Identification of long non-protein-coding RNAs collaborating with PRC2 in MM
Multiple myeloma (MM) despite becoming increasingly treatable remains to date incurable and accounts for the second most common hematological cancer. This warrants innovative approaches into tackling the disease overtly complex genetic background. Our research aims is to identify interacting partners with the Polycomb group proteins by RNA immunoprecipitation coupled with next generation sequencing (RIP-seq, CLIP seq or iCLIP) in MM cell lines and primary MM samples. We also aim to identify the genome-specific binding of lncRNAs of interest by chromatin isolation via RNA precipitation (ChIRP) assays coupled to next generation sequencing and mass spectrometry.
In this project we will investigate Polycomb-collaborating non-protein-coding RNAs, which remain poorly understood in MM. These investigations will be performed in well-characterized MM cell lines, primary MM cells and age matched normal bone marrow plasma cells, in order to identify developmental and malignant regulators. Our ultimate goal is to provide a solid pre-clinical evaluation of novel targets for treatment of MM.
Multiple myeloma (MM) is characterized by the accumulation and expansion of malignant plasma cells within the bone marrow. Large sequencing efforts have reported an extensive genetic heterogeneity within and among patients. Despite advances in treatment protocols MM patients often develop drug resistance and eventually relapse. We were first to focus on investigating common underlying patterns of epigenetic dysregulation in MM. As the epigenome is known to safeguard the genetic integrity this is an especially interesting target for innovative therapy. By identifying a common among patients underexpressed gene profile in large cohorts of MM patients we established gene targeting by the Polycomb repressor complex 2 (PRC2) as a common denominator in MM.
We further established that its enrichment correlated with advanced stages of MM and poor disease outcome. Notably, we deciphered a level of gene regulation involving the aberrant Polycomb silencing of non-protein-coding regulatory microRNAs, which act as tumor-suppressors by targeting well-described MM oncogenes. In addition, we recently provided evidence that targeting collaborators to the PRC2 complex, in this case PRC1, holds a novel anti-myeloma potential. At the pre-clinical level, it is now essential to further evaluate the potential of targeting Polycomb in a relevant immunocompetent animal disease model, such as the 5T syngeneic murine models of MM, proven to provide solid grounds for pharmacological investigations. Furthermore, with increased screening of EZH2 inhibition among MM cell lines and primary cells have identified a proportion of cells that are resistant to several independent EZH2 inhibitors. Our most current data points towards interplay between PRC2 and other epigenetic machineries as supporting this resistance.
Previous studies have identified multiple lncRNAs to interact with the PRC2 and play a critical role in fine tuning the transcriptional and translational network in embryonic stem cells. In MM, lncRNA has in a few cases, such as MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) and MEG3 (Maternally Expressed Gene 3), recently also been suggested as both prognostic markers and therapeutic target. Although specific signatures of lncRNAs have partially been characterized in MM, the function of most lncRNAs yet remains largely unknown. It is clear that by investigating the complex partnership between lncRNA and the PRC2, we may unveil novel biological functions and provide an explanation to the seemingly repressed genomic landscape of MM. As a result, continuous analysis of the lncRNA expression profiles in MM and the biological function of lncRNAs associated with the PRC2 could provide novel therapeutic targets and improve patient survival.
Our initial investigation will focus on identifying lncRNAs that are physically interacting with the catalytic core component, EZH2, of PRC2 by RNA immunoprecipitation sequencing (RIP-seq). By using an EZH2 specific antibody for protein pull down and annotate the sequence attached to the complex will provide insight into what lncRNAs are associated with PRC2. Once a candidate list has been generated, chromatin isolation by RNA purification (ChIRP-seq) can be performed to evaluate lncRNA binding site within the genome of MM. This will be achieved by the use of biotin-labeled oligonucleotide probes for the lncRNAs of interest. Verification of histone mark and EZH2 binding enrichment to these identified regions will be accomplished by chromatin immunoprecipitation qPCR (ChIP-qPCR). To validate the ChIRP-seq platforms performance, ChIRP-qPCR will be performed, based on the biotin-streptavidin concept previously presented. As a proof of concept of that our hypothesis is correct and can be validated within the time frame of the project our initial experiments in MM cell lines show overexpression of a lncRNA e.g. PVT1, a lncRNA previously associated with EZH2 and preventing PRC2 interaction with anti-tumor suppressive genes. Our preliminary data also support a novel physical interaction between PVT1 and EZH2 in MM, not previously reported, and potential DNA binding sites in which many of these overlap with, by our group, identified unique H3K27me3 and bivalent PRC2 targets. These data now provide a proof-of-concept for continuing to investigate lncRNAs as part of a complex epigenetic machinery and evaluating the biological relevance and function of disease associated lncRNAs.
If you are a driven and highly motivated student with an interests in epigenetics and cancer biology please don’t hesitate to contact us.
Prof. Helena Jernberg-Wiklund, PI.
Experimental and Clinical Oncology / IGP
Patrick Nylund, PhD Student
Experimental and Clinical Oncology / IGP