Multiple myeloma (MM) is an incurable and biologically diverse blood cancer that begins with the uncontrolled growth of plasma cells in the bone marrow.
One of plasma’s main roles is to produce immunoglobulins, or antibodies, which are essential to immune system function. In multiple myeloma, plasma cells begin producing non-functional monoclonal immunoglobulin proteins (M proteins), while malignant myeloma cells crowd out the bone marrow’s healthy cells that produce red blood cells and other vital components.
Multiple myeloma is preceded by a premalignant condition known as monoclonal gammopathy of undetermined significance (MGUS), which is defined by the accumulation of M proteins.
MGUS may develop into an intermediate stage referred to as smoldering multiple myeloma (SMM), eventually progressing to the symptomatic stage of multiple myeloma, including end-organ damage.
MM cells may also proliferate beyond the confines of the bone marrow, resulting in more aggressive forms, including plasma cell leukemia (PCL) or extramedullary multiple myeloma (EMM).
More research is needed into the transition from MGUS to MM, but genetic alterations are typically implicated in disease progression. Cells within the bone marrow microenvironment also play a key role in MM advancement.
Bone marrow stromal cells, endothelial cells, and other hematopoietic cells secrete molecules such as chemokines that interact with MM cells, promoting migration to the bone marrow and enabling their proliferation and survival.
The disease is linked to an unfavorable prognosis, including disease relapse and treatment resistance. Available therapeutic options for MM include chemotherapy, stem cell transplantation, and targeted drug therapy—all of which aim to improve patients’ quality of life and extend survival time.
Image Credit: Norgen Biotek Corp.
cfRNA as a biomarker for multiple myeloma
Early detection of cancer continues to offer the highest chance of improving long-term patient survival, despite ongoing advances in cancer treatment. Even with blood cancers like MM, 95 % of patients are diagnosed when cancer has already spread systemically.
This late diagnosis leads to at least a 20% decrease in 5-year survival rates compared to detection at earlier stages.2 Because of this, non-invasive, affordable, and dependable cancer diagnostic assays like liquid biopsies could offer significant benefits to patients by increasing access to early cancer screening.
Cell-free RNA (cfRNA) circulating in the bloodstream originates from the cell itself, either through active secretion or via apoptosis and necrosis. The analysis of cfRNA in plasma could serve as a useful indicator of phenotypic changes occurring in both localized cancer sites and the systemic host response, as well as helping to identify the tissue of origin.
One recent study used Norgen's Plasma/Serum Circulating and Exosomal RNA Purification Kit to extract cfRNA and perform RNA sequencing. Researchers sequenced cfRNA from plasma samples of MM patients, patients with precancerous conditions (MGUS), and non-cancerous (NC) donors.3
Following sequence analysis, machine learning models were used to identify combinations of discriminating genes, enabling the separation of cancerous and non-cancerous individuals. A set of 10 genes was identified, with expression levels used to classify plasma samples as NC, MGUS, or MM. These genes demonstrated a gradual increase in cfRNA levels from NC donors through MGUS to MM.
Using this three-group classification model, the gene set achieved a classification accuracy of 86.8 % (18/20 NC, 6/8 MGUS, and 9/10 MM). Because 8 of these 10 genes are already known to exhibit higher expression in bone marrow, this supports the biological validity of the machine learning model.
This study proves that a panel of cfRNA sequences could be used to classify a patient's stage of multiple myeloma, offering a minimally invasive and cost-effective method for early cancer detection. Liquid biopsies can also detect exosomal RNA and proteins that may serve as useful biomarkers for MM.4
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The role of ncRNA in multiple myeloma
Epigenetic dysregulation has been identified as an important factor in MM pathogenesis, including DNA methylation, alterations in histone modification, and microRNA (miRNA) activity.
miRNAs are non-coding short RNAs whose core function is to inhibit mRNA translation into protein by binding to complementary sequences on RNA molecules. miRNAs significantly impact various biological processes, including cellular differentiation, proliferation, metabolism, and apoptosis.
Dysregulation of miRNAs can disrupt key cellular functions, contributing to the initiation and progression of cancer and other diseases.
Image Credit: Norgen Biotek Corp.
miRNAs in multiple myeloma
One known example of this phenomenon is the influence of miRNAs on MM through the EGFR signaling pathway. The epidermal growth factor receptor (EGFR) signaling pathway is a fundamental mechanism governing cell growth, proliferation, survival, and differentiation in mammalian cells. It has also been implicated in the development and progression of many cancer types.
Vascular endothelial growth factor (VEGF) is a key growth factor involved in forming new blood vessels, a process called angiogenesis. EGFR and VEGF share common downstream signaling pathways, with research showing that miR-15a/16 may contribute to MM tumor development by regulating angiogenesis via VEGF-A targeting.5
This research has been applied to other cancers, with miR-16 mimics now in a Phase I clinical trial for patients with malignant pleural mesothelioma. Minicells loaded with a miR-16-based mimic and targeted to EGFR have been designed to address the loss of miR-15 and miR-16 family miRNAs in tumor cells.6
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lncRNAs in multiple myeloma
Understanding the impact of miRNAs on cancer becomes increasingly complex as knowledge of long non-coding RNAs (lncRNAs) is integrated. Recent research has highlighted a network of interacting lncRNAs, miRNAs, and cancer pathways.
