Molecular Mechanisms Underlying Disease Progression Research Articles

NRF2 activation by cysteine as a survival mechanism for triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a very aggressive and heterogeneous group of tumors. In order to develop effective therapeutic strategies, it is therefore essential to identify the subtype-specific molecular mechanisms underlying disease progression and resistance to chemotherapy. TNBC cells are highly dependent on exogenous cystine, provided by overexpression of the cystine/glutamate antiporter SLC7A11/xCT, to fuel glutathione synthesis and promote an oxidative stress response consistent with their high metabolic demands. Here we show that TNBC cells of the mesenchymal stem-like subtype (MSL) utilize forced cystine uptake to induce activation of the transcription factor NRF2 and promote a glutathione-independent mechanism to defend against oxidative stress. Mechanistically, we demonstrate that NRF2 activation is mediated by direct cysteinylation of the inhibitor KEAP1. Furthermore, we show that cystine-mediated NRF2 activation induces the expression of important genes involved in oxidative stress response, but also in epithelial-to-mesenchymal transition and stem-like phenotype. Remarkably, in survival analysis, four upregulated genes (OSGIN1, RGS17, SRXN1, AKR1B10) are negative prognostic markers for TNBC. Finally, expression of exogenous OSGIN1, similarly to expression of exogenous NRF2, can prevent cystine depletion-dependent death of MSL TNBC cells. The results suggest that the cystine/NRF2/OSGIN1 axis is a potential target for effective treatment of MSL TNBCs.

Abstract LB392: Transformative pathomics in oncology: Harnessing FFPE tissue slide imagery for clinically relevant gene expression prediction in invasive breast carcinoma

Abstract Purpose: Pathologists routinely analyze H&E-stained FFPE tissue slides microscopically to arrive at diagnoses for patients. Pathomics, the study of quantitative imaging from such samples, aims to elevate diagnostic accuracy by revealing intricate tissue and cellular details. Our research evaluates how pathomic features from tissue imagery can predict gene expression, as determined by RNA sequencing, offering advancements in the molecular profiling and targeted therapy for invasive breast carcinoma (IBC). Method: We conducted an analysis of 90 regions of interest (ROIs) on FFPE tissue slide images from the TCGA registry for patients with IBC. Using HistomicsTK software, we extracted nearly 300 pathomic features depicting cell morphometry, intensity, and gradient from these regions of interest (ROIs). We then assessed the intra-correlation of the pathomic features within the ROIs of the same tissue slide to identify the most heterogeneous and highly correlating features (n=20) across categories—morphometry, intensity, and gradient—with a Pearsonʼs Correlation coefficient (r) greater than 0.9 and a FDR adjusted p-value less than 0.05. Subsequently, gene expression data in the form of FPKM were obtained for the corresponding tissues from TCGA. We trained a Multitask Elastic Net (MTEN) model on these pathomic features using an 80:20 split for the training and testing sets, with a three-fold cross-validation applied to the training set utilizing ImaGene software. The model's performance was evaluated by measuring the AUC and R² value for the predicted gene expression on the testing set. Result and Biological significance: The testing of the MTEN model using a testing set predicted the expression of three genes identified in the literature as prognostic markers in breast cancer, namely ALDH1L2, MFAP5, and MXRA8, with an AUC greater than 0.8 and an R² above 0.5 at a p-value of less than 0.002. According to the literature, expression of genes from the ALDH1 superfamily in breast cancer correlates with the stage of the disease, triple-negative status, and response to neoadjuvant therapy. The upregulation of MFAP5 in invasive breast carcinomas has been associated with high risk prognostic features, such as higher tumor grade and stage and increased angiogenesis, and poorer outcomes, such as lymph node metastasis. Furthermore, MXRA8 is involved in modulating the progression of human triple-negative breast cancer, likely through its influence on the interactions of tumor cells with their microenvironment. Conclusion: The present study predicts the expression of clinically relevant genes in IBC using a heterogeneous set of pathomic features extracted from FFPE tissue slide imagery. By linking cell and tissue-based morphometric, intensity, and gradient features with gene expression, researchers can gain insights into the molecular mechanisms underlying disease progression. Digital pathology and pathomics may reduce the need for additional genetic testing in critical patient-cases by providing predictive information from routinely acquired pathology slides. Bridging the gap between phenotypic tissue data and molecular data, the predictive capabilities of pathomic features represent a significant advancement in the field of precision medicine. Citation Format: Shrey S. Sukhadia, Digvijay Yadav, Kristen E. Muller. Transformative pathomics in oncology: Harnessing FFPE tissue slide imagery for clinically relevant gene expression prediction in invasive breast carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB392.

