Targeting Myeloma Stem Cells through Simultaneous Inhibition of Wnt and Hedgehog (Hh) Signaling Pathways

Abstract 615

Cancer stem cells (CSC) have been identified in a variety of tumor types, including multiple myeloma (MM). Different signaling pathways, such as Wnt, Hedgehog (Hh), Notch and Bmi, are up-regulated in CSC. MM stem cells (MMSC) are enriched in the side population and have increased aldehyde dehydrogenase 1 A1 (ALDH1A1) activity. They are non-cycling and resistant to drugs typically used to treat MM. In this study, MMSC were isolated from 10 MM cell lines (CD138− fraction) and 4 primary MM patients by selecting the CD19+ CD138⁻ cells from bone marrows heavily involved with MM. We confirmed that the CD19+/CD138− cells were part of the malignant clone by comparing expression levels of spiked genes, such as MAF-B and Cyclin D3, in MMSC and CD138+ MM cells. We also verified that CD138⁻ from MM cell lines and the primary CD138−CD19+ had stem cell characteristics by clonogenic in vitro assays and tumor formation in NOD/SCID mice. GEP and real-time PCR analyses indicated that RARa2 levels were significantly higher levels in MM cell line-derived and primary MMSC than in CD138+ bulk MM cells. We also showed that over-expression of RARα2 in low-expressing MM cell lines resulted in increased Wnt and Hh activity, as evidenced by higher levels of nuclear β-catenin, Cyclin D1, TCF4, LEF1, Smo and GLI1. Expression levels of these proteins were also higher in MMSC than in CD138+ MM cells. We subsequently evaluated the efficacy of inhibition of Wnt (CAY10404, a COX-2 inhibitor) and Hh (cyclopamine and itraconazole) signaling on MMSC in vitro and also in vivo using the 5TGM1 mouse model. These drugs given separately induced potent cell death and growth inhibition in MMSC. Quantitative PCR and western blot revealed that CAY10404 decreased nuclear β-catenin and Gli1 expression, while cyclopamine and itraconazole inhibited nuclear GLI1 expression. The efficacy of CAY10404 and cyclopamine was further evaluated in the 5TGM1 mouse model. The CD138− fraction of this cell line has many of the same characteristics as human CD138− MMSC. After injection of 0.5 M cells into the C57BL/KaLwRij mice through the tail vein and allowing growth for 1 week, CAY10404 (20mg/kg, I.P.) and cyclopamine (20mg/kg, I.P) were given separately three times per week. Interestingly, we observed that the mice injected with untreated CD138+ cells survived longer than untreated mice receiving CD138− cells (P < 0.05). Treatment with CAY10404 and cyclopamine significantly reduced the tumor burden measured by idiotype IgG2 protein levels and dramatically prolonged survival (P < 0.05). We conclude that activation of Wnt and Hh pathway maintains the “stemness” features of MMSC. In MMSC, these pathways are activated by increased RARα2 expression, which we have shown to be related to drug resistance. MMSC can be eradicated by a combination of Wnt and Hh inhibitors.