Phenotypic and molecular states of IDH1 mutation-induced CD24-positive glioma stem-like cells.

Mutations in IDH1 and IDH2 drive the development of gliomas. These genetic alterations promote tumor cell renewal, disrupt differentiation states, and induce stem-like properties. Understanding how this phenotypic reprogramming occurs remains an area of high interest in glioma research. Previously, we showed that IDH mutation results in the development of a CD24-positive cell population in gliomas. Here, we demonstrate that this CD24-positive population possesses striking stem-like properties at the molecular and phenotypic levels. We found that CD24 expression is associated with stem-like features in IDH-mutant tumors, a patient-derived gliomasphere model, and a neural stem cell model of IDH1-mutant glioma. In orthotopic models, CD24-positive cells display enhanced tumor initiating potency compared to CD24-negative cells. Furthermore, CD24 knockdown results in changes in cell viability, proliferation rate, and gene expression that closely resemble a CD24-negative phenotype. Our data demonstrate that induction of a CD24-positive population is one mechanism by which IDH-mutant tumors acquire stem-like properties. These findings have significant implications for our understanding of the molecular underpinnings of IDH-mutant gliomas.

CD97 is a critical regulator of acute myeloid leukemia stem cell function.

Despite significant efforts to improve therapies for acute myeloid leukemia (AML), clinical outcomes remain poor. Understanding the mechanisms that regulate the development and maintenance of leukemic stem cells (LSCs) is important to reveal new therapeutic opportunities. We have identified CD97, a member of the adhesion class of G protein-coupled receptors (GPCRs), as a frequently up-regulated antigen on AML blasts that is a critical regulator of blast function. High levels of CD97 correlate with poor prognosis, and silencing of CD97 reduces disease aggressiveness in vivo. These phenotypes are due to CD97's ability to promote proliferation, survival, and the maintenance of the undifferentiated state in leukemic blasts. Collectively, our data credential CD97 as a promising therapeutic target on LSCs in AML.

Tobacco Smoking-Associated Alterations in the Immune Microenvironment of Squamous Cell Carcinomas.

Background: Tobacco smoking creates DNA damage, inducing mutations and potentially altering the tumor immune microenvironment. These types of genetic and immune microenvironment alterations are critical factors known to affect tumor response to immunotherapy. Here we analyze the association between the mutational signature of tobacco smoking, tumor mutational load, and metrics of immune activity in squamous cell carcinomas arising in the head and neck and lung. Methods: Using RNA and DNA sequencing data from The Cancer Genome Atlas head and neck (HNSC; n = 287) and lung (LUSC; n = 130) squamous cell carcinoma data sets and two independent gene expression data sets (HNSC, n = 136; LUSC, n = 75), we examined associations between the mutational smoking signature, mutation count, immune cell infiltration, cytolytic activity, and interferon-γ signaling. Results: An increasing mutational smoking signature was associated with statistically significantly increased overall mutational load in both HNSC (ρ = .33, P = 1.01 × 10-7) and LUSC (ρ = .49, P = 2.80 × 10-9). In HNSC, a higher mutational smoking signature was associated with lower levels of immune infiltration (ρ = -.37, P = 1.29 × 10-10), cytolytic activity (ρ = -.28, P = 4.07 × 10-6), and interferon-γ pathway signaling (ρ = .39, P = 3.20 × 10-11). In LUSC, these associations were reversed (ρ = .19, P = .03; ρ = .20, P = .02; and ρ = .18, P = .047, respectively). Differentially expressed genes between smoking-high and smoking-low tumors revealed broad tobacco-induced immunosuppression in HNSC, in contrast to a tumor-inflamed microenvironment in smokers with LUSC. Conclusions: In squamous cell carcinomas, the genetic smoking signature is associated with higher mutational load, but variable effects on tumor immunity can occur, depending on anatomic site. In HNSC, smoking is predominantly immunosuppressive; in LUSC, more pro-inflammatory. Both tumor mutation load and immune microenvironment affect clinical response to immunotherapy. Thus, the mutational smoking signature is likely to have relevance for immunotherapeutic investigation in smoking-associated cancers.

