Andrea L. Webber

Dual role of Nr2e3 in photoreceptor development and maintenance

Nr2e3 is a photoreceptor-specific nuclear receptor believed to play a role in photoreceptor development, differentiation, and survival. Much research has focused on the interaction of Nr2e3 with other transcription factors in determining the milieu of target gene expression in photoreceptors of the neonatal and adult retina. To investigate the downstream targets of Nr2e3 and thereby shed light on the functional pathways relevant to photoreceptor development and maintenance, expression profiling was performed on retinas from two different mouse knockout lines, one containing a targeted disruption of the Nr2e3 gene (Nr2e3 -/-), the other containing a spontaneous null allele of the Nr2e3 locus (rd7). Using whole genome microarrays, mRNA expression profiles of retinas from the two mutant strains were compared to those of wildtype C57BL/6 mice over a time course that ranged from postnatal day (p) 2 to 6months of age (p180). Additionally, expression profiling was performed on retinal explants treated with a putative NR2E3 agonist. The molecular profiling of Nr2e3 -/- and rd7/rd7 retinas identified 281 putative Nr2e3-dependent genes that were differentially expressed between wildtype and mutant retinas during at least one time point. Consistent with previous reports that Nr2e3 is necessary for the repression of cone-specific genes, increased expression of cone-specific genes was observed in the mutant samples, thereby providing proof-of-concept for the microarray screen. Further annotation of these data sets revealed ten predominant functional classes involved in the Nr2e3-mediated development and/or maintenance of photoreceptors. Interestingly, differences in the expression of Nr2e3-dependent genes exhibited two distinct temporal patterns. One group of genes showed a sustained difference in expression as compared to wildtype over the entire time course of the study, whereas a second group showed only transient differences which were largest around p10. Comparison of gene expression changes in Nr2e3 -/- and rd7/rd7 retinas with those uncovered by treating retinal explants with a putative NR2E3 agonist revealed four genes that were down-regulated in mutant retinas that lack Nr2e3 function but were up-regulated in agonist-treated explants. These results strongly suggest that the four genes may be direct targets of Nr2e3. Our identification of two sets of Nr2e3-regulated genes provides further evidence of a dual role for Nr2e3 in specification of photoreceptor fate during development as well as photoreceptor maintenance in the adult.

Pembrolizumab in patients with advanced hepatocellular carcinoma previously treated with sorafenib (KEYNOTE-224): a non-randomised, open-label phase 2 trial

BACKGROUNDnImmune checkpoint blockade therapy has shown promising results in patients with advanced hepatocellular carcinoma. We aimed to assess the efficacy and safety of pembrolizumab in this patient population.nnnMETHODSnKEYNOTE-224 is a non-randomised, multicentre, open-label, phase 2 trial that is set in 47 medical centres and hospitals across ten countries. Eligible patients had pathologically confirmed hepatocellular carcinoma; had previously been treated with sorafenib and were either intolerant to this treatment or showed radiographic progression of their disease after treatment; an Eastern Cooperative Oncology Group performance status of 0-1; adequate organ function, and were Child-Pugh class A. Participants received 200 mg pembrolizumab intravenously every 3 weeks for about 2 years or until disease progression, unacceptable toxicity, patient withdrawal, or investigator decision. The primary endpoint was objective response, defined as the proportion of patients with complete or partial response in all patients who received at least one dose of pembrolizumab, which was radiologically confirmed by use of the Response Evaluation Criteria in Solid Tumors version 1.1 by central review. Safety was also assessed in all treated patients. This trial is ongoing but closed to enrolment and is registered with ClinicalTrials.gov number NCT02702414.nnnFINDINGSnBetween June 7, 2016, and Feb 9, 2017, we screened 169 patients with advanced hepatocellular carcinoma, of whom 104 eligible patients were enrolled and treated. As of data cutoff on Feb 13, 2018, 17 (16%) patients were still receiving pembrolizumab. We recorded an objective response in 18 (17%; 95% CI 11-26) of 104 patients. The best overall responses were one (1%) complete and 17 (16%) partial responses; meanwhile, 46 (44%) patients had stable disease, 34 (33%) had progressive disease, and six (6%) patients who did not have a post-baseline assessment on the cutoff date were considered not to be assessable. Treatment-related adverse events occurred in 76 (73%) of 104 patients, which were serious in 16 (15%) patients. Grade 3 treatment-related events were reported in 25 (24%) of the 104 patients; the most common were increased aspartate aminotransferase concentration in seven (7%) patients, increased alanine aminotransferase concentration in four (4%) patients, and fatigue in four (4%) patients. One (1%) grade 4 treatment-related event of hyperbilirubinaemia occurred. One death associated with ulcerative oesophagitis was attributed to treatment. Immune-mediated hepatitis occurred in three (3%) patients, but there were no reported cases of viral flares.nnnINTERPRETATIONnPembrolizumab was effective and tolerable in patients with advanced hepatocellular carcinoma who had previously been treated with sorafenib. These results indicate that pembrolizumab might be a treatment option for these patients. This drug is undergoing further assessment in two phase 3, randomised trials as a second-line treatment in patients with hepatocellular carcinoma.nnnFUNDINGnMerck & Co, Inc.

