Multiple Myeloma Cancer Stem Cells Research Articles

The TGF- β Family Member Growth Differentiation Factor 15 (GDF15) Regulates the Self-Renewal of Multiple Myeloma Cancer Stem Cells

Abstract 2954Multiple myeloma (MM) cancer stem cells (CSCs) possess both enhanced tumorigenic potential and relative drug resistance suggesting they play a major role in disease relapse and progression. Therefore, a better understanding of the processes regulating MM CSCs may lead to the development of novel therapies that prevent tumor regrowth and improve long-term outcomes. Normal stem cells are tightly regulated by factors within the local microenvironment that include both soluble factors and direct contact with accessory cells. However, external factors regulating MM CSCs have not been identified. Recent studies have demonstrated that stromal cells in the MM bone marrow microenvironment secrete growth differentiation factor 15 (GDF15), a member of the TGF-b family. We initially studied the role of this cytokine in the pathogenesis of MM by examining circulating GDF15 levels in MM patients. Compared to healthy volunteers, we found that median GDF15 levels were significantly increased in MM patients (821 vs. 390 pg/ml; n=16; p<0.05) and increased with disease stage (Stage II=585 pg/ml, Stage III=1, 004 pg/ml). To examine the functional effects of GDF15 on MM cells, we cultured human MM cell lines (NCI-H929, RPMI 8226) with recombinant GDF15 and found that it induced the expansion of isolated CD138neg MM CSCs in a dose-dependent manner but had little impact on the growth of CD138+ plasma cells (Fig). Furthermore, GDF15 enhanced clonogenic myeloma growth as evidenced by increased colony formation that was maintained during serial replating, a surrogate for self-renewal. This effect appeared to be GDF15 specific since it could be blocked using anti-GDF15 antibody. Similarly, GDF15 treatment increased the in vitro clonogenic growth of MM CSCs from primary clinical bone marrow specimens. We also investigated the down-stream cellular pathways potentially mediating the effects of GDF15 and found that it activates the AKT signaling pathway known to improve the self-renewal of embryonic (ES) and normal hematopoietic stem cells. GDF15 also induced expression of the SOX2 transcription factor known to be upregulated in CD138neg MM CSCs. Since SOX2 is required for the self-renewal of ES cells and the generation of induced pluripotent stem (iPS) cells, its induction by GDF15 may also increase the self-renewal of MM CSCs. GDF15 is the first soluble factor identified that regulates MM CSCs, and its effects are mediated by the activation of highly conserved self-renewal programs. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract C131: Two-step strategy for efficient killing of multiple myeloma stem cells using Auger-electrons emitting nucleoside analogue 5-Iod-4'-thio-2'-desoxyuridin (I-125-ITdU).

Abstract Background: Despite advances in treatment, multiple myeloma (MM) remains an incurable disease. Tumor relapse is at least in part due to the existence of drug resistant cancer stem cells (CSCs). A new therapeutic paradigm that effectively eradicates bulk MM cells and CSC needs to be developed. Auger-electrons emitting nucleoside analogues are attractive for nano-irradiation therapy when incorporated into the DNA. For this proposal, the quiescent CSCs need to be awaked. Although dormant hematopoietic stem cells (HSC) are mostly highly resistant to therapeutic regiments, they can be activated by pro-drug 5-FU to enter active cell cycle and to generate proliferative, therapy sensitive progenitor and mature cells. In analogy to HSC, the activation of highly resistant dormant CSC may present the crucial step for achieving long-term cure of cancers. Therefore in this study we evaluated the therapeutic potential of I-125-ITdU for targeting of multiple myeloma CSCs after priming with a potent thymidylate synthase (TS) inhibitor FdUrd, an active metabolite of 5-FU. Methods: For CSCs isolation, KMS12BM cells were incubated with CD138 and CD27 MicroBeads. The CD Marker expression was investigated by flow cytometry. For FdUrd treatment, cells were incubated with 0.1 μM FdUrd for 2 d. The expression level of aldehyde dehydrogenase (ALDH) was evaluated using Aldefluor assay by flow cytometry. Cell cycle was investigated by Nicoletti. The uptake and DNA incorporation of I-125-ITdU (50kBq/2*10∘4 cells) were assessed after 4 d using gamma counter. Apoptotic cells were identified using Annexin-V by flow cytometry. Results: The purity of isolated CSC was more than 97% (CD27+CD138−). FdUrd treatment of CD27+CD138− cells yielded a decreased ALDH activity (65.2% vs. 29.8%). Furthermore, FdUrd induced CSC to enter the cell cycle (48.5% vs. 18.7% and 29.2% vs. 47.7% of cells in G0/G1 and S phase, respectively) and increased the CD138 expression. The treatment with a non-toxic dose of FdUrd was essential for effective incorporation of ITdU and inducing of irreparable DNA damage. The activation with FdUrd increased the cellular uptake by a factor of 25. KMS12BM CSC incorporated 46.3% ± 2.8% of internalized activity into DNA. Most important, I-125-ITdU showed a potent antimyeloma effect on isolated CSC. More than 95% of treated cells were detected as apoptotic. Conclusion: This is the first report to demonstrate that DNA is a promising target for endo-radio-therapy of CSC of multiple myeloma using two-step-strategy: FdUrd for activating and Auger-radiation emitting thymidine analogue ITdU for eliminating of malign cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C131.