Multi-dimensional genomic analysis of myoepithelial carcinoma identifies prevalent oncogenic gene fusions.

Myoepithelial carcinoma (MECA) is an aggressive salivary gland cancer with largely unknown genetic features. Here we comprehensively analyze molecular alterations in 40 MECAs using integrated genomic analyses. We identify a low mutational load, and high prevalence (70%) of oncogenic gene fusions. Most fusions involve the PLAG1 oncogene, which is associated with PLAG1 overexpression. We find FGFR1-PLAG1 in seven (18%) cases, and the novel TGFBR3-PLAG1 fusion in six (15%) cases. TGFBR3-PLAG1 promotes a tumorigenic phenotype in vitro, and is absent in 723 other salivary gland tumors. Other novel PLAG1 fusions include ND4-PLAG1; a fusion between mitochondrial and nuclear DNA. We also identify higher number of copy number alterations as a risk factor for recurrence, independent of tumor stage at diagnosis. Our findings indicate that MECA is a fusion-driven disease, nominate TGFBR3-PLAG1 as a hallmark of MECA, and provide a framework for future diagnostic and therapeutic research in this lethal cancer.

Tumor and Microenvironment Evolution during Immunotherapy with Nivolumab.

The mechanisms by which immune checkpoint blockade modulates tumor evolution during therapy are unclear. We assessed genomic changes in tumors from 68 patients with advanced melanoma, who progressed on ipilimumab or were ipilimumab-naive, before and after nivolumab initiation (CA209-038 study). Tumors were analyzed by whole-exome, transcriptome, and/or T cell receptor (TCR) sequencing. In responding patients, mutation and neoantigen load were reduced from baseline, and analysis of intratumoral heterogeneity during therapy demonstrated differential clonal evolution within tumors and putative selection against neoantigenic mutations on-therapy. Transcriptome analyses before and during nivolumab therapy revealed increases in distinct immune cell subsets, activation of specific transcriptional networks, and upregulation of immune checkpoint genes that were more pronounced in patients with response. Temporal changes in intratumoral TCR repertoire revealed expansion of T cell clones in the setting of neoantigen loss. Comprehensive genomic profiling data in this study provide insight into nivolumab's mechanism of action.

NF-κB c-Rel Is Crucial for the Regulatory T Cell Immune Checkpoint in Cancer.

Regulatory T cells (Tregs) play a pivotal role in the inhibition of anti-tumor immune responses. Understanding the mechanisms governing Treg homeostasis may therefore be important for development of effective tumor immunotherapy. We have recently demonstrated a key role for the canonical nuclear factor κB (NF-κB) subunits, p65 and c-Rel, in Treg identity and function. In this report, we show that NF-κB c-Rel ablation specifically impairs the generation and maintenance of the activated Treg (aTreg) subset, which is known to be enriched at sites of tumors. Using mouse models, we demonstrate that melanoma growth is drastically reduced in mice lacking c-Rel, but not p65, in Tregs. Moreover, chemical inhibition of c-Rel function delayed melanoma growth by impairing aTreg-mediated immunosuppression and potentiated the effects of anti-PD-1 immunotherapy. Our studies therefore establish inhibition of NF-κB c-Rel as a viable therapeutic approach for enhancing checkpoint-targeting immunotherapy protocols.

An Integrated Systems Biology Approach Identifies TRIM25 as a Key Determinant of Breast Cancer Metastasis.

At the root of most fatal malignancies are aberrantly activated transcriptional networks that drive metastatic dissemination. Although individual metastasis-associated genes have been described, the complex regulatory networks presiding over the initiation and maintenance of metastatic tumors are still poorly understood. There is untapped value in identifying therapeutic targets that broadly govern coordinated transcriptional modules dictating metastatic progression. Here, we reverse engineered and interrogated a breast cancer-specific transcriptional interaction network (interactome) to define transcriptional control structures causally responsible for regulating genetic programs underlying breast cancer metastasis in individual patients. Our analyses confirmed established pro-metastatic transcription factors, and they uncovered TRIM25 as a key regulator of metastasis-related transcriptional programs. Further, in vivo analyses established TRIM25 as a potent regulator of metastatic disease and poor survival outcome. Our findings suggest that identifying and targeting keystone proteins, like TRIM25, can effectively collapse transcriptional hierarchies necessary for metastasis formation, thus representing an innovative cancer intervention strategy.

