Jessica Silva-Fisher, Ph.D.
FINAL REPORT
Chemotherapy remains one of the most effective treatments for cancer, yet its benefits are frequently accompanied by systemic toxicities that extend beyond tumor tissue. Among these, skeletal and cardiac muscle loss are increasingly recognized as major determinants of morbidity, treatment intolerance, and long-term functional decline. Anthracyclines such as doxorubicin are well known to cause cardiotoxicity, but they also induce a broader catabolic state characterized by muscle atrophy, altered substrate utilization, and impaired regenerative capacity. These effects are particularly consequential in older individuals, in whom baseline muscle mass and metabolic flexibility are already diminished.
Muscle loss during cancer therapy intersects biologically with aging. Both processes involve dysregulated nutrient sensing, mitochondrial stress, inflammatory signaling, and impaired proteostasis. As lifespan increases and more patients survive cancer into older age, preventing therapy-induced muscle decline has become central not only to oncology outcomes but also to long-term healthspan. Interventions that modulate metabolism, such as fasting, macronutrient manipulation, or pharmacologic targeting of nutrient-sensing pathways, are increasingly promoted to mitigate chemotherapy toxicity, yet their effects on cardiac and skeletal muscle remain incompletely defined.
In this context, our project examined whether dietary interventions could modify doxorubicin-induced muscle atrophy and cardiac remodeling. We found that intermittent fasting induced marked adipose tissue remodeling that unexpectedly accelerated cardiac wasting in the setting of doxorubicin exposure. Rather than protecting against chemotherapy-associated muscle loss, fasting altered systemic metabolic signaling, thereby amplifying cardiomyocyte atrophy. In contrast, a high-fat diet prevented doxorubicin-induced cardiac muscle atrophy and preserved cardiomyocyte size under stress conditions. Mechanistic studies identified lysosomal lipolysis as a critical regulator of cardiomyocyte growth and survival, a pathway not previously recognized as central to chemotherapy-associated cardiac wasting. Unexpectedly, high protein feeding failed to rescue cardiac muscle mass and, in some settings, exacerbated atrophy, refining prevailing assumptions regarding protein supplementation during catabolic stress.
Although the original proposal focused on chemotherapy-associated muscle loss, this line of investigation ultimately led to the downstream identification of a novel therapeutic strategy that prevents muscle loss in the broader context of weight reduction. This discovery extends beyond anthracycline cardiotoxicity and has potential implications for aging-associated sarcopenia, obesity treatment, and intentional weight loss. Further translational development and grant submissions are in progress. Specific molecular components and therapeutic details are withheld pending intellectual property protection and considerations.
ABSTRACT:
Multiple myeloma (MM) is one the most common hematologic malignancies that accounts for about 13% of all hematologic malignancies and 1% of overall cancer. Despite advances in treatments, MM is still incurable and the lack of reliable biomarkers to predict development of MM is a critical barrier. MM is always preceded by a premalignant phase called monoclonal gammopathy of undetermined significance (MGUS) and can then progress to smoldering multiple myeloma (SMM) and/or malignant MM. Although survival of MM patients has improved with new treatments, most patients suffer fatal relapse. Long non-coding RNAs (lncRNAs) are longer than 200 base pairs, have important regulatory functions by binding to proteins, and have proven to play roles in promoting cancer. Due to tissue specificity of lncRNAs, they show promise as prognostic and diagnostic biomarkers for MM.
