IntroductionMultiple myeloma (MM), a malignancy hallmarked by accumulation of malignant plasma cells in the bone marrow, remains largely incurable despite the use of conventional and novel therapies. 1 The bone marrow (BM) microenvironment promotes tumor cell growth, survival, and confers drug resistance against conventional agents. 2 Although currently available anti-MM strategies have been effective in targeting the bulk of tumor cells, it has been postulated that a tumor-initiating subpopulation or cancer stem cell persists, which may be responsible for eventual relapses. 3 Side population (SP) cells are an enriched source of cancer-initiating cells with stem cell properties, which have been identified in solid tumors, as well as in hematopoietic malignancies. [4][5][6][7][8] The SP cells show a distinct ''low-staining pattern" with the Hoechst 33342 dye. 9 Importantly, SP cells possess the ability to generate non-SP cells both in vitro and in vivo, and are associated with chemoresistance and tumorigenicity in vivo. 4,10 The prevalence and biologic function of SP cells in MM are not fully defined.In the late 1990s, thalidomide was introduced to the treatment of relapsed/refractory MM; however, its effect in patients is associated with dose-and duration-dependent side effects. 11,12 Since then, more potent immunomodulatory drugs (IMiDs), such as lenalidomide, have been introduced. Lenalidomide has been approved for the treatment of both myelodysplasia with deletion of chromosome 5q and for relapsed MM, specifically in combination with dexamethasone. 12,13 Although IMiDs act directly on tumor cells, block adherence to bone marrow stromal cells (BMSCs), modulate angiogenesis and cytokines, and up-regulate host antitumor immunity, the molecular mechanism for their action remains largely undefined, and it is unclear whether they target SP cells in MM. [14][15][16][17][18] In this study, we identified SP cells in MM cell lines as well as in primary MM tumor cells by flow cytometry-based Hoechst 33342 staining, and showed heterogeneity in the percentage of SP cells, as well as the lack of strict correlation between SP fraction and CD138 Ϫ status. SP cells exhibited clonogenic and tumorigenic potential; and importantly, lenalidomide significantly decreased the percentage and clonogenicity of SP cells at clinically relevant concentrations. Moreover, lenalidomide only slightly altered expression of drug-resistant transporter ABCG2 with no effect on functional activity of BCRP1 efflux pump. Modulation of diverse signaling cascades in SP cells by lenalidomide, including changes in Akt, GSK-3␣/␤, MEK1, c-Jun, p53, and p70S6K phosphorylation was observed. Adherence to BMSCs increased the percentage, viability, and proliferation potential of SP cells. Interestingly, both lenalidomide and thalidomide attenuated this stimulatory effect of BMSCs by significantly decreasing SP cell percentages. Therefore, our studies provide insight toward developing novel strategies Submitted February 5, 2010; accepted October 10, 2010. Prepub...

Malignant cells have a higher nicotinamide adenine dinucleotide (NAD ؉) IntroductionMultiple myeloma (MM) is a clonal B-cell malignancy characterized by excessive bone marrow plasma cells in association with monoclonal protein. 1 The therapeutics currently available improve patients' survival and quality of life, but resistance to therapy and disease progression remain unsolved issues. Therefore, the definition of new aspects of MM biology that can be targeted and exploited from a therapeutic perspective remains a major basic and clinical research goal.Autophagy is a conserved process of normal cell turnover by regulating degradation of its components, which is characterized by the formation of autophagosomes, double-membrane cytoplasmic vesicles engulfing intracellular material including protein, lipids, as well as organelles, such as mitochondria and endoplasmic reticulum. Subsequently autophagosomes fuse with lysosomes, and their contents are degradated by lysosomal enzymes. 2 This selfcannibalization event is a highly conserved response to metabolic stress, in which cellular components are degraded for the maintenance of homeostasis. 3 Intriguingly, the waste removal function of autophagy appears as to be a double-edged sword, because it can either lead to cell survival or death. 4 A series of molecular mechanisms coordinate the autophagy machinery. Specifically, the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is the major intracellular hub for integrating autophagy-related signals. 5 Upstream of mTORC1 is the cellular energy-sensing pathway. 6 Regulation of autophagy also occurs through the transcription factors EB (TFEB) and forkhead box (FOXO), whose activation leads to transcription of Atg genes. 7,8 Although apoptosis induction has been the major focus of research in novel MM therapies, a recent study documented a pivotal role for autophagy as a prosurvival mechanism in MM cells, suggesting its potential as an additional target for novel therapeutics. 9,10 Intracellular nicotinamide adenine nucleotide (NAD ϩ ) plays a major role in the regulation of several cellular processes. 11,12 In mammals, NAD ϩ is replenished from nicotinamide (Nam), tryptophan or nicotinic acid (NA), with Nam as the most important and widely available precursor. 13 Nicotinamide phosphoribosyltransferase (NAMPT), pre-B colony enhancing factor, is the ratelimiting enzyme in NAD ϩ synthesis from Nam. 14 The expression of this enzyme is up-regulated in activated immune cells, 15 in differentiated myeloid cells, 16 during the circadian clock, 17 in glucose-restriction impaired skeletal myoblast differentiation, 18 and during cytokine production in immune cells. 19 Importantly, Nampt is also overexpressed in cancer cells, which exhibit a significant dependence on NAD ϩ to support their rapid cell proliferation. 20 Importantly, a specific chemical inhibitor of Nampt FK866, also called APO866 or WK175, exhibits a broad antitumor activity both in vitro and in vivo against cell lines derived from several tumors, with a favorabl...