Bi-allelic alterations in DNA repair genes underpin homologous recombination DNA repair defects in breast cancer.

Homologous recombination (HR) DNA repair-deficient (HRD) breast cancers have been shown to be sensitive to DNA repair targeted therapies. Burgeoning evidence suggests that sporadic breast cancers, lacking germline BRCA1/BRCA2 mutations, may also be HRD. We developed a functional ex vivo RAD51-based test to identify HRD primary breast cancers. An integrated approach examining methylation, gene expression, and whole-exome sequencing was employed to ascertain the aetiology of HRD. Functional HRD breast cancers displayed genomic features of lack of competent HR, including large-scale state transitions and specific mutational signatures. Somatic and/or germline genetic alterations resulting in bi-allelic loss-of-function of HR genes underpinned functional HRD in 89% of cases, and were observed in only one of the 15 HR-proficient samples tested. These findings indicate the importance of a comprehensive genetic assessment of bi-allelic alterations in the HR pathway to deliver a precision medicine-based approach to select patients for therapies targeting tumour-specific DNA repair defects. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The head and neck cancer immune landscape and its immunotherapeutic implications.

Recent clinical trials have demonstrated a clear survival advantage in advanced head and neck squamous cell carcinoma (HNSCC) patients treated with immune checkpoint blockade. These emerging results reveal that HNSCC is one of the most promising frontiers for immunotherapy research. However, further progress in head and neck immuno-oncology will require a detailed understanding of the immune infiltrative landscape found in these tumors. We leveraged transcriptome data from 280 tumors profiled by The Cancer Genome Atlas (TCGA) to comprehensively characterize the immune landscape of HNSCC in order to develop a rationale for immunotherapeutic strategies in HNSCC and guide clinical investigation. We find that both HPV+ and HPV- HNSCC tumors are among the most highly immune-infiltrated cancer types. Strikingly, HNSCC had the highest median Treg/CD8+ T cell ratio and the highest levels of CD56dim NK cell infiltration, in our pan-cancer analysis of the most immune-infiltrated tumors. CD8+ T cell infiltration and CD56dim NK cell infiltration each correlated with superior survival in HNSCC. Tumors harboring genetic smoking signatures had lower immune infiltration and were associated with poorer survival, suggesting these patients may benefit from immune agonist therapy. These findings illuminate the immune landscape of HPV+ and HPV- HNSCC. Additionally, this landscape provides a potentially novel rationale for investigation of agents targeting modulators of Tregs (e.g., CTLA-4, GITR, ICOS, IDO, and VEGFA) and NK cells (e.g., KIR, TIGIT, and 4-1BB) as adjuncts to anti-PD-1 in the treatment of advanced HNSCC.

Recurrent SERPINB3 and SERPINB4 mutations in patients who respond to anti-CTLA4 immunotherapy.

Immune checkpoint blockade has shown significant promise as an anticancer treatment, yet the determinants of response are not completely understood. Here we show that somatic mutations in SERPINB3 and SERPINB4 are associated with survival after anti-CTLA4 immunotherapy in two independent cohorts of patients with melanoma (n = 174). Interestingly, serpins are homologs of the well-known ovalbumin antigen and are associated with autoimmunity. Our findings have implications for the personalization of immunotherapy.

Integrated Genomics for Pinpointing Survival Loci within Arm-Level Somatic Copy Number Alterations.

The identification of driver loci underlying arm-level somatic copy number alterations (SCNAs) in cancer has remained challenging and incomplete. Here, we assess the relative impact and present a detailed landscape of arm-level SCNAs in 10,985 patient samples across 33 cancer types from The Cancer Genome Atlas (TCGA). Furthermore, using chromosome 9p loss in lower grade glioma (LGG) as a model, we employ a unique multi-tiered genomic dissection strategy using 540 patients from three independent LGG datasets to identify genetic loci that govern tumor aggressiveness and poor survival. This comprehensive approach uncovered several 9p loss-specific prognostic markers, validated existing ones, and redefined the impact of CDKN2A loss in LGG.

