Cell Adhesion-mediated Drug Resistance Research Articles

The Monoclonal Antibody nBT062 Conjugated to Cytotoxic Maytansinoids Has Potent and Selective Cytotoxicity against CD138 Positive Multiple Myeloma Cells in Vitro and in Vivo.

CD138 is expressed on differentiated plasma cells and is involved in the development and/or proliferation of multiple myeloma (MM), for which it is a primary diagnostic marker. In this study, we report that immunoconjugates comprised of the murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives (nBT062-SMCC-DM1, nBT062-SPDB-DM4 and nBT062-SPP-DM1) showed cytotoxic activity against CD138-positive MM cells both in vitro and in vivo. These agents demonstrated cytotoxicity against OPM1 and RPMI8226 (CD138-positive MM cell lines) in a dose and time-dependent fashion and were also cytotoxic against primary tumor cells from MM patients. Minimal cytotoxicity was noted in CD138-negative cell lines and no activity was observed against peripheral blood mononuclear cells from healthy volunteers, suggesting that CD138-targeting is important for immunoconjugate-mediated cytotoxicity. Examination of the mechanism of action whereby these immunoconjugates induced cytotoxicity in MM cells demonstrated that treatment triggered G2/M cell cycle arrest, followed by apoptosis associated with cleavage of PARP and caspase-3, -8 and -9. Neither interleukin-6 nor insulin-like growth factor-I could overcome the apoptotic effect of these agents. The level of soluble (s)CD138 in the BM plasma from 15 MM patients was evaluated to determine the potential impact of sCD138 on immunoconjugate function. The sCD138 level in BM plasma was found to be significantly lower than that present in MM cell culture supernatants where potent in vitro cytotoxicity was observed, suggesting that sCD138 levels in MM patient BM plasma would not interfere with immunoconjugate activity. Because adhesion to bone marrow stromal cells (BMSCs) triggers cell adhesion mediated drug resistance to conventional therapies, we next examined the effects of the conjugates on MM cell growth in the context of BMSC. Co-culture of MM cells with BMSCs, which protects against dexamethasoneinduced death, had no impact on the cytotoxicity of the immunoconjugates. The in vivo efficacy of these immunoconjugates was also evaluated in SCID mice bearing established CD138-positive MM xenografts and in a SCID-human bone xenograft model of myeloma. Significant tumor growth delay or regressions were observed at immunoconjugate concentrations that were well tolerated in all models tested. The ability of these agents to mediate bystander killing of proximal CD138-negative cells was also evaluated. While nBT062-SPDB-DM4 was inactive against CD138-negative Namalwa cells cultured alone, significant killing of these CD138-negative cells by nBT062-SPDB-DM4 was observed when mixed with CD138-positive OPM2 cells. This bystander killing may contribute to the eradication of MM tumors by disrupting the tumor microenvironment and/or killing CD138-negative MM tumor cells, such as the putative CD138 negative myeloma stem cells. These studies demonstrate strong evidence of in vitro and in vivo selective cytotoxicity of these immunoconjugates and provide the preclinical framework supporting evaluation of nBT062-based immunoconjugates in clinical trials to improve patient outcome in MM.

β1 Integrin Adhesion Enhances IL-6 Mediated STAT3 Signaling in Multiple Myeloma Cells: Implications for Microenvironment Influence on Tumor Cell Survival and Proliferation.

