Pre-encoded responsiveness to type I interferon in the peripheral immune system defines outcome of PD1 blockade therapy.

Type I interferons (IFN-Is) are central regulators of anti-tumor immunity and responses to immunotherapy, but they also drive the feedback inhibition underlying therapeutic resistance. In the present study, we developed a mass cytometry approach to quantify IFN-I-stimulated protein expression across immune cells and used multi-omics to uncover pre-therapy cellular states encoding responsiveness to inflammation. Analyzing peripheral blood cells from multiple cancer types revealed that differential responsiveness to IFN-Is before anti-programmed cell death protein 1 (PD1) treatment was highly predictive of long-term survival after therapy. Unexpectedly, IFN-I hyporesponsiveness efficiently predicted long-term survival, whereas high responsiveness to IFN-I was strongly associated with treatment failure and diminished survival time. Peripheral IFN-I responsive states were not associated with tumor inflammation, identifying a disconnect between systemic immune potential and 'cold' or 'hot' tumor states. Mechanistically, IFN-I responsiveness was epigenetically imprinted before therapy, poising cells for differential inflammatory responses and dysfunctional T cell effector programs. Thus, we identify physiological cell states with clinical importance that can predict success and long-term survival of PD1-blocking immunotherapy.

The transcription factor IRF2 drives interferon-mediated CD8+ T cell exhaustion to restrict anti-tumor immunity.

Type I and II interferons (IFNs) stimulate pro-inflammatory programs that are critical for immune activation, but also induce immune-suppressive feedback circuits that impede control of cancer growth. Here, we sought to determine how these opposing programs are differentially induced. We demonstrated that the transcription factor interferon regulatory factor 2 (IRF2) was expressed by many immune cells in the tumor in response to sustained IFN signaling. CD8+ T cell-specific deletion of IRF2 prevented acquisition of the T cell exhaustion program within the tumor and instead enabled sustained effector functions that promoted long-term tumor control and increased responsiveness to immune checkpoint and adoptive cell therapies. The long-term tumor control by IRF2-deficient CD8+ T cells required continuous integration of both IFN-I and IFN-II signals. Thus, IRF2 is a foundational feedback molecule that redirects IFN signals to suppress T cell responses and represents a potential target to enhance cancer control.

Tryptophan-derived microbial metabolites activate the aryl hydrocarbon receptor in tumor-associated macrophages to suppress anti-tumor immunity.

The aryl hydrocarbon receptor (AhR) is a sensor of products of tryptophan metabolism and a potent modulator of immunity. Here, we examined the impact of AhR in tumor-associated macrophage (TAM) function in pancreatic ductal adenocarcinoma (PDAC). TAMs exhibited high AhR activity and Ahr-deficient macrophages developed an inflammatory phenotype. Deletion of Ahr in myeloid cells or pharmacologic inhibition of AhR reduced PDAC growth, improved efficacy of immune checkpoint blockade, and increased intra-tumoral frequencies of IFNγ+CD8+ T cells. Macrophage tryptophan metabolism was not required for this effect. Rather, macrophage AhR activity was dependent on Lactobacillus metabolization of dietary tryptophan to indoles. Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα+IFNγ+CD8+ T cells; provision of dietary indoles blocked this effect. In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype, suggesting conservation of this regulatory axis in human disease.

KSR1 Knockout Mouse Model Demonstrates MAPK Pathway's Key Role in Cisplatin- and Noise-induced Hearing Loss.

Hearing loss is a major disability in everyday life and therapeutic interventions to protect hearing would benefit a large portion of the world population. Here we found that mice devoid of the protein kinase suppressor of RAS 1 (KSR1) in their tissues (germline KO mice) exhibit resistance to both cisplatin- and noise-induced permanent hearing loss compared with their wild-type KSR1 littermates. KSR1 is a scaffold protein that brings in proximity the mitogen-activated protein kinase (MAPK) proteins BRAF, MEK1/2 and ERK1/2 and assists in their activation through a phosphorylation cascade induced by both cisplatin and noise insults in the cochlear cells. KSR1, BRAF, MEK1/2, and ERK1/2 are all ubiquitously expressed in the cochlea. Deleting the KSR1 protein tempered down the MAPK phosphorylation cascade in the cochlear cells following both cisplatin and noise insults and conferred hearing protection of up to 30 dB SPL in three tested frequencies in male and female mice. Treatment with dabrafenib, an FDA-approved oral BRAF inhibitor, protected male and female KSR1 wild-type mice from both cisplatin- and noise-induced hearing loss. Dabrafenib treatment did not enhance the protection of KO KSR1 mice, providing evidence dabrafenib works primarily through the MAPK pathway. Thus, either elimination of the KSR1 gene expression or drug inhibition of the MAPK cellular pathway in mice resulted in profound protection from both cisplatin- and noise-induced hearing loss. Inhibition of the MAPK pathway, a cellular pathway that responds to damage in the cochlear cells, can prove a valuable strategy to protect and treat hearing loss.

