Irene Cherni

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.

STAT3 is activated by JAK2 independent of key oncogenic driver mutations in non-small cell lung carcinoma.

Constitutive activation of STAT3 is a common feature in many solid tumors including non-small cell lung carcinoma (NSCLC). While activation of STAT3 is commonly achieved by somatic mutations to JAK2 in hematologic malignancies, similar mutations are not often found in solid tumors. Previous work has instead suggested that STAT3 activation in solid tumors is more commonly induced by hyperactive growth factor receptors or autocrine cytokine signaling. The interplay between STAT3 activation and other well-characterized oncogenic “driver” mutations in NSCLC has not been fully characterized, though constitutive STAT3 activation has been proposed to play an important role in resistance to various small-molecule therapies that target these oncogenes. In this study we demonstrate that STAT3 is constitutively activated in human NSCLC samples and in a variety of NSCLC lines independent of activating KRAS or tyrosine kinase mutations. We further show that genetic or pharmacologic inhibition of the gp130/JAK2 signaling pathway disrupts activation of STAT3. Interestingly, treatment of NSCLC cells with the JAK1/2 inhibitor ruxolitinib has no effect on cell proliferation and viability in two-dimensional culture, but inhibits growth in soft agar and xenograft assays. These data demonstrate that JAK2/STAT3 signaling operates independent of known driver mutations in NSCLC and plays critical roles in tumor cell behavior that may not be effectively inhibited by drugs that selectively target these driver mutations.

Plant-derived human butyrylcholinesterase, but not an organophosphorous-compound hydrolyzing variant thereof, protects rodents against nerve agents

The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.

Biochemical and immunological characterization of the plant-derived candidate human immunodeficiency virus type 1 mucosal vaccine CTB-MPR649-684.

Plants are potentially the most economical platforms for the large-scale production of recombinant proteins. Thus, plant-based expression of subunit human immunodeficiency virus type 1 (HIV-1) vaccines provides an opportunity for their global use against the acquired immunodeficiency syndrome pandemic. CTB-MPR(649-684)[CTB, cholera toxin B subunit; MPR, membrane proximal (ectodomain) region of gp41] is an HIV-1 vaccine candidate that has been shown previously to induce antibodies that block a pathway of HIV-1 mucosal transmission. In this article, the molecular characterization of CTB-MPR(649-684) expressed in transgenic Nicotiana benthamiana plants is reported. Virtually all of the CTB-MPR(649-684) proteins expressed in the selected line were shown to have assembled into pentameric, GM1 ganglioside-binding complexes. Detailed biochemical analyses on the purified protein revealed that it was N-glycosylated, predominantly with high-mannose-type glycans (more than 75%), as predicted from a consensus asparagine-X-serine/threonine (Asn-X-Ser/Thr) N-glycosylation sequon on the CTB domain and an endoplasmic reticulum retention signal attached at the C-terminus of the fusion protein. Despite this modification, the plant-expressed protein retained the nanomolar affinity to GM1 ganglioside and the critical antigenicity of the MPR(649-684) moiety. Furthermore, the protein induced mucosal and serum anti-MPR(649-684) antibodies in mice after mucosal prime-systemic boost immunization. Our data indicate that plant-based expression can be a viable alternative for the production of this subunit HIV-1 vaccine candidate.

Transgenic plants as a source for the bioscavenging enzyme, human butyrylcholinesterase.

Organophosphorous pesticides and nerve agents inhibit the enzyme acetylcholinesterase at neuronal synapses and in neuromuscular junctions. The resulting accumulation of acetylcholine overwhelms regulatory mechanisms, potentially leading to seizures and death from respiratory collapse. While current therapies are only capable of reducing mortality, elevation of the serum levels of the related enzyme butyrylcholinesterase (BChE) by application of the purified protein as a bioscavenger of organophosphorous compounds is effective in preventing all symptoms associated with poisoning by these toxins. However, BChE therapy requires large quantities of enzyme that can easily overwhelm current sources. Here, we report genetic optimization, cloning and high-level expression of human BChE in plants. Plant-derived BChE is shown to be biochemically similar to human plasma-derived BChE in terms of catalytic activity and inhibitor binding. We further demonstrate the ability of the plant-derived bioscavenger to protect animals against an organophosphorous pesticide challenge.

Plant-derived human acetylcholinesterase-R provides protection from lethal organophosphate poisoning and its chronic aftermath

Therapeutically valuable proteins are often rare and/or unstable in their natural context, calling for production solutions in heterologous systems. A relevant example is that of the stress‐induced, normally rare, and naturally unstable “read‐through” human acetylcho‐linesterase variant, AChE‐R. AChE‐R shares its active site with the synaptic AChE‐S variant, which is the target of poisonous organophosphate anticholinesterase insecticides such as the parathion metabolite paraoxon. Inherent AChE‐R overproduction under organophosphate intoxication confers both short‐term protection (as a bioscavenger) and long‐term neuromuscular damages (as a regulator). Here we report the purification, characterization, and testing of human, endoplasmic reticulum‐retained AChE‐RER produced from plant‐optimized cDNA in Nicotiana benthamiana plants. AChE‐RER purified to homogeneity showed indistinguishable biochemical properties, with IC50 = 10 −7 M for the organophosphate paraoxon, similar to mammalian cell culture‐derived AChE. In vivo titration showed dose‐dependent protection by intravenously injected AChE‐RER of FVB/N male mice challenged with a lethal dose of paraoxon, with complete elimination of short‐term clinical symptoms at near molar equivalence. By 10 days postexposure, AChE‐R prophylaxis markedly limited postexposure increases in plasma murine AChE‐R levels while minimizing the organophos‐phate‐induced neuromuscular junction dismorphology. Our findings present plant‐produced AChE‐RER as a bimodal agent, conferring both short‐ and long‐term protection from organophosphate intoxication.—Evron T., Geyer, B. C., Cherni, I., Muralidharan, M., Kilbourne, J., Fletcher, S. P., Soreq, H., Mor T. S. Plant‐derived human acetylcholinesterase‐R provides protection from lethal organophosphate poisoning and its chronic aftermath. FASEB J. 21, 2961–2969 (2007)