Multiple Myeloma (MM) is a B cell malignancy characterized by the monoclonal expansion of plasma cells. Although numerous genetic alterations have been implicated in MM pathogenesis, it is widely hypothesized that the bone marrow (BM) microenvironment contributes to MM cell pathogenesis. The BM microenvironmental components, interleukin (IL)-6 and fibronectin (FN), have individually been shown to influence the proliferation and survival of MM cells; however, in vivo these effectors most likely work together. We examined signaling events, cell cycle progression, and levels of drug response in MM cells either adhered to FN via β1 integrins, stimulated with IL-6, or with the two combined. IL-6 and FN adhesion have been demonstrated to protect cells from a host of cytotoxic stimuli suggesting co-stimulation of MM cell lines with IL-6 and FN-adhesion may confer a greater protection against chemotherapeutics than either effector alone. However, MTT cytotoxicity assays demonstrate that although adhesion to FN provides significant protection against treatment with mitoxantrone or doxorubicin (p=0.0002 and p=<0.0001 respectively), the addition of IL-6 provides no further protection. These findings were corroborated by analysis of drug-mediated apoptosis using FCM by Annexin-V/7-AAD. In regards to cell cycle kinetics, our laboratory has previously demonstrated that adhesion of the 8226 MM cell line to FN mediated a p27Kip1 dependent G0/G1 cell cycle arrest. As predicted, BrdU/PI analysis of 8226 cells adhered to FN for 24 hours results in an increased number of cells in G0/G1 relative to cells maintained in suspension (p=0.0028). In contrast, when cells were adhered to FN in the presence of IL-6 no accumulation of cells in G0/G1 was observed, with levels similar to that observed in cells maintained in suspension with or without stimulation by IL-6. Our studies demonstrated that the G1/S cell cycle arrest associated with FN adhesion of MM cell lines was overcome when IL-6 was added; however, the cell adhesion mediated drug resistance (CAM-DR) was maintained in the presence of IL-6. Investigation of the biochemical signaling following concomitant exposure of MM cells to IL-6 and FN adhesion revealed a synergistic increase in STAT3 phosphorylation, nuclear translocation and DNA-binding as compared to either IL-6 or FN-adhesion alone in four MM cell lines. STAT3 phosphorylation was increased in cells adhered to FN in an IL-6 dose dependent manner. Electrophoretic mobility shift assay demonstrated a parallel 3-fold increase in STAT3/DNA complexes in cells adhered to FN relative to cells in suspension. To further characterize the receptor proximal affects of FN adhesion on IL-6 signaling we immunoprecipitated the IL-6R complex with antisera to gp130. Immunoprecipitation of gp130 revealed enhanced tyrosine phosphorylation of the gp130/Jak family complexes following stimulation FN-adhered RPMI 8226 MM cells with IL-6. Consistent with increased phosphorylation of the receptor complex, increased levels of phospho-STAT3 were identified associated with gp130 under co-stimulatory conditions relative to IL-6 or FN adhesion alone. Interestingly, immunoprecipitation with gp130 antibodies also revealed an association between STAT3 (non-phosphorylated) and gp130 in the absence of IL-6 stimulation in cells adhered to FN. These results suggest that adhesion to FN facilitates an IL-6-independent association between gp130 and STAT3, resulting in enhanced STAT3 signaling. Taken together, these data demonstrate a novel mechanism by which collaborative signaling by β1 integrin and gp130 confer an increased survival advantage to MM cells.

Influence of Targeted Therapy in Redefining High-Risk Myeloma.