Analysis of gene expression datasets from MM patients identified one highly differentially expressed circular RNA (circRNA)—circPSAP—which was further validated in a larger cohort by analyzing bone marrow specimens using qRT-PCR.
This study showed that circPSAP was significantly overexpressed in MM patients compared to healthy controls and that its expression correlated with predicted survival rates based on clinical pathology.
Bioinformatic analysis also identified a miRNA with a complementary sequence to circPSAP—miR-331-3p. Norgen's Cytoplasmic and Nuclear RNA Purification Kit was used to confirm the cytoplasmic location of this miRNA, with further experiments validating histone deacetylase 4 (HDAC4) as its target.
The HDAC family is critical in MM development and Bortezomib (BTZ) resistance. CircPSAP functions as an effective miR-331-3p sponge, upregulating HDAC4 and helping regulate the proliferation, apoptosis, and BTZ sensitivity of human MM cells.7
lncRNAs in drug resistance
Other research has highlighted a similar triad mechanism in which lncRNA H19 functions as a competitive endogenous RNA (ceRNA) sponge for miR-29b-3p to promote MCL-1 expression.
The myeloid cell leukemia sequence 1 protein (MCL-1) is central to the survival of cancer cells during treatment and is frequently associated with drug resistance.
H19 inhibited apoptosis in MM cells and promoted BTZ resistance by regulating MCL-1 translation through miR-29b-3p sponging. This suggests that the H19/miR-29b-3p/MCL-1 axis could be a promising therapeutic target for overcoming drug resistance in MM.8
Single-cell RNA sequencing
Research undertaken at single-cell resolution has highlighted considerable inter-patient heterogeneity across the disease spectrum—from MGUS to active MM—as well as at the relapsed/refractory multiple myeloma (RRMM) stage.
Single-cell RNA sequencing (scRNA-seq) has confirmed that MM exhibits high intra-tumor heterogeneity, comprising a mixture of clones and diverse transcriptional programs. These represent both opportunities and obstacles for myeloma therapy.
Researchers recently applied scRNA-seq to individuals before and after two rounds of proteasome inhibitor (PI) treatment. After two treatment cycles, each patient showed a different extent of tumor cell reduction.
Analysis of the scRNA-seq data revealed transcriptional changes in four cellular programs common among MM patients: unfolded protein response (UPR), stress-associated, metabolic-associated, and immune-reactive programs.
MM patients who did not respond to PI treatment also had the lowest scores in immune-reactive programs, and tumor cells with lower immune-reactive profiles exhibited an immune escape phenotype.
Further characterization of specific cell types in the tumor microenvironment allowed researchers to identify a transcription factor—YBX1 (Y-box binding protein-1)—that was upregulated in myeloma cells with low immune-reactive status.
These findings suggest that elevated YBX1 levels could induce a ‘cold’ tumor immune phenotype, marked by attenuated immune cell activation, reduced leukocyte infiltration, and the emergence of immunosuppressive signaling. This highlights YBX1’s potential as a promising therapeutic target.
Image Credit: Norgen Biotek Corp.
RNA therapeutics and CAR T-cell therapy for the treatment of multiple myeloma
Conventional treatments like chemotherapy, radiation therapy, and surgery remain the first line of treatment for most cancers, but new immune-based therapies are showing significant promise.
One especially promising immunotherapy is chimeric antigen receptor (CAR) T-cell therapy. This involves drawing blood from a patient, isolating their T cells, and genetically engineering them to express chimeric antigen receptors (CARs) before re-infusing the CAR-expressing T cells back into the patient.
CARs are fusion proteins that selectively target and destroy antigens on the surface of cancer cells.9 The US FDA has approved six CAR T-cell therapies since 2017, targeting various blood cancers, including leukemia, lymphomas, and MM.
Despite promising initial outcomes, recent long-term clinical follow-up shows that primary resistance affects 10–20 % of patients, and relapse occurs in approximately 30–50 %.10
One common cause of CAR T-cell failure is the high expression of multiple inhibitory immune checkpoint receptors (ICRs). Ongoing research is exploring how small non-coding RNAs (sncRNAs) can be used to downregulate or silence these ICRs, with the goal of improving CAR T-cell therapy outcomes.
A wide range of sncRNAs is involved in gene silencing, and researchers are actively working to harness them. For example, short hairpin RNA (shRNA or hairpin vector) is an engineered RNA molecule with a hairpin structure. In cells, shRNA is processed into siRNA, which suppresses gene expression via RNA interference (RNAi).
Recent studies have explored the use of a dual shRNA-based strategy to simultaneously downregulate two ICRs in CAR T-cells. Various combinations were tested, revealing differing effects on CAR T-cell function, with some combinations proving deleterious.
However, simultaneous downregulation of PD-1 and TIGIT (a T-cell immunoreceptor with Ig and ITIM domains) significantly enhanced the performance of CAR T-cells derived from both NHL patients and healthy donors.