Activating Mutations Affecting the Dbl Homology Domain of SOS2 Cause Noonan Syndrome.

The RASopathies constitute a family of autosomal-dominant disorders whose major features include facial dysmorphism, cardiac defects, reduced postnatal growth, variable cognitive deficits, ectodermal and skeletal anomalies, and susceptibility to certain malignancies. Noonan syndrome (NS), the commonest RASopathy, is genetically heterogeneous and caused by functional dysregulation of signal transducers and regulatory proteins with roles in the RAS/extracellular signal-regulated kinase (ERK) signal transduction pathway. Mutations in known disease genes account for approximately 80% of affected individuals. Here, we report that missense mutations altering Son of Sevenless, Drosophila, homolog 2 (SOS2), which encodes a RAS guanine nucleotide exchange factor, occur in a small percentage of subjects with NS. Four missense mutations were identified in five unrelated sporadic cases and families transmitting NS. Disease-causing mutations affected three conserved residues located in the Dbl homology (DH) domain, of which two are directly involved in the intramolecular binding network maintaining SOS2 in its autoinhibited conformation. All mutations were found to promote enhanced signaling from RAS to ERK. Similar to NS-causing SOS1 mutations, the phenotype associated with SOS2 defects is characterized by normal development and growth, as well as marked ectodermal involvement. Unlike SOS1 mutations, however, those in SOS2 are restricted to the DH domain.

Dabrafenib Alters MDSC Differentiation and Function by Activation of GCN2.

The effect of targeted therapeutics on anticancer immune responses is poorly understood. The BRAF inhibitor dabrafenib has been reported to activate the integrated stress response (ISR) kinase GCN2, and the therapeutic effect has been partially attributed to GCN2 activation. Because ISR signaling is a key component of myeloid-derived suppressor cell (MDSC) development and function, we measured the effect of dabrafenib on MDSC differentiation and suppressive activity. Our data showed that dabrafenib attenuated MDSC ability to suppress T-cell activity, which was associated with a GCN2-dependent block of the transition from monocytic progenitor to polymorphonuclear (PMN)-MDSCs and proliferative arrest resulting in PMN-MDSC loss. Transcriptional profiling revealed that dabrafenib-driven GCN2 activation altered metabolic features in MDSCs enhancing oxidative respiration, and attenuated transcriptional programs required for PMN development. Moreover, we observed a broad downregulation of transcriptional networks associated with PMN developmental pathways, and increased activity of transcriptional regulons driven by Atf5, Mafg, and Zbtb7a. This transcriptional program alteration underlies the basis for PMN-MDSC developmental arrest, skewing immature MDSC development toward monocytic lineage cells. In vivo, we observed a pronounced reduction in PMN-MDSCs in dabrafenib-treated tumor-bearing mice suggesting that dabrafenib impacts MDSC populations systemically and locally, in the tumor immune infiltrate. Thus, our data reveal transcriptional networks that govern MDSC developmental programs, and the impact of GCN2 stress signaling on the innate immune landscape in tumors, providing novel insight into potentially beneficial off-target effects of dabrafenib. SIGNIFICANCE: An important, but poorly understood, aspect of targeted therapeutics for cancer is the effect on antitumor immune responses. This article shows that off-target effects of dabrafenib activating the kinase GCN2 impact MDSC development and function reducing PMN-MDSCs in vitro and in vivo. This has important implications for our understanding of how this BRAF inhibitor impacts tumor growth and provides novel therapeutic target and combination possibilities.

An Immune Gene Expression Risk Score for Distant Metastases after Radiotherapy for Cervical Cancer.