Doxorubicin downregulates autophagy to promote apoptosis-induced dilated cardiomyopathy via regulating the AMPK/mTOR pathway

The broad-spectrum antineoplastic drug doxorubicin (DOX) has one of the most serious chronic side effects on the heart, dilated cardiomyopathy, but the precise molecular mechanisms underlying disease progression subsequent to long latency periods remain puzzling. Here, we established a model of DOX-induced dilated cardiomyopathy. In a cardiac cytology exploration, we found that differentially expressed genes in the KEGG signaling pathway enrichment provided a novel complex network of mTOR bridging autophagy and oxidative stress. Validation results showed that DOX caused intracellular reactive oxygen species accumulation in cardiomyocytes, disrupted mitochondria, led to imbalanced intracellular energy metabolism, and triggered cardiomyocyte apoptosis. Apoptosis showed a negative correlation with DOX-regulated cardiomyocyte autophagy. To evaluate whether the inhibition of mTOR could upregulate autophagy to protect cardiomyocytes, we used rapamycin to restore autophagy depressed by DOX. Rapamycin increased cardiomyocyte survival by easing the autophagic flux blocked by DOX. In addition, rapamycin reduced oxidative stress, prevented mitochondrial damage, and restored energy metabolic homeostasis in DOX-treated cardiomyocytes. In vivo, we used metformin (Met) which is an AMPK activator to protect cardiac tissue to alleviate DOX-induced dilated cardiomyopathy. In this study, Met significantly attenuated the oxidative stress response of myocardial tissue caused by DOX and activated cardiomyocyte autophagy to maintain cardiomyocyte energy metabolism and reduce cardiomyocyte apoptosis by downregulating mTOR activity. Overall, our study revealed the role of autophagy and apoptosis in DOX-induced dilated cardiomyopathy and demonstrated the potential role of regulation of the AMPK/mTOR axis in the treatment of DOX-induced dilated cardiomyopathy.

Smoking Cessation in Mice Does Not Switch off Persistent Lung Inflammation and Does Not Restore the Expression of HDAC2 and SIRT1.

Once COPD is established, pulmonary lesions can only progress and smoking cessation by itself is not sufficient to switch off persistent lung inflammation. Similarly, in former-smoker mice, neutrophil inflammation persists and lung lesions undergo progressive deterioration. The molecular mechanisms underlying disease progression and the inefficiency of smoking cessation in quenching neutrophilic inflammation were studied in male C57 Bl/6 mice after 6 months of rest from smoking cessation. As compared with the mice that continued to smoke, the former-smoker mice showed reduced expression of histone deacetylases HDAC2 and SIRT1 and marked expression of p-p38 MAPK and p-Ser10. All these factors are involved in corticosteroid insensitivity and in perpetuating inflammation. Former-smoker mice do show persistent lung neutrophilic influx and a high number of macrophages which account for the intense staining in the alveolar structures of neutrophil elastase and MMP-9 (capable of destroying lung scaffolding) and 8-OHdG (marker of oxidative stress). “Alarmins” released from necrotic cells together with these factors can sustain and perpetuate inflammation after smoking cessation. Several factors and mechanisms all together are involved in sustaining and perpetuating inflammation in former-smoker mice. This study suggests that a better control of COPD in humans may be achieved by precise targeting of the various molecular mechanisms associated with different phenotypes of disease by using a cocktail of drug active toward specific molecules.

Identification of Unique Transcriptomic Signatures and Hub Genes Through RNA Sequencing and Integrated WGCNA and PPI Network Analysis in Nonerosive Reflux Disease.

PurposeTranscriptomic studies on gastroesophageal reflux disease are scarce, and gene expression signatures in nonerosive reflux disease (NERD) remain elusive. The aim of the study was to identify gene expression profiles and potential hub genes in NERD.Patients and MethodsWe performed RNA sequencing on biopsy samples from nine consecutive patients with NERD and six healthy controls. Differentially expressed genes (DEGs) were analysed with the DESeq2 R package. A DEG-based protein–protein interaction (PPI) network was constructed to filter hub genes using Cytoscape. Weighted gene coexpression network analysis (WGCNA) was conducted to identify the coexpression relationships of all modules and explore the relationship between gene sets and clinical traits.ResultsIn total, 1195 DEGs were identified, including 649 upregulated and 546 downregulated genes involved in regulating the inflammatory response and epithelial cell differentiation. Overlap of the PPI and WGCNA networks identified five shared genes, namely, THY1, BMP2, LOX, KDR and MMP9, as candidate hub genes in NERD. Quantitative PCR analysis of the expression of these five genes confirmed the sequencing results. Receiver operating characteristic analyses indicated that these hub genes had diagnostic potential for NERD patients. Gene set enrichment analysis confirmed that each hub gene was closely associated with the pathophysiological processes of NERD. In addition, a regulatory network comprising 42 transcription factors (TFs), 28 miRNAs and 5 hub genes was established.ConclusionThe five core genes may be promising biomarkers of NERD. The TF/miRNA/hub gene network can improve the understanding of the molecular mechanisms underlying disease progression.