Abstract Multiple myeloma is a disease that typically responds to initial treatment; however, the disease is not cured by chemotherapy, and drug resistance ultimately develops. Most studies investigating the problem of drug resistance have focused on acquired resistance or resistance that occurs after response to prior therapy as a result of residual disease. Intrinsic factors, such as reduced drug uptake, enhanced damage response (i.e., DNA repair), altered drug metabolism, or inhibition of programmed cell death pathways are known to contribute to acquired drug resistance. For example, it was recently reported that the acquired melphalan resistant phenotype in myeloma cell lines was associated with over-expression of the Fanconi anemia (FA)/BRCA pathway genes. Enhanced interstrand cross-link (ICL) repair via the FA/BRCA pathway was causally related to melphalan resistance and disruption of this pathway using knock-down techniques reversed drug resistance. Furthermore, bortezomib (Velcade) has been reported to enhance melphalan treatment, and recent pre-clinical data has shown that bortezomib reduces FA/BRCA gene expression and function. Clinical trials are necessary to determine the role of the FA/ BRCA pathway in acquired drug resistance for myeloma patients and whether targeting this pathway enables prevention of or the ability to overcome acquired melphalan resistance in myeloma patients. Conversely, factors that promote tumor cell survival and drug resistance that are external to the tumor cell itself might exist. Evidence supporting the importance of understanding the influence of the tumor microenvironment on drug sensitivity has been reported by several investigators. The tumor microenvironment for hematologic malignancies, including myeloma, is principally the bone marrow. The bone marrow contains candidate components that contribute to reduced drug activity, minimal residual disease, and emergence of drug resistant cells. Cell adhesion molecules expressed by myeloma cells, including the β integrins, bind to fibronectin and other extracellular matrix components of the bone marrow, and this interaction contributes to a reversible, de novo drug resistance phenotype called “cell adhesion mediated drug resistance” or CAMDR. Adhesion via integrins is known to activate a network of signal transduction pathways that influence cell survival, growth, and differentiation. Several targets that are influenced by integrin adhesion and may contribute to CAM-DR include the following: reduced proapoptotic Bim levels, alterations in nuclear topoisomerase II levels, increased p27 kip1 levels, and changes in FLIP1 levels. In addition, myeloma cell adhesion to bone marrow stroma (BMS) involves other adhesion molecules and signaling events that promote CAMDR. For example, Notch1 receptors expressed on multiple myeloma cells when stimulated by Jagged causes growth arrest and protection from drug-induced apoptosis. Recently, approaches to inhibit integrin and Notch signaling associated with CAM-DR have been examined pre-clinically. Clinical trials are necessary to determine if these approaches will prevent or overcome CAM-DR in patients.

Pan-Selectin Inhibitor GMI-1070 Blocks Cell Adhesion of Acute Myelogenous Leukemia Cell Lines

Most patients with acute myelogenous leukemia (AML) initially respond to chemotherapy but later relapse and die from the disease (50–70%). One mechanism by which AML cells resist treatment with anti-proliferative drugs is by cell adhesion in protective microenvironments in areas such as the bone marrow. Here, we investigate the effects of GMI-1070 on the mechanism of adhesion of an AML cell line (MV-4-11) derived from biphenotypic myelomonocytic cells. GMI-1070 is a small molecule rationally designed pan-selectin antagonist with particularly strong activity for E-selectin (IC50 = 3.4 μM). Over 95% of these AML cells express ligands for E and P-selectins as determined by fluorescence-activated cell sorter analysis using E or P-selectin/hIg chimeras. Virtually all of these cells strongly express the specific E-selectin ligands CD65 and FH-6 using flow cytometry. The ability of these AML cells to roll and adhere to E or P-selectin expressed on monolayers of human endothelial cells (HUVECs) under the shear forces of normal blood flow was determined in vitro using flow chambers. The data was captured by videomicroscopy and processed using digital imaging. AML cells rolled and adhered to endothelium expressing E or P-selectin and both forms of adhesion are inhibited by GMI-1070. In contrast, much less rolling and adhesion was observed by multiple myeloma cell line U-266 only on endothelial cells expressing P-selectin although these specific interactions are also inhibited by GMI-1070. To analyze the adhesion, homing and infiltration of this AML cell line in vivo, recently developed NOD-scid IL2rgnull mice were used as hosts for in vivo experimentation. These mice lack adaptive immune function, are completely deficient in host natural killer cells and support long term growth of primary human leukemias (AML, CML, and ALL). Mice were injected i.v. with MV-4-11 AML cells (1 × 106) and blood samples were taken from the retro-orbital plexus at 1 and 2 weeks post injection. Blood counts were performed using an ADVIA Hematology Analyzer. At intervals post injection, the presence of AML cells in the bone marrow (BM), spleen, and peripheral blood was determined by flow cytometry of cells co-expressing human CD45 and human CD33. At 3 weeks, AML cells represented 26% of cells in the bone marrow and 2.3% of cells in the spleen. At 6 weeks, high percentages of AML cells were found in both tissues as well as in the blood (bone marrow, 83%; spleen, 69%; blood, 36%). Histological analyses showed accumulations of AML cells in the BM, spleen, lungs, liver, kidneys, and ovaries. At these later stages, hematopoietic cells in the BM cavity were largely replaced by sheets of leukemic cells. This model proved to be effective in studying the homing of an AML cell line to protective environments in the bone marrow and dissemination of disease to other organs and is being used to determine the effects on cell adhesion-mediated drug resistance (CAM-DR) by selectin-mediated inhibition of adhesion using small molecule antagonist, GMI-1070. GMI-1070 is currently in Phase I clinical trials.

