Related Experiment Video
Updated: Sep 21, 2025
Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models
Published on: December 9, 2016
Single-Cell Proteomics and Tumor RNAseq Identify Novel Pathways Associated With Clofazimine Sensitivity in PI- and
Harish Kumar1, Suman Mazumder1,2, Neeraj Sharma3
1Department of Drug Discovery and Development, Harrison College of Pharmacy, Auburn University, Auburn, AL, United States.
Abstract:
Multiple myeloma (MM) is an incurable plasma cell malignancy with dose-limiting toxicities and inter-individual variation in response/resistance to the standard-of-care/primary drugs, proteasome inhibitors (PIs), and immunomodulatory derivatives (IMiDs). Although newer therapeutic options are potentially highly efficacious, their costs outweigh the effectiveness. Previously, we have established that clofazimine (CLF) activates peroxisome proliferator-activated receptor-γ, synergizes with primary therapies, and targets cancer stem-like cells (CSCs) in drug-resistant chronic myeloid leukemia (CML) patients. In this study, we used a panel of human myeloma cell lines as in vitro model systems representing drug-sensitive, innate/refractory, and clonally-derived acquired/relapsed PI- and cereblon (CRBN)-negative IMiD-resistant myeloma and bone marrow-derived CD138+ primary myeloma cells obtained from patients as ex vivo models to demonstrate that CLF shows significant cytotoxicity against drug-resistant myeloma as single-agent and in combination with PIs and IMiDs. Next, using genome-wide transcriptome analysis (RNA-sequencing), single-cell proteomics (CyTOF; Cytometry by time-of-flight), and ingenuity pathway analysis (IPA), we identified novel pathways associated with CLF efficacy, including induction of ER stress, autophagy, mitochondrial dysfunction, oxidative phosphorylation, enhancement of downstream cascade of p65-NFkB-IRF4-Myc downregulation, and ROS-dependent apoptotic cell death in myeloma. Further, we also showed that CLF is effective in killing rare refractory subclones like side populations that have been referred to as myeloma stem-like cells. Since CLF is an FDA-approved drug and also on WHO's list of safe and effective essential medicines, it has strong potential to be rapidly re-purposed as a safe and cost-effective anti-myeloma drug.
Insights
Clofazimine (CLF) demonstrates significant anti-myeloma activity, particularly against drug-resistant multiple myeloma (MM) cells. This FDA-approved drug shows potential for cost-effective repurposing as a safe and effective MM treatment.
Area of Science:
- Hematology
- Oncology
- Pharmacology
Background:
- Multiple myeloma (MM) is an incurable plasma cell malignancy with limited treatment options and significant inter-individual variability in response to standard therapies.
- Current treatments, including proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs), face challenges with dose-limiting toxicities and emerging drug resistance.
- Novel therapeutic strategies are needed, but their high cost can outweigh their effectiveness, necessitating the exploration of repurposed, cost-effective agents.
Purpose of the Study:
- To evaluate the efficacy of clofazimine (CLF) as a single agent and in combination with PIs and IMiDs against drug-resistant multiple myeloma (MM) models.
- To identify the molecular pathways and mechanisms underlying CLF's anti-myeloma activity.
- To assess CLF's potential for repurposing as a safe and cost-effective treatment for MM.
Main Methods:
- Utilized a panel of human myeloma cell lines (sensitive, refractory, and relapsed) and primary patient-derived myeloma cells (ex vivo).
- Employed genome-wide transcriptome analysis (RNA-sequencing), single-cell proteomics (CyTOF), and Ingenuity Pathway Analysis (IPA) to investigate molecular mechanisms.
- Assessed CLF's cytotoxicity as a single agent and in combination with PIs and IMiDs.
Main Results:
- CLF exhibited significant cytotoxicity against both drug-sensitive and drug-resistant MM cell lines and primary patient cells.
- CLF demonstrated efficacy as a single agent and synergized with PIs and IMiDs in resistant myeloma models.
- Identified key pathways involved in CLF's efficacy, including ER stress induction, autophagy, mitochondrial dysfunction, and ROS-dependent apoptosis, leading to downregulation of p65-NFkB-IRF4-Myc.
- CLF effectively targeted rare refractory subclones, including myeloma stem-like cells.
Conclusions:
- Clofazimine (CLF) is a potent anti-myeloma agent effective against drug-resistant and refractory multiple myeloma, including stem-like cells.
- CLF exerts its effects through novel mechanisms including ER stress, autophagy, mitochondrial dysfunction, and apoptosis.
- Given its FDA approval and established safety profile, CLF holds significant promise for cost-effective repurposing in MM treatment.
