Teresa Calimeri

A unique three-dimensional SCID-polymeric scaffold (SCID-synth-hu) model for in vivo expansion of human primary multiple myeloma cells.

Multiple myeloma (MM) cells home to the bone marrow (BM) and adhere to extracellular matrix (ECM) proteins and to bone marrow stromal cells (BMSCs). The close cross talk between MM cells and cells of the non-tumor compartment within the BM has a key role in supporting tumor growth, survival and development of drug resistance. This biological scenario has led to a growing interest in novel drugs, targeting MM cells and/or interfering with their human BM milieu (HuBMM).1, 2 Based on this, appropriate in vivo models that recapitulate the complex interactions occurring between MM and its HuBMM are required for preclinical evaluation of new anti-MM agents. To date, the in vivo study of MM pathobiology and the validation of therapeutic anti-MM agents has been carried out using a variety of models of murine MM or human MM xenografts in immunocompromised mice.3 These models, however, do not replicate the HuBMM. The development of the SCID (severe combined immunodeficiency)-hu model has been an important advance, as it was the first model to recapitulate a HuBMM in mice.4, 5, 6 However, although the SCID-hu system remains a highly relevant model for preclinical investigation, it does have important limitations: (i) restricted availability of human fetal bone chips; (ii) the allogeneic nature of the fetal BM milieu versus MM cells; and (iii) the significant heterogeneity of implanted human bone chips, collected from different individuals at different gestational age, that may produce experimental variability.

Nanotechnologies to use bisphosphonates as potent anticancer agents: the effects of zoledronic acid encapsulated into liposomes.

UNLABELLED Zoledronic acid (ZOL) is a potent amino-bisphosphonate used for the treatment of bone metastases with recently reported antitumor activity. However, the short plasma half-life and rapid accumulation in bone limits the use of ZOL as an antitumor agent in extraskeletal tissues. Therefore, we developed stealth liposomes encapsulating ZOL (LipoZOL) to increase extraskeletal drug availability. Compared to free ZOL, LipoZOL induced a stronger inhibition of growth of a range of different cancer cell lines in vitro. LipoZOL also caused significantly larger inhibition of tumor growth and increased the overall survival in murine models of human prostate cancer and multiple myeloma, in comparison with ZOL. Moreover, a strong inhibition of vasculogenetic events without evidence of necrosis in the tumor xenografts from prostate cancer was recorded after treatment with LipoZOL. We demonstrated both antitumor activity and tolerability of LipoZOL in preclinical animal models of both solid and hematopoietic malignancies, providing a rationale for early exploration of use of LipoZOL as a potential anticancer agent in cancer patients. FROM THE CLINICAL EDITOR The short plasma half-life and rapid accumulation in bone limits the use of zoledronic acid as an antitumor agent in extraskeletal tissues. Therefore, stealth liposomes encapsulating ZOL (LipoZOL) have been developed to increase extraskeletal drug availability.

Targeting NAD salvage pathway induces autophagy in multiple myeloma cells via mTORC1 and extracellular signal-regulated kinase (ERK1/2) inhibition

Malignant cells have a higher nicotinamide adenine dinucleotide (NAD(+)) turnover rate than normal cells, making this biosynthetic pathway an attractive target for cancer treatment. Here we investigated the biologic role of a rate-limiting enzyme involved in NAD(+) synthesis, Nampt, in multiple myeloma (MM). Nampt-specific chemical inhibitor FK866 triggered cytotoxicity in MM cell lines and patient MM cells, but not normal donor as well as MM patients PBMCs. Importantly, FK866 in a dose-dependent fashion triggered cytotoxicity in MM cells resistant to conventional and novel anti-MM therapies and overcomes the protective effects of cytokines (IL-6, IGF-1) and bone marrow stromal cells. Nampt knockdown by RNAi confirmed its pivotal role in maintenance of both MM cell viability and intracellular NAD(+) stores. Interestingly, cytotoxicity of FK866 triggered autophagy, but not apoptosis. A transcriptional-dependent (TFEB) and independent (PI3K/mTORC1) activation of autophagy mediated FK866 MM cytotoxicity. Finally, FK866 demonstrated significant anti-MM activity in a xenograft-murine MM model, associated with down-regulation of ERK1/2 phosphorylation and proteolytic cleavage of LC3 in tumor cells. Our data therefore define a key role of Nampt in MM biology, providing the basis for a novel targeted therapeutic approach.

Canonical and noncanonical Hedgehog pathway in the pathogenesis of multiple myeloma.

The Hedgehog (Hh) pathway is required for cell-fate determination during the embryonic life, as well as cell growth and differentiation in the adult organism, where the inappropriate activation has been implicated in several cancers. Here we demonstrate that Hh signaling plays a significant role in growth and survival of multiple myeloma (MM) cells. We observed that CD138(+) MM cells express Hh genes and confirmed Smoothened (Smo)-dependent Hh signaling in MM using a novel synthetic Smo inhibitor, NVP-LDE225 (Novartis), which decreased MM cell viability by inducing specific down-regulation of Gli1 and Ptch1, hallmarks of Hh activity. In addition, we detected a nuclear localization of Gli1 in MM cells, which is completely abrogated by Forskolin, a Gli1-modulating compound, confirming Smo-independent mechanisms leading to Hh activation in MM. Finally, we identified that bone marrow stromal cells are a source of the Shh ligand, although they are resistant to the Hh inhibitor because of defective Smo expression and Ptch1 up-regulation. Further in vitro as well as in vivo studies showed antitumor efficacy of NVP-LDE225 in combination with bortezomib. Altogether, our data demonstrate activation of both canonical and noncanonical Hh pathway in MM, thus providing the rationale for testing Hh inhibitors in clinical trials to improve MM patient outcome.

Loss of BRCA1 function increases the antitumor activity of cisplatin against human breast cancer xenografts in vivo.

Background: Previous reports suggested a central role of BRCA1 in DNA-damage repair mechanisms elicited by cell exposure to anti-tumour agents. Here we studied if BRCA1-defective HCC1937 or BRCA1–reconstituted HCC1937/WTBRCA1 human breast cancer xenografts (HBCXs) generated in SCID mice were differentially sensitive to cisplatin (CDDP) in vivo and we investigated potential molecular correlates of this effect. Results: CDDP induced almost complete growth inhibition of BRCA1-defective HBCXs, while BRCA1-reconstituted HBCXs were only partially inhibited. Cell cycle analysis showed a significant S- and G2/M blockade in BRCA1-defective as compared with parental BRCA1-reconstituted cells. Comparative gene expression profiling of HCC1937 and HCC1937/WTBRCA1 showed upregulation of RAD52 and XRCC4, whereas ERCC1 and RRM1 were downregulated. Pathway finder analysis of gene arrays data indicated perturbations of major proliferation and survival pathways suggesting that BRCA1 is mostly involved in G2/M but also in G1/S-phase checkpoints as well as in several important signaling pathways, including IGF, VEGF, oestrogen receptor, PI3K/AKT and EGF. Methods: HCC1937 or HCC1937/WTBRCA1 HBCXs were generated in SCID mice. Animals were then weekly treated with 5 mg/kg CDDP i.p. or with vehicle for 4 weeks. Tumour volume and mice survival were evaluated. Tumours were retrieved from animals 12 hours after the last treatment with CDDP or vehicle treatment and the cell suspension underwent cell cycle analysis. Differential gene expression and pathway modulation between HCC1937 and HCC1937/WTBRCA1 cells were also studied. Conclusion: Our data suggest that BRCA1-defective in vivo HBCXs express a molecular scenario predictive of high sensitivity to platinum-derived compounds strongly supporting the rationale for prospective tailored clinical trials in hereditary breast cancer.

miR-29b induces SOCS-1 expression by promoter demethylation and negatively regulates migration of multiple myeloma and endothelial cells

Epigenetic silencing of tumor suppressor genes frequently occurs and may account for their inactivation in cancer cells. We previously demonstrated that miR-29b is a tumor suppressor microRNA (miRNA) that targets de novo DNA methyltransferases and reduces the global DNA methylation of multiple myeloma (MM) cells. Here, we provide evidence that epigenetic activity of miR-29b leads to promoter demethylation of suppressor of cytokine signaling-1 (SOCS-1), a hypermethylated tumor suppressor gene. Enforced expression of synthetic miR-29b mimics in MM cell lines resulted in SOCS-1 gene promoter demethylation, as assessed by Sequenom MassARRAY EpiTYPER analysis, and SOCS-1 protein upregulation. miR-29b-induced SOCS-1 demethylation was associated with reduced STAT3 phosphorylation and impaired NFκB activity. Downregulation of VEGF-A and IL-8 mRNAs could be detected in MM cells transfected with miR-29b mimics as well as in endothelial (HUVEC) or stromal (HS-5) cells treated with conditioned medium from miR-29b-transfected MM cells. Notably, enforced expression of miR-29b mimics increased adhesion of MM cells to HS-5 and reduced migration of both MM and HUVEC cells. These findings suggest that miR-29b is a negative regulator of either MM or endothelial cell migration. Finally, the proteasome inhibitor bortezomib, which induces the expression of miR-29b, decreased global DNA methylation by a miR-29b-dependent mechanism and induced SOCS-1 promoter demethylation and protein upregulation. In conclusion, our data indicate that miR-29b is endowed with epigenetic activity and mediates previously unknown functions of bortezomib in MM cells.

In vivo anti-myeloma activity and modulation of gene expression profile induced by valproic acid, a histone deacetylase inhibitor.

Valproic acid (VPA) is a well‐tolerated anticonvulsant that exerts anti‐tumour activity as a histone deacetylase inhibitor. This study investigated the in vitro and in vivo activity of VPA against multiple myeloma (MM) cells. In vitro exposure of interleukin‐6‐dependent or ‐independent MM cells to VPA inhibited cell proliferation in a time‐ and dose‐dependent manner and induced apoptosis. In a cohort of severe combined immunodeficiency mice bearing human MM xenografts, VPA induced tumour growth inhibition and survival advantage in treated animals versus controls. Flow cytometric analysis performed on MM cells from excised tumours showed increase of G0–G1 and a decreased G2/M‐ and S‐phase following VPA treatment, indicating in vivo effects of VPA on cell cycle regulation. Gene expression profiling of MM cells exposed to VPA showed downregulation of genes involved in cell cycle progression, DNA replication and transcription, as well as upregulation of genes implicated in apoptosis and chemokine pathways. Pathfinder analysis of gene array data identified cell growth, cell cycle, cell death, as well as DNA replication and repair as the most important signalling networks modulated by VPA. Taken together, our data provide the preclinical rationale for VPA clinical evaluation as a single agent or in combination, to improve patient outcome in MM.

A peroxisome proliferator-activated receptor gamma (PPARG) polymorphism is associated with zoledronic acid-related osteonecrosis of the jaw in multiple myeloma patients: analysis by DMET microarray profiling

The aminobisphosphonate zoledronic acid (ZA) is the most important antiresorptive agent for the treatment of multiple myeloma (MM)-related bone disease (BD). Osteonecrosis of the jaw (ONJ) is an important complication of ZA-treated MM patients (Vannucchi et al, 2005; Filleul et al, 2010). So far, the mechanism of ONJ pathogenesis has not been clearly elucidated. Recently, a genetic susceptibility to ONJ has been suggested and a polymorphism of the cytochrome P450 CYP2C8 has been associated with ZA-related ONJ in MM (Sarasquete et al, 2008). To further investigate the genetic bases of ONJ, we genotyped in a case-control study a cohort of 19 MM patients treated with ZA who developed [nine cases, median age 66 years (range: 63–79)] or not [10 matched controls, median age 69 years (range: 63–84)] ONJ. We used the novel Affymetrix DMET™ plus platform (Affymetrix, Santa Clara, CA, USA), which interrogates 1936 genetic variations in 225 genes associated with phase I–II drug metabolism, disposition and transport (Deeken, 2009). The study protocol was approved by our University Hospital Bioethical Committee and informed consent was obtained from each patient. All patients received ZA according to the conventional dose and administration schedule; the ONJ group received 20 ± standard deviation (SD) 5·1 treatment courses and the control group underwent 15·1 ± 4·2 courses. MM patients were homogeneous on clinical and pathological characteristics at diagnosis and on their response to treatment. ONJ was diagnosed by clinical examination and imaging, including radiographs and/or computed tomography or magnetic resonance imaging. Peripheral blood was collected and used for DNA extraction. Genotypes were determined for each single nucleotide polymorphism (SNP) site of the 1931 of all interrogated SNPs and for the five Copy Number Variations (CNVs) included in DMET™ Plus GeneChip. Pharmacogenomic profiles were generated by Affymetrix DMET™ Console software®. Statistical analysis was performed by two-tailed Fisher’s exact test. No correction for multiple comparisons was performed. Results are therefore to be interpreted as hypothesis generating. Eight SNPs were significantly (P ≤ 0·05) associated with ONJ occurrence. Table I shows these SNPs, the reference and variant allele and the genotype and allele frequencies. All alleles were in Hardy-Weinberg equilibrium. The four genes correlated to the eight statistically relevant SNPs were PPARG (peroxisome proliferator-activated receptor gamma), ABP1{amiloride binding protein 1 [amine oxidase (copper-containing)]}, CHST11 [carbohydrate (chondroitin 4) sulfotransferase 11] and CROT (carnitine O-octanoyltransferase). The different distribution of SNP alleles and genotypes between ONJ patients and control cases are reported in Table II. The SNP rs1152003, mapping in PPARG, showed the strongest association with ONJ. We detected a highly significant (P = 0·0055) differential occurrence of the C/C homozygous (HOM) genotype in 77·7% of ONJ cases (7/9) versus only 10% of controls (1/10) (Fig 1A). Moreover, homozygous and heterozygous genotypes for the C variant were differently distributed between ONJ patients and the control group (Table II). The frequency of the C variant allele in the PPARG SNP underlines a highly significant association of the C allele with the ONJ group (P = 0·0064, Table II). No clinical association has been previously reported for these SNPs. Table I SNP polymorphisms associated with ONJ in MM patients. Table II Allele and genotype frequencies of polymorphisms in MM patients. Fig 1 SNP rs1152003 genotype clustering of MM patients. (A) The red colour symbols are MM patients with ONJ. Blue colour symbols are matched MM control patients. Genotypes are identified as homozygote reference allele , heterozygote (•) and homozygote ... Direct nucleotide sequencing was carried out on patient specimens to further confirm the presence of genetic variations, using an Applied Biosystems ABI 3100 Genetic Analyser. We found a concordance rate of 100% between DMET genotyping and sequence analysis (Fig 1B). The rs1152003 SNP maps in the 3′UTR region of PPARG, at position 12477055 of chromosome 3 (Genome Build 37.1). Although no clinical correlation has been reported for the rs1152003 variant, polymorphisms in PPARG have been associated with increased risk of a variety of diseases (Dallongeville et al, 2009). PPARG is located in the human chromosome 3, band 3p25. Chromosomal abnormalities, such as 3p deletion, have been identified in several hematologic malignancies. PPARG is involved in adipocyte differentiation and in angiogenesis (Rosen & Spiegelman, 2001). Recently, the PPARG pathway has been recognized as key mechanism for bone remodelling. It acts on mesenchymal stem cell differentiation by increasing adipogenesis but also inhibiting osteoblast and osteoclast formation. Moreover, PPARG polymorphisms correlate with the bone mass density (Ackert-Bicknell et al, 2008). However, a recent study on a wide cohort of Korean individuals, with idiopathic, steroid-induced or alcohol-induced osteonecrosis of the femoral head, failed to demonstrate a significant correlation with three common PPARG polymorphisms (Kim et al, 2007). Interestingly, modulation of PPARG activity within the bone marrow microenvironment has been recently shown to interfere with cytokines such as IL6, which is involved with a central role in the pathogenesis of MM (Wang et al, 2004), suggesting also that PPARG may represent a valuable therapeutic target in MM (Garcia-Bates et al, 2008). The present study also showed that three SNPs identified in ABP1 were associated with ONJ and were in linkage disequilibrium (data not shown). ABP1 encodes a membrane glycoprotein that is expressed in many epithelial and haematopoietic tissues. Moreover, a further three ONJ-associated SNPs map to CHST11, which was recently described as a factor required for proper chondroitin sulfation and cartilage morphogenesis. Expression of the chondroitin sulfotransferase genes is crucial for the correct mammalian bone morphogenesis. Finally, the ONJ-associated rs2097937 maps to CROT, whose protein is involved in the trans-esterification of acyl-CoA molecules. Our findings indicate that genetic polymorphisms are involved in the pathogenesis of ONJ in MM patients. The highly significant association of ONJ with the rs1152003 SNP polymorphism in PPARG strongly suggests this genetic variant as candidate biomarker for the identification of MM patients at risk of ONJ if treated with ZA. In fact, the C/C genotype demonstrated an odds ratio of 31·5 (95% confidence interval, 2·35–422·32) for developing ONJ following ZA treatment. Differently from the recent report (Sarasquete et al, 2008), where the study was based on the 500K Affymetrix high density array, we used the DMET platform that interrogates only highly selective SNPs associated with drug toxicity and has the advantage of avoiding an extremely high number of comparisons, which requires statistical corrections and large patient cohorts. We propose the rs1152003 C/C genotype as a candidate genetic biomarker for ONJ, which warrants validation in larger series.

Intracellular NAD+ depletion enhances bortezomib-induced anti-myeloma activity

We recently demonstrated that Nicotinamide phosphoribosyltransferase (Nampt) inhibition depletes intracellular NAD⁺ content leading, to autophagic multiple myeloma (MM) cell death. Bortezomib has remarkably improved MM patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here we observed higher Nampt messenger RNA levels in bortezomib-resistant patient MM cells, which correlated with decreased overall survival. We demonstrated that combining the NAD⁺ depleting agent FK866 with bortezomib induces synergistic anti-MM cell death and overcomes bortezomib resistance. This effect is associated with (1) activation of caspase-8, caspase-9, caspase-3, poly (ADP-ribose) polymerase, and downregulation of Mcl-1; (2) enhanced intracellular NAD⁺ depletion; (3) inhibition of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities; (4) inhibition of nuclear factor κB signaling; and (5) inhibition of angiogenesis. Furthermore, Nampt knockdown significantly enhances the anti-MM effect of bortezomib, which can be rescued by ectopically overexpressing Nampt. In a murine xenograft MM model, low-dose combination FK866 and Bortezomib is well tolerated, significantly inhibits tumor growth, and prolongs host survival. Taken together, these findings indicate that intracellular NAD⁺ level represents a major determinant in the ability of bortezomib to induce apoptosis in MM cells and provide proof of concept for the combination with FK866 as a new strategy to enhance sensitivity or overcome resistance to bortezomib.

Eph-B2/Ephrin-B2 Interaction Plays a Major Role in the Adhesion and Proliferation of Waldenstrom's Macroglobulinemia

Purpose: The ephrin receptors (Eph) are found in a wide range of cancers and correlate with metastasis. In this study, we characterized the role of Eph-B2 receptor in the interaction of Waldenstroms macroglobulinemia (WM) cells with the bone marrow microenvironment. Experimental Design: We screened the activity of different receptor tyrosine kinases in WM patients and found that Eph-B2 was overexpressed compared with control. Also, we tested the expression of ephrin-B2 ligand on endothelial cells and bone marrow stromal cells (BMSC) isolated from WM patients. We then tested the role of Eph-B2/Ephrin-B2 interaction in the adhesion of WM cells to endothelial cells and BMSCs; the cell signaling induced by the coculture in both the WM cells and the endothelial cells; WM cell proliferation, apoptosis, and cell cycle in vitro and tumor progression in vivo; and in angiogenesis. Results: Eph-B2 receptor was found to be activated in WM patients compared with control, with a 5-fold increase in CD19+ WM cells, and activated cell adhesion signaling, including focal adhesion kinase, Src, P130, paxillin, and cofilin, but decreased WM cell chemotaxis. Ephrin-B2 ligand was highly expressed on endothelial cells and BMSCs isolated from WM patients and on human umbilical vein endothelial cells and induced signaling in the endothelial cells promoting adhesion and angiogenesis. Blocking of ephrin-B2 or Eph-B2 inhibited adhesion, cytoskeletal signaling, proliferation, and cell cycle in WM cells, which was induced by coculture with endothelial cells and decreased WM tumor progression in vivo. Conclusion: Ephrin-B2/Eph-B2 axis regulates adhesion, proliferation, cell cycle, and tumor progression in vivo through the interaction of WM with the cells in the bone marrow microenvironment. Clin Cancer Res; 18(1); 91–104. ©2011 AACR.