Migratory Activity and Stromal Cell Adhesion of Mantle Cell Lymphoma Cells Can Be Diminished by Blocking of VLA-4 and CXCR4 Receptors

Mantle cell lymphoma (MCL) is an aggressive lymphoma that generally is associated with a rapid dissemination of the malignant B-cells and a high risk of relapse. Chemokine receptors and adhesion molecules play key roles in normal B cell migration and homing to distinct microenvironments, but their expression and function in MCL is largely unknown. In this study, we profiled the expression and function of chemokine receptors and adhesion molecules (CXCR4, CXCR5, CXCR3, CD49d/VLA-4, CD44, and CD62L) in MCL cell lines (SP-53, MINO, JeKo-1 and Granta-519) and primary MCL cells. Except for the EBV-positive cell line Granta 519, all MCL lines displayed high levels of CXCR4, CXCR5, CD49d and CD44. Primary MCL cells from different patients (n=6) displayed a similar immunophenotype. We then analyzed chemotaxis of MCL cells towards CXCL12 (200 ng/ml) and CXCL13 (1μg/ml) in transwell assays. 35.7±5.7% of input SP-53, 25.8±2.8% of MINO, and 6.7±0.9% of JeKo-1 cells migrated towards CXCL12 within 3 hours (mean±SEM, triplicates). 43.4±9.8% of input SP-53, 16.7±2.8% of MINO, and 4.3±0.5% of JeKo-1 cells migrated toward CXCL13. Granta-519 did not demonstrate any significant chemotaxis. Pre-treating the cells with a CXCR4 antagonist (AMD3100, Plerixafor) effectively blocked chemotaxis towards CXCL12. Marrow stromal cells (MSC) constitutively secrete CXCL12 and provide ligands for VLA-4 integrins. These two molecules now can be targeted clinically using CXCR4 antagonists or monoclonal antibodies (mAbs), respectively. In co-cultures with MSC, SP-53 and MINO displayed abundant spontaneous migration beneath MSC (pseudoemperipolesis/PEP). Therefore we examined whether blocking of CXCR4 or VLA-4 affects the PEP of MCL cells. Pre-incubation of SP-53 or MINO cells with AMD3100 reduced PEP to levels that were 49.4±1.5% (p<.07) or 11.7±1.7% (p<.04) of controls. Pretreatment with anti-VLA-4 antibodies (Natalizumab) also resulted in significant decrease of PEP to levels that were 5.8±1.4% (p<.02) in SP-53 and 2.2±0.6% (p<.01) in MINO cells. Pre-incubation with a cyclic peptide inhibitor with the minimal VLA-4 binding motif “LDV” also significantly reduced PEP of MCL cells to 22.1±4.0% (p<.006) in SP-53 and 7.1±1.9% (p<.005) in MINO cells. MCL cells treated with 10 μM fludarabine (F-ara-A) in suspension cultures resulted in high levels of apoptosis in MCL cells within 24 to 72 hrs. We found that co-culture with MSC significantly reduced F-ara-A-induced apoptosis, a primary drug resistance mechanism termed cell adhesion-mediated drug resistance (CAM-DR). Interestingly, the MCL cell fraction that had migrated into the stromal cell layer was particularly protected from the cytotoxic effect of F-ara-A. As such, clinical targeting of CXCR4 and VLA-4 integrins in MCL may not only antagonize the migration and homing associated with the dissemination of the disease, but also disrupt the adhesion to stromal cells, help to overcome CAM-DR, and thus make MCL cells more accessible to conventional drugs. Collectively, our studies provide a rationale to further explore the efficacy of combinations of CXCR4- and/or VLA-4 antagonists with conventional drugs in patients with this disease. This approach may lead to new therapeutic avenues for MCL patients.

