Development of New Treatment Strategies Targeting Clonogenic and Tumorigenic Myeloma Side Population.

Recurrence of multiple myeloma (MM) after establishment of complete remission indicates the presence of a small fraction of resistant tumor cells which persists after treatment and expresses stem cell-like characteristics.

In our previous studies, we confirmed the existence of a MM population with “stem-like” features, known as side population (SP) cells. SP cells exhibit substantial heterogeneity not only in MM cell lines, but also in primary samples. Importantly, SP cells manifest significant clonogenic potential in vitro compared to the main population (MP). In our current study, we examined whether the clonogenicity of SP cells in vitro is associated with tumorigenic potential of SP cells in vivo.

To evaluate tumorigenicity of SP cells, several dilutions of sorted SP and MP cells were injected into immunocompromised mice. In CB17/SCID mice, sorted SP cells (range 10⁵ - 10⁶ cells/mouse) from OPM1 and KMS11 cell lines induced significantly greater tumor growth compared to MP cells (90-70% less tumor mass than SP tumors). At lower innocula of cells (10 × 10³ cells/mouse), SP cells resulted in tumors, whereas no tumors were produced by MP cells. In NOD/SCID mice, lower numbers of SP cells (1 × 10³ cells/mouse) produced tumor. These data strongly support the tumorigenic potential of SP sub-population and provide the rationale for targeting these cells in novel therapeutic strategies in MM.

Indeed, our previous studies show that lenalidomide significantly decreased the percentage and clonogenicity of SP cells. We therefore expanded these studies to next examine whether other novel agents target SP cells. Novel agents pomalidomide (1 μM; decreased by 97%) and histone deacetylase (HDAC) inhibitor vorinostat (0.4 μM; decreased by 86%) significantly affected SP cells. Natural plant compound sulforaphane (2 μM; decreased by 86%), but not isothiocyanate PEITC, also decreased percentage of SP cells.

We similarly evaluated the anti-SP effect of compounds known or proposed to target crucial signaling pathways in hematopoietic/cancer stem cells including cantharidin (terpenoid inhibitor of PPA2) and its demethylated analog norcantharin, deguelin (PI3K/Akt inhibitor), plumbagin (quinonoid substrate of ABCG2) and ionophore salinomycin. All these compounds significantly decreased or completely eliminated SP cells at low nontoxic doses in flow cytometry-based Hoechst 33342 staining assays. However, clonogenicity of SP cells treated by salinomycin was only slightly affected compared to control SP cells by clonogenic assay in vitro.

In addition, Hedgehog pathway inhibitor cyclopamine (10 μM; decreased by 82%), as well as its analog tomatidine (10 μM; decreased by 64%), significantly decreased the percentage of SP cells, while sonic hedgehog agonist purmorphamine (0.8 μM; decreased by 17%) only modestly decreased the percentage of SP cells. To study these substantial inhibitory effects of tested compounds, the clonogenic assays were performed in vitro. Finally, the effect of drugs and compounds against SP cells was also evaluated in co-culture with bone marrow stromal cells, which confer growth and survival in MM. Importantly, the SP fraction was decreased by these agents, whereas no effect of hedgehog agonist and plumbagin was detected.

Our studies confirmed the tumorigenic potential of SP cells isolated from established MM cell lines and identify the impact of targeted therapies against these cells, providing the rationale for further studies to evaluate the clinical potential of therapeutic targeting of SP cells in MM.

Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.