Bone Marrow Niche Down-Regulates Mir-30 In Multiple Myeloma Cells to Promote Cancer Progression and Cancer Initiation by Targeting BCL9/Wnt Pathway.

AbstractAbstract 1569“Niche” is a specialized microenvironment which controls the fate specification and development of stem and progenitor cells. The bone marrow (BM) niche is composed of osteoblasts, osteoclasts, bone marrow endothelial cells, stromal cells, adipocytes, and extracellular matrix proteins (ECM). These elements provide an optimal growth environment for multiple hematological malignancies, including multiple myeloma (MM) cancer stem cells (CSCs). For example, the MM bone marrow stromal cells (BMSCs) confer survival and chemoresistance of MM cells to current therapies. A better and more detailed understanding of the neoplastic MM niche will therefore provide a model for identifying and validating novel targeted therapies directed against MM. Our previous data in 78 MM patient samples showed that miR-30 family members were more weakly expressed in patients samples compared with normal plasma cells. In our present study, we showed that miR-30 is downregulated by co-culture of either MM cell line or MM patients sample with BMSCs. Bioinformatics analysis showed that Bcl9 is a common target of miR-30, with two different binding sites in 3'UTR region of Bcl9 mRNA predicted by 3 different web-based softwares. Importantly, we confirmed Bcl9 as a direct target of miR-30 in MM cells by both gain of function and loss of function studies. Specifically, ectopic expression of miR-30 by using HIV based lentivirus (V-miR-30) downregulates Bcl9 gene expression by mRNA degradation in either 293T or H929 cells compared with GFP control cells (V-GFP). Conversely, knockdown of miR-30 family expression upregulates Bcl9 mRNA and protein level in MMS1 cells, which have relatively higher miR-30 and lower Bcl9 levels. To prove a direct interaction between miR-30 and the 3'UTR of Bcl9, two wild type Bcl9-3'UTR reporter vectors (pmiR-Bcl9-30-1W and pmiR-Bcl9-30-2W) and two mutant Bcl9-3'UTR reporter vectors (pmiR-Bcl9-30-1M and pmiR-Bcl9-30-2M) were cotransfected into H929 cells, together with V-miR-30 or V-GFP. Luciferase activity of wild type, but not mutant, was significantly decreased with V-miR-30 with respect to V-GFP. Downregulation of miR-30 induced overexpression of Bcl9 in MM cell line or patients samples, which in turn transcriptionally activates WNT pathway downstream target genes such as Axin2 and CD44. As expected, WNT pathway TOP/FOP luciferase activity was induced by knockdown miR-30 in MMS1 cells and suppressed by ectopic expression of miR-30 in H929 cells. In addition, the stem cell population of H929 V-miR-30 dramatically decreased, identified using functional Hoechst 33342 stem cell staining assay. Moreover, in experiments using stem cell medium to culture sorted CSCs, the sphere number and size is less in H929 V-miR-30c CSCs compared with H929 V-GFP control CSCs. In addition, proliferation and tumor formation were inhibited in vitro and in vivo in H929 V-miR-30 stable cells compared with H929 V-GFP cells. Finally, H929 V-miR-30 stable cells were more sensitive to bortezomib treatment than GFP control cells in the presence or absence of BMSCs. Our studies therefore demonstrate that miR-30 regulates the WNT pathway by targeting Bcl9 in MM cells in the BM milieu and represents a promising novel therapeutic target in MM. Disclosures:Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Telomerase Inhibition Targets Clonogenic Multiple Myeloma Cells through Telomere Length-Dependent and Independent Mechanisms

