We are a translational research laboratory interested in basic mechanisms of development and oncogenesis in the hematolymphoid system. A central question in our research is how non-traditional mechanisms of gene regulation effect fundamental aspects of development and how these mechanisms go awry in cancer. Our research program spans the spectrum from discovery of these novel regulators in in disease states to understanding their biological significance to developing rational therapeutic approaches to these diseases.
One of the key discoveries in the field has been that microRNA function is highly dependent on context. For example the transcriptional program of a given cell can greatly influence which transcripts are available for a given microRNA to target. To get towards a comprehensive understanding of microRNA function, we propose single cell studies.However, the changes induced by miR-146a expression may be heterogeneous across a population, and this heterogeneity can cause effects at the single cell level to specify cell fate decisions. Even when a tissue contains cells of the same type, the expression of a particular protein can show stochastic cell-to-cell variation as well as intracellular temporal fluctuations. Sometimes these fluctuations can be dramatic- up to ~1000 fold between the low expressing and high expressing cells at a given point in time. Recent reports suggest that such non genetic heterogeneity might be generated by miRNA-mediated effects on gene expression, and that this depends on the number of miRNA binding sites in the 3'UTR, the level of target mRNA in the cell, and the level of miRNA in the cell. The net effect of these is to cause a threshold effect- and that our conventional thinking about miRNA- partial repression of gene expression- may only hold for intermediate concentrations of the miRNA. In experiments with miRNA over expression or knock down, most group0s usually collect the RNA from an entire population of cells, revealing a snapshot of the average gene expression in the cells. This certainly doesn't tell us the entire story-different cells with differing unperturbed target mRNA levels would have different levels of change, depending on the mRNA threshold. Therefore, single cell expression studies are necessary to study cell-to-cell variation in response to miRNA's.
Developmental processes occur as a result of coordinated changes in gene expression, and dysregulated gene expression is at the heart of oncogenic transformation. Simplistically, gene expression can be regulated transcriptionally, at the level of chromatin and transcription factors, and post-transcriptionally, at the level of ribonucleic acid (RNA). The latter element of regulation is now understood to be highly complex and dynamic, and involves the interplay of sequences intrinsic to the regulated RNA molecules and extrinsic factors such as RNA binding proteins (RBPs), microRNAs, and other non-coding RNAs. RBPs have recently emerged as key regulators of post-transcriptional gene regulation, and as such, are likely to have quintessential roles in development and disease. The key to understanding the mechanisms of RBP regulation lies in assessment of the RNA binding repertoires of these RBPs, and meticulously connecting such data to specific points in development and oncogenesis.
Insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) is an oncofetal RBP with roles in mRNA translation, stabilization and localization. In an effort to understand post-transcriptional regulation of oncogenesis in the B-cell lineage, we undertook a study in B-lymphoblastic leukemia (B-ALL), finding upregulation of IGF2BP3 in cases that carry a translocation of the BLL gene, which portends a bad prognosis. Interestingly, it is dynamically expressed during B-cell development, suggesting a developmental function, and knockdown of IGFBP3 causes decreased growth in B-ALL cell lines. We are currently characterizing the role of this protein in hematopoeitic development and disease.
We have previously published studies on two tumor suppressive microRNAs, miR-34a and miR-146a, and how they influence B-cell and myeloid development respectively. These two miRNAs lie within central signaling pathways in cancer, and are induced by p53 and NF-kB, respectively. We are therefore very interested to understand if they are dysregulated in B-cell malignancies in particular, which have previously been shown to have disruptions in these pathways. In particular, we are;will define the status of these two miRNAs in a large cohort of clinical lymphoma samples, determine the contribution of these miRNAs to oncogenesis in mouse models, and explore the mechanism of their action using high-throughput sequencing techniques and bioinformatics to define regulated targets and cellular pathways. These studies will likely determine novel interconnections of the p53 and NF-kB pathways and turn up new targets to pursue as potential targets in oncogenesis.