Reciprocal Regulation of BCL6 and MLL Expression As a Vulnerability in MLL -Rearranged Acute Lymphoblastic Leukemia

Background: Chromosomal translocations involving the Mixed Lineage Leukemia gene MLL account for ~70% of infant acute lymphoblastic leukemias (ALLs; Ayton and Cleary, 2001) and are associated with dismal prognosis. These findings underscore the importance of understanding the molecular mechanisms underlying disease progression of MLL-rearranged (MLLr) leukemia for the development of novel therapeutics. Recently, an integrative epigenomic analysis has identified the transcriptional repressor and proto-oncogene BCL6 as a transcriptional target in MLL-AF4 -driven pre-B ALL (Geng et al., 2012). BCL6 functions as a mediator of drug resistance to tyrosine kinase inhibitors in Ph+ ALL (Duy et al., 2011). Here, we tested the hypothesis that BCL6 is required for MLLr leukemia and represents a therapeutic target in MLLr pre-B ALL.Results: Western blot analysis of a panel of patient-derived pre-B ALL cells (MLLr : n= 7; ETV6-RUNX1 : n= 2; BCR-ABL1 : n= 1; not otherwise specified: n= 4) and normal CD19+ B cells (n= 3) revealed that BCL6 levels were elevated in MLL r ALL samples. Furthermore, immunohistochemistry staining in bone marrow biopsies from ALL patients (n= 72) showed that BCL6 levels were upregulated in MLLr cases. Expression of MLL-ENL or MLL-AF4 fusion protein increased Bcl6 protein levels (~10-fold) in murine pre-B cells. Moreover, ChIP-seq analysis of human MLLr pre-B ALL cell lines using antibodies against MLL, AF4 and ENL demonstrated direct binding to the BCL6 promoter. Studying methylation status revealed that the BCL6 promoter was hypomethylated in patients with MLLr pre-B ALL compared to patients with other subtypes of pre-B ALL (including BCR-ABL1, ETV6-RUNX 1 and hyperdiploid) as well as normal pre-B cells from healthy donors (St. Jude, n= 132, Figueroa, 2014). Consistent with these observations, multivariate analysis of high risk pre-B ALL patients showed that high BCL6 levels in combination with MLL rearrangements were associated with the worst clinical outcome (COG P9906). Collectively, our findings demonstrated that BCL6 levels are elevated in MLLr ALL, and that expression levels of MLL and BCL6 can serve as predictors for risk stratification in ALL.Interestingly, expression of wild-type MLL was positively correlated with that of BCL6 in both pediatric (COG P9906) and adult (ECOG E2993) pre-B ALL. Furthermore, multivariate analysis comparing MLLHighBCL6High patients with MLLLowBCL6Low patients showed that high expression levels of both genes correlated with poor clinical outcome (COG P9906). These findings prompted us to study whether MLL regulates BCL6 expression, and vice versa . Cre-mediated deletion of Mll abrogated imatinib-mediated induction of Bcl6 in BCR-ABL1 -driven pre-B ALL, indicating that Mll is required for upregulation of Bcl6 expression. While genetic inactivation of Bcl6 as well as treatment with the BCL6-specific retro-inverso peptide-inhibitor (RI-BPI) decreased Mll expression, doxycycline-induced expression of Bcl6 upregulated Mll expression. Combining chromatin immunoprecipitation and DNA microarray (ChIP-on-chip), we found that BCL6 was recruited to the MLL promoter in pre-B ALL. Taken together, these findings support our proposed scenario of reciprocal regulation of MLL and BCL6 expression.Notably, ChIP-seq analyses of MLLr pre-B ALL cells showed that there are 268 common gene targets of both MLL and BCL6. Among the common targets include cell cycle checkpoint regulators (CDKN1B, ETS2, BMI1, RB1), apoptosis facilitator BIM, and tumor suppressor TET2, indicating that BCL6 is a key regulator of the transcriptional program in MLLr pre-B ALL. In support of an essential role of BCL6 in MLLr ALL, expression of a dominant negative BCL6 mutant in patient-derived MLLr pre-B ALL cells induced rapid depletion from cell culture in competitive-growth assays. Furthermore, RI-BPI treatment resulted in cell cycle arrest, impaired colony forming ability and inhibited leukemia initiation in transplant recipient mice. Importantly, RI-BPI in combination with standard chemotherapy Vincristine exerted more potent effects on killing MLLr pre-B ALL cells than either agent alone.Conclusions: Our findings suggest that aberrant BCL6 expression levels are important for disease progression, and that pharmacological inhibition of BCL6 augments responsiveness to standard chemotherapy in the treatment of MLLr pre-B ALL. DisclosuresHurtz:Incyte: Research Funding. Armstrong:Cyteir: Consultancy; Imago: Consultancy; C4 Therapeutics: Consultancy; Epizyme: Consultancy.

The Regulatory Functions of lncRNAs on Angiogenesis Following Ischemic Stroke.

Ischemic stroke is one of the leading causes of global mortality and disability. It is a multi-factorial disease involving multiple factors, and gene dysregulation is considered as the major molecular mechanisms underlying disease progression. Angiogenesis can promote collateral circulation, which helps the restoration of blood supply in the ischemic area and reduces ischemic necrosis following ischemic injury. Aberrant expression of long non-coding RNAs (lncRNAs) in ischemic stroke is associated with various biological functions of endothelial cells and serves essential roles on the angiogenesis of ischemic stroke. The key roles of lncRNAs on angiogenesis suggest their potential as novel therapeutic targets for future diagnosis and treatment. This review elucidates the detailed regulatory functions of lncRNAs on angiogenesis following ischemic stroke through numerous mechanisms, such as interaction with target microRNAs, downstream signaling pathways and target molecules.

