Introduction Multiple myeloma (MM) remains incurable despite novel therapies, highlighting the need for further identification of factors mediating disease progression and resistance. One such factor is the development of a drug-resistant stem cell-like subpopulation. Previous studies have shown that MM side population cells (MM-SPs) exhibit stem-cell like features and contribute to relapse of MM. Recent research efforts, which have focused the biology of MM stem-like cells in order to derive specific therapy, have shown that stem-cell transcription factor Oct-4 is linked to stemness and chemoresistance. Here we examined the effect of enforced expression of Oct4 in MM cells and MM-SPs on the development of stem cell-like characteristics and drug-resistance in MM. These studies allow us to establish stable MM cell lines with characteristic stem-cell like features, which in turn facilitate screening of novel agents that effectively target this cell population in MM.

Methods MM-SPs were isolated from RPMI-8226 cells by flow-cytometry based Hoechst 33342 staining. RPMI-8226 and RPMI-8226-SP cells were transfected with a phOct4-GFP construct (Gerrard et al., Stem Cell 2005, 23:124-133), and selected with G418 (0.5 mg/ml) to derive stable RPMI-8226-Oct4 and RPMI-8226-SP-Oct4 cell lines. Oct-4 expression was confirmed using FACS. Cell viability was analyzed by WST assays. SL-401 is a targeted therapy directed to IL-3Rα/CD123, comprised of recombinant human IL-3 fused to truncated diphtheria toxin. Drug and reagent source: SL-401 was obtained from Stemline Therapeutics; Bortezomib and flow antibodies were purchased from Selleck Chemicals and BD Biosciences, respectively. Statistical significance was derived using GraphPad Prism.

Results1) RPMI-8226 and RPMI-8226-Oct4 cells were analyzed for the expression of surface markers associated with stem cells (CD123/IL-3Rα, CD133 and CD27) by multicolor flow analysis. Oct-4 transfection does not affect the overall CD123 expression in RPMI-8226 cells, as the % MFI-CD123hi in RPMI-8226 versus RPMI-8226-Oct4 remains unchanged. However, the stable selection makes RPMI-8226-Oct4 more clonal in nature (%CD123hi : RPMI8226; 14.9% vs RPMI-8226-Oct4; 60%). 2) A significant increase in the frequency of CD133+ve cells was observed in RPMI-8226-SP-Oct4-tranfected cells versus either RPMI-8226-SP cells or RPMI-8226-Oct-4 cells [RPMI-8226-SP: 6.3%; RPMI-8226-Oct4: 27.8%; RPMI-8226-SP-Oct4: 40%; p< 0.05]. 3) Analysis of CD27 surface marker showed highest expression in RPMI-8226-SP-Oct4 cells compared to RPMI-8226-Oct4, RPMI-8226-SP, or RPMI-8226 cells (% MFI: RPMI-8226-SP-Oct4 > RPMI-8226-Oct4 > RPMI-8226-SP > RPMI-8226 cells). 4) Treatment of RPMI-8226 and RPMI-8226-Oct4 cells with proteasome inhibitor bortezomib decreased the viability of RPMI-8226 cells; in contrast, bortezomib did not significantly alter the viablity of RPMI-8226-Oct4 cells [% Viability after bortezomib: RPMI-8226; <50% versus RPMI-8226-Oct4; 95%]. Finally, 5) SL-401 significantly decreased the viability of RPMI-8226-Oct4 cells [IC50: RPMI-8226-Oct4 cells: 75 pM; RPMI-8226-SP cells: 350 pM; RPMI-8226 cells: 1367 pM]. We have previously shown anti-MM activity of SL-401 by an additional mechanism of targeting IL-3Rα-expressing plasmacytoid dendritic cells (pDCs) localized in the tumor microenvironment and blocking pDC-induced MM cell growth.

Conclusions Our data show that stem-like cells in MM are relatively resistant to proteasome inhibitor therapy. Importantly, a novel agent SL-401 effectively targets these cells. Oct4-driven stable RPMI-8226 MM cell line serves as a novel tool to screen and develop newer agents targeting stem-like cells in MM. Overall, we show the ability of SL-401 to target a drug-resistant stem-like cell population in MM, and provide an additional rationale for clinical evaluation of SL-401 to improve patient outcome. A clinical trial of SL-401 in MM is currently ongoing (NCT02661022).

Disclosures

Macri:Stemline Therapeutics, Inc.: Employment. Chen:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Chauhan:Oncopeptide AB: Consultancy; Epicent Rx: Consultancy; C4 Therapeutics: Equity Ownership; Stemline Therapeutics, Inc.: Consultancy. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Sonofi Aventis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Gilead: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

Author notes

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Asterisk with author names denotes non-ASH members.

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