Hatice Gulcin Ozer

Resistance Mechanisms for the Bruton's Tyrosine Kinase Inhibitor Ibrutinib

BACKGROUND Ibrutinib is an irreversible inhibitor of Brutons tyrosine kinase (BTK) and is effective in chronic lymphocytic leukemia (CLL). Resistance to irreversible kinase inhibitors and resistance associated with BTK inhibition have not been characterized. Although only a small proportion of patients have had a relapse during ibrutinib therapy, an understanding of resistance mechanisms is important. We evaluated patients with relapsed disease to identify mutations that may mediate ibrutinib resistance. METHODS We performed whole-exome sequencing at baseline and the time of relapse on samples from six patients with acquired resistance to ibrutinib therapy. We then performed functional analysis of identified mutations. In addition, we performed Ion Torrent sequencing for identified resistance mutations on samples from nine patients with prolonged lymphocytosis. RESULTS We identified a cysteine-to-serine mutation in BTK at the binding site of ibrutinib in five patients and identified three distinct mutations in PLCγ2 in two patients. Functional analysis showed that the C481S mutation of BTK results in a protein that is only reversibly inhibited by ibrutinib. The R665W and L845F mutations in PLCγ2 are both potentially gain-of-function mutations that lead to autonomous B-cell-receptor activity. These mutations were not found in any of the patients with prolonged lymphocytosis who were taking ibrutinib. CONCLUSIONS Resistance to the irreversible BTK inhibitor ibrutinib often involves mutation of a cysteine residue where ibrutinib binding occurs. This finding, combined with two additional mutations in PLCγ2 that are immediately downstream of BTK, underscores the importance of the B-cell-receptor pathway in the mechanism of action of ibrutinib in CLL. (Funded by the National Cancer Institute and others.).

JNK3 Perpetuates Metabolic Stress Induced by Aβ Peptides

Although Aβ peptides are causative agents in Alzheimers disease (AD), the underlying mechanisms are still elusive. We report that Aβ42 induces a translational block by activating AMPK, thereby inhibiting the mTOR pathway. This translational block leads to widespread ER stress, which activates JNK3. JNK3 in turn phosphorylates APP at T668, thereby facilitating its endocytosis and subsequent processing. In support, pharmacologically blocking translation results in a significant increase in Aβ42 in a JNK3-dependent manner. Thus, JNK3 activation, which is increased in human AD cases and a familial AD (FAD) mouse model, is integral to perpetuating Aβ42 production. Concomitantly, deletion of JNK3 from FAD mice results in a dramatic reduction in Aβ42 levels and overall plaque loads and increased neuronal number and improved cognition. This reveals AD as a metabolic disease that is under tight control by JNK3.

Dysregulation of miR-31 and miR-21 induced by zinc deficiency promotes esophageal cancer

Zinc deficiency (ZD) increases the risk of esophageal squamous cell carcinoma (ESCC). In a rat model, chronic ZD induces an inflammatory gene signature that fuels ESCC development. microRNAs regulate gene expression and are aberrantly expressed in cancers. Here we investigated whether chronic ZD (23 weeks) also induces a protumorigenic microRNA signature. Using the nanoString technology, we evaluated microRNA profiles in ZD esophagus and six additional tissues (skin, lung, pancreas, liver, prostate and peripheral blood mononuclear cells [PBMC]). ZD caused overexpression of inflammation genes and altered microRNA expression across all tissues analyzed, predictive of disease development. Importantly, the inflammatory ZD esophagus had a distinct microRNA signature resembling human ESCC or tongue SCC miRNAomes with miR-31 and miR-21 as the top-up-regulated species. Circulating miR-31 was also the top-up-regulated species in PBMCs. In ZD esophagus and tongue, oncogenic miR-31 and miR-21 overexpression was accompanied by down-regulation of their respective tumor-suppressor targets PPP2R2A and PDCD4. Importantly, esophageal miR-31 and miR-21 levels were directly associated with the appearance of ESCC in ZD rats, as compared with their cancer-free Zn-sufficient or Zn-replenished counterparts. In situ hybridization analysis in rat and human tongue SCCs localized miR-31 to tumor cells and miR-21 to stromal cells. In regressing tongue SCCs from Zn-supplemented rats, miR-31 and miR-21 expression was concomitantly reduced, establishing their responsiveness to Zn therapy. A search for putative microRNA targets revealed a bias toward genes in inflammatory pathways. Our finding that ZD causes miR-31 and miR-21 dysregulation associated with inflammation provides insight into mechanisms whereby ZD promotes ESCC.