In Situ Studies of Fibrillar Polymorphs in Alzheimer's Disease

Aggregates of Aβ peptides and tau protein are defining features of Alzheimer's disease (AD) but the contribution of these structures to the etiology of disease remains uncertain. High resolution structures of fibrillar polymorphs have provided a basis for studies of disease, but fall short of defining the molecular mechanisms underlying disease progression. Evidence is accumulating that the peptide conformation within fibrils correlates with disease subtype and suggests that protofilament conformation represents the physical embodiment of a ‘strain’ of AD. Within a single strain the fold of peptides may be invariant. But the fibrils are capable of polymorphism that includes variation in the packing of protofilaments into fibrils; the pitch of the resultant fibrils and the higher order organization of plaques into which they aggregate. These intra-strain polymorphisms are divided by low-energy barriers allowing multiple configurations to coexist within a single preparation or tissue. Clinical presentation of different strains may be determined by variation in the way different protofilament structures generate relevant toxic species, be they monomers, oligomers or higher order structures. To better understand their role in disease, in situ studies of fibril structure have been carried out on histological thin sections of human brain tissue. Scanning x-ray microdiffraction is providing information on the variation of fibrillar polymorphism at sub-cellular length scales and makes possible mapping the distribution of polymorphs within tissue. Scattering in the wide-angle regime produces information on fibrillar twist. Scattering in the small-angle regime can be used to generate the cross-sectional shape of fibrils. Mapping the variation of these structural parameters across and among plaques and tangles in different regions of the brain provides insight into the development and evolution of neurodegenerative lesions.

Diet-Induced Rodent Models of Diabetic Peripheral Neuropathy, Retinopathy and Nephropathy.

Unhealthy dietary habits are major modifiable risk factors for the development of type 2 diabetes mellitus, a metabolic disease with increasing prevalence and serious consequences. Microvascular complications of diabetes, namely diabetic peripheral neuropathy (DPN), retinopathy (DR), and nephropathy (DN), are associated with high morbidity rates and a heavy social and economic burden. Currently, available therapeutic options to counter the evolution of diabetic microvascular complications are clearly insufficient, which strongly recommends further research. Animal models are essential tools to dissect the molecular mechanisms underlying disease progression, to unravel new therapeutic targets, as well as to evaluate the efficacy of new drugs and/or novel therapeutic approaches. However, choosing the best animal model is challenging due to the large number of factors that need to be considered. This is particularly relevant for models induced by dietary modifications, which vary markedly in terms of macronutrient composition. In this article, we revisit the rodent models of diet-induced DPN, DR, and DN, critically comparing the main features of these microvascular complications in humans and the criteria for their diagnosis with the parameters that have been used in preclinical research using rodent models, considering the possible need for factors which can accelerate or aggravate these conditions.

Patient-derived cell line models revealed therapeutic targets and molecular mechanisms underlying disease progression of high grade serous ovarian cancer.

High grade serous ovarian cancer (HGSOC) is the most frequent type of ovarian cancer. Most patients have primary response to platinum-based chemotherapy but frequently relapse, which leads to patient death. A lack of well documented and characterized patient-derived HGSOC cell lines is so far a major barrier to define tumor specific therapeutic targets and to study the molecular mechanisms underlying disease progression. We established 34 patient-derived HGSOC cell lines and characterized them at cellular and molecular level. Particularly, we demonstrated that a cancer-testis antigen PRAME and Estrogen Receptor could serve as therapeutic targets. Notably, data from the cell lines did not demonstrate acquired resistance due to tumor recurrence that matched with clinical observations. Finally, we presented that all HGSOC had no or very low CDKN1A (p21) expression due to loss of wild-type TP53, suggesting that loss of cell cycle control is the determinant for tumorigenesis and progression. In conclusion, patient-derived cell lines reveal that PRAME is a potential tumor specific therapeutic target in HGSOC and counteracting the down-regulation of p21 caused by loss of wild-type TP53 might be the key to impede disease progression.

Assessment of protein extraction and digestion efficiency of well-established shotgun protocols for heart proteomics

Ischemic heart disease is the leading cause of deaths worldwide. Thus, understanding the molecular mechanisms underlying disease progression is needed. Due to heart importance and lack of studies evaluating different sample preparation methods for heart proteomics, we compared three well-established protocols in shotgun proteomics using dimethyl label quantitation to allow relative quantitation. The tested methods for the analysis of left ventricle (LV) tissue were: i) in-solution digestion (ISD); ii) on-pellet digestion (OPD); and iii) on-filter digestion (OFD). Protein extraction was done using SDS-containing buffer for OPD and OFD while this step was under urea-containing buffer for ISD. We used an optimized one-step reaction for reduction of disulfide bonds and alkylation of thiol groups in ISD and OPD. Using the same amount of tissue, we observed that OFD and ISD extracted significantly higher amount of protein than OPD. ISD outperformed OFD and OPD in the number of proteins identified. We did not observe significant bias related to physicochemical features of the identified proteins when comparing the three protocols. ISD was more efficient to identify low abundant proteins and yielded more proteins per protocol duration. Thus, we concluded that the optimized ISD suited better for heart proteomics than OFD and OPD.

Sphingolipid Metabolism Is Dysregulated at Transcriptomic and Metabolic Levels in the Spinal Cord of an Animal Model of Amyotrophic Lateral Sclerosis.

