(PA-175) Comprehensive Characterization and Pharmacological Targeting of Stem‐like Side Population Compartment in Multiple Myeloma

Multiple myeloma (MM) is sustained by a discrete subpopulation of stem-like cells that drive tumor propagation, foster therapeutic resistance, and precipitate relapse. These tumor‐initiating cells are characterized by high drug‐efflux capacity, prolonged quiescence, and robust self‐renewal and confers enhanced clonogenicity and tumorigenicity on side population (SP)‐enriched MM cells compared to their non‐SP counterparts.

Methods:   

We performed flow cytometry to quantify and isolate SP cells from MM cell lines and patient‐derived bone marrow samples, applied mass cytometry and immunoblotting to delineate molecular signatures in SP versus non‐SP subsets.

Results:   

In this study, western blot analysis of key components of the Wnt/β-catenin, SHH-GLI-NANOG (Ptch1, Ptch2, Gli1, Gli2, Sufu), and Notch1 pathways in SP-enriched MM cell lines revealed no significant differential protein expression compared to non-SP cells, with the only exception being a modest change in β-catenin levels. Furthermore, through combined flow cytometry (Nanog, Nestin, Oct3/4, Sox2, CD24, CXCR4) and immunoblotting (Sox2, KLF-4, Oct3/4, ABCG2/BCRP1), we confirmed that SP and non-SP cells share analogous stemness and pluripotency marker profiles, underscoring the need for advanced techniques to resolve phenotypic distinctions.

Nevertheless, the frequency, molecular features, and functional properties of SP in patient-derived MM samples remain poorly characterized. To address this, we quantified SP frequency within malignant plasma cell populations in clinically annotated bone marrow specimens from MGUS, newly diagnosed MM, and relapsed/refractory MM patients, revealing marked heterogeneity across disease stages. To define the cellular and molecular signatures that distinguish SP from non-SP cells, we employ mass cytometry to profile stemness and pluripotency factors alongside key developmental pathways, disease-specific signaling pathways, and plasma cell-associated antigens, and will present the resulting data. Furthermore, we pharmacologically targeted SP-enriched MM cells by evaluating small-molecule inhibitors, including vismodegib (Hedgehog pathway inhibitor), plumbagin (PI3K/Akt/mTOR pathway suppressor), deguelin (oxidative stress inducer via SOD and GSH upregulation), parthenolide (NF-κB pathway inhibitor), and the polyether ionophores salinomycin and monensin, to assess their capacity to deplete SP cells and elucidate their mechanisms of action.

 

Conclusions:   

Systematic interrogation of this stem-like SP thus provides a compelling strategy for biomarker discovery and preclinical evaluation of novel therapeutics, with the ultimate goal of eradicating relapse-initiating cells and substantially improving patient outcomes.

This study was supported by the Scientific Grant Agency VEGA 2/0088/23 (JJ), VEGA 2/0087/23 (DC), the SRDA grants APVV-20-0183 (JJ), APVV-19-0212 (DC), APVV-21-0215 (JJ), APVV-23-0482 (DC) and the NextGenerationEU project No.09I03-03-V04-00451 (JJ) and No. 09I03-03-V02-00031 (DC & KS).