miRNA profiling for biomarker discovery in Multiple Sclerosis: from microarray to deep sequencing

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. miRNAs are highly expressed in cells of the immune and nervous system, attesting to their importance in Neuroimmunology. Besides their involvement in modulation of physiological and pathological processes, miRNAs hold high promise as disease biomarkers, therapeutic agents and/or drug targets. Several studies have recently explored the involvement of miRNAs in Multiple Sclerosis (MS) using a variety of miRNA profiling techniques. In this review, we discuss basic miRNA biology and nomenclature, the techniques available for miRNA profiling research and recent miRNA profiling studies in Multiple Sclerosis.

Using gene co-expression network analysis to predict biomarkers for chronic lymphocytic leukemia

BackgroundChronic lymphocytic leukemia (CLL) is the most common adult leukemia. It is a highly heterogeneous disease, and can be divided roughly into indolent and progressive stages based on classic clinical markers. Immunoglobin heavy chain variable region (IgVH) mutational status was found to be associated with patient survival outcome, and biomarkers linked to the IgVH status has been a focus in the CLL prognosis research field. However, biomarkers highly correlated with IgVH mutational status which can accurately predict the survival outcome are yet to be discovered.ResultsIn this paper, we investigate the use of gene co-expression network analysis to identify potential biomarkers for CLL. Specifically we focused on the co-expression network involving ZAP70, a well characterized biomarker for CLL. We selected 23 microarray datasets corresponding to multiple types of cancer from the Gene Expression Omnibus (GEO) and used the frequent network mining algorithm CODENSE to identify highly connected gene co-expression networks spanning the entire genome, then evaluated the genes in the co-expression network in which ZAP70 is involved. We then applied a set of feature selection methods to further select genes which are capable of predicting IgVH mutation status from the ZAP70 co-expression network.ConclusionsWe have identified a set of genes that are potential CLL prognostic biomarkers IL2RB, CD8A, CD247, LAG3 and KLRK1, which can predict CLL patient IgVH mutational status with high accuracies. Their prognostic capabilities were cross-validated by applying these biomarker candidates to classify patients into different outcome groups using a CLL microarray datasets with clinical information.

Characterization of CLL exosomes reveals a distinct microRNA signature and enhanced secretion by activation of BCR signaling

Multiple studies show that chronic lymphocytic leukemia (CLL) cells are heavily dependent on their microenvironment for survival. Communication between CLL cells and the microenvironment is mediated through direct cell contact, soluble factors, and extracellular vesicles. Exosomes are small particles enclosed with lipids, proteins, and small RNAs that can convey biological materials to surrounding cells. Our data herein demonstrate that CLL cells release significant amounts of exosomes in plasma that exhibit abundant CD37, CD9, and CD63 expression. Our work also pinpoints the regulation of B-cell receptor (BCR) signaling in the release of CLL exosomes: BCR activation by α-immunoglobulin (Ig)M induces exosome secretion, whereas BCR inactivation via ibrutinib impedes α-IgM-stimulated exosome release. Moreover, analysis of serial plasma samples collected from CLL patients on an ibrutinib clinical trial revealed that exosome plasma concentration was significantly decreased following ibrutinib therapy. Furthermore, microRNA (miR) profiling of plasma-derived exosomes identified a distinct exosome microRNA signature, including miR-29 family, miR-150, miR-155, and miR-223 that have been associated with CLL disease. Interestingly, expression of exosome miR-150 and miR-155 increases with BCR activation. In all, this study successfully characterized CLL exosomes, demonstrated the control of BCR signaling in the release of CLL exosomes, and uncovered a disease-relevant exosome microRNA profile.