Lipid metabolism is drastically dysregulated in amyotrophic lateral sclerosis and impacts prognosis of patients. Animal models recapitulate alterations in the energy metabolism, including hypermetabolism and severe loss of adipose tissue. To gain insight into the molecular mechanisms underlying disease progression in amyotrophic lateral sclerosis, we have performed RNA-sequencing and lipidomic profiling in spinal cord of symptomatic SOD1G86R mice. Spinal transcriptome of SOD1G86R mice was characterized by differential expression of genes related to immune system, extracellular exosome, and lysosome. Hypothesis-driven identification of metabolites showed that lipids, including sphingomyelin(d18:0/26:1), ceramide(d18:1/22:0), and phosphatidylcholine(o-22:1/20:4) showed profound altered levels. A correlation between disease severity and gene expression or metabolite levels was found for sphingosine, ceramide(d18:1/26:0), Sgpp2, Sphk1, and Ugt8a. Joint-analysis revealed a significant enrichment of glycosphingolipid metabolism in SOD1G86R mice, due to the down-regulation of ceramide, glucosylceramide, and lactosylceramide and the overexpression of genes involved in their recycling in the lysosome. A drug-gene interaction database was interrogated to identify potential drugs able to modulate the dysregulated genes from the signaling pathway. Our results suggest that complex lipids are pivotally changed during the first phase of motor symptoms in an animal model of amyotrophic lateral sclerosis.

Progression in Patients with with Low- and Intermediate-1 Risk Del(5q) MDS Is Predicted By a Limited Subset of Mutations

▪A high proportion of lower-risk del(5q) MDS patients will respond to treatment with lenalidomide. The estimated duration of transfusion-independence is 2 years including some long-lasting responses, but almost 40% of patients progress to acute leukemia by 5 years after start of treatment. As the molecular mechanisms underlying disease progression in del(5q) MDS remain to be elucidated, we do not know how to predict disease progression or how to monitor patients during lenalidomide treatment. We previously reported that small TP53 mutated subclones predict for an unfavorable outcome in del(5q) patients, and that these subclones expand with disease progression. However, whether or not other somatic mutations or factors related to the bone marrow microenvironment also contribute to disease progression has not been comprehensively assessed.We studied a longitudinal cohort of 35 low- and intermediate-1-risk del(5q) patients treated with lenalidomide (n=22), or other treatments including stem cell transplantation (n=13) by flow cytometric surveillance of hematopoietic stem and progenitor cells (HSPC) subsets, targeted sequencing of mutational patterns, and changes in the bone marrow microenvironment. In our cohort, 13 of 35 patients progressed to either higher-risk MDS (n=4) or leukemia (n=9), 12 of whom were treated with lenalidomide. Progression was associated with the detection of a restricted subset of new recurrent mutations, either alone or in combination: TP53 (n=9, p=0.0004), TET2 (n=6, p=0.006), RUNX1 (n=3, p=0.044), and PTPN11 (n=1). Regardless of whether the three mutations (TP53, TET2 and RUNX1) were present in the initial sample or whether they subsequently developed, testing positive for any of them carried a high probability (13/16, 81%) for predicting progression. For 11 out of 13 patients the new mutations were detected prior to the time point of clinical progression and the median time from detection of the mutation to clinical evidence of progression was 42 months (range 0-83.9). Thus, we were able to detect the mutation in the majority of cases well before clinical signs of disease progression. Seven of the nine patients who developed leukemia carried a TP53 mutation. Based on a median sequencing depth of 370 reads, the mutation was considered present pre-treatment in one of these patients and to have developed under treatment in the other six. Using flow cytometry for surveillance of HSPC subsets in lenalidomide-treated patients, we found that neither lenalidomide treatment nor the acquisition of additional mutations led to any uniform profound changes in the hematopoietic hierarchy unless the patient showed clinical signs of progression. Microarray analysis of mesenchymal stromal cells (MSC) exhibited an expression footprint consistent with MSC with high expression of typical MSC markers and absence of hematopoietic gene signatures. However, we observed only minor differences in gene expression between pre- treatment del(5q) and healthy MSC.In conclusion, while flow cytometric analysis of HSPC populations or analysis of the microenvironment had limited predictive value in this cohort of lower-risk del(5q) MDS, all patients who progressed to either higher-risk MDS or leukemia were identified by harboring recurrent mutations in a limited number of genes, i.e., TP53, RUNX1, TET2, and PTPN11. Based on our data, we advocate for conducting a prospective study aimed at investigating in a larger number of del(5q) MDS cases pre- and post-lenalidomide treatment, whether the detection of such mutations can guide clinical decision making, such as suggesting which patients should undergo hematopoietic cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

Developmental origins of neurotransmitter and transcriptome alterations in adult female zebrafish exposed to atrazine during embryogenesis