This project aims to address the critical gap of understanding the malignant evolution of MM by identifying and characterizing the mechanisms of lncRNAs for use as biomarkers for prognosis of disease progression. Thereby, we compared single-cell RNA sequencing data from plasma and B cells from a publicly available dataset of normal (n=11), MGUS (n=7), SMM (n=6), and MM (n=12) patient samples and a validation cohort of 18 MM patient samples from the Multiple Myeloma Research Foundation’s CoMMpass Study. We identified five differentially expressed lncRNAs comparing normal to MGUS samples, nine lncRNAs comparing MGUS to SMM samples, 26 lncRNAs comparing SMM to MM, and 25 lncRNAs comparing normal to MM, (27 unique lncRNAs) which we term Multiple Myeloma Progression associated lncRNAs (MMPals). We focused on the top 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. MMPal1 expression is lowly expressed in SMM, however increases significantly comparing SMM to MM, and in MM compared to normal. MMPal1 is also significantly expressed only in Mature B Cell Neoplasm cell lines as compared to a panel of leukemia cells. Silencing MMPal1 expression in U266B1 MM cells, 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 transplant. Melphalan sensitive MM1.S 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 determined that indeed MMPal1 binds to CBX4. Lastly, we show that MMPal1 binds to CBX4 only in Melphalan resistant cells when treated with Melphalan and not in Melphalan sensitive cells. We hypothesize that MMPal1 may play a role as a master epigenetic regulator to promote MM progression and resistance indicating it as a potential biomarker. Our hypothesis will be tested in three specific aims. Aim 1 will validate the expression of MMPal1 as a prognostic biomarker. We will perform multiplexed Fluorescent RNA In situ Hybridization of MMPal1 in a larger cohort of 12 normal and 24 bone marrow aspirates each from normal, MGUS, SMM, and MM patient samples using RNAScope. Aim 2 will characterize MMPal1-dependant CBX4 regulation. We will generate a U266B1 MMPal1 CRISPR/Cas9 knock-out (CRISPR KO) cell line to validate MMPal1 function. We will then characterize changes in gene promoter occupancy of CBX4 in U266B1 wild type, MMPal1 CRISPR KO, and Melphalan treated cells via chromatin immunoprecipitation sequencing and assess expression of gene targets via RNA sequencing. Aim 3 will identify functionally important lncRNAs associated with promoting MM. A CRISPR interference (CRISPRi) platform targeting all known 16,401 lncRNAs will be used to assess a genome-wide view of cell viability, cytotoxicity, and apoptosis of lncRNA sgRNA transduced U266B1 GFP-luciferase cells compared to negative control sgRNAs. DNA isolated from transduced cells will be sequenced to identify specific sgRNAs causing loss of function. Overall, this proposal will be the first to assess MMPal1 as a potential biomarker and provide a functional understanding of lncRNAs in MM progression. This proposal will fill a knowledge gap for the clinical significance of lncRNAs and have translational impact by evaluating lncRNAs as diagnostics and prognostics to improve survival and longevity.
LAY SUMMARY:
The overall research goal of this proposal is to understand why and how multiple myeloma (MM) progresses to address the critical need for better prognostic biomarkers, improve diagnoses, and create targeted therapies.
MM is a type of cancer that forms from plasma cells and accumulates in the bone marrow. Despite advances in treatment for MM, there is a lack of reliable biomarkers to predict disease development and direct further therapies. MM proceeds by the abnormal growth of plasma cells called asymptomatic condition monoclonal gammopathy of undetermined significance (MGUS). MGUS enters a more aggressive stage called smoldering multiple myeloma (SMM) and finally becomes the malignant form of active MM. Why normal plasma cells become abnormal and progress into MM is not known. Many studies have focused on the 2% of genes in the human genome that are protein-coding genes, but our proposal will analyze long non-protein coding RNAs (lncRNAs) that are part of the larger remaining 98% of the human genome that is often ignored. lncRNAs are not translated into proteins but have been found to play important roles in all levels of regulation, as they are capable of binding to proteins, DNA, and other small RNAs to promote cancer. Our lab’s research has shown that lncRNAs bind to proteins for inducing tumorigenesis, metastasis, and drug resistance in metastatic colon cancer and late-stage relapse breast cancer.
To better understand the role of lncRNAs in MM progression, we analyzed single-cell RNA sequencing data from plasma and B Cells in normal, MGUS, SMM, and MM patient samples. We found 27 unique deregulated lncRNAs when comparing each stage of progression, we term Multiple Myeloma Progression associated lncRNAs (MMPals). Notably, the top up-regulated candidate, MMPal1, (i) shows increasing expression comparing normal to MGUS, SMM to MM, and normal to MM, (ii) has specific expression in mature B cell neoplasm cells and is highly expressed in MM cancer cell lines, (iii) decreases proliferation and viability, (iv) is more highly expressed in melphalan-resistant cell lines, standard treatment commonly used followed by autologous stem cell transplant for transplant-eligible patients with MM, and (v) binds to CBX4 in melphalan treated resistant cells. These key findings provide strong premise for our hypothesis that MMPal1 is a prognostic biomarker that interacts with CBX4 to regulate downstream genes and promote MM progression.
To test this hypothesis, Aim 1 will assess and validate RNA expression of MMPal1 in a larger cohort of patient samples using Fluorescent RNA In situ Hybridization. Aim 2 will characterize MMPal1-dependant CBX4 gene network regulation. Aim 3 will identify all functionally important lncRNAs associated with promoting MM using a CRISPR/Cas9 knock-out global screen. This proposal will provide a functional understanding of MMPal1 as a master epigenetic regulator to promote MM progression and will have a direct translational impact for the utility of lncRNAs as improved diagnostic and prognostic markers to improve patient survival and longevity.