Heterogeneous Molecular Mechanisms Research Articles

Interplay of genetic and epigenetic factors in regulating immune‐related pathways in Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease (AD) is a major cause of dementia and among the most complex of diseases, featuring various heterogeneous molecular mechanisms. Not only genetic but also non‐genetic factors (i.e., epigenetics) contribute to progression in the human brain; dysregulation of gene expression by single nucleotide polymorphisms (SNPs) and altered DNA methylation is often observed in AD patients. Although genetic and epigenetic regulators have each independently received intensive study for their roles in AD progression, their interactive contributions to expression regulation are not yet fully understood. In this study, we aimed to elucidate the interplay of SNPs and DNA methylation in determining transcript‐level expression status in AD.MethodWe performed integrative analysis of whole‐genome sequencing (WGS), RNA‐seq, and methylation data from the dorsolateral prefrontal cortex in AD patients (n=495), obtained from the ROSMAP project. We tested a set of 160,126 SNPs having minor allele frequency (MAP) > 0.05 for interaction with 208,911 methylation loci within promoter regions (+3000 to ‐200 bp) related to expression status of 81,715 transcripts having transcripts per million (TPM) values. Likelihood ratio tests (LRTs) were used to determine the statistical significance of differences between linear models with and without the interaction term, with false discovery rate (FDR) < 0.05 for significance criteria.ResultWe identified 511 SNP‐methylation probe pairs as having interactions with statistically significant impacts on the expression of 76 transcripts (56 unique genes). Genes in the HLA family (e.g., HLA‐A, HLA‐B, HLA‐C, HLA‐DPA1, HLA‐DRB1, and HLA‐DMA) were found to be especially associated with the interplay of SNPs and methylation. For example, the SNP rs1126511 and DNA methylation locus cg13581859 interact in regulating expression of the HLA‐DPA1 transcript ENST0000419277: neither the SNP nor the methylation locus alone impacted its expression, but transcript levels tended to be significantly decreased under the combination of SNP allele T and low methylation. In addition, gene‐set enrichment analysis found that interaction‐regulated genes were significantly enriched in immune‐related pathways.ConclusionOur study provides a new molecular insight into the crosstalk between genetic and epigenetic elements that are involved in transcript expression status in AD and suggests that immune‐related pathways may especially be regulated by such interactions.

PrecISE: Precision Medicine in Severe Asthma: An adaptive platform trial with biomarker ascertainment

Severe asthma accounts for almost half the cost associated with asthma. Severe asthma is driven by heterogeneous molecular mechanisms. Conventional clinical trial design often lacks the power and efficiency to target subgroups with specific pathobiological mechanisms. Furthermore, the validation and approval of new asthma therapies is a lengthy process. Alarge proportion of that time is taken by clinical trials to validate asthma interventions. The National Institutes of Health Precision Medicine in Severe and/or Exacerbation Prone Asthma (PrecISE) program was established with the goal of designing and executing a trial that uses adaptive design techniques to rapidly evaluate novel interventions in biomarker-defined subgroups of severe asthma, while seeking to refine these biomarker subgroups, and to identify early markers of response to therapy. The novel trial design is an adaptive platform trial conducted under a single master protocol that incorporates precision medicine components. Furthermore, it includes innovative applications of futility analysis, cross-over design with use of shared placebo groups, and early futility analysis to permit more rapid identification of effective interventions. The development and rationale behind the study design are described. The interventions chosen for the initial investigation and the criteria used to identify these interventions are enumerated. The biomarker-based adaptive design and analytic scheme are detailed as well as special considerations involved in the final trial design.