Atrazine is an herbicide applied to agricultural crops and is indicated to be an endocrine disruptor. Atrazine is frequently found to contaminate potable water supplies above the maximum contaminant level of 3μg/L as defined by the U.S. Environmental Protection Agency. The developmental origin of adult disease hypothesis suggests that toxicant exposure during development can increase the risk of certain diseases during adulthood. However, the molecular mechanisms underlying disease progression are still unknown. In this study, zebrafish embryos were exposed to 0, 0.3, 3, or 30μg/L atrazine throughout embryogenesis. Larvae were then allowed to mature under normal laboratory conditions with no further chemical treatment until 7 days post fertilization (dpf) or adulthood and neurotransmitter analysis completed. No significant alterations in neurotransmitter levels was observed at 7dpf or in adult males, but a significant decrease in 5-hydroxyindoleacetic acid (5-HIAA) and serotonin turnover was seen in adult female brain tissue. Transcriptomic analysis was completed on adult female brain tissue to identify molecular pathways underlying the observed neurological alterations. Altered expression of 1928, 89, and 435 genes in the females exposed to 0.3, 3, or 30μg/L atrazine during embryogenesis were identified, respectively. There was a high level of overlap between the biological processes and molecular pathways in which the altered genes were associated. Moreover, a subset of genes was down regulated throughout the serotonergic pathway. These results provide support of the developmental origins of neurological alterations observed in adult female zebrafish exposed to atrazine during embryogenesis.

Helicobacter pylori and microRNAs: Relation with innate immunity and progression of preneoplastic conditions.

The accepted paradigm for intestinal-type gastric cancer pathogenesis is a multistep progression from chronic gastritis induced by Helicobacter pylori (H. pylori) to gastric atrophy, intestinal metaplasia, dysplasia and ultimately gastric cancer. The genetic and molecular mechanisms underlying disease progression are still not completely understood as only a fraction of colonized individuals ever develop neoplasia suggesting that bacterial, host and environmental factors are involved. MicroRNAs are noncoding RNAs that may influence H. pylori-related pathology through the regulation of the transcription and expression of various genes, playing an important role in inflammation, cell proliferation, apoptosis and differentiation. Indeed, H. pylori have been shown to modify microRNA expression in the gastric mucosa and microRNAs are involved in the immune host response to the bacteria and in the regulation of the inflammatory response. MicroRNAs have a key role in the regulation of inflammatory pathways and H. pylori may influence inflammation-mediated gastric carcinogenesis possibly through DNA methylation and epigenetic silencing of tumor suppressor microRNAs. Furthermore, microRNAs influenced by H. pylori also have been found to be involved in cell cycle regulation, apoptosis and epithelial-mesenchymal transition. Altogether, microRNAs seem to have an important role in the progression from gastritis to preneoplastic conditions and neoplastic lesions and since each microRNA can control the expression of hundreds to thousands of genes, knowledge of microRNAs target genes and their functions are of paramount importance. In this article we present a comprehensive review about the role of microRNAs in H. pylori gastric carcinogenesis, identifying the microRNAs downregulated and upregulated in the infection and clarifying their biological role in the link between immune host response, inflammation, DNA methylation and gastric carcinogenesis.

Next Generation Sequencing Identifies DNA Methylation Patterns Indicative of Disease Progression in Ph+ CML

Ph+ chronic myeloid leukemia (CML) is a stem cell malignancy characterized by the BCR-ABL1 oncoprotein and leukemic expansion of myeloid progenitor cells. In the chronic phase (CP) of CML, clonal cells undergo myeloid differentiation and respond well to BCR-ABL1 inhibitors. In the accelerated phase (AP) and blast phase (BP) of CML, however, neoplastic cells are immature and often resistant against most BCR-ABL1-targeting drugs which is a challenge in clinical hematology. So far, little is known about molecular mechanisms underlying disease progression in CML. Methylation of CG sites around the transcriptional start site of various cancer-related genes including diverse tumor suppressor genes (referred to as methylation) is a frequently occurring epigenetic feature in neoplastic cells resulting in silencing of these genes. Although methylation is considered a critical factor in the pathogenesis of various malignant diseases including myeloid neoplasms, no comprehensive studies on the impact of methylation in the pathogenesis of CML have been conducted so far. We hypothesized that methylation may be an important mechanism regulating the transcriptional gene activity in CML cells during disease progression. Therefore, we investigated the methylome and the transcriptome of neoplastic cells in patients with CML in various phases of the disease (CP, n=15; AP, n=5; BP, n=7). Genome-wide methylation and gene expression patterns were analysed by next generation sequencing approaches using bone marrow (BM) or peripheral blood (PB) mononuclear cells (MNC) obtained from patients with CML and BM or PB MNC from control individuals. Methylation was analysed by reduced representation bisulfite sequencing (RRBS), and mRNA expression was determined by RNA-sequencing (RNA-seq). By comparing the methylome of patients who were initially diagnosed with CP-CML and who relapsed several months later (AP-CML, n=1; BC-CML, n=3), we identified a large number of genes which were methylated around their transcriptional start site in leukemic cells in patients at the time of progression compared to the time of CP-CML (range in the 4 patients: 423-1209 genes, adjusted p<0.05). These methylated genes were found to be less methylated or not methylated in BM or PB MNC of control individuals. When the methylome of all patients in all cohorts was examined and compared, more genes were found to be methylated in AP-CML and BC-CML compared to CP-CML (CP-CML, n=200; AP-CML, n=311; BC-CML, n=570). In addition, we identified several genes that were less methylated or not methylated in AP-CML and BC-CML cells compared to cells in CP-CML samples (range: 16-541 genes). Moreover, we analysed and compared the transcriptome of CP-CML, AP-CML and BC-CML samples and identified a large number of genes whose expression is downregulated in AP-CML and BC-CML samples compared to CP-CML (range: 187-382 genes). By correlating RRBS results and RNA-seq data, we found that expression of >100 of the methylated genes is downregulated in AP-CML/BC-CML compared to CP-CML suggesting that these genes may be regulated by methylation. Expression of the majority of these genes was detected in BM or PB MNC of control individuals. In silico pathway analyses and gene network analyses revealed that some methylated genes are involved in the regulation of apoptosis (e.g. CYP1B1, ZBTB16), negative regulation of cell proliferation (e.g. BTG3, VSX2) or regulation of Wnt signalling (e.g. SFRP1). Currently, a large number of CML patients are analysed gene-specifically for methylation by methylation-sensitive high resolution melting PCR and for expression of selected genes by RT-PCR in order to define prognostic patterns in CML. In summary, our results demonstrate that methylation changes are frequent events accompanying disease progression in patients with CML. These results may contribute to the identification of clinically relevant methylation patterns in CML and thus may improve prognostication. In addition, our data may reveal new epigenetic targets of therapy and may help to develop new treatment strategies for high risk or relapsing patients with CML. DisclosuresValent:Novartis: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; BMS: Honoraria.