Pre-Clinical Evaluation of a Small Molecule Inhibitor of CDK1 as Potential Therapy for MYC Expressing Mutiple Myeloma Tumors

Through the recent elucidation of molecular and cellular processes in multiple myeloma (MM) pathogenesis that effect well-defined proliferative and survival pathways, a number of molecular targets for anti-cancer agents have emerged. This includes proteins involved in cell cycle regulation. A recent model of early MM pathogenesis proposes that at least one of the cyclin D genes is dysregulated in all myeloma tumors facilitating activation of cyclin dependent kinase (CDK)4 (or CDK6), and transition from G1 to S phase. It is hypothesized that this renders myeloma cells more responsive to bone marrow (BM) derived proliferative stimuli including IL-6 that upregulates MYC expression in MM cells and cooperates with cyclin D to promote transit to S-phase. In addition, recent studies have established a causative role of MYC dysreguation in progression from monoclonal gammopathy to myeloma. Thus an approach targeting deregulated cell cycle progression and MYC may prove effective MM therapy. Purvalanol is a potent and selective inhibitor of CDK1, an important regulator of cell cycle progression and has been reported to induce MYC-dependent apoptosis. Activity of purvalanol against a panel of 14 standardized, annotated myeloma cell lines was measured in a 48 hour MTT viability assay. IC50-s of all 14 cell lines ranged between 5 uM to 7.5 uM. Western blot and real-time PCR analysis revealed variability of MYC protein and mRNA levels between the 14 myeloma cell lines. We compared potential therapeutic activity of purvalanol against 3 myeloma cell lines (U266, H929, KMS12PE) with low, intermediate, and high levels of c-MYC expression, respectively. All three cell lines demonstrated G2-M growth arrest however marked apoptosis as determined by PI/annexin V staining was observed only in the cell lines with the highest level of MYC expression. Exposure of MM patient-derived BM mononuclear cells to purvalanol preferentially induced apoptosis of CD138+ MM cells. In contrast, purvalanol was minimally cytotoxic to the non-myeloma cell fraction and to non-transformed fibroblast cell lines (MRC5, IMR90 and W138) and failed to inhibit normal bone marrow-derived CD34 colony formation. Recent studies have demonstrated that the BM microenvironment offers protection of myeloma cells from chemotherapeutic agents by common mechanisms. Myeloma cell lines were cultured in 3 different conditions to mimic the tumor's protective microenvironment. Soluble factors produced by the BM, IL6 and IGF-1 induced a modest degree of resistance to purvalanol while co-culture on BM stroma cells was completely protective. Studies evaluating the in vivo efficacy of purvalanol in a novel Vk*myc transgenic mouse model that spontaneously develops myeloma with a low proliferative index are ongoing and will be presented. The CDK1 inhibitor, purvalanol demonstrates broad single agent activity against myeloma cells with enhanced activity against MYC overexpressing cells. However, the strong protective effect of the BM microenvironment suggest that combination with agents that can reverse cell-adhesion mediated drug resistance may prove beneficial in optimizing the efficacy of this class of drugs for the treatment of MM.

Bortezomib overcomes cell adhesion-mediated drug resistance through downregulation of VLA-4 expression in multiple myeloma