PURPOSE: To develop an immune-based gene expression risk score to identify patients with cervical cancer at increased risk of distant metastases (DM). EXPERIMENTAL DESIGN: Tumor biopsies were obtained from 81 patients prior to chemoradiotherapy. Whole-transcriptome RNA sequencing was performed (Illumina NextSeq500). Beginning with 4,723 immune-related genes, a 55-gene risk score for DM was derived using Cox modeling and principal component analysis. It was validated in independent cohorts of 274 patients treated at the Norwegian Radium Hospital (NRH) and 206 patients from The Cancer Genome Atlas (TCGA). RESULTS: The risk score was predictive of DM (HR, 2.7; P < 0.0001) and lower cause-specific survival (CSS) by univariate analysis (HR, 2.0; P = 0.0003) and multivariate analysis adjusted for clinical factors (DM HR, 3.0; P < 0.0001; CSS HR, 2.2; P = 0.0004). The risk score predicted DM (HR, 1.4; P = 0.05) and CSS (HR, 1.48; P = 0.013) in the NRH cohort and CSS (HR, 1.4; P = 0.03) in TCGA cohort. Higher risk scores were associated with lower CIBERSORT estimates of tumor-infiltrating immune cells, including CD8 T cells and M1 and M2 macrophages (all P < 0.001). Higher risk scores were associated with lower expression (all P < 0.001) of important chemokines (CXCL12, CXCR4), IFN-regulated genes (IRF1, STAT1, IDO1), and immune checkpoint regulators (PD-1, PD-L1, CTLA-4). CONCLUSIONS: The immune metastatic risk score addresses important challenges in the treatment of cervical cancer-identifying patients at high risk of DM after radiotherapy. The findings of this study indicate that high tumor mutational burden and a "cold," immune-excluded tumor microenvironment influence distant metastatic recurrence. Further validation of the risk score is needed.

In silico transcriptome screens identify epidermal growth factor receptor inhibitors as therapeutics for noise-induced hearing loss.

Noise-induced hearing loss (NIHL) is a common sensorineural hearing impairment that lacks U.S. Food and Drug Administration-approved drugs. To fill the gap in effective screening models, we used an in silico transcriptome-based drug screening approach, identifying 22 biological pathways and 64 potential small molecule treatments for NIHL. Two of these, afatinib and zorifertinib [epidermal growth factor receptor (EGFR) inhibitors], showed efficacy in zebrafish and mouse models. Further tests with EGFR knockout mice and EGF-morpholino zebrafish confirmed their protective role against NIHL. Molecular studies in mice highlighted EGFR's crucial involvement in NIHL and the protective effect of zorifertinib. When given orally, zorifertinib was found in the perilymph with favorable pharmacokinetics. In addition, zorifertinib combined with AZD5438 (a cyclin-dependent kinase 2 inhibitor) synergistically prevented NIHL in zebrafish. Our results underscore the potential for in silico transcriptome-based drug screening in diseases lacking efficient models and suggest EGFR inhibitors as potential treatments for NIHL, meriting clinical trials.

Dabrafenib alters MDSC differentiation and function by activation of GCN2.

The effect of targeted therapeutics on anti-cancer immune responses is poorly understood. The BRAF inhibitor dabrafenib has been reported to activate the integrated stress response (ISR) kinase GCN2, and the therapeutic effect has been partially attributed to GCN2 activation. Since ISR signaling is a key component of myeloid-derived suppressor cell (MDSC) development and function, we measured the effect of dabrafenib on MDSC differentiation and suppressive activity. Our data showed that dabrafenib attenuated MDSC ability to suppress T cell activity, which was associated with a GCN2-dependent block of the transition from monocytic progenitor to polymorphonuclear (PMN)-MDSCs and proliferative arrest resulting in PMN-MDSC loss. Transcriptional profiling revealed that dabrafenib-driven GCN2 activation altered metabolic features in MDSCs enhancing oxidative respiration, and attenuated transcriptional programs required for PMN development. Moreover, we observed a broad downregulation of transcriptional networks associated with PMN developmental pathways, and increased activity of transcriptional regulons driven by Atf5 , Mafg , and Zbtb7a . This transcriptional program alteration underlies the basis for PMN-MDSC developmental arrest, skewing immature MDSC development towards monocytic lineage cells. In vivo , we observed a pronounced reduction in PMN-MDSCs in dabrafenib-treated tumor-bearing mice suggesting that dabrafenib impacts MDSC populations systemically and locally, in the tumor immune infiltrate. Thus, our data reveals transcriptional networks that govern MDSC developmental programs, and the impact of GCN2 stress signaling on the innate immune landscape in tumors, providing novel insight into potentially beneficial off target effects of dabrafenib.