Background Previously, we validated capability of human adipose tissuederived mesenchymal stem cells (AT-MSC) to serve as cellular vehicles for gene-directed enzyme prodrug molecular chemotherapy. Yeast fusion cytosine deaminase : uracil phosphoribosyltransferase expressing AT-MSC (CD y -AT-MSC) combined with systemic 5-fluorocytosine (5FC) significantly inhibited growth of human colon cancer xenografts. We aimed to determine the cytotoxic efficiency to other tumour cells both in vitro and in vivo.

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Histone deacetylases (HDACs) represent novel molecular targets for the treatment of various types of cancers, including multiple myeloma (MM). Many HDAC inhibitors have already shown remarkable anti-tumor activities in the preclinical setting; however, their clinical utility is limited due to unfavorable toxicities associated with their broad range HDAC inhibitory effects. Isoform-selective HDAC inhibition may allow for MM cytotoxicity without attendant side effects. In this study, we demonstrated that HDAC3 knockdown and a small molecule HDAC3 inhibitor BG45 trigger significant MM cell growth inhibition via apoptosis, evidenced by caspase and PARP cleavage. Importantly, HDAC3 inhibition downregulates phosphorylation (tyrosine 705 and serine 727) of STAT3. Neither IL-6 nor bone marrow stromal cells overcome this inhibitory effect of HDAC3 inhibition on p-STAT3 and MM cell growth. Moreover, HDAC3 inhibition also triggers hyperacetylation of STAT3, suggesting crosstalk signaling between phosphorylation and acetylation of STAT3. Importantly, inhibition of HDAC3, but not HDAC1 or HDAC2, significantly enhances bortezomib-induced cytotoxicity. Finally, we confirm that BG45 alone and in combination with bortezomib trigger significant tumor growth inhibition in vivo in a murine xenograft model of human MM. Our results indicate that HDAC3 represents a promising therapeutic target, and validate a prototype novel HDAC3 inhibitor BG45 in MM.

IntroductionThe Hedgehog (Hh) pathway regulates multiple processes involved in development and differentiation of tissues and organs during embryonic life. 1 Recently, it has become evident that Hh signaling retains some activity even during adult life: in mature tissues, it regulates tissue homeostasis and repair and, in those tissues undergoing constant renewal, such as skin, colon, liver, and blood, it is also implicated in maintaining a stem/progenitor cell compartment, 2,3 explaining how the Hh pathway deregulation may cause developmental defects during the embryonic life. 1,4 Its abnormal activity can also lead to tumorigenesis during adult life either by stem cell pool expansion 2,3 or mutations affecting the normal growth-regulatory mechanisms. [5][6][7] An aberrant expression of developmental genes from Wnt and Hh pathways has been reported during malignant transformation of multiple myeloma (MM) cells. 8 Several findings support a role of Hh signaling in regulating a stem cell niche also in MM 9 and in modulating clinical response to conventional and novel therapeutic agents. 10 Indeed, Hh ligands produced by murine bone marrow stromal cells (BMSCs) support growth and survival of human primary CD19 ϩ lymphoma and CD138 ϩ MM cells, demonstrating a role of the Hh pathway both in lymphoma and in terminally differentiated MM cells. 11 Finally, we recently showed ciliary protein overexpression as a possible cause of constitutive and noncanonical Hh pathway activation, suggesting a cilia-dependent mode of Hh signaling in MM. 12 Aberrant Hh signaling has been described in almost all tumors and is associated with 3 possible mechanisms: genetic alterations, autocrine and/or paracrine Hh activity, and alternative and synergistic pathways leading to Hh gene activation. 13,14 Sonic (Shh), Desert (Dhh), and Indian (Ihh) hedgehog are the ligands for the pathway. The signaling is triggered by binding of endogenously or exogenously produced ligand to Patched1 (Ptch1) on target cells. This leads to inhibition of Ptch1 via cellular internalization and Smoothened (Smo) localization on the cell surface, both by a ciliummediated mechanism. 15 Smo activation leads to nuclear translocation of Glioma (Gli) transcription factors followed by expression of Gli target genes, including Ptch1 and Gli1, in a negative and positive feedback loop, respectively. The biologic effect is cell proliferation, with deregulation contributing to tumorigenesis. Abnormal Hh pathway activation occurs not only through liganddependent or receptor-induced signaling, also known as canonical Hh signaling, but also by mechanisms of activation downstream to Smo known as noncanonical or ligand-independent Hh signaling. Genetic alterations or ciliary protein overexpression leading to functional redundancy of Gli transcription factors, crosstalk between Hh signaling and unrelated pathways, are all causes of noncanonical Hh signaling activation.In the present study, we first show Hh gene overexpression in CD138 ϩ plasma cells (PCs) from persons with monoclon...