BackgroundPlasma cells constitute the majority of tumor cells in multiple myeloma (MM) but lack the potential for sustained clonogenic growth. In contrast, clonotypic B cells can engraft and recapitulate disease in immunodeficient mice suggesting they serve as the MM cancer stem cell (CSC). These tumor initiating B cells also share functional features with normal stem cells such as drug resistance and self-renewal potential. Therefore, the cellular processes that regulate normal stem cells may serve as therapeutic targets in MM. Telomerase activity is required for the maintenance of normal adult stem cells, and we examined the activity of the telomerase inhibitor imetelstat against MM CSC. Moreover, we carried out both long and short-term inhibition studies to examine telomere length-dependent and independent activities.Methodology/Principal FindingsHuman MM CSC were isolated from cell lines and primary clinical specimens and treated with imetelstat, a specific inhibitor of the reverse transcriptase activity of telomerase. Two weeks of exposure to imetelstat resulted in a significant reduction in telomere length and the inhibition of clonogenic MM growth both in vitro and in vivo. In addition to these relatively long-term effects, 72 hours of imetelstat treatment inhibited clonogenic growth that was associated with MM CSC differentiation based on expression of the plasma cell antigen CD138 and the stem cell marker aldehyde dehydrogenase. Short-term treatment of MM CSC also decreased the expression of genes typically expressed by stem cells (OCT3/4, SOX2, NANOG, and BMI1) as revealed by quantitative real-time PCR.ConclusionsTelomerase activity regulates the clonogenic growth of MM CSC. Moreover, reductions in MM growth following both long and short-term telomerase inhibition suggest that it impacts CSC through telomere length-dependent and independent mechanisms.

Multiple Myeloma: A Paradigm for Translation of the Cancer Stem Cell Hypothesis

Despite recent advances in drug development, multiple myeloma (MM) remains incurable for the majority of patients due to relapse and disease progression. The cancer stem cell (CSC) hypothesis may provide an explanation for these clinical findings. It suggests that the long-term proliferative potential responsible for disease initiation, maintenance, and relapse is contained within specific subpopulations of biologically distinct tumor cells. Data in MM suggest that CSCs represent a rare cell population phenotypically resembling normal memory B cells. Compared to MM plasma cells, MM CSCs also appear to be relatively resistant to a wide variety of standard anti-cancer agents suggesting they may persist following treatment and mediate tumor re-growth and relapse. A unique property CSCs share with their normal counterparts is the potential for self-renewal that likely maintains the malignant clone over time. The development of therapeutic strategies targeting the signaling elements contributing to cancer cell self-renewal has been limited primarily because the cellular processes involved are poorly understood. However, it is common that the signaling pathway components regulating normal stem cell self-renewal are aberrantly activated in human cancers and may serve as potential therapeutic targets. One class of shared regulatory pathways are those active during normal embryonic patterning and organ formation such as Hedgehog (Hh), Notch and Wingless (Wnt), and emerging data suggest that these may play a role in CSCs. Here we review the identification and characterization of MM CSCs, the role of Hh in MM, and issues to be considered during the early clinical testing of CSC targeting agents.

Cancer stem cells of multiple myeloma:recent progress

Cancer stem cells(CSCs) are a small proportion of tumor cells with the property of stem cells in the tumor tissue;they are capable of self-renewal,multi-lineage differentiation and serve as the source of tumor cells and tumor tissues.The discovery of multiple myeloma CSCs and the study on its relationship with side population cells and niche provide a new interpretation on the pathogenesis of multiple myeloma.