KSR1 knockout mouse model demonstrates MAPK pathway's key role in cisplatin- and noise-induced hearing loss.

Hearing loss is a major disability in everyday life and therapeutic interventions to protect hearing would benefit a large portion of the world population. Here we found that mice devoid of the protein kinase suppressor of RAS 1 (KSR1) in their tissues (germline KO mice) exhibit resistance to both cisplatin- and noise-induced permanent hearing loss compared to their wild-type KSR1 littermates. KSR1 is expressed in the cochlea and is a scaffold protein that brings in proximity the mitogen-activated protein kinase (MAPK) proteins BRAF, MEK and ERK and assists in their activation through a phosphorylation cascade induced by both cisplatin and noise insults in the cochlear cells. Deleting the KSR1 protein tempered down the MAPK phosphorylation cascade in the cochlear cells following both cisplatin and noise insults and conferred hearing protection of up to 30 dB SPL in three tested frequencies in mice. Treatment with dabrafenib, an FDA-approved oral BRAF inhibitor, downregulated the MAPK kinase cascade and protected the KSR1 wild-type mice from both cisplatin- and noise-induced hearing loss. Dabrafenib treatment did not enhance the protection of KO KSR1 mice, as excepted, providing evidence dabrafenib works primarily through the MAPK pathway. Thus, either elimination of the KSR1 gene expression or drug inhibition of the MAPK cellular pathway in mice resulted in profound protection from both cisplatin- and noise-induce hearing loss. Inhibition of the MAPK pathway, a cellular pathway that responds to damage in the cochlear cells, can prove a valuable strategy to protect and treat hearing loss.

In Silico Transcriptome-based Screens Identify Epidermal Growth Factor Receptor Inhibitors as Therapeutics for Noise-induced Hearing Loss.

Noise-Induced Hearing Loss (NIHL) represents a widespread disease for which no therapeutics have been approved by the Food and Drug Administration (FDA). Addressing the conspicuous void of efficacious in vitro or animal models for high throughput pharmacological screening, we utilized an in silico transcriptome-oriented drug screening strategy, unveiling 22 biological pathways and 64 promising small molecule candidates for NIHL protection. Afatinib and zorifertinib, both inhibitors of the Epidermal Growth Factor Receptor (EGFR), were validated for their protective efficacy against NIHL in experimental zebrafish and murine models. This protective effect was further confirmed with EGFR conditional knockout mice and EGF knockdown zebrafish, both demonstrating protection against NIHL. Molecular analysis using Western blot and kinome signaling arrays on adult mouse cochlear lysates unveiled the intricate involvement of several signaling pathways, with particular emphasis on EGFR and its downstream pathways being modulated by noise exposure and Zorifertinib treatment. Administered orally, Zorifertinib was successfully detected in the perilymph fluid of the inner ear in mice with favorable pharmacokinetic attributes. Zorifertinib, in conjunction with AZD5438 - a potent inhibitor of cyclin dependent kinase 2 - produced synergistic protection against NIHL in the zebrafish model. Collectively, our findings underscore the potential application of in silico transcriptome-based drug screening for diseases bereft of efficient screening models and posit EGFR inhibitors as promising therapeutic agents warranting clinical exploration for combatting NIHL. Highlights: In silico transcriptome-based drug screens identify pathways and drugs against NIHL.EGFR signaling is activated by noise but reduced by zorifertinib in mouse cochleae.Afatinib, zorifertinib and EGFR knockout protect against NIHL in mice and zebrafish.Orally delivered zorifertinib has inner ear PK and synergizes with a CDK2 inhibitor.

Chronic cochlear implantation with and without electric stimulation in a mouse model induces robust cochlear influx of CX3CR1+/GFP macrophages.