Comprehensive Molecular Characterization of Salivary Duct Carcinoma Reveals Actionable Targets and Similarity to Apocrine Breast Cancer.

PURPOSE: Salivary duct carcinoma (SDC) is an aggressive salivary malignancy, which is resistant to chemotherapy and has high mortality rates. We investigated the molecular landscape of SDC, focusing on genetic alterations and gene expression profiles. EXPERIMENTAL DESIGN: We performed whole-exome sequencing, RNA sequencing, and immunohistochemical analyses in 16 SDC tumors and examined selected alterations via targeted sequencing of 410 genes in a second cohort of 15 SDCs. RESULTS: SDCs harbored a higher mutational burden than many other salivary carcinomas (1.7 mutations/Mb). The most frequent genetic alterations were mutations in TP53 (55%), HRAS (23%), PIK3CA (23%), and amplification of ERBB2 (35%). Most (74%) tumors had alterations in either MAPK (BRAF/HRAS/NF1) genes or ERBB2 Potentially targetable alterations based on supportive clinical evidence were present in 61% of tumors. Androgen receptor (AR) was overexpressed in 75%; several potential resistance mechanisms to androgen deprivation therapy (ADT) were identified, including the AR-V7 splice variant (present in 50%, often at low ratios compared with full-length AR) and FOXA1 mutations (10%). Consensus clustering and pathway analyses in transcriptome data revealed striking similarities between SDC and molecular apocrine breast cancer. CONCLUSIONS: This study illuminates the landscape of genetic alterations and gene expression programs in SDC, identifying numerous molecular targets and potential determinants of response to AR antagonism. This has relevance for emerging clinical studies of ADT and other targeted therapies in SDC. The similarities between SDC and apocrine breast cancer indicate that clinical data in breast cancer may generate useful hypotheses for SDC. Clin Cancer Res; 22(18); 4623-33. ©2016 AACR.

The role of neoantigens in response to immune checkpoint blockade.

Immune checkpoint blockade has demonstrated substantial promise for the treatment of several advanced malignancies. These agents activate the immune system to attack tumor cells. For example, agents targeting CTLA4 and programmed cell death 1 (PD-1) have resulted in impressive response rates and, in some cases, durable remissions. Neoantigens are mutations that encode immunologically active proteins that can cause the immune system to recognize the affected cell as foreign. Recent data have made it clear that these mutations are, in large part, the functional targets of immune checkpoint blockade. This review summarizes the key discoveries leading up to this important conclusion and discusses possible applications of neoantigens in cancer therapy.

Pan-cancer analysis of intratumor heterogeneity as a prognostic determinant of survival.

As tumors accumulate genetic alterations, an evolutionary process occurs in which genetically distinct subclonal populations of cells co-exist, resulting in intratumor genetic heterogeneity (ITH). The clinical implications of ITH remain poorly defined. Data are limited with respect to whether ITH is an independent determinant of patient survival outcomes, across different cancer types. Here, we report the results of a pan-cancer analysis of over 3300 tumors, showing a varied landscape of ITH across 9 cancer types. While some gene mutations are subclonal, the majority of driver gene mutations are clonal events, present in nearly all cancer cells. Strikingly, high levels of ITH are associated with poorer survival across diverse types of cancer. The adverse impact of high ITH is independent of other clinical, pathologic and molecular factors. High ITH tends to be associated with lower levels of tumor-infiltrating immune cells, but this association is not able to explain the observed survival differences. Together, these data show that ITH is a prognostic marker in multiple cancers. These results illuminate the natural history of cancer evolution, indicating that tumor heterogeneity represents a significant obstacle to cancer control.

Cancer Neoantigens and Applications for Immunotherapy.

Recent advances in immune checkpoint blockade therapy have revolutionized the treatment of cancer. Tumor-specific antigens that are generated by somatic mutation, neoantigens, can influence patient response to immunotherapy and contribute to tumor shrinkage. Recent evidence demonstrating the success of checkpoint blockade immunotherapy in boosting T-cell reactivity against patient-specific neoantigens constitutes a strong rationale for the development of personalized vaccines against these nonself peptides. With the decreasing cost of next-generation sequencing, peptide manufacturing, and improvement of in silico prediction of peptide immunogenicity, it is increasingly important to evaluate the potential use of neoantigens in both diagnosis and treatment. Specifically, these neoantigens could be useful both as predictors of immune checkpoint blockade therapy response and/or incorporated in therapeutic vaccination strategies.

Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses.

We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model.

Diet-induced hypoxia responsive element demethylation increases CEACAM6 expression, favouring Crohn's disease-associated Escherichia coli colonisation.

OBJECTIVE: Adherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohn's disease (CD) ileal mucosa. AIEC strains adhere to enterocytes via interaction between type 1 pili and CEACAM6 receptors abnormally expressed on CD ileal mucosa, leading to gut inflammation. We analysed whether epigenetic mechanisms are involved in the upregulation of CEACAM6 expression in intestinal epithelial cells (IECs). DESIGN: Methylation of CEACAM6 promoter was analysed using bisulfite sequencing and site-specific methylation by SnapShot. pCpGfree reporter system was used to analyse CEACAM6 promoter activity. Transgenic mice expressing human CEACAM6 fed either standard food or a low-methyl diet (LMD) were orally challenged with 10(9) AIEC LF82. After 3 days, gut-associated AIEC and proinflammatory cytokines were quantified. RESULTS: Analysis of CEACAM6 gene promoter revealed potentially methylated dinucleotide CpGs within HIF-1-responsive elements (HREs). Methylation levels of CpG within CEACAM6 promoter were inversely correlated with CEACAM6 expression in IEC expressing various levels of CEACAM6. We show the critical role of HRE methylation and transcription factor HIF-1 in the regulation of CEACAM6 gene in IEC. This was confirmed in transgenic mice expressing human CEACAM6 fed a LMD. LMD-dependent HRE demethylation led to abnormal gut expression of CEACAM6, favouring AIEC colonisation and subsequent inflammation. CONCLUSIONS: HRE hypomethylation in CEACAM6 promoter correlates with high expression in IEC. Our findings suggest that abnormal DNA methylation leading to CEACAM6 increased expression and AIEC-mediated gut inflammation can be related to changes in nutritional habits, such as low intake in methyl donor molecules, leading to abnormal epigenetic marks in mouse model mimicking CD susceptibility.

Genetic basis for clinical response to CTLA-4 blockade in melanoma.

BACKGROUND: Immune checkpoint inhibitors are effective cancer treatments, but molecular determinants of clinical benefit are unknown. Ipilimumab and tremelimumab are antibodies against cytotoxic T-lymphocyte antigen 4 (CTLA-4). Anti-CTLA-4 treatment prolongs overall survival in patients with melanoma. CTLA-4 blockade activates T cells and enables them to destroy tumor cells. METHODS: We obtained tumor tissue from patients with melanoma who were treated with ipilimumab or tremelimumab. Whole-exome sequencing was performed on tumors and matched blood samples. Somatic mutations and candidate neoantigens generated from these mutations were characterized. Neoantigen peptides were tested for the ability to activate lymphocytes from ipilimumab-treated patients. RESULTS: Malignant melanoma exomes from 64 patients treated with CTLA-4 blockade were characterized with the use of massively parallel sequencing. A discovery set consisted of 11 patients who derived a long-term clinical benefit and 14 patients who derived a minimal benefit or no benefit. Mutational load was associated with the degree of clinical benefit (P=0.01) but alone was not sufficient to predict benefit. Using genomewide somatic neoepitope analysis and patient-specific HLA typing, we identified candidate tumor neoantigens for each patient. We elucidated a neoantigen landscape that is specifically present in tumors with a strong response to CTLA-4 blockade. We validated this signature in a second set of 39 patients with melanoma who were treated with anti-CTLA-4 antibodies. Predicted neoantigens activated T cells from the patients treated with ipilimumab. CONCLUSIONS: These findings define a genetic basis for benefit from CTLA-4 blockade in melanoma and provide a rationale for examining exomes of patients for whom anti-CTLA-4 agents are being considered. (Funded by the Frederick Adler Fund and others.).