More recently, our independent work has shown that the microRNA miR-146a seems to work through the Early Growth response-1 gene (Egr1). This manuscript was recently published, connecting a tumor suppressive microRNA with a novel pathway. The net result of this deletion of miR-146a was to cause more differentiated B-cell tumors in mice with Myc overexpression. We believe that this effect was exerted via derepression of Egr1 expression which controls the differentiation of B-cells. Depicted below are some of the gene expression parameters that we measured and their normal distribution of expression during the various stages of B-cell maturation.
Genomic level analyses (tiling arrays and high throughput sequencing) have shown the extent of transcription of non-coding RNA. Estimates vary- about 1-2% of the genome produces protein-coding RNA transcripts; while 30-50% of the genome is transcribed into non-coding transcripts! We are very interested to find out whether non-coding RNA, including but not limited to microRNAs, are important in the pathogenesis of leukemia. We are actively pursuing these studies, and with the help of bioinformatics collaborators and clinical colleagues in Los Angeles and elsewhere, are making significant progress towards defining non-coding RNAs in both pathologic states such as leukemia and lymphoma, but also in normal development. These studies involve a significant computational component, and we are actively working with bioinformatics and computational biology experts to resolve these important questions in how to recognize novel transcripts.
The recently discovered novel class of non-coding RNA, termed long intergenic non-coding RNA (lincRNA), play important roles in gene expression regulation, development and cancer. However, their roles in cancer have only begun to be explored. We have initiated an ambitious research study, using gene expression profiling to characterize the expression of lincRNA in B-lymphoblastic leukemia (B-ALL). We have discovered a set of lincRNAs that are differentially regulated in leukemia and particularly in poor-prognosis B-ALL with translocations of the mixed lineage leukemia (MLL) gene. One of these lincRNAs, BALR-2, was chosen for further study based on their correlation with clinicopathologic parameters and conservation of their genomic loci in mammals. We have made significant progress in understanding the cellular functions of BALR-2, finding that it promotes survival of leukemic cells. BALR-2 knockdown leads to activation of apoptosis pathways through the pro-apoptotic protein BIM. Further studies to characterize the role of BALR-2 in malignant transformation of hematopoietic cells and its utility as a therapeutic are actively being pursued in the laboratory.
Moreover, we are now beginning to study additional long non-coding RNA molecules in B-cell development and leukemia. We recently published a paper characterizing the role of BALR-6 in MLL-translocated leukemia; and we are now investigating the long non-coding RNA, CASC15, in B-cell leukemia and B-cell development.
In 2004, the first papers detailing the roles of microRNA in hematopoiesis were published by the labs of David Bartel and Harvey Lodish. Since then, there have been a plethoraof discoveries of how microRNAs influence hematopoiesis, and this has led to a better understanding not only of hematopoeisis but of how microRNAs may function in development in general. Some emerging themes include the idea that some microRNAs act primarily through a single target at critical points of development, or that microRNAs may serve as organizing loci for complex developmental pathways of, finally, that microRNAs connect disparate cellular pathways whose interconnections were previously unrecognized. Our current research has come to focus on microRNAs that are involved in hematopoiesis and the immune response. One such microRNA, miR-146a, is found to be involved in the regulation of developmental processes in a variety of hematopoietic cells. One angle we are focused on is to understand how NF-kappa B dysregulation may result in the range of myeloid and hematopoeitic phenotypes that we are observing. To do so, we are collaborating with the laboratory of Alex Hoffmann. Secondly, we are very interested in understanding the role of miR-146a in the immune response. We have recently published a paper on defective marginal zone B-cell development in miR-146a deficient mice, and collaborated on a manuscript that describes roles for miR-146a in regulating T-cell activation. Studies of how miR-146a regulates B-cell activation and autoimmunity are future focuses of this area of research in the laboratory.