BACKGROUND: Cochlear implantation is an effective auditory rehabilitation strategy for those with profound hearing loss, including those with residual low frequency hearing through use of hybrid cochlear implantation techniques. Post-mortem studies demonstrate the nearly ubiquitous presence of intracochlear fibrosis and neo-ossification following cochlear implantation. Current evidence suggests post-implantation intracochlear fibrosis is associated with delayed loss of residual acoustic hearing in hybrid cochlear implant (CI) recipients and may also negatively influence outcomes in traditional CI recipients. This study examined the contributions of surgical trauma, foreign body response and electric stimulation to intracochlear fibrosis and the innate immune response to cochlear implantation and the hierarchy of these contributions. METHODS: Normal hearing CX3CR1+/GFP mice underwent either round window opening (sham), acute CI insertion or chronic CI insertion with no, low- or high-level electric stimulation. Electric stimulation levels were based on neural response telemetry (NRT), beginning post-operative day 7 for 5 h per day. Subjects (n=3 per timepoint) were sacrificed at 4 h, 1,4,7,8,11,14 and 21 days. An unoperated group (n=3) served as controls. Cochleae were harvested at each time-point and prepared for immunohistochemistry with confocal imaging. The images were analyzed to obtain CX3CR1+ macrophage cell number and density in the lateral wall (LW), scala tympani (ST) and Rosenthal's canal (RC). RESULTS: A ST peri-implant cellular infiltrate and fibrosis occurred exclusively in the chronically implanted groups starting on day 7 with a concurrent infiltration of CX3CR1+ macrophages not seen in the other groups. CX3CR1+ macrophage infiltration was seen in the LW and RC in all experimental groups within the first week, being most prominent in the 3 chronically implanted groups during the second and third week. CONCLUSIONS: The cochlear immune response was most prominent in the presence of chronic cochlear implantation, regardless of electric stimulation level. Further, the development of intracochlear ST fibrosis was dependent on the presence of the indwelling CI foreign body. An innate immune response was evoked by surgical trauma alone (sham and acute CI groups) to a lesser degree. These data suggest that cochlear inflammation and intrascalar fibrosis after cochlear implantation are largely dependent on the presence of a chronic indwelling foreign body and are not critically dependent on electrical stimulation. Also, these data support a role for surgical trauma in inciting the initial innate immune response.

Cochlear implant material effects on inflammatory cell function and foreign body response.

OBJECTIVES: The objectives of this study were to assess the effects of cochlear implant (CI) biomaterials on the function of macrophages and fibroblasts, two key mediators of the foreign body response (FBR) and to determine how these materials influence fibrous tissue growth and new bone formation within the cochlea. METHODS: Macrophages and fibroblasts were cultured on polydimethylsiloxane (PDMS) and platinum substrates and human CI electrodes in vitro. Cell count, cell proliferation, cytokine production, and cell adhesion were measured. CI electrodes were implanted into murine cochleae for three weeks without electrical stimulation. Implanted cochleae were harvested for 3D X-ray microscopy with the CI left in-situ. The location of new bone growth within the scala tympani (ST) with reference to different portions of the implant (PDMS vs platinum) was quantified. RESULTS: Cell counts of macrophages and fibroblasts were significantly higher on platinum substrates and platinum contacts of CI electrodes. Fibroblast proliferation was greater on platinum relative to PDMS, and cells grown on platinum formed more/larger focal adhesions. 3D X-ray microscopy showed neo-ossification in the peri­implant areas of the ST. Volumetric quantification of neo-ossification showed a trend toward greater bone formation adjacent to the platinum electrodes compared to areas opposite or away from the platinum electrode bearing surfaces. CONCLUSIONS: Fibrotic reactions are biomaterial specific, as demonstrated by the differences in cell adhesion, proliferation, and fibrosis on platinum and PDMS. The inflammatory reaction to platinum contacts on CI electrodes likely contributes to fibrosis to a greater degree than PDMS, and platinum contacts may influence the deposition of new bone, as demonstrated in the in vivo data. This information can potentially be used to influence the design of future generations of neural prostheses.

Mutations in Noncoding Cis-Regulatory Elements Reveal Cancer Driver Cistromes in Luminal Breast Cancer.

