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(1) Discovering lncRNA-dependent interactomes in cancers

Long non-coding RNAs (lncRNAs) are emerging as important regulators of tissue physiology and disease processes, especially cancers. Although dysregulated lncRNA expression has been associated with cancer progression, the contribution of lncRNAs to oncogenesis is poorly understood because their molecular and biological functions are obscure. We recently identified a novel lncRNA and its interacting proteins important for melanoma invasion. Projects are available to understand how this lncRNA functions at the molecular level, which may be important for determining why more males than females die from melanomas.

More broadly, the Novina lab is attempting to understand lncRNA biology and its roles in oncogenesis by systematically identifying lncRNA-associated proteins. It is virtually impossible to bioinformatically predict lncRNA function (or interacting proteins) by sequence analysis because (1) lncRNAs are poorly conserved and (2) proteins bind to RNAs by a poorly understood combination of RNA sequence and secondary structure. Traditional methods to define lncRNA-protein interactions focus on immunoprecipitating one candidate RNA with an RNA tag or antisense DNA oligos, or incubating cell lysates with biotinylated in vitro transcribed RNAs followed by mass spectrometry or western blotting. Not only are these methods low throughput, but the protein identification step is extremely challenging due to limited biomass from low-efficiency immunoprecipitations, loss of transient and weak interactors, or isolation of promiscuous RNA binding proteins. We are beginning to define lncRNA-dependent interactomes through development of a lncRNA-based yeast three hybrid (Y3H) platform. Projects are available to systematically define lncRNA-protein interactomes for a set of cancer-promoting lncRNAs through implementation of RATA and Y3H systems.

(2) Molecular pathogenesis of the ribosomopathies SDS and DBA

We discovered that reduced ribosome biogenesis and function decreases microRNA activity. This led us study the molecular basis of inherited bone marrow failure syndromes, which are caused by germline mutations in ribosomal pathway genes (e.g. Shwachman Diamond Syndrome (SDS) and Diamond Blackfan Anemia (DBA)). Though these diseases were characterized more than 40 years ago, their molecular pathogenesis has remained obscure in part due to the rarity and heterogeneity of the affected bone marrow progenitors.

Dysfunction in rare or heterogeneous cell types contributes to human disease. Recent technological advancements have enabled robust analysis of single eukaryotic cells. Single cell RNA-sequencing on primary CD34+ hematopoietic progenitors from normal and SDS bone marrows identified dysregulation of TGF-beta target genes in SDS hematopoietic stem cells and multipotent progenitors, but not in lineage committed progenitors. Proteomic analysis of primary SDS patient plasma identified increased TGF-beta family ligand production. Treatment of SDS patient BM with a TGFBR1 inhibitor increased hematopoietic colony formation, supporting a causative role for TGF-beta signaling through its receptor as a mechanism of SDS pathogenesis (manuscript under review). Projects are available to perturb specific effectors in the TGF-beta signaling network in selected cell populations in SDS bone marrows. These studies might enable translation of insights from single cell biology into a novel SDS therapy.

(3) Targeted DNA methylation and epigenetic regulation of gene expression

Regulatory RNAs are just one of many regulatory mechanisms that coordinate gene expression in normal and disease contexts. MicroRNA and lncRNA genes themselves are developmentally regulated and demonstrate altered epigenetic marks such as aberrant promoter hypo- and hyper-methylation, especially in cancers. Altered microRNA expression has been correlated with the tissue of origin, prognosis, and drug sensitivity of cancers and other diseases.

We recently described a novel tool for targeted DNA methylation by tethering a “split-fusion” methyltransferase to an endonuclease-deficient mutant Cas9. Our split-fusion approach minimizes off-target effects by ensuring that enzyme activity is specifically reconstituted at the targeted locus. We are also developing gRNA screening strategies to fine-tune targeting within each locus. How are epigenetic marks set, maintained, spread and inherited? How do establishing DNA marks relate to establishing histone marks? These fundamentally important questions must be answered to realize the full potential of epigenetic engineering in the clinic. The epigenetic engineering tools developed in this project can be applied to other projects in the Novina Lab.

Projects are available to (1) reprogram methylation states at disease-relevant loci for therapy, in particular for cancer immunotherapy; (2) compare methylation-dependent changes in microRNA transcription to oncogenic phenotypes; and (3) relate differential promoter methylation to changes in chromatin architecture and transcription factor binding at promoters.

(4) Engineering T cells for immunotherapy

T cells play a central role in our immune system’s responses to infections. Recent clinical studies have shown tremendous promise that the immune system can be “taught” to reject tumors as if the tumors were infections. One way to teach T cells is to “engineer” them to bind specific proteins found on the surface of tumors. An especially exciting example of this strategy is the use of “chimeric antigen receptor” (CAR) T cells. Clinical trials have shown that CAR T cells can selectively bind to a protein found on only antibody producing cells (B cells) and on B cell cancers (leukemias). However, patients who receive such CAR T cells often show complete remission of their leukemia as well as a dramatic loss of their normal B cells. This approach is limited and therefore not used to treat patients with solid tumors partly because few proteins are found only on tumors but not on normal tissues.

The Novina Lab is developing technologies that broaden the application of CAR T cell technology to attack solid tumors by selectively unmasking artificial antigens on tumors or through direct engineering of T cell receptors. We are working with clinical and industry collaborators to engineer CAR T cells that mount robust immune responses at tumors with minimal effects on normal tissues. Projects are available to engineer autologous T cells for ovarian cancer, neuroendocrine and brain cancer immunotherapy. We are also exploring the use of RNA-based strategies to engineer T cells to resist the anti-inflammatory environment which limits immune responses to tumors.