Pharmacological Validation of Candidate Causal Sleep Genes Identified in an N2 Cross

Abstract: Despite the substantial impact of sleep disturbances on human health and the many years of study dedicated to understanding sleep pathologies, the underlying genetic mechanisms that govern sleep and wake largely remain unknown. Recently, the authors completed large-scale genetic and gene expression analyses in a segregating inbred mouse cross and identified candidate causal genes that regulate the mammalian sleep-wake cycle, across multiple traits including total sleep time, amounts of rapid eye movement (REM), non-REM, sleep bout duration, and sleep fragmentation. Here the authors describe a novel approach toward validating candidate causal genes, while also identifying potential targets for sleep-related indications. Select small-molecule antagonists and agonists were used to interrogate candidate causal gene function in rodent sleep polysomnography assays to determine impact on overall sleep architecture and to evaluate alignment with associated sleep-wake traits. Significant effects on sleep architecture were observed in validation studies using compounds targeting the muscarinic acetylcholine receptor M3 subunit (Chrm3) (wake promotion), nicotinic acetylcholine receptor alpha4 subunit (Chrna4) (wake promotion), dopamine receptor D5 subunit (Drd5) (sleep induction), serotonin 1D receptor (Htr1d) (altered REM fragmentation), glucagon-like peptide-1 receptor (Glp1r) (light sleep promotion and reduction of deep sleep), and calcium channel, voltage-dependent, T type, alpha 1I subunit (Cacna1i) (increased bout duration of slow wave sleep). Taken together, these results show the complexity of genetic components that regulate sleep-wake traits and highlight the importance of evaluating this complex behavior at a systems level. Pharmacological validation of genetically identified putative targets provides a rapid alternative to generating knock out or transgenic animal models, and may ultimately lead towards new therapeutic opportunities.