CD200 Is Preferentially Expressed in CD138 dim Multiple Myeloma Cells: Implications for a Myeloma Stem Cell Subpopulation with Potential Immune-Resistance.

Abstract 4877 IntroductionThe CD138negCD20+ cell population has been suggested as the putative multiple myeloma (MM) cancer stem cells, both in MM cell lines and in patient specimens. CD200 is an immunosuppressive membrane glycoprotein reported to be co-expressed with other stem-cell markers in prostate, breast, brain, and colon cancers. Also, CD200 is a negative prognostic factor for MM. It remains unknown whether CD200 is expressed in the CD138negCD20+ MM cell population, the putative MM cancer stem cells. Here we addressed this question by assessing CD200 expression in different subpopulations of MM cells according to their CD138 expression levels. MethodsTwo MM cell lines (RPMI8226 and NCI-H929) were co-stained with FITC-labeled anti-CD200, PE-labeled anti-CD20, and APC-labeled anti-CD138 antibodies for multicolor flow cytometry analysis. The cells were gated into 4 subpopulations (h1>h2>h3>h4 corresponding to CD138 expression levels: high>dim>low>negative). In each gated cell population, CD200 expression was assessed, and the CD200+ and CD200neg cell subpopulations were further gated for analysis of their CD20 expression levels. ResultsIn the RPMI 8226 cell line, the distribution of gated h1, h2, h3, and h4 populations was 87.51%, 5.86%, 5.26%, and 1.29%, respectively. In the h2 (CD138dim) population, 22.88% of the cells were CD200+. In contrast, CD200 was expressed at much lower levels in the other three populations, ranging from 7.28% (h1), 6.65% (h3), to 0.48% (h4). In CD200+ cells from the gated h1, h2, h3, and h4 population, CD20+ cells were 19.50%, 23.03%, 27.67%, and 18.75%, respectively, while in the CD200neg cells, CD20+ cells were 20.79%, 22.50%, 25.08%, and 28.02%, respectively. In the NCI-H929 cell line, 18.59% of the cells in the h2 population were CD200+, whereas only 1.61%, 1.69%, and 0.47% of the cells in the h1, h3, and h4 populations were CD200+. In each population, there were more CD20+ cells in CD200+ cells than in CD200neg cells, which were 24.52% vs 0.04% (h1), 41.94% vs 11.34% (h2), 11.11% vs 3.06% (h3), and 50.00% vs 2.25% (h4), respectively. ConclusionsThese results demonstrate that the immunosuppressive molecule CD200 is preferentially expressed in a CD138dim subpopulation of multiple myeloma cells. Depending on cell line, the putative myeloma stem cell marker CD20 is either preferentially, or not preferentially, expressed in the CD200+ cells, suggesting that an immune-resistant subpopulation of MM stem cells might exist, which may have important implications for designing immunotherapeutic approaches to treat this disease. DisclosuresGoldenberg: Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Multiple Myeloma Cancer Stem Cell Animal Model.