Whole-genome sequencing of primary breast tumors enabled the identification of cancer driver genes and noncoding cancer driver plexuses from somatic mutations. However, differentiating driver from passenger events among noncoding genetic variants remains a challenge. Herein, we reveal cancer-driver cis-regulatory elements linked to transcription factors previously shown to be involved in development of luminal breast cancers by defining a tumor-enriched catalogue of approximately 100,000 unique cis-regulatory elements from 26 primary luminal estrogen receptor (ER)+ progesterone receptor (PR)+ breast tumors. Integrating this catalog with somatic mutations from 350 publicly available breast tumor whole genomes, we uncovered cancer driver cistromes, defined as the sum of binding sites for a transcription factor, for ten transcription factors in luminal breast cancer such as FOXA1 and ER, nine of which are essential for growth in breast cancer with four exclusive to the luminal subtype. Collectively, we present a strategy to find cancer driver cistromes relying on quantifying the enrichment of noncoding mutations over cis-regulatory elements concatenated into a functional unit. IMPLICATIONS: Mapping the accessible chromatin of luminal breast cancer led to discovery of an accumulation of mutations within cistromes of transcription factors essential to luminal breast cancer. This demonstrates coopting of regulatory networks to drive cancer and provides a framework to derive insight into the noncoding space of cancer.

An Integrative DNA Sequencing and Methylation Panel to Assess Mismatch Repair Deficiency.

Clinical testing for mismatch repair (MMR) deficiency often entails serial testing of tumor and constitutional DNA using multiple assays. To minimize cost and specimen requirements of MMR testing, we developed an integrated targeted sequencing protocol (termed MultiMMR) that tests for promoter methylation, mutations, copy number alterations, copy neutral loss of heterozygosity, and microsatellite instability from a single aliquot of DNA. Hybrid capture of DNA-sequencing libraries constructed with methylated adapters was performed on 142 samples (60 tumors and 82 constitutional samples) from 82 patients with MMR-associated colorectal, endometrial, and brain cancers as well as a synthetic DNA mix with 11 known mutations. The captured material was split to enable parallel bisulfite and conventional sequence analysis. The panel targeted microsatellite regions and 13 genes associated with MMR, hypermutation, and hereditary colorectal cancer. MultiMMR recapitulated clinical testing results in 23 of 24 cases, was able to explain MMR loss in an additional 29 of 48 patients with incomplete or inconclusive testing, and identified all 11 MMR variants within the synthetic DNA mix. Promoter methylation and microsatellite instability analysis found 95% and 97% concordance with clinical testing, respectively. We report the feasibility for amalgamation of the current stepwise and complex clinical testing workflow into an integrated test for hereditary and somatic causes of MMR deficiency.

Assessment of Genetic Drift in Large Pharmacogenomic Studies.

Genomic instability affects the reproducibility of experiments that rely on cancer cell lines. However, measuring the genomic integrity of these cells throughout a study is a costly endeavor that is commonly forgone. Here, we validate the identity of cancer cell lines in three pharmacogenomic studies and screen for genetic drift within and between datasets. Using SNP data from these datasets encompassing 1,497 unique cell lines and 63 unique pharmacological compounds, we show that genetic drift is widely prevalent in almost all cell lines with a median of 4.5%-6.1% of the total genome size drifted between any two isogenic cell lines. This study highlights the need for molecular profiling of cell lines to minimize the effects of passaging or misidentification in biomedical studies. We developed the CCLid web application, available at www.cclid.ca, to allow users to screen the genomic profiles of their cell lines against these datasets. A record of this paper's transparent peer review process is included in the Supplemental Information.

Rare driver mutations in head and neck squamous cell carcinomas converge on NOTCH signaling.

In most human cancers, only a few genes are mutated at high frequencies; most are mutated at low frequencies. The functional consequences of these recurrent but infrequent "long tail" mutations are often unknown. We focused on 484 long tail genes in head and neck squamous cell carcinoma (HNSCC) and used in vivo CRISPR to screen for genes that, upon mutation, trigger tumor development in mice. Of the 15 tumor-suppressor genes identified, ADAM10 and AJUBA suppressed HNSCC in a haploinsufficient manner by promoting NOTCH receptor signaling. ADAM10 and AJUBA mutations or monoallelic loss occur in 28% of human HNSCC cases and are mutually exclusive with NOTCH receptor mutations. Our results show that oncogenic mutations in 67% of human HNSCC cases converge onto the NOTCH signaling pathway, making NOTCH inactivation a hallmark of HNSCC.

Centromeric cohesion failure invokes a conserved choreography of chromosomal mis-segregations in pancreatic neuroendocrine tumor.