Most importantly, phenotypic and functional analysis showed that reduced PD-1 expression boosted short-term effector function, while TIGIT downregulation helped maintain a less differentiated, less exhausted state. These findings offer a potential mechanism for this observed synergy.
When derived from T-cells of patients with diffuse large B-cell lymphoma, CAR T-cells with PD-1 and TIGIT downregulation demonstrated strong antitumor activity and improved in vivo persistence. A clinical trial is now assessing the safety and efficacy of CD19-targeting CAR T-cells with this dual ICR knockdown in adult patients with relapsed or refractory large B-cell lymphoma.11
Modulating the gut microbiome offers another avenue for improving CAR T-cell therapy outcomes. A growing body of research has shown that microbiome composition, probiotics, and prebiotics can enhance the efficacy of immunotherapies such as anti-CTLA-4 and anti-PD-1.12,13
Recent work suggests that the gut microbiome similarly affects CAR T-cell therapy efficacy. Metagenomic sequencing of patients undergoing CAR T therapy revealed that Bifidobacterium longum and peptidoglycan biosynthesis were closely correlated with CAR T-cell response and long-term survival.14
Machine-learning algorithms based on microbiome data can reliably distinguish long-term responders from non-responders. Bacterial genera, including Bacteroides, Eubacterium, Ruminococcus, and Akkermansia, have proven key in determining CAR T responsiveness.
References and further reading
- Cancer. Key Statistics for Multiple Myeloma. (online) Available at: https://www.cancer.org/cancer/types/multiple-myeloma/about/key-statistics.html.
- SEER. (2018). SEER Cancer Statistics Review, 1975-2018. (online) Available at: https://seer.cancer.gov/csr/1975_2018/index.html (Accessed 2 Apr. 2025).
- Breeshey Roskams-Hieter, et al. and Thuy (2022). Plasma cell-free RNA profiling distinguishes cancers from pre-malignant conditions in solid and hematologic malignancies. npj precision oncology, 6(1). https://doi.org/10.1038/s41698-022-00270-y.
- Menu, E. and Vanderkerken, K. (2022). Exosomes in Multiple Myeloma: from bench to bedside. Blood, 140(23). https://doi.org/10.1182/blood.2021014749.
- Sun, C., et al. (2013). miR-15a and miR-16 affect the angiogenesis of multiple myeloma by targeting VEGF. Carcinogenesis, 34(2), pp.426–435. https://doi.org/10.1093/carcin/bgs333.
- Alipoor, S.D. and Chang, H. (2023). Exosomal miRNAs in the Tumor Microenvironment of Multiple Myeloma. Cells, 12(7), pp.1030–1030. https://doi.org/10.3390/cells12071030.
- Ma, H., et al. (2022). Circular RNA circPSAP functions as an efficient miR-331-3p sponge to regulate proliferation, apoptosis and bortezomib sensitivity of human multiple myeloma cells by upregulating HDAC4. Journal of pharmacological sciences, (online) 149(1), pp.27–36. https://doi.org/10.1016/j.jphs.2022.01.013.
- Pan, Y., et al. (2019). LncRNA H19 overexpression induces bortezomib resistance in multiple myeloma by targeting MCL-1 via miR-29b-3p. Cell Death & Disease, 10(2). https://doi.org/10.1038/s41419-018-1219-0.
- Sheykhhasan, M., Ahmadieh-Yazdi, A., Vicidomini, R., Poondla, N., Tanzadehpanah, H., Dirbaziyan, A., Mahaki, H., Manoochehri, H., Kalhor, N. and Dama, P. (2024). CAR T Therapies in Multiple myeloma: Unleashing the Future. Cancer Gene Therapy, (online) 31, pp.1–20. https://doi.org/10.1038/s41417-024-00750-2.
- Park, J.H., et al. (2018). Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia. New England Journal of Medicine, 378(5), pp.449–459. https://doi.org/10.1056/nejmoa1709919.
- Lee, Y.-H., et al. (2022). PD-1 and TIGIT downregulation distinctly affect the effector and early memory phenotypes of CD19-targeting CAR T cells. Molecular therapy, 30(2), pp.579–592. https://doi.org/10.1016/j.ymthe.2021.10.004.
- Zhang, M., Liu, J. and Xia, Q. (2023). Role of gut microbiome in cancer immunotherapy: from predictive biomarker to therapeutic target. Experimental Hematology & Oncology, 12(1). https://doi.org/10.1186/s40164-023-00442-x.
- Ansaldo, E. and Belkaid, Y. (2021). How microbiota improve immunotherapy. Science, 373(6558), pp.966–967. https://doi.org/10.1126/science.abl3656.
- Stein-Thoeringer, C.K., et al. (2023). A non-antibiotic-disrupted gut microbiome is associated with clinical responses to CD19-CAR-T cell cancer immunotherapy. Nature Medicine, (online) pp.1–11. https://doi.org/10.1038/s41591-023-02234-6.
Acknowledgements
Produced from materials originally authored by Norgen Biotek Corporation.
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