Abstract 1848Poster Board I-874Multiple myeloma (MM) is the second most common hematologic malignancy and characterized by clonal proliferation of CD138+ bone marrow plasma cells. Despite various treatment options few patients with MM have been cured. Furthermore, high relapse rates and recent evidence from xenogeneic transplantation models and primary MM marrow samples indicate that a rare population of cells or MM cancer stem cells (MM CSCs) within the marrow regenerates itself and may be responsible for drug resistance. These MM CSCs are phenotypically similar to memory B cells (CD138- CD34-CD19+) but differ in that they have the capacity to regenerate themselves or self-renewal. However, most of the reports on MM CSC animal models are established in NOD/SCID mice that require a larger number (1 – 10 × 106) of bead sorted cells for each animal. In addition, the latency of MM induction (4 – 6 months) in NOD/SCID mouse models and lack of in vivo tracking of the malignant clone preclude robust pre-clinical testing of novel therapeutic strategies that target MM CSC.Mononuclear cells were isolated from autologous mobilized peripheral blood of at least four primary MM patients after Ficoll gradient centrifugation followed by immunomagnetic bead depletion of CD34+ and CD138+ cells and/or further sorted using a FACSAria. The CD138-CD34- population was transduced with lentiviral luciferase GFP (GLF) and transplanted (10,000 to 106 cells per mouse) intrahepatically into neonatal RAG2-/- gamma chain-/- (RAG2-/-gc-/-) mice. Engraftment was compared to mice transplanted with either CD34+ or CD138+ cells. Mice were imaged with an in vivo imaging system (IVIS) to detect bioluminescent engraftment. Results showed that a relatively rare CD138- CD27+ population, resembling memory B cells (∼1.2%), persists in MM autografts and can engraft immunocompromised mice more rapidly and effectively than the CD138+ (Lin+) population of mature plasma cells. This data supports the persistence of CSCs despite high dose chemotherapy further underscoring the need for CSC targeted therapy. Bioluminescence was detected in live mice transplanted with as little as 60,000 cells of CD138- CD34- population and as soon as 4 weeks after transplantation. FACS analysis of these mice demonstrated successful engraftment with the presence of CD45+ and CD138+ population in bone marrow, spleen and liver and bioluminescence was also detected in the secondary transplantation of cells from MMCSC primary engraftment demonstrating the self-renewal capacity of this rare CD138- CD27+ population. Our results suggest that by utilizing a lentiviral GFP-luciferase system in a highly immunocompromised mouse strain fewer cells will be required to monitor MM engraftment and perhaps hasten disease development. Further studies to confirm the expression of selected IgG genes from myeloma cells and to characterize the self-renewal capacity with genes involved in developmental signaling such as sonic hedgehog and wnt pathways are underway. Disclosures:Goff:Coronado Biosciences: Research Funding.

Bortezomib Resistant Multiple Myeloma Cells Are Tumor Initiating Cells That Have a Stem Cell-Like Genetic Signature.

Abstract 2805Poster Board II-781Bortezomib, a proteasome inhibitor, effectively suppresses survival and growth of multiple myeloma (MM) cells. It can inhibit activation of nuclear factor-kB (NF-kB) and has been used in the treatment of MM. However, recent studies reported that patients with relapsed or refractory MM acquired resistance against bortezomib during disease progression. Therefore, we need to elucidate the molecular mechanism by which resistance emerges and this is crucial for improving treatment outcome by potentiating therapeutic efficacy of bortezomib in MM. Here, we established bortezomib resistant MM cells (U266/vel R) using soft-agar assay and identified the major molecular events that are related with acquired resistance against bortezomib. MTT assay showed that U266/Vel R cells have a growth advantage over bortezomib and the endogenous levels of pERK and pSTAT-3, -5 in U266/vel R were higher than those in parental cells. The expression of a set of genes associated with cancer stem cells was up-regulated in U266/vel R. FACS analysis showed that side population was gradually increased when U266 cells were serially exposed to bortezomib. In addition, increasing number of CD138 negative U266 cells were found in U266/ vel R cell population. It suggested that surviving cells in the presence of bortezomib could be MM cancer stem cells. Our data strongly suggest that biologic characteristics of bortezomib resistant MM cells are mediated by these potential cancer stem cells. Disclosures:No relevant conflicts of interest to declare.