Proinflammatory status-stratified blood transcriptome profiling of civilian women with PTSD

Etiology of posttraumatic stress disorder (PTSD) remains largely unknown. Studies have shown that a significant subset of patients with PTSD exhibit increased inflammation, suggesting that the understanding of this disorder could be facilitated by classifying these patients by inflammatory status. Here we performed a microarray-based blood transcriptome analysis on proinflammatory status-stratified Japanese civilian women with PTSD most of whom developed the disorder after experiencing interpersonal violence. By utilizing our previously identified cut-off serum interleukin-6 (IL-6) level that approximately corresponded to the median IL-6 level of our PTSD patients, we classified patients into those with high IL-6 levels and those with normal IL-6 levels (n = 16 for each). Transcriptome profiles of these 2 groups were compared with the profile of 16 age-matched healthy control women. Differentially expressed genes between high IL-6 patients and controls showed significant enrichment in a number of gene ontology terms and pathways primarily involved in immune/inflammatory responses, and their protein-protein interaction network was significantly enriched. In contrast, differentially expressed genes between normal IL-6 patients and controls showed significant enrichment in several gene ontology terms related to ion transport and neural function. The microarray data were confirmed by reverse transcription quantitative PCR. These findings illustrate the heterogeneous molecular mechanisms of PTSD within this relatively homogeneous sample in terms of sex, trauma type, and ethnicity, suggesting that peripheral proinflammatory status such as IL-6 levels could be a useful subtyping marker for this disorder. With further research, it is hoped that our findings will be translated into personalized medicine.

Abstract 74: Heterogeneity response to afatinib in gastric cancer patient with uncommon EGFR mutations

Abstract Introduction: Gastric cancer is the third leading cause of cancer mortality worldwide. Gatric cancer based on driver gene mutation detection may benefit patients by facilitating molecular targeted drugs development and improving survival in gastric cancer patients. On the other hand, the complex and heterogeneous molecular mechanisms of gastric cancer also plays an essential role in the drug resistance. Methods: Whole exome sequencing (WES) was performed, revealing uncommon EGFR ex21 L861Q and ex18 G719S mutation of formalin-fixed paraffin-embedded sections from primary tumor and different metastatic lesions in a gastric cancer patient. The periodic circulating tumor DNA (ctDNA) was also determined by next generation sequencing (NGS). Stable gastric cancer cell and NIH-3T3 cell lines harboring the mutations were established to investigate the effect and mechanisms. Results: Tumors with compond EGFR L861Q/G719S mutations and EGFR gene amplfication are sensitive to afatinib, which caused tumor progression in short time. However, the lung metastatic lesion, which lacked EGFR gene amplification demonstrated primary resistance to afatinib. A dramatic increase of MET gene copy number may collectively related to the patient’s rapid progression. Periodic mutation profiling of patient’s ctDNA by NGS correspondingly revealed compond EGFR L861Q/G719S mutations, and a consitent increase of MET gene amplification. In in vitro studies, afatinib treatment reduced proliferation and inhibited EGFR phosphorylation in L861Q/G719S and L861Q mutant cells. Conclusions: Afatinib may be a beneficial therapeutic option for a subset of gastric cancer patients with rare EGFR mutations in their tumors. Our results also illustrated the great potential of ctDNA profiling for treatment decision-making to patients with gastric cancer. Note: This abstract was not presented at the meeting. Citation Format: Qin Liu, Jia Wei, Yang Yang, Yue Wang, Baorui Liu, Yang Shao. Heterogeneity response to afatinib in gastric cancer patient with uncommon EGFR mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 74.

Circulating tumor DNA profiling by next generation sequencing reveals heterogeneity of crizotinib resistance mechanisms in a gastric cancer patient with MET amplification.

Crizotinib has been used to counter MET gene amplification in a number of different human malignancies. Transient response to crizotinib in MET-amplified gastric cancer has been reported, but the mechanisms of resistance are not well studied. Here, we reported a stage IV gastric cancer patient with high levels of MET amplification. The implementation of crizotinib treatment led to significant symptomatic improvement in the first 2 months, but was followed by rapid disease progression. Periodic mutation profiling of patient's circulating tumor DNA (ctDNA) by next generation sequencing (NGS) revealed a number of genetic alterations including re-occurrence of MET amplification, multiple secondary MET mutations, a dramatic increase of FGFR2 gene relative copy number as well as mutations in other downstream and bypassing elements, which may collectively related to the patient's cancer progression. Our results illustrate the complex and heterogeneous molecular mechanisms for crizotinib resistance in this patient, and demonstrate the great potential of ctDNA profiling for treatment decision-making and prognosis in clinical practice.

