Targeting Wnt and Hh signaling induces myeloma cell apoptosis in vitro and in the 5TGM1 myeloma mouse model. (A) Clonogenic assay shows the effect of ATRA, Wnt, and Hh inhibitors in RARα2 overexpressing OCI-MY5 and ARP1 cells (magnification ×40). (B) Real-time PCR shows that the expression of TCF4, LEF1, CD44, CCND1, c-Myc, SMO, and Gli1 in CD138⁻ cells and CD138⁺ cells derived from ARK and KMS11 cell lines. (C) Cell growth and viability were evaluated in CD138⁻ cells derived from KMS11 and ARK lines treated with ATRA, Wnt, and Hh inhibitors. Results are expressed as means ± SD of 3 independent experiments. (D) Kaplan-Meier curves show the 5TGM1 C57BL/KaLwRij mouse survival treated with CAY10404, cyclopamine, and itraconazole. (E) Tumor burden was examined idiotype lgG2b levels by ELISA in the 5TGM1 C57BL/KaLwRij mice treated with CAY10404, cyclopamine, and itraconazole. (F) The model of our working hypothesis shows potential mechanisms by which RARα2 maintains myeloma stem cell features.