Purpose Bortezomib is an important agent in multiple myeloma treatment, but resistance in cell lines and patients has been described. The main mechanisms of resistance described in cancer fall into one of two categories, pharmacokinetic resistance (PK), e.g. over expression of drug efflux pumps and pharmacodynamic resistance, e.g. apoptosis resistance or altered survival pathways, where the agent reaches an appropriate concentration, but this fails to propagate an appropriate cell death response. Of the known pump mechanisms, P-glycoprotein (P-gp) is the best studied and considered to be the most important in contributing to general PK drug resistance. Resistance to bortezomib is multifactorial and there are conflicting indications that cellular overexpression of P-gp may contribute to resistance agent. Hence, better characterization of the interactions of this drug with classical resistance mechanisms should identify improved treatment applications. Methods Cell lines with different P-gp expression levels were used to determine the relationship between bortezomib and P-gp. Coculture system with stromal cells was used to determine the effect of the local microenvironment on the bortezomib–elacridar combination. To further assess P-gp function, intracellular accumulation of P-gp probe rhodamine-123 was utilised. Results In the present study, we show that bortezomib is a substrate for P-gp, but not for the other drug efflux transporters. Bortezomib activity is affected by P-gp expression and conversely, the expression of P-gp affect bortezomib’s ability to act as a P-gp substrate. The local microenvironment did not alter the cellular response to bortezomib. We also demonstrate that bortezomib directly affects the expression and function of P-gp. Conclusions Our findings strongly support a role for P-gp in bortezomib resistance and, therefore, suggest that combination of a P-gp inhibitor and bortezomib in P-gp positive myeloma would be a reasonable treatment combination to extend efficacy of this important drug.

The phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway mediates proliferation, survival, and drug resistance in multiple myeloma (MM) cells. Here, we tested the anti-MM activity of NVP-BEZ235 (BEZ235), which inhibits PI3K/Akt/mTOR signaling at the levels of PI3K and mTOR. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric survival assays showed that MM cell lines exhibited dose-and time-dependent decreased viability after exposure to BEZ235 (IC 50 , 25-800 nmol/L for 48 hours). MM cells highly sensitive (IC 50 , <25 nmol/L) to BEZ235 (e.g., MM.1S, MM.1R, Dox40, and KMS-12-PE) included both lines sensitive and resistant to conventional (dexamethasone, cytotoxic chemotherapeutics) agents. Pharmacologically relevant BEZ235 concentrations (25-400 nmol/L) induced rapid commitment to and induction of MM.1S and OPM-2 cell death. Furthermore, normal donor peripheral blood mononuclear cells were less sensitive (IC 50 , >800 nmol/L) than the majority of MM cell lines tested, suggesting a favorable therapeutic index. In addition, BEZ235 was able to target MM cells in the presence of exogenous interleukin-6, insulin-like growth factor-1, stromal cells, or osteoclasts, which are known to protect against various anti-MM agents. Molecular profiling revealed that BEZ235 treatment decreased the amplitude of transcriptional signatures previously associated with myc, ribosome, and proteasome function, as well as high-risk MM and undifferentiated human embryonic stem cells. In vivo xenograft studies revealed significant reduction in tumor burden (P = 0.011) and survival (P = 0.028) in BEZ235-treated human MM tumor-bearing mice. Combinations of BEZ235 with conventional (e.g., dexamethasone and doxorubicin) or novel (e.g., bortezomib) anti-MM agents showed lack of antagonism. These results indicate that BEZ235 merits clinical testing, alone and in combination with other agents, in