Pan-tumor genomic biomarkers for PD-1 checkpoint blockade–based immunotherapy

Mining immunotherapy clinical trials Clinical trial data can provide a wealth of information about how drugs work. Yet such information often belongs to pharmaceutical companies and is rarely accessible to the scientific community at large. Cristescu et al. provide exploratory analysis of a cancer genomics dataset, collected from four separate clinical trials of Mercks PD-1 immunotherapy drug, pembrolizumab. This informative public resource examines more than 300 patient samples representing 22 different tumor types. Two widely used signatures that currently predict immunotherapy response are tumor mutational burden and a “hot” T cell–inflamed microenvironment. The study analyzed these two proposed biomarkers in combination to see what predictive clinical utility they may hold. Science, this issue p. eaar3593 Genomic biomarkers will help to elucidate which cancer patients will benefit from PD-1 blockade immunotherapy. INTRODUCTION Immunotherapy targeting the programmed cell death protein–1 (PD-1) axis elicits durable antitumor responses in multiple cancer types. However, clinical responses vary, and biomarkers predictive of response may help to identify patients who will derive the greatest therapeutic benefit. Clinically validated biomarkers predictive of response to the anti–PD-1 monoclonal antibody pembrolizumab include PD-1 ligand 1 (PD-L1) expression in specific cancers and high microsatellite instability (MSI-H) regardless of tumor type. Tumor mutational burden (TMB) and T cell–inflamed gene expression profile (GEP) are emerging predictive biomarkers for pembrolizumab. Both PD-L1 and GEP are inflammatory biomarkers indicative of a T cell–inflamed tumor microenvironment (TME), whereas TMB and MSI-H are indirect measures of tumor antigenicity generated by somatic tumor mutations. However, the relationship between these two categories of biomarkers is not well characterized. RATIONALE This study assessed the potential for TMB and a T cell–inflamed GEP to jointly predict clinical response to pembrolizumab in >300 patient samples with advanced solid tumors and melanoma across 22 tumor types from four KEYNOTE clinical trials. To assess the individual and joint clinical utility of TMB and GEP, patients were stratified in four biomarker-defined clinical response groups [GEP low and TMB low (GEPlo TMBlo), GEP low and TMB high (GEPlo TMBhi), GEPhi TMBlo, and GEPhi TMBhi] based on predefined cutoffs for TMB and GEP. These patient-defined biomarker groups were further used to guide transcriptome and exome analyses of tumors in a large molecular database [The Cancer Genome Atlas (TCGA)] (n = 6384 tumors) to identify targetable patterns of biology that may modulate response and resistance. RESULTS TMB and GEP exhibited only modest correlation and were independently predictive of response across the KEYNOTE clinical datasets. We found that objective response rates were strongest in patients with GEPhi TMBhi (37 to 57%), moderate in those with GEPhi TMBlo (12 to 35%) and GEPlo TMBhi (11 to 42%), and reduced or absent in those with GEPlo TMBlo (0 to 9%) (see the figure). Additionally, longer progression-free survival times were seen in patients with higher levels of both TMB and GEP. Findings were comparable when TMB and PD-L1 expression were jointly assessed. Within TCGA database, GEP and TMB again had a low correlation, demonstrating the potential to jointly stratify transcriptomic and genomic features across cancer types. Specific gene expression patterns reflective of TME biology showed significant associations with TMB, GEP, or both. In particular, gene set enrichment analysis identified proliferative and stromal, myeloid, and vascular biology corresponding to specific TMB-defined subgroups within GEPhi tumors. In TMBhi tumors, indication-dependent somatic DNA alterations in key cancer driver genes showed a strong negative association with GEP. CONCLUSION This analysis shows that TMB and inflammatory biomarkers (T cell–inflamed GEP and PD-L1 expression) can jointly stratify human cancers into groups with different clinical responses to pembrolizumab monotherapy and identify patterns of underlying, targetable biology related to these groups. TMB and inflammatory biomarkers independently predict response and may capture distinct features of neoantigenicity and T cell activation, respectively. This approach may provide a precision medicine framework for rationally constructing and evaluating anti–PD-1– and/or –PD-L1–based combination therapy regimens. Biomarker-defined responses to pembrolizumab monotherapy identify targetable-resistance biology. (A) Tumors have low TMB and low neoantigenicity and lack a T cell–inflamed TME. (B) Tumors can evade the immune response despite high TMB and high neoantigenicity

Abstract LB-339: Biomarkers predictive of response to pembrolizumab in head and neck cancer (HNSCC)

Background: Biomarkers predictive of response to anti-PD-1 therapy include tumor mutational burden (TMB) and inflammatory biomarkers such as PD-L1 expression and T-cell activated gene expression profile (GEP). This study evaluated relationships between TMB, PD-L1 expression, and GEP and response to pembrolizumab in HNSCC patients in the KEYNOTE (KN)-012 and KN-55 trials. Methods: Data were combined from 261 HNSCC patients in KN-012 (NCT01848834, subsets of B1 [PD-L1 + , ≥1%, modified proportion score or interface pattern, QualTek IHC; n=34] and B2 [n=73] cohorts) and KN-055 (NCT02255097, platinum/cetuximab resistant, n=154) who had TMB data available by whole exome sequencing. Of these, 258 patients had PD-L1 data (PD-L1 IHC using 22C3 pharmDx, CPS readout) and 240 patients had GEP score (RNA analyzed on NanoString nCounter). HPV +/- status was assessed by p16 IHC and WES methods. Statistical testing of relationships between biomarkers and best overall response (BOR) by logistic regression and progression free survival (PFS) by Cox regression was prespecified and performed in a blinded manner. Results: TMB, PD-L1 CPS and GEP were each significantly associated with BOR in the overall population (p - and 80 HPV + patients. TMB and GEP were each highly associated with BOR regardless of HPV - (p=0.0034 and 0.0084) and HPV + (p=0.0446 and 0.0008) status, respectively. PD-L1 CPS showed similar associations with BOR in HPV - (p=0.0649) and HPV + (p=0.0003) patients. TMB showed no correlation with PD-L1 (r = -0.047) and GEP (r = -0.135); whereas PD-L1 CPS and GEP were moderately correlated (r = 0.469), consistent with an interferon-γ induced T-cell activated microenvironment including PD-L1 induction. TMB, PD-L1 CPS and GEP remained significantly predictive for BOR when assessed individually in joint models of TMB and PD-L1, and TMB and GEP (all p - (n=201) and HPV + (n=58) patients identified by p16 IHC, indicating the feasibility of using the WES method. Conclusion: TMB, PD-L1 and T-cell inflamed GEP were independently predictive of response to pembrolizumab in HNSCC patients, in general regardless of HPV status. When used alone or jointly, these biomarkers may have utility in characterizing responses to anti PD-1 therapies and novel cancer regimens in HNSCC. Citation Format: Tanguy Y. Seiwert, Robert Haddad, Joshua Bauml, Jared Weiss, David G. Pfister, Shilpa Gupta, Ranee Mehra, Iris Gluck, Hyunseok Kang, Francis Worden, J. Paul Eder, Makoto Tahara, Barbara Burtness, Stephen V. Liu, Andrea Webber, Lingkang Huang, Robin Mogg, Razvan Cristescu, Jonathan Cheng, Laura Q. Chow. Biomarkers predictive of response to pembrolizumab in head and neck cancer (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-339.