Characterization of Cancer Stem Cells in Multiple Myeloma

Cancer stem cells persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Development of specific therapies targeted at cancer stem cells gives hope for improvement in the survival and quality life of cancer patients. Multiple myeloma (MM) is a cancer characterized by clonal expansion of terminally differentiated B cells. In order to characterize whether cancer stem cells can be identified in these patients, fresh bone marrow biopsies with 90% MM cells from MM patients were implanted into the superficial gluteal muscle of C.B-17 severe combined immunodeficient (SCID) mice. The tumors were excised from donor mice two months following implantation, and digested with proteinase-E to produce a single cell suspension. These cells were analyzed using flow cytometry to identify specific cellular phenotypes within the tumor population. Approximately 13% of the tumor cells were CD138+ cells, 1–2% CD20+ cells and 2–3% CD133+ cells. To examine gene expression within these populations, we isolated the tumor cells using immunomagnetic bead selection. Cells (1X108) were incubated with 200ml of anti-CD138 microbeads and either anti-CD133 or CD20 microbeads. The cell suspension was applied to the magnetic column and unbound cells were passed through the column by washing followed by centrifugation, and finally resuspended. Total RNA was purified from the cells and gene expression of each population was examined using RT-PCR analysis of specific previously identified stem cell-related transcription factors. β-catenin plays a critical role in stem cell development; and, furthermore, the Wnt-β-catenin signaling pathway is important for maintaining the balance of proliferation versus differentiation in the stem cell population. The gene expression of KLT-4, Oct-4, SOX2, and C-myc has recently been shown to convert nonterminally differentiated B cells into a pluripotent stem cell state. In our studies, we found that the CD20+/CD138− and CD133+/CD138− subpopulations both expressed high levels of β-catenin, KLT-4, Oct-4, SOX2, and C-myc. These small populations of tumor cells are likely to represent MM cancer stem cells as they express genes consistently identified in cancer stem cells identified in other types of cancers. We unexpectedly found that CD138+ cells also expressed β-catenin, KLT-4, Oct-4, SOX2, and C-myc. This population of cells might be a “premature” tumor cell in MM at a middle stage of tumor cell differentiation which ultimately differentiates into a mature MM cell. Only CD20−/CD138− cells showed no expression of β-catenin, KLT-4 and SOX2 and markedly reduced Oct-4 gene expression whereas the amount of C-myc gene expression was similar to the levels in the other tumor cell subtypes. Only CD133−/CD138− cells lost β-catenin and showed a reduction in Oct-4 gene expression but still expressed the KLT-4, SOX2, and C-myc genes. To further examine these cancer stem cell and mature tumor cell populations in terms of growth in vivo, we have injected subcutaneously CD20+/CD138−, CD133+/CD138−, CD20−/CD138−, and CD133−/CD138− tumor cell subpopulations back into SCID mice. We will assess growth of cells from these subtypes in vivo as determined by changes in tumor volume and Ig protein levels. We also will determine the sensitivity of these subtypes in vivo to treatment with a variety of agents with anti-MM activity including bortezomib, lenalidomide, melphalan, and Doxil. These studies have uncovered specific subpopulations within the tumor clone of MM and identified differences in expression of genes known to be involved in stem cell function. Further work should lead to specific treatments that can effectively treat these different subpopulations within the tumor clone in MM.

Cancer Stem Cell Activity in Mantle Cell Lymphoma Is Inhibited by TLR9 Activation

Patients with Mantle Cell Lymphoma (MCL) often respond to initial cytotoxic therapy, but invariably relapse. This clinical pattern suggests that standard therapies fail to eliminate a population of chemoresistant cells responsible for tumor regrowth and disease relapse. Studies in myeloid leukemias and multiple myeloma have suggested that cancer stem cells (CSC) are relatively quiescent and that this property promotes drug resistance. Therefore, the induction of CSC proliferation may increase their susceptibility to anti-cancer treatment. Unmethylated CpG DNA sequences induce the activation and differentiation of normal B cells through toll like receptor 9 (TLR9), and synthetic CpG oligonucleotides (ODN) are currently undergoing clinical trials in MCL and other B cell non-Hodgkin's lymphomas. We initially examined the effects of TLR9 agonists on the clonogenic growth of the human MCL cell lines Granta 519, Jeko-1, and Rec-1 and found that the unmethylated CpG ODN 2006 (5ug/mL, 48 hours) significantly inhibited the ability of each of these cell lines to form colonies in methylcellulose (p<0.01). We previously demonstrated that clonogenic CSC in multiple myeloma and Hodgkin's lymphoma may be identified based on the relative activity of the intracellular detoxification enzyme aldehydedehydrogenase (ALDH). Similarly, each of the MCL cell lines contained small populations (0.5−2.3%) of ALDH+ cells that were enriched for in vitro clonogenic growth upon primary and secondary plating in methylcellulose. Following treatment with CpG ODN 2006, the population of ALDH+ cells was significantly reduced in each cell line (p<0.05). The self-renewal potential of normal and cancer stem cells is lost through the process of differentiation, and treatment of the MCL cell lines with CpG ODN 2006 resulted in the loss of CD19 and CD20 expression and induction of the plasma cell antigen CD138 by FACS. Similarly, RT-PCR for regulators of B cell differentiation revealed decreased PAX-5 and BCL-6 expression coupled with increased transcription of BLIMP-1 and XBP-1. We hypothesized that the plasma cell differentiation induced by TLR9 agonists may improve the efficacy of anti-tumor agents with known activity against plasma cells, specifically the proteosome inhibitor bortezomib. Treatment with CpG ODN 2006 or bortezomib (10–100nM, 48 hours) alone induced apoptosis in a small percentage of cells within each MCL line, but the combination of these agents synergistically increased the proportion of Annexin V+ cells. In summary, cellular activation by CpG ODN 2006 reduces MCL clonogenicity and stem cell function through the induction of differentiation. In turn, plasma cell maturation appears to increases the sensitivity of MCL to bortezomib and suggests a novel mechanism for the anti-tumor activity of CpG ODN.