Mesenchymal Stem Cells Support Survival and Proliferation of Primary Human Acute Myeloid Leukemia Cells through Heterogeneous Molecular Mechanisms.

Acute myeloid leukemia (AML) is a bone marrow malignancy, and various bone marrow stromal cells seem to support leukemogenesis, including osteoblasts and endothelial cells. We have investigated how normal bone marrow mesenchymal stem cells (MSCs) support the in vitro proliferation of primary human AML cells. Both MSCs and primary AML cells show constitutive release of several soluble mediators, and the mediator repertoires of the two cell types are partly overlapping. The two cell populations were cocultured on transwell plates, and MSC effects on AML cells mediated through the local cytokine/soluble mediator network could thus be evaluated. The presence of normal MSCs had an antiapoptotic and growth-enhancing effect on primary human AML cells when investigating a group of 51 unselected AML patients; this was associated with increased phosphorylation of mTOR and its downstream targets, and the effect was independent of cytogenetic or molecular-genetic abnormalities. The MSCs also supported the long-term proliferation of the AML cells. A subset of the patients also showed an altered cytokine network with supra-additive levels for several cytokines. The presence of cytokine-neutralizing antibodies or receptor inhibitors demonstrated that AML cells derived from different patients were heterogeneous with regard to effects of various cytokines on AML cell proliferation or regulation of apoptosis. We conclude that even though the effects of single cytokines derived from bone marrow MSCs on human AML cells differ among patients, the final cytokine-mediated effects of the MSCs during coculture is growth enhancement and inhibition of apoptosis.

Heterogeneous Molecular Mechanisms Confer Chemoresistance in Ovarian Cancer

Abstract Gene breakage, reversion mutations, and promoter fusions drive chemotherapy resistance in HGSC.

Mechanism of the All-α to All-β Conformational Transition of RfaH-CTD: Molecular Dynamics Simulation and Markov State Model.

The C-terminal domain of the bacterial transcription antiterminator RfaH undergoes a dramatic all-α-helix to all-β-barrel transition when released from its N-terminal domain. These two distinct folding patterns correspond to different functions: the all-α state acts as an essential regulator of transcription to ensure RNA polymerase binding, whereas the all-β state operates as an activator of translation by interacting with the ribosomal protein S10 and recruits ribosomal mRNA. Accordingly, this drastic conformational change enables RfaH to physically couple the transcription and translation processes in gene expression. To understand the mechanism behind this extraordinary functionally relevant structural transition, we constructed Markov state models using an adaptive seeding method. The constructed models highlight several parallel folding pathways with heterogeneous molecular mechanisms, which reveal the folding kinetics and atomic details of the conformational transition.

Challenging paradigms: long non-coding RNAs in breast ductal carcinoma in situ (DCIS)

OVERVIEW The continuing challenges to the treatment of breast ductal carcinoma in situ (DCIS) warrant a new avenue of investigation. In parallel, a startling revelation of the first post-genomic decade has been the rising prominence of long non-coding RNAs (lncRNAs) as key gene regulators, including in breast cancer. High-throughput experimental surveys of the human transcriptome show that lncRNAs are abundant. In contrast to microRNAs (miRNAs), lncRNAs possess multiple, diverse, heterogeneous molecular mechanisms of action making them high-level master regulators of gene regulatory networks and pathways in cancer. In particular, lncRNAs directly regulate the activity of transcription proteins and epigenetic modifiers, which, in turn, govern their own downstream sets of target genes. Therefore, provided that such lncRNA regulatory activities are functional in cancer, including DCIS, targeting the lncRNAs or the regulatory networks which they mediate can represent an important new avenue of treatment. Development of lncRNAtargeting therapies may emerge into a key innovation generated in clinical and translational research by the innovative use of the latest human genome annotation datasets combined with high throughput transcriptome analyses. We propose a paradigm shift: direct interrogation of lncRNAs in the molecular characterization of DCIS subtypes, including the idea that lncRNAs may be the trigger for progression from in situ to invasive cancer.

