Many agents are active in multiple myeloma, but the majority of patients relapse. This clinical pattern suggests most cancer cells are eliminated, but cells with the clonogenic potential to mediate tumor regrowth are relatively chemoresistant. Our previous data suggested that CD138 + multiple myeloma plasma cells cannot undergo long-term proliferation but rather arise from clonogenic CD138 neg B cells. We compared the relative sensitivity of these distinct cell types to clinical antimyeloma agents and found that dexamethasone, lenadilomide, bortezomib, and 4-hydroxycyclophosphamide inhibited CD138+ multiple myeloma plasma cells but had little effect on CD138 neg precursors in vitro. We further characterized clonogenic multiple myeloma cells and stained cell lines using the Hoechst side population and Aldefluor assays. Each assay identified CD138 neg cells suggesting that they possess high drug efflux capacity and intracellular drug detoxification activity. We also found that multiple myeloma cells expressing the memory B-cell markers CD20 and CD27 could give rise to clonogenic multiple myeloma growth in vitro and engraft immunodeficient nonobese diabetes/ severe combined immunodeficient mice during both primary and secondary transplantation. Furthermore, both the side population and Aldefluor assays were capable of identifying circulating clonotypic memory B-cell populations within the peripheral blood of multiple myeloma patients. Our results suggest that circulating clonotypic B-cell populations represent multiple myeloma stem cells, and the relative drug resistance of these cells is mediated by processes that protect normal stem cells from toxic injury. [Cancer Res 2008;68(1):190-7]

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The Hedgehog (Hh) pathway is essential for normal embryonic development and tissue repair. The role of Hh signaling in hematopoiesis has been studied primarily by modulating the activity of Patched and Smoothened, but results have been conflicting. Some studies demonstrate a requirement for pathway activity in hematopoiesis, whereas others report that it is dispensable. Hh activity converges on the Gli transcription factors, but the specific role of these downstream effectors in hematopoiesis has not been reported. We have analyzed hematopoietic stem cell (HSC) and progenitor function in mice with a homozygous deletion of Gli1 (Gli1 null ). Gli1 null mice have more longterm HSCs that are more quiescent and show increased engraftment after transplantation. In contrast, myeloid development is adversely affected with decreased in vitro colony formation, decreased in vivo response to granulocyte colonystimulating factor (G-CSF), and impaired leukocyte recovery after chemotherapy. Levels of the proto-oncogene Cyclin D1 are reduced in Gli1 null mice and may explain the loss of proliferation seen in HSCs and progenitor cells. These data demonstrate that Gli1 regulates normal and stress hematopoiesis. Moreover, they suggest that Gli1 and Smoothened may not be functionally redundant, and direct GLI1 inhibitors may be needed to effectively block HH/GLI1 activity in human disease. (Blood. 2010;115:2391-2396) IntroductionThe proliferation of hematopoietic stem cells (HSCs) and progenitors is tightly regulated during normal homeostasis. HSCs are normally quiescent in the adult mouse but they can be induced to proliferate in response to stress or cytokine stimulation. In contrast, progenitors are highly proliferative to maintain a constant supply of infection-fighting white blood cells. Precisely how HSC and progenitor proliferation are regulated is not completely understood, but recent data have implicated a role for developmental signaling pathways such as Wnt and Notch in the regulation of stem cell proliferation, self-renewal, and differentiation. [1][2][3][4] The Hedgehog (Hh) signaling pathway in mammals consists of 3 closely related ligands, Sonic Hh (Shh), Indian Hh (Ihh), and Desert Hh (Dhh), that can each bind to the transmembrane protein Patched (Ptch). Upon ligand binding, Ptch inhibition of the positive effector Smoothened (Smo) is released and signaling is transduced. Three zinc finger transcription factors, Gli1, Gli2, and Gli3, lie downstream of Smo and mediate Hh's effects. Gli1 is a positive effector of signaling, Gli3 is predominantly a transcriptional inhibitor, and Gli2 can function in both roles. 5 The precise role of Hh signaling in normal hematopoiesis, however, is not known and the literature is contradictory. One group has reported that loss of Smo activity leads to a severe defect in HSC function, 6 whereas others have reported a more modest phenotype, 7 or none at all. 8,9 All of these studies have focused primarily on the upstream modulators of pathway activity, Ptch and Smo. To better understand ...