Role of ChIP-seq in the discovery of transcription factor binding sites, differential gene regulation mechanism, epigenetic marks and beyond

Many biologically significant processes, such as cell differentiation and cell cycle progression, gene transcription and DNA replication, chromosome stability and epigenetic silencing etc. depend on the crucial interactions between cellular proteins and DNA. Chromatin immunoprecipitation (ChIP) is an important experimental technique for studying interactions between specific proteins and DNA in the cell and determining their localization on a specific genomic locus. In recent years, the combination of ChIP with second generation DNA-sequencing technology (ChIP-seq) allows precise genomic functional assay. This review addresses the important applications of ChIP-seq with an emphasis on its role in genome-wide mapping of transcription factor binding sites, the revelation of underlying molecular mechanisms of differential gene regulation that are governed by specific transcription factors, and the identification of epigenetic marks. Furthermore, we also describe the ChIP-seq data analysis workflow and a perspective for the exciting potential advancement of ChIP-seq technology in the future.

Chromatin modification by SUMO-1 stimulates the promoters of translation machinery genes

SUMOylation of transcription factors and chromatin proteins is in many cases a negative mark that recruits factors that repress gene expression. In this study, we determined the occupancy of Small Ubiquitin-like MOdifier (SUMO)-1 on chromatin in HeLa cells by use of chromatin affinity purification coupled with next-generation sequencing. We found SUMO-1 localization on chromatin was dynamic throughout the cell cycle. Surprisingly, we observed that from G1 through late S phase, but not during mitosis, SUMO-1 marks the chromatin just upstream of the transcription start site on many of the most active housekeeping genes, including genes encoding translation factors and ribosomal subunit proteins. Moreover, we found that SUMO-1 distribution on promoters was correlated with H3K4me3, another general chromatin activation mark. Depletion of SUMO-1 resulted in downregulation of the genes that were marked by SUMO-1 at their promoters during interphase, supporting the concept that the marking of promoters by SUMO-1 is associated with transcriptional activation of genes involved in ribosome biosynthesis and in the protein translation process.

Down-regulation of homeobox genes MEIS1 and HOXA in MLL-rearranged acute leukemia impairs engraftment and reduces proliferation

Rearrangements of the MLL (ALL1) gene are very common in acute infant and therapy-associated leukemias. The rearrangements underlie the generation of MLL fusion proteins acting as potent oncogenes. Several most consistently up-regulated targets of MLL fusions, MEIS1, HOXA7, HOXA9, and HOXA10 are functionally related and have been implicated in other types of leukemias. Each of the four genes was knocked down separately in the human precursor B-cell leukemic line RS4;11 expressing MLL-AF4. The mutant and control cells were compared for engraftment in NOD/SCID mice. Engraftment of all mutants into the bone marrow (BM) was impaired. Although homing was similar, colonization by the knockdown cells was slowed. Initially, both types of cells were confined to the trabecular area; this was followed by a rapid spread of the WT cells to the compact bone area, contrasted with a significantly slower process for the mutants. In vitro and in vivo BrdU incorporation experiments indicated reduced proliferation of the mutant cells. In addition, the CXCR4/SDF-1 axis was hampered, as evidenced by reduced migration toward an SDF-1 gradient and loss of SDF-1–augmented proliferation in culture. The very similar phenotype shared by all mutant lines implies that all four genes are involved and required for expansion of MLL-AF4 associated leukemic cells in mice, and down-regulation of any of them is not compensated by the others.

Targeting Leukemia Stem Cells in vivo with AntagomiR-126 Nanoparticles in Acute Myeloid Leukemia

Current treatments for acute myeloid leukemia (AML) are designed to target rapidly dividing blast populations with limited success in eradicating the functionally distinct leukemia stem cell (LSC) population, which is postulated to be responsible for disease resistance and relapse. We have previously reported high miR-126 expression levels to be associated with a LSC-gene expression profile. Therefore, we hypothesized that miR-126 contributes to ‘stemness’ and is a viable target for eliminating the LSC in AML. Here we first validate the clinical relevance of miR-126 expression in AML by showing that higher expression of this microRNA (miR) is associated with worse outcome in a large cohort of older (⩾60 years) cytogenetically normal AML patients treated with conventional chemotherapy. We then show that miR-126 overexpression characterizes AML LSC-enriched cell subpopulations and contributes to LSC long-term maintenance and self-renewal. Finally, we demonstrate the feasibility of therapeutic targeting of miR-126 in LSCs with novel targeting nanoparticles containing antagomiR-126 resulting in in vivo reduction of LSCs likely by depletion of the quiescent cell subpopulation. Our findings suggest that by targeting a single miR, that is, miR-126, it is possible to interfere with LSC activity, thereby opening potentially novel therapeutic approaches to treat AML patients.