The Transcriptomic Signature Of Disease Development And Progression Of Nonalcoholic Fatty Liver Disease

A longitudinal molecular model of the development and progression of nonalcoholic fatty liver disease (NAFLD) over time is lacking. We have recently validated a high fat/sugar water-induced animal (an isogenic strain of C57BL/6u2009J:129S1/SvImJ mice) model of NAFLD that closely mimics most aspects of human disease. The hepatic transcriptome of such mice with fatty liver (8 weeks), steatohepatitis with early fibrosis (16–24 weeks) and advanced fibrosis (52 weeks) after initiation of the diet was evaluated and compared to mice on chow diet. Fatty liver development was associated with transcriptional activation of lipogenesis, FXR-RXR, PPAR-α mediated lipid oxidation and oxidative stress pathways. With progression to steatohepatitis, metabolic pathway activation persisted with additional activation of IL-1/inhibition of RXR, granulocyte diapedesis/adhesion, Fc macrophage activation, prothrombin activation and hepatic stellate cell activation. Progression to advanced fibrosis was associated with dampening of metabolic, oxidative stress and cell stress related pathway activation but with further Fc macrophage activation, cell death and turnover and activation of cancer-related networks. The molecular progression of NAFLD involves a metabolic perturbation which triggers subsequent cell stress and inflammation driving cell death and turnover. Over time, inflammation and fibrogenic pathways become dominant while in advanced disease an inflammatory-oncogenic profile dominates.

Identification of proximal biomarkers of PKC agonism and evaluation of their role in HIV reactivation.

Design: The HIV latent CD4+ T cell reservoir is broadly recognized as a barrier to HIV cure. Induction of HIV expression using protein kinase C (PKC) agonists is one approach under investigation for reactivation of latently infected CD4+ T cells (Beans et al., 2013; Abreu et al., 2014; Jiang et al., 2014; Jiang and Dandekar, 2015). We proposed that an increased understanding of the molecular mechanisms of action of PKC agonists was necessary to inform on biological signaling and pharmacodynamic biomarkers. RNA sequencing (RNA Seq) was applied to identify genes and pathways modulated by PKC agonists. Methods: Human CD4+ T cells were treated ex vivo with Phorbol 12‐myristate 13‐acetate, prostatin or ingenol‐3‐angelate. At 3 h and 24 h post‐treatment, cells were harvested and RNA‐Seq was performed on RNA isolated from cell lysates. The genes differentially expressed across the PKC agonists were validated by quantitative RT‐PCR (qPCR). A subset of genes was evaluated for their role in HIV reactivation using siRNA and CRISPR approaches in the Jurkat latency cell model. Results: Treatment of primary human CD4+ T cells with PKC agonists resulted in alterations in gene expression. qPCR of RNA Seq data confirmed upregulation of 24 genes, including CD69, Egr1, Egr2, Egr3, CSF2, DUSP5, and NR4A1. Gene knockdown of Egr1 and Egr3 resulted in reduced expression and decreased HIV reactivation in response to PKC agonist treatment, indicating a potential role for Egr family members in latency reversal. Conclusion: Overall, our results offer new insights into the mechanism of action of PKC agonists, biomarkers of pathway engagement, and the potential role of EGR family in HIV reactivation. HighlightsRNASeq of PKC agonists in human CD4 T cells reveals altered signalling pathways and time‐dependent changes in gene expression.Several biomarkers of PKC agonisms were identified and validated in human CD4+ T cells and in in vivo rat studies.Egr family members have a role in PKC mediated latent HIV‐1 reactivation.