome), whose abundance or expression is altered during infection would identify existing kinase inhibitors that could be repurposed as new antivirals. To this end, we applied a kinase capture technique, multiplexed kinase inhibitor bead-mass spectrometry (MIB-MS) kinome, to quantitatively measure perturbations in >240 cellular kinases simultaneously in cells infected with a laboratory-adapted (AD169) or clinical (TB40E) HCMV strain. MIB-MS profiling identified time-dependent increases and decreases in MIB binding of multiple kinases including cell cycle kinases, receptor tyrosine kinases, and mitotic kinases. Based on the kinome data, we tested the antiviral effects of kinase inhibitors and other compounds, several of which are in clinical use or development. Using a novel flow cytometry-based assay and a fluorescent reporter virus we identified three compounds that inhibited HCMV replication with IC50 values of 3 log decrease in virus replication. These results show the utility of MIB-MS kinome profiling for identifying existing kinase inhibitors that can potentially be repurposed as novel antiviral drugs.

The Cancer Genome Atlas (TCGA) identified four integrated clusters for endometrial cancer (EC): POLE, MSI, CNL and CNH. We evaluated differences in gene expression profiles of obese and non-obese women with EC and examined the association of body mass index (BMI) within the clusters identified in TCGA.TCGA RNAseq data was used to identify genes related to increasing BMI among ECs. The POLE, MSI and CNL clusters were composed mostly of endometrioid EC. Patient BMI was compared between these three clusters with one-way ANOVA. Association between gene expression and BMI was also assessed while adjusting for confounding effects of potential confounding factors. p-Values testing the association between gene expression and BMI were adjusted for multiple hypothesis testing over the 20,531 genes considered.Mean BMI was statistically different between the ECs in the CNL (35.8) versus POLE (29.8) cluster (p=0.006) and approached significance for the MSI (33.0) versus CNL (35.8) cluster (p=0.05). 181 genes were significantly up- or down-regulated with increasing BMI in endometrioid EC (q-value0.01), including LPL, IRS-1, IGFBP4, IGFBP7 and the progesterone receptor. DAVID functional annotation analysis revealed significant enrichment in "cell cycle" (adjusted p-value=1.5E-5) and "DNA metabolic processes" (adjusted p-value=1E-3) for the identified genes.Obesity related genes were found to be upregulated with increasing BMI among endometrioid ECs. Patients with POLE tumors have the lowest median BMI when compared to MSI and CNL. Given the heterogeneity among endometrioid EC, consideration should be given to abandoning the Type I and II classification of EC tumors.

Significance The mammalian circadian clock is controlled by a transcription-translation feedback loop consisting of transcriptional activators circadian locomotor output cycles kaput (CLOCK)–brain and muscle Arnt-like protein-1 (BMAL1), which function as a complex at E/E'-box elements, and repressors Cryptochrome 1 (CRY1)/CRY2 and PER1/PER2. CRYs repress upon binding as CRY–CLOCK–BMAL1–E-box complexes. Period proteins (PERs) repress by removing the heterotrimeric complexes from the E-box. We report here that in the Cry1 promoter, the CRY1–CLOCK–BMAL1–E-box complex represses a transcriptional activator acting in cis, and removal of the heterotrimeric complex by PER2 de-represses the transcriptional activator. ChIP-seq and RNA-seq experiments identified other genes also de-repressed by PER2. These data clarify the role of PER2 and reveal the level of complexity in regulation of Cry1 and other circadian-controlled genes.

Our recent ERK inhibitor analyses in pancreatic ductal adenocarcinoma (PDAC) demonstrated that MYC protein loss is a marker of inhibitor sensitivity, and indicated that resistant cell lines possess KRASdependent, ERK-independent mechanisms maintaining MYC protein levels. In seeking to identify these mechanisms, we first determined the signaling networks by which mutant KRAS regulates MYC. Acute KRAS suppression caused rapid proteasome-dependent loss of MYC protein, through both ERK1/2-dependent and -independent mechanisms. Surprisingly, this MYC degradation was independent of both PI3K-AKTGSK3β signaling and the E3 ligase FBWX7. We then established and applied a high-throughput screen for MYC protein degradation, and performed a kinome-wide proteomics screen. We identified an ERK1/2- inhibition-induced feed-forward mechanism dependent on EGFR and SRC, leading to ERK5 activation and phosphorylation of MYC at S62, preventing MYC degradation. We further determined that concurrent inhibition of ERK1/2 and ERK5 disrupted this feed-forward compensatory mechanism, synergistically causing loss of MYC and suppressing PDAC growth.

Multiple myeloma is an incurable plasma cell malignancy with a complex and incompletely understood molecular pathogenesis. Here we use whole-exome sequencing, copy-number profiling and cytogenetics to analyse 84 myeloma samples. Most cases have a complex subclonal structure and show clusters of subclonal variants, including subclonal driver mutations. Serial sampling reveals diverse patterns of clonal evolution, including linear evolution, differential clonal response and branching evolution. Diverse processes contribute to the mutational repertoire, including kataegis and somatic hypermutation, and their relative contribution changes over time. We find heterogeneity of mutational spectrum across samples, with few recurrent genes. We identify new candidate genes, including truncations of SP140, LTB, ROBO1 and clustered missense mutations in EGR1. The myeloma genome is heterogeneous across the cohort, and exhibits diversity in clonal admixture and in dynamics of evolution, which may impact prognostic stratification, therapeutic approaches and assessment of disease response to treatment., Multiple myeloma is a malignant plasma cell disorder with a complex molecular pathogenesis. Here, the authors perform whole-exome sequencing, copy-number profiling and cytogenetic analysis in 84 myeloma samples and highlight the diversity and evolution of the mutational profile underlying the disease.

The subcutaneous and systemic injection of serotonin reduces cutaneous and visceral pain thresholds and increases responses to noxious stimuli. Different subtypes of 5-hydroxytryptamine (5-HT) receptors are suggested to be associated with different types of pain responses. Here we show that serotonin also inhibits catechol Omethyltransferase (COMT), an enzyme that contributes to modultion the perception of pain, via non-competitive binding to the site bound by catechol substrates with a binding affinity comparable to the binding affinity of catechol itself ( Ki=44 μM). Using computational modeling, biochemical tests and cellular assays we show that serotonin actively competes with the methyl donor S-adenosyl-L-methionine (SAM) within the catalytic site. Binding of serotonin to the catalytic site inhibits the access of SAM, thus preventing methylation of COMT substrates. The results of in vivo animal studies show that serotonin-induced pain hypersensitivity in mice is reduced by either SAM pretreatment or by the combined administration of selective antagonists for β2- and β3-adrenergic receptors, which have been previously shown to mediate COMT-dependent pain signaling. Our results suggest that inhibition of COMT via serotonin binding contributes to pain hypersensitivity, providing additional strategies for the treatment of clinical pain conditions.

ZINBA (Zero-Inflated Negative Binomial Algorithm) identifies genomic regions enriched in a variety of ChIP-seq and related next-generation sequencing experiments (DNA-seq), calling both broad and narrow modes of enrichment across a range of signal-to-noise ratios. ZINBA models and accounts for factors that co-vary with background or experimental signal, such as G/C content, and identifies enrichment in genomes with complex local copy number variations. ZINBA provides a single unified framework for analyzing DNA-seq experiments in challenging genomic contexts. Software website: http://code.google.com/p/zinba/