Single-Cell Proteomics and Tumor RNAseq Identify Novel Pathways Associated With Clofazimine Sensitivity in PI- and IMiD- Resistant Myeloma, and Putative Stem-Like Cells

Harish Kumar; Suman Mazumder; Neeraj Sharma; Sayak Chakravarti; Mark D Long; Nathalie Meurice; Joachim Petit; Song Liu; Marta Chesi; Sabyasachi Sanyal; A Keith Stewart; Shaji Kumar; Leif Bergsagel; S Vincent Rajkumar; Linda B Baughn; Brian G Van Ness; Amit Kumar Mitra

doi:10.3389/fonc.2022.842200

Single-Cell Proteomics and Tumor RNAseq Identify Novel Pathways Associated With Clofazimine Sensitivity in PI- and IMiD- Resistant Myeloma, and Putative Stem-Like Cells

Front Oncol. 2022 May 11:12:842200. doi: 10.3389/fonc.2022.842200. eCollection 2022.

Authors

Harish Kumar¹, Suman Mazumder^{1

2}, Neeraj Sharma³, Sayak Chakravarti¹, Mark D Long⁴, Nathalie Meurice⁵, Joachim Petit⁵, Song Liu⁴, Marta Chesi⁵, Sabyasachi Sanyal⁶, A Keith Stewart⁵, Shaji Kumar⁷, Leif Bergsagel⁵, S Vincent Rajkumar⁷, Linda B Baughn³, Brian G Van Ness⁸, Amit Kumar Mitra^{1

2}

Affiliations

¹ Department of Drug Discovery and Development, Harrison College of Pharmacy, Auburn University, Auburn, AL, United States.
² Center for Pharmacogenomics and Single-Cell Omics (AUPharmGx), Harrison College of Pharmacy, Auburn University, Auburn, AL, United States.
³ Division of Laboratory Genetics, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, United States.
⁴ Department of Biostatistics and Bioinformatics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, United States.
⁵ Division of Hematology/Oncology, Mayo Clinic Arizona, Scottsdale, AZ, United States.
⁶ Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow, India.
⁷ Division of Hematology, Department of Internal Medicine, Mayo Clinic, Rochester, MN, United States.
⁸ Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 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.

Keywords: CyTOF; RNAseq; clofazimine; human myeloma cell lines (HMCLs); immunomodulatory drugs (IMiDs); myeloma; proteasome inhibitors (PI); side population (SP).

Abstract

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