Multiple Myeloma Cancer Stem Cells

Multiple myeloma is characterized by the clonal expansion of neoplastic plasma cells within the bone marrow, elevated serum immunoglobulin, and osteolytic bone disease. The disease is highly responsive to a wide variety of anticancer treatments including conventional cytotoxic chemotherapy, corticosteroids, radiation therapy, and a growing number of agents with novel mechanisms of action. However, few if any patients are cured with these modalities and relapse remains a critical issue. A better understanding of clonogenic multiple myeloma cells is essential to ultimately improving long-term outcomes, but the nature of the cells responsible for myeloma regrowth and disease relapse is unclear. We review evidence that functional heterogeneity exists in multiple myeloma and discuss potential strategies and clinical implications of the stem-cell model of cancer in this disease.

Cancer Stem Cell Targeting in Multiple Myeloma by GRN163L, a Novel and Potent Telomerase Inhibitor.

Telomerase activity (TA) is required within normal cells capable of long-term replication, including stem cells and is upregulated in many cancers. In the absence of TA or presence of TA inhibitors, the progressive shortening of telomeres ultimately results in cellular senescence and/or apoptosis. These observations support that TA inhibitors represent a novel class of anti-tumor agents. Much evidence suggests that human cancers display a hierarchical cellular organization that mirrors normal tissues. Cancer stem cells (CSC) are derived from the malignant transformation of normal stem cells and progenitors and retain the capacity to self-renew. Moreover, CSC give rise to differentiated tumor cells that form the bulk of the tumor mass, but have little or no capacity for long-term proliferation. We recently demonstrated that the malignant CD138+ plasma cells in multiple myeloma (MM) have limited replicative potential; instead they arise from the differentiation of clonogenic CSC that resemble normal memory B cells (CD138negCD19+CD27+). In addition, several groups have demonstrated that telomerase inhibitors are active against human MM cell lines in vitro and in vivo. We examined TA in CD138+ plasma cells and CD138neg precursors, and studied the effects of telomerase inhibition against both cell populations. We isolated CD138+ and CD138neg cells by FACS from three human MM cell lines (RPMI 8225, NCI-H929, and U266) and measured TA using a PCR-based assay of activity. For each cell line, TA was detectable within both the CD138neg and CD138+ cell populations. GRN163L is a lipid conjugated 13 nucleotide thio-phosphoramidate oligonucleotide that acts as a potent and specific active site inhibitor of telomerase. We found that treatment with GRN163L (0.1–5μM) markedly reduced TA within 48 hours. To examine the effects of telomerase inhibition on clonogenic growth, we continuously cultured CD138+ and CD138neg RPMI 8226 cells with GRN163L (1μM). Cells were collected weekly, washed to remove GRN163L, and then plated in methylcellulose to assess colony formation. We found that GRN163L was active against both CD138+ and CD138neg cells and eliminated the colony forming potential of both by 5 weeks. Similarly, we found that GRN163L inhibited the in vitro clonogenic growth of CD138neg MM CSC isolated from the bone marrow aspirates of patients with MM. These data demonstrate that TA is detectable within both immature MM CSC and mature MM plasma cells, and that CSC from both cell lines and primary clinical samples are targeted by the telomerase inhibitor GRN163L. Therefore, this agent may offer a novel therapeutic approach to myeloma as well as other diseases in which CSC have been identified.