Multiple myeloma (MM) is incurable, mainly because of cell adhesion-mediated drug resistance (CAM-DR). In this study, we performed functional screening using short hairpin RNA (shRNA) to define the molecule(s) responsible for CAM-DR of MM. Using four bona fide myeloma cell lines (KHM-1B, KMS12-BM, RPMI8226 and U266) and primary myeloma cells, we identified CD29 (beta1-integrin), CD44, CD49d (alpha4-integrin, a subunit of VLA-4), CD54 (intercellular adhesion molecule-1 (ICAM-1)), CD138 (syndecan-1) and CD184 (CXC chemokine receptor-4 (CXCR4)) as major adhesion molecules expressed on MM. shRNA-mediated knockdown of CD49d but not CD44, CD54, CD138 and CD184 significantly reversed CAM-DR of myeloma cells to bortezomib, vincristine, doxorubicin and dexamethasone. Experiments using blocking antibodies yielded almost identical results. Bortezomib was relatively resistant to CAM-DR because of its ability to specifically downregulate CD49d expression. This property was unique to bortezomib and was not observed in other anti-myeloma drugs. Pretreatment with bortezomib was able to ameliorate CAM-DR of myeloma cells to vincristine and dexamethasone. These results suggest that VLA-4 plays a critical role in CAM-DR of MM cells. The combination of bortezomib with conventional anti-myeloma drugs may be effective in overcoming CAM-DR of MM.

HSP70 inhibition reverses cell adhesion mediated and acquired drug resistance in multiple myeloma

Heat shock proteins (HSPs) are a super family of highly conserved molecular chaperone proteins, which are induced in response to stress. HSP70 has been demonstrated to inhibit apoptosis induced by a number of chemotherapeutic agents. Previous investigations have suggested the development of drug resistance in multiple myeloma (MM) cells after adhesion to stroma. This study used MM cell lines and primary plasma cells to determine if HSP70 had a role in development of chemo resistance. Adhesion of MM cells to either bone marrow stromal cells or fibronectin (FN) enhanced HSP70 expression. Inhibition of the HSP70 expression decreased 8226 cell adhesion to stroma or FN and induced more apoptosis in FN-adhered 8226 cells than in suspension cultures at 24 h. Further, HSP70 inhibitors enhanced melphalan-induced apoptosis and reversed melphalan-induced cell adhesion-mediated drug resistance (CAM-DR) phenotype. In addition, compared to parental cells, KNK-437, a heat shock factor inhibitor caused more apoptosis in melphalan-resistant 8226/LR5 cells and sensitized them to melphalan. Primary CD138 positive cells showed high expression of HSPA4 mRNA, and KNK-437 caused apoptosis in these cells. In conclusion, our data suggest inhibition of HSP70, reduced adhesion and caused apoptosis of both acquired and de novo drug resistant MM cells.

Identification of a novel switch in the dominant forms of cell adhesion-mediated drug resistance in glioblastoma cells

The failure of malignant cells to undergo apoptosis is a major obstacle in cancer therapy, and thus identifying the underlining molecules involved therein is imperative for improving patient survival. An important mechanism of drug resistance is cell adhesion-mediated drug resistance (CAM-DR). In this study we identify a novel switch by which glioblastoma multiforme (GBM) cells alter the mode of CAM-DR. In the absence of a microenvironmental cue provided by components of the extracellular matrix (ECM), GBM cells are able to employ an alternative, but equally effective, mode of CAM-DR by forming spheres via cell-cell interactions. Intriguingly, when inhibiting cell-cell interactions in the absence of ECM components, either by low cell density or by inhibition of gap junctions (intercellular connexin tunnels) through chemical inhibition with carbenoxyolone or co-incubation with the connexin-mimicking Gap 27 Cx37,43 peptide, GBM cells were sensitized to tumor necrosis factor-related apoptosis-inducing ligand- and CD95-induced apoptosis. By demonstrating that GBM cells can alternate from one form of CAM-DR (cell-substrate tethering) to another (homocellular cell-cell adhesion) and that inhibition of both forms is necessary for apoptosis sensitization, our findings not only have important implications for novel approaches to restore defective apoptosis programs, but also reveal a novel role of gap junctions in GBM.