A HIERARCHICAL MODELING APPROACH OF HIPPOCAMPUS LOCAL CIRCUIT

The modeling and simulation of a realistic nervous tissue are difficult because of the number of implied cell types (neuronal and glial), the topology of the networks, and the various heterogeneous molecular mechanisms. The MTIP (Mathematical Theory of Integrative Physiology) is used as a new modeling approach based on a representation in terms of functional interactions and a formalism (S-Propagator) related to n-level field theory. This work presents the passage from a theoretical description of the biological system to a computing implementation in the general case. The specific case of the hippocampus is presented, as well as how a drug allows learning and memory improvement in the local circuit of the CA1 area of the hippocampus. This in silico result is used to experimentally predict the drug effect in vitro to confirm the accuracy of MTIP.

ZD6474, a Novel Tyrosine Kinase Inhibitor of Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor, Inhibits Tumor Growth of Multiple Nervous System Tumors

Primary central nervous system (CNS) tumors represent a diverse group of tumor types with heterogeneous molecular mechanisms that underlie their formation and maintenance. CNS tumors depend on angiogenesis and often display increased activity of ErbB-associated pathways. Current nonspecific therapies frequently have poor efficacy in many of these tumor types, so there is a pressing need for the development of novel targeted therapies. ZD6474 is a novel, orally available low molecular weight inhibitor of the kinase activities associated with vascular endothelial growth factor receptor-2 and epidermal growth factor receptor. We hypothesized that ZD6474 may provide benefit in the treatment of several CNS tumor types. In mice bearing established s.c. tumor xenografts of CNS tumors (malignant glioma and ependymoma) or rhabdomyosarcoma, a limited course of ZD6474 treatment produced significant tumor growth delays and a high rate of partial tumor regression in most models examined. Mice with i.c. malignant glioma xenografts treated with ZD6474 experienced a significant prolongation of survival. Tumors from mice treated with ZD6474 displayed a lower proliferative index and disrupted tumor vascularity. Notably, some of these models are insensitive to low molecular weight kinase inhibitors targeting only vascular endothelial growth factor receptor-2 or epidermal growth factor receptor functions, suggesting that the combined disruption of both epidermal growth factor receptor and vascular endothelial growth factor receptor-2 activities may significantly increase tumor control. In conclusion, ZD6474 shows significant activity against xenograft models of several primary human CNS tumor types. Consideration for clinical development in this disease setting seems warranted.

Protein Phosphatase-1 Regulation in the Induction of Long-Term Potentiation: Heterogeneous Molecular Mechanisms

Protein phosphatase inhibitor-1 (I-1) has been proposed as a regulatory element in the signal transduction cascade that couples postsynaptic calcium influx to long-term changes in synaptic strength. We have evaluated this model using mice lacking I-1. Recordings made in slices prepared from mutant animals and also in anesthetized mutant animals indicated that long-term potentiation (LTP) is deficient at perforant path-dentate granule cell synapses. In vitro, this deficit was restricted to synapses of the lateral perforant path. LTP at Schaffer collateral-CA1 pyramidal cell synapses remained normal. Thus, protein phosphatase-1-mediated regulation of NMDA receptor-dependent synaptic plasticity involves heterogeneous molecular mechanisms, in both different dendritic subregions and different neuronal subtypes. Examination of the performance of I-1 mutants in spatial learning tests indicated that intact LTP at lateral perforant path-granule cell synapses is either redundant or is not involved in this form of learning.