Sensitive detection of pathway perturbations in cancers

BackgroundThe normal functioning of a living cell is characterized by complex interaction networks involving many different types of molecules. Associations detected between diseases and perturbations in well-defined pathways within such interaction networks have the potential to illuminate the molecular mechanisms underlying disease progression and response to treatment.ResultsIn this paper, we present a computational method that compares expression profiles of genes in cancer samples to samples from normal tissues in order to detect perturbations of pre-defined pathways in the cancer. In contrast to many previous methods, our scoring function approach explicitly takes into account the interactions between the gene products in a pathway. Moreover, we compute the sub-pathway that has the highest score, as opposed to merely computing the score for the entire pathway. We use a permutation test to assess the statistical significance of the most perturbed sub-pathway. We apply our method to 20 pathways in the Netpath database and to the Global Cancer Map of gene expression in 18 cancers. We demonstrate that our method yields more sensitive results than alternatives that do not consider interactions or measure the perturbation of a pathway as a whole. We perform a sensitivity analysis to show that our approach is robust to modest changes in the input data. Our method confirms numerous well-known connections between pathways and cancers.ConclusionsOur results indicate that integrating differential gene expression with the interaction structure in a pathway is a powerful approach for detecting links between a cancer and the pathways perturbed in it. Our results also suggest that even well-studied pathways may be perturbed only partially in any given cancer. Further analysis of cancer-specific sub-pathways may shed new light on the similarities and differences between cancers.

Differential expression of cartilage and bone-related proteins in pediatric and adult diseased aortic valves

Approximately 5million people are affected with aortic valve disease (AoVD) in the United States. The most common treatment is aortic valve (AoV) replacement surgery, however, replacement valves are susceptible to failure, necessitating additional surgeries. The molecular mechanisms underlying disease progression and late AoV calcification are not well understood. Recent studies suggest that genes involved in bone and cartilage development play an active role in osteogenic-like calcification in human calcific AoVD (CAVD). In an effort to define the molecular pathways involved in AoVD progression and calcification, expression of markers of valve mesenchymal progenitors, chondrogenic precursors, and osteogenic differentiation was compared in pediatric non-calcified and adult calcified AoV specimens. Valvular interstitial cell (VIC) activation, extracellular matrix (ECM) disorganization, and markers of valve mesenchymal and skeletal chondrogenic progenitor cells were observed in both pediatric and adult AoVD. However, activated BMP signaling, increased expression of cartilage and bone-type collagens, and increased expression of the osteogenic marker Runx2 are observed in adult diseased AoVs. They are not observed in the majority of pediatric diseased valves, representing a marked distinction in the molecular profile between pediatric and adult diseased AoVs. The combined evidence suggests that an actively regulated osteochondrogenic disease process underlies the pathological changes affecting AoVD progression, ultimately resulting in stenotic AoVD. Both pediatric and adult diseased AoVs express protein markers of valve mesenchymal and chondrogenic progenitor cells while adult diseased AoVs also express proteins involved in osteogenic calcification. These findings provide specific molecular indicators of AoVD progression, which may lead to identification of early disease markers and the development of potential therapeutics.