Hedgehog signaling pathway molecules and ALDH1A1 expression in early-stage non-small cell lung cancer

INTRODUCTION The Hedgehog Signaling Pathway (HHSP) has been implicated in the development of multiple cancers. HHSP activation may primarily be hedgehog ligand-dependent in non-small cell lung cancer (NSCLC); while a subset may be ligand-independent. In this study NSCLC primary tumors were used to identify correlations between multiple biomarkers thought to be involved in the HHSP and the clinical outcomes of patients with NSCLC. Identification of such correlations could be used to aid in NSCLC treatment and predicting patient prognosis. METHODS A tissue microarray representing 248 clinically annotated stage I-II NSCLC cases was stained using immunohistochemistry (IHC) and scored for HHSP proteins namely, SHH, PTCH1, SMO, GLI1, and GLI2; as well as, ALDH1A1, a putative cancer stem cell marker. Data was analyzed for correlation between IHC staining, EGFR and KRAS mutations, and clinical characteristics including relapse-free survival (RFS) and overall survival (OS). RESULTS In adenocarcinoma, there were significant correlations of IHC expression between SHH and downstream HHSP receptor SMO (p=0.017) and transcription factor GLI1 (p=0.001), while SMO correlated with GLI1 (p=0.007). In squamous cell carcinoma, SHH significantly correlated with GLI2 protein expression (p=0.003). After multiple testing correction, there was no significant correlation between any of the six markers and RFS or OS. CONCLUSIONS Key downstream components of the HHSP show correlation with sonic hedgehog ligand (SHH) expression, suggesting that ligand-dependent signaling is more prevalent in primary NSCLC tumors. Surprisingly, in early-stage NSCLC, there were no significant correlations between HHSP proteins or ALDH1A1 and RFS or OS.

miRNAs in lung cancer: large roles for small players

miRNAs play an important role in the regulation of a wide assortment of cellular processes by sequestering target mRNAs and inhibiting translation of the proteins that they encode. Multiple miRNAs can regulate single mRNA molecules and, alternatively, a single miRNA can act on a number of mRNA targets. Dysfunctional miRNAs are commonly found in a variety of solid cancers and are attractive candidates for next-generation therapeutics. This article highlights miRNA signatures proposed for lung cancer classification and diagnosis, chemo- and radio-therapy resistance, metastasis and prediction of treatment outcome and survival.

Paired Tumor and Normal Whole Genome Sequencing of Metastatic Olfactory Neuroblastoma

Background Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal tract with little molecular characterization. We performed whole genome sequencing (WGS) on paired normal and tumor DNA from a patient with metastatic-ONB to identify the somatic alterations that might be drivers of tumorigenesis and/or metastatic progression. Methodology/Principal Findings Genomic DNA was isolated from fresh frozen tissue from a metastatic lesion and whole blood, followed by WGS at >30X depth, alignment and mapping, and mutation analyses. Sanger sequencing was used to confirm selected mutations. Sixty-two somatic short nucleotide variants (SNVs) and five deletions were identified inside coding regions, each causing a non-synonymous DNA sequence change. We selected seven SNVs and validated them by Sanger sequencing. In the metastatic ONB samples collected several months prior to WGS, all seven mutations were present. However, in the original surgical resection specimen (prior to evidence of metastatic disease), mutations in KDR, MYC, SIN3B, and NLRC4 genes were not present, suggesting that these were acquired with disease progression and/or as a result of post-treatment effects. Conclusions/Significance This work provides insight into the evolution of ONB cancer cells and provides a window into the more complex factors, including tumor clonality and multiple driver mutations.

DNA Methylation in Multiple Myeloma Is Weakly Associated with Gene Transcription

Previous studies have now demonstrated that both genic and global hypomethylation characterizes the multiple myeloma (MM) epigenome. Whether these methylation changes are associated with global and corresponding increases (or decreases) in transcriptional activity are poorly understood. The purpose of our current study was to correlate DNA methylation levels in MM to gene expression. We analyzed matching datasets generated by the GoldenGate methylation BeadArray and Affymetrix gene expression platforms in 193 MM samples. We subsequently utilized two independent statistical approaches to identify methylation-expression correlations. In the first approach, we used a linear correlation parameter by computing a Pearson correlation coefficient. In the second approach, we discretized samples into low and high methylation groups and then compared the gene expression differences between the groups. Only methylation of 2.1% and 25.3% of CpG sites on the methylation array correlated to gene expression by Pearson correlation or the discretization method, respectively. Among the genes with methylation-expression correlations were IGF1R, DLC1, p16, and IL17RB. In conclusion, DNA methylation may directly regulate relatively few genes and suggests that additional studies are needed to determine the effects of genome-wide methylation changes in MM.