Jessica Silva-Fisher, Ph.D. (Year 2)
Lay Summary (Final Progress Report)
Multiple myeloma is a type of blood cancer that accounts for about 13% of blood cancers and 1% of all cancers. It is always preceded by an earlier condition called MGUS (monoclonal gammopathy of undetermined significance), which can progress to smoldering myeloma and then to full-blown myeloma. Although new treatments have improved survival rates, myeloma remains incurable, and most patients eventually relapse. A major challenge in treating myeloma is the lack of reliable tools to predict when and how the disease will progress.
In our research, we focused on long non-coding RNAs (lncRNAs), a special type of RNA that doesn’t produce proteins but instead helps control how cells function by interacting with other molecules. Because lncRNAs are specific to certain tissues, they have great potential as early warning signs or targets for treating diseases like myeloma.
We studied patient samples to find lncRNAs that change as the disease progresses. We identified a group of these RNAs, which we call “Multiple Myeloma Progression-associated lncRNAs” (MMPals), and focused on one in particular, called MMPal1. We found that MMPal1 levels increase as myeloma develops and that blocking MMPal1 in cancer cells reduces their growth and survival. We also discovered that MMPal1 interacts with a protein called CBX4 in drug-resistant myeloma cells, which might explain why some patients don’t respond to certain treatments.
To understand MMPal1’s role in drug resistance, we tested cells with a common chemotherapy drug, melphalan, and found that resistant cells had higher levels of MMPal1. We also developed tools to target MMPal1 directly, which showed promise in reducing cancer cell survival and increasing their sensitivity to treatment.
This study is the first to show how MMPal1 works in myeloma progression and drug resistance. In the future, this research could help improve early detection of myeloma, predict which patients are at risk for drug resistance, and lead to new treatments that target this RNA to improve outcomes for patients.
Abstract
This project aims to address the critical gap in understanding the malignant evolution of myeloma by identifying and characterizing the mechanisms of lncRNAs for use as biomarkers for prognosis of disease progression. Thereby, we compared singlecell RNA sequencing data from plasma and B cells from a publicly available dataset of normal (n=11), MGUS (n=?), SMM (n=6), and myeloma (n=12) patient samples and a validation cohort of 18 myeloma patient samples from the Multiple Myeloma Research Foundation’s CoMMpass Study. We identified six differentially expressed lncRNAs comparing MGUS to SMM samples, 14 lncRNAs comparing SMM to myeloma, and 19 lncRNAs comparing normal to myeloma, which we term Multiple Myeloma Progression-associated lncRNAs (MMPals).
We focused on the top most differentially expressed lncRNA, MMPal1, also known as NEAT1. We detected little to no MMPal1 expression in normal samples, and saw an increase of expression in MGUS patient samples to myeloma samples. Silencing MMPal1 expression with silencer RNAs shows a decrease in proliferation and viability.
To determine if MMPal1 is associated with drug resistance, we treated cells with melphalan, a chemotherapy drug used as the conditioning agent in autologous stem cell transplantation. Melphalan-sensitive MM.1S cells showed less MMPal1 expression when compared to melphalan resistant U266B1 cells. Next, we assessed if MMPal1 binds to Chromobox 4 (CBX4) protein, due to its similar cellular location in nuclear speckles, epigenetic regulation, and known binding to lncRNAs. We conducted RNA immunoprecipitation and individual-nucleotide resolution cross-linking immunoprecipitation (iCLIP) qPCR to determine that indeed CBX4 binds to MMPal1. We show that MMPal1-CBX4 interaction only occurs in melphalan-resistant cells when treated with melphalan and not in melphalan-sensitive cells.
More recently, to determine clinical importance, we used multiplexed Fluorescent RNA In situ Hybridization (mFISH) to detect MMPal1 in cells and patient samples. Additionally, we created locked nucleic acid antisense oligonucleotides (LNA ASOs) targeting MMPal1 and saw a decrease in viability and increase in cytotoxicity and apoptosis with decreased MMPal1 expression. Our preliminary data serves as strong rationale for our hypothesis that MMPal1 binding to CBX4 plays a role as a master epigenetic regulator to promote myeloma progression and that lncRNAs can be utilized as biomarkers for prognosis of myeloma disease progression.
Our hypothesis will be tested in three specific aims.
- Aim 1 will assess interaction of MMPa/1 RNA and CBX4 protein in patient and melphalan-treated cells. We hypothesize that MMPal1 expression increases and interacts with CBX4 in melphalan-treated cells. To date, we have optimized detection of MMPal1 expression in myeloma cells and patient bone marrow aspirate samples using mFISH. Next, we will use mFISH combined with immunohistochemistry to assess MMPal1 RNA and CBX4 protein expression simultaneously in cells treated with and without melphalan and in myeloma patient samples.
- Aim 2 will identify CBX4-lncRNA interactions and their clinical importance in We discovered binding of MMPal1 RNA to CBX4 protein and hypothesize that other RNAs may also bind to CBX4 thereby promoting myeloma. Thus, we will conduct CBX4 iCLIP sequencing to identify all bound RNAs targets in vivo.
- Aim 3. MMPal1 CRISPR and CBX4 knockdown, overexpression, or LNA ASOs, will be assessed to determine cell viability, cytotoxicity, and apoptosis using the ApoTox-Glo Triplex Assay. RNA will be isolated from respective cells for sequencing to identify gene regulation.
Overall, this proposal will be the first to assess the MMPal1-CBX4 interaction in understanding multiple myeloma progression. Further, this proposal will fill a knowledge gap for the clinical significance of lncRNAs and have translational impact by evaluating lncRNAs as diagnostics and therapies to improve survival and longevity.
To read the full progress report, click here.