A Comprehensive Deep-Sequencing Study of Blast Crisis Chronic Myeloid Leukemia (CML) Reveals New Insights Into Molecular Heterogeneity and Detects Mutations In 12 Different Genes In 82.5% of Cases

AbstractAbstract 884Blast crisis is the terminal phase of chronic myeloid leukemia (CML) with a short median survival of approximately six months. At present, little is known about molecular mechanisms underlying disease progression. We hypothesized that mutations occurring in other myeloid and lymphatic malignancies are acquired during disease progression from chronic phase to blast crisis. Here, in total 40 blast crisis CML cases (n=25 myeloid, n=10 lymphoid, n=5 not specified) were analyzed, all diagnosed between 9/2005 and 7/2009. First, all cases were investigated for IKZF1 deletions by PCR using specific primer pairs for the common intragenic deletions spanning from exon 2–7, or exon 4–7 as published by Iacobucci et al. (Blood, 114:2159-67, 2009). In total, in 17.5% (7/40) of cases intragenic IKZF1 deletions were detected. Secondly, next-generation deep-sequencing (454 Life Sciences, Branford, CT) was used to investigate 11 candidate genes in all 40 patients for a broad molecular screening. Known hotspot regions were sequenced for CBL (exons 8 and 9), NRAS (exons 2 and 3), KRAS (exons 2 and 3), IDH1 (exon 4), IDH2 (exon 4), and NPM1 (exon 12). Complete coding regions were analyzed for RUNX1, TET2, WT1, and TP53. To perform this comprehensive study, amplicon-based deep-sequencing was applied using the small volume Titanium chemistry assay. To cope with the great number of amplicons, in total 59, 48.48 Access Arrays were applied (Fluidigm, South San Francisco, CA), amplifying and barcode-tagging 48 amplicons across 48 samples in one single array (2,304 reactions). In median, 430 reads per amplicon were obtained, thus yielding sufficient coverage for detection of mutations with high sensitivity. Further, ASXL1 exon 12 aberrations were investigated by Sanger sequencing. In summary, after excluding known polymorphisms and silent mutations in 33/40 patients 53 mutations were identified: RUNX1 (16/40; 40.0%), ASXL1 (12/40; 30.0%), WT1 (6/40; 15.0%), NRAS (2/40; 5.0%), KRAS (2/40; 5.0%), TET2 (3/40; 7.5%), CBL (1/40; 2.5%), TP53 (1/40; 2.5%), IDH1 (3/40; 7.5%), IDH2 (0/40), and NPM1 (0/40). Thus, 82.5% of blast crisis CML patients harbored at least one molecular aberration. In median, one affected gene per patient was observed (range 1–5). In detail, RUNX1 was associated with additional mutations in other genes, i.e. 9/16 cases were harboring additional mutations in combination with RUNX1. Similarly, in 8/12 patients with ASXL1 mutations additional aberrations were detected. With respect to myeloid or lymphoid features ASXL1 mutations (n=11) were exclusively observed in patients with myeloid blast crisis (n=1 not specified), in contrast 5/7 IKZF1 cases were detected in cases with lymphoid features (n=1 myeloid, n=1 not specified). Interestingly, besides IKZF1 (n=5) and RUNX1 (n=3) alterations there was no other mutated gene occurring in lymphoid blast crisis CML. In addition, no aberration was detected in NPM1, and in contrast to published data, in our cohort only one patient harbored a mutation in TP53. Moreover, for 8 patients with mutations in IKZF1 (n=3), RUNX1 (n=3), ASXL1 (n=1), WT1 (n=2), and IDH1 (n=2), matched DNA from the initial diagnosis at chronic state was available. In these specimens respective IKZF1 deletions, RUNX1, and ASXL1 mutations were not detectable indicating that IKZF1, RUNX1, and ASXL1 mutations had been developed during disease progression and act as driver mutations in these cases. WT1 and IDH1 mutations occurred at first diagnosis in one case each, indicating these genes would constitute passenger mutations. In conclusion, this comprehensive study on 12 molecular markers enabled to characterize for the first time that 82.5% of blast crisis CML cases harbor specific molecular mutations. IKZF1 and RUNX1 alterations were identified as important markers of disease progression from chronic state to blast crisis. Moreover, technically, a novel combination of a high-throughput sample preparation assay for targeted PCR-based next-generation deep-sequencing was developed and allowed to broaden our molecular understanding in blast crisis CML. Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Wille:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding.

The Y-box binding protein YB-1 is associated with progressive disease and mediates survival and drug resistance in multiple myeloma

AbstractCurrent knowledge about molecular mechanisms underlying disease progression and drug resistance in multiple myeloma (MM) is still limited. Here, we analyzed the potential pathogenetic role of the Y-box binding protein YB-1 in MM. YB-1 is a member of the cold-shock domain protein superfamily and involved in various cellular functions such as proliferation. Immunohistochemical analyses revealed that neither normal bone marrow (BM) plasma cells (PCs), premalignant PCs of patients with monoclonal gammopathy of unknown significance (MGUS), nor MM cells with a mature morphology showed expression of YB-1 in situ. In contrast, YB-1 was strongly expressed in situ in normal PC precursor blasts as well as in a MM subset and in vitro in all of the evaluated MM cell lines. The YB-1–expressing MM cells were characterized by an immature morphology and a highly proliferative phenotype as defined by Ki 67 expression. We observed that siRNA-mediated knockdown of YB-1 decreased proliferation and induced apoptosis in MM cells even in the presence of BM stromal cells. Furthermore, we found that overexpression of YB-1 mediated resistance toward doxorubicin-induced apoptosis in MM cells. Thus, YB-1 contributes to disease progression, survival, and drug resistance in MM and might therefore provide an attractive therapeutic target.