The Bone Marrow Microenvironment as a Tumor Sanctuary and Contributor to Drug Resistance

The bone marrow microenvironment facilitates the survival, differentiation, and proliferation of hematopoietic cells. These cells are supported by fibroblast-like bone marrow stromal cells, osteoblasts, and osteoclasts which secrete soluble factors and extracellular matrix proteins that mediate these functions. This rich environment serves as a safe haven not only for normal and malignant hematopoietic cells, but also for epithelial tumor cells that metastasize to bone, offering protection from chemotherapeutic agents by common mechanisms. Soluble factors produced in the bone marrow, such as stromal cell-derived factor-1 and interleukin-6, mediate homing, survival, and proliferation of tumor cells, and integrin-mediated adhesion sequesters tumor cells to this protective niche. Environment-mediated drug resistance includes a combination of soluble factors and adhesion, and can be subdivided into soluble factor-mediated drug resistance and cell adhesion-mediated drug resistance. Because it is induced immediately by the microenvironment and is independent of epigenetic or genetic changes caused by the selective pressure of drug exposure, environment-mediated drug resistance is a form of de novo drug resistance. In this form of drug resistance, tumor cells are transiently and reversibly protected from apoptosis induced by both chemotherapy and physiologic mediators of cell death. This protection allows tumor cells to survive the insult of chemotherapy, leading to minimal residual disease, and thereby increases the probability for the development of acquired drug resistance.

Wnt3/RhoA/ROCK Signaling Pathway Is Involved in VLA6-Dependent Adhesion-Mediated Drug Resistance of Multiple Myeloma.

Adhesion of myeloma cells to BM stromal cells is now considered to play a critical role in chemo-resistance. However, little is known about the molecular mechanism of cell adhesion mediated drug resistance (CAM-DR) in multiple myeloma. In this study, we focused on relationship between drug resistance and expression of Wnts, the factor regulating the cell adhesion and proliferation, in myeloma cells. To gain insight into involvement of Wnt signaling in CAM-DR, we first screened the expression of Wnt family in myeloma cell lines (RPMI8226, ARH77, KMS-5 and MM1S) by reverse transcription-polymerase chain reaction analysis. Although the mRNAs of Wnt2b, Wnt7a and Wnt10b were variably expressed in some of myeloma cell lines, Wnt3 mRNA was detected in all the myeloma cells examined. KMS-5 and ARH77, which highly expressed Wnt3 protein, tightly adhered to human BM stromal cells and accumulation of β-catenin and GTP-bounded RhoA was observed in these myeloma cell lines. Conversely, RPMI8226 and MM1S, which modestly expressed Wnt3 protein, rather weakly adhered to human BM stromal cells. We then examined the relevance of Wnt3 expression to adhesive property to stromal cells and to CAM-DR of myeloma cells. KMS-5 and ARH-77 exhibited apparent CAM-DR against Doxorubicin. This CAM-DR was significantly reduced by anti-integrinβ1 antibody, anti- integrinα6 antibody and a Wnt-receptor competitor, secreted Frizzled related protein-1 and Rho kinase inhibitor (Y27632 and OH-fasudil), but not by the specific inhibitor of canonical signaling (DKK-1), indicating that Wnt-mediated CAM-DR which is dependent on integrinα6/β1 (VLA-6)-mediated attachment to stromal cells is induced by Wnt/RhoA-Rho kinase (ROCK) pathway signal. This CAM-DR for doxorubicin was also significantly reduced by Wnt3 siRNA transfer to KMS-5 and further augmented by addition of Wnt3 conditioned medium. These results indicate that Wnt3 contributes to VLA-6-mediated CAM-DR via the Wnt/RhoA/ROCK pathway of myeloma cells. Thus, the Wnt3/RhoA/ROCK signaling pathway could be a promising molecular target to overcome CAM-DR.

The Effect of Statin Use at the Time of Autologous Transplant on Response and Survival in Patients with Multiple Myeloma.