BACKGROUND: Pancreatic neuroendocrine tumors (PANETs) are rare, slow growing cancers that often present with local and distant metastasis upon detection. PANETS contain distinct karyotypes, epigenetic dysregulation, and recurrent mutations in MEN1, ATRX, and DAXX (MAD+); however, the molecular basis of disease progression remains uncharacterized. METHODS: We evaluated associations between aneuploidy and the MAD+ mutational state of 532 PANETs from 11 published genomic studies and 19 new cases using a combination of exome, targeted panel, shallow WGS, or RNA-seq. We mapped the molecular timing of MAD+ PANET progression using cellular fractions corrected for inferred tumor content. RESULTS: In 287 PANETs with mutational data, MAD+ tumors always exhibited a highly recurrent signature of loss of heterozygosity (LOH) and copy-number alterations affecting 11 chromosomes, typically followed by genome doubling upon metastasis. These LOH chromosomes substantially overlap with those that undergo non-random mis-segregation due to ectopic CENP-A localization to flanking centromeric regions in DAXX-depleted cell lines. Using expression data from 122 PANETs, we found decreased gene expression in the regions immediately adjacent to the centromere in MAD+ PANETs. Using 43 PANETs from AACR GENIE, we inferred this signature to be preceded by mutations in MEN1, ATRX, and DAXX. We conducted a meta-analysis on 226 PANETs from 8 CGH studies to show an association of this signature with metastatic incidence. Our study shows that MAD+ tumors are a genetically diverse and aggressive subtype of PANETs that display extensive chromosomal loss after MAD+ mutation, which is followed by genome doubling. CONCLUSIONS: We propose an evolutionary model for a subset of aggressive PANETs that is initiated by mutation of MEN1, ATRX, and DAXX, resulting in defects in centromere cohesion from ectopic CENP-A deposition that leads to selective loss of chromosomes and the LOH phenotype seen in late-stage metastatic PANETs. These insights aid in disease risk stratification and nominate potential therapeutic vulnerabilities to treat this disease.

Intracochlear fibrosis and the foreign body response to cochlear implant biomaterials.

OBJECTIVE: To report current knowledge on the topic of intracochlear fibrosis and the foreign body response following cochlear implantation (CI). METHODS: A literature search was performed in PubMed to identify peer-reviewed articles. Search components included "cochlear implant," "Foreign body response (FBR)," and "fibrosis." Original studies and review articles relevant to the topic were included. RESULTS: Ninety peer-reviewed articles describing the foreign body response or intracochlear fibrosis following CI were included. CONCLUSIONS: Intracochlear fibrosis following CI represents a significant limiting factor for the success of CI users. Several strategies have been employed to mitigate the foreign body response within the cochlea including drug delivery systems and modifications in surgical technique and electrode design. A better understanding of the FBR has the potential to improve CI outcomes and the next generation of cochlear prostheses.

A mouse model of cochlear implantation with chronic electric stimulation.

OBJECTIVES: Cochlear implants provide an effective treatment option for those with severe hearing loss, including those with preserved low frequency hearing. However, certain issues can reduce implant efficacy including intracochlear tissue response and delayed loss of residual acoustic hearing. We describe a mouse model of cochlear implantation with chronic electric stimulation that can be used to study cochlear implant biology and related pathologies. METHODS: Twelve normal hearing adult CBA/J mice underwent unilateral cochlear implantation and were evenly divided into one group receiving electric stimulation and one not. Serial impedance and neural response telemetry (NRT) measurements were made to assess implant functionality. Functionality was defined as having at least one electrode with an impedance ≤ 35 kOhms. Mouse cochleae were harvested for histology and 3D x-ray microscopy 21 days post-operatively, or, in case the implant was still functional, at a later time point when the implant failed. A separate experiment measured the hearing preservation rate in 7 adult CBA/J mice undergoing unilateral cochlear implantation with serial auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE). RESULTS: Implants maintained functionality for a mean of 35 days in the non-stimulated group and 19.8 days in the stimulated group. Reliable NRT and behavioral responses to electric stimulation were recorded. A robust intracochlear peri-implant tissue response with neo-ossification was seen in all cochleae. Six of seven mice maintained intact low frequency hearing up to 6 weeks following cochlear implantation. CONCLUSIONS: We demonstrate the feasibility of cochlear implantation and behaviorally significant electric stimulation in the mouse, with the potential for hearing preservation. This model may be combined with established mouse models of hearing loss and the large genetic and molecular research toolkit unique to the mouse for mechanistic and therapeutic investigations of cochlear implant biology.