Background : Statins (HMG-CoA reductase inhibitors) prevent the synthesis of mevalonic acid and ultimately this interferes with the isoprenylation of proteins involved in the cell cycle, namely Ras, Rac, and Rho A kinases, sometimes resulting in caspase-3 dependent apoptosis. Lovastatin has been shown to have antimyeloma activity in myeloma cells (van de Donk, Leukemia, 2002) and simvastatin restores chemosensitivity to myeloma cells exhibiting cell adhesion mediated drug resistance (Schmidmaier, Blood, 2004). Simvastatin when added to bortezomib or bendamustine in 6 patients with relapsed myeloma improved responses (Schmidmaier, EJH, 2007). Because autologous hematopoietic stem cell transplantation (HSCT) following high-dose melphalan is a standard treatment and statins may serve as sensitizing agents, we hypothesized that statin use at the time of autologous HSCT may increase the response rate to transplant, improve progression free survival (PFS), and prolong overall survival (OS).Methods : 146 patients with MM underwent autologous HSCT after melphalan 200mg/m2 at Ohio State University between June 1999 and December 2006. We reviewed their records and divided the patients into those that received (n=28) or did not receive (n=118) statins (≥ 20mg/day) for at least one month before and after transplant. Response was assessed according to the International Myeloma Working Group (IMWG) uniform response criteria (Durie BG et al. 2006).Results : The median age of the study population was 58 years (range 35–74yrs) with 86 male and 58 female patients. The two groups had similar baseline characteristics including age, serum cholesterol, international stage, disease status at the time of transplantation, number of prior therapies, and percentage of patients with high-risk cytogenetics (defined by the presence of t[4;14], t[14;16], deletion 17p, karyotypic del 13, del 13q w/14q32 rearrangement, or any tetrasomy). Stem cell mobilization in patients using statins yielded a mean of 8.3 x 106 CD34+ cells/kg versus 6.5 in patients not taking statins (p=0.4). The rate of complete response (CR) plus very good partial response (VGPR) in patients using statins (43%) was comparable to the CR + VGPR rate (45%) in patients not using statins (p=0.84). However the overall response rate (CR + VGPR + PR) was higher in the statin group (93% vs. 78%; p=0.07). In a subset analysis of patients with high-risk cytogenetics, the overall response rate in the statin group (n=5) was 100% versus 78% for non-statin group (n=14). Median overall survival (OS) for the statin vs. non-statin groups were 25.7 and 22 months respectively (P 0.65), and the progression free survivals were 19.5 and 14.8 months respectively (p=0.97). Three year PFS in the statin versus no statin groups were 32% and 30% respectively. No differences in terms of hepatic toxicity, myositis, infectious complications, and fatal cardiovascular events between groups were noted.Conclusion: Patients using statins at the time of transplant showed a trend for a better overall response rate (93% vs. 78%; p=0.07) that did not translate to improved PFS or OS.

Combination therapy of an anticancer drug with the FNIII14 peptide of fibronectin effectively overcomes cell adhesion-mediated drug resistance of acute myelogenous leukemia

We investigated whether FNIII14, a 22-mer peptide derived from fibronectin (FN) that potently impairs interaction of FN with beta1-integrin, could overcome cell adhesion-mediated drug resistance (CAM-DR) induced by very late antigen (VLA)-4-to-FN interaction in acute myelogenous leukemia (AML). Two AML cell lines, U937 cells and HL-60 cells, and fresh leukemic cells from six AML patients with high alpha4-integrin expression exhibited CAM-DR to cytosine arabinoside (Ara C) through VLA-4-to-FN interaction, while fresh leukemic cells from two AML patients with low alpha4-integrin expression did not display CAM-DR to Ara C. FNIII14 impaired VLA-4-to-FN interaction and restored sensitivity to Ara C in the CAM-DR leukemic cells. In these CAM-DR leukemic cells, upregulation of Bcl-2, which was induced through the focal adhesion kinase/Akt signal pathway upon VLA-4-to-FN interaction, was inhibited by FNIII14 treatment. In a mouse model of minimal residual disease (MRD) in bone marrow, 100% survival was achieved by combining FNIII14 with Ara C, whereas Ara C alone prolonged survival only slightly. The myelosuppression induced by Ara C was not augmented by the combination of FNIII14 in mouse experiments. Thus, the combination of anticancer drugs and FNIII14 holds promise to eradicate MRD in bone marrow after chemotherapy.

The association of up‐regulation of X‐linked inhibitor of apoptosis protein with cell adhesion‐mediated drug resistance in U937 cells

An increasing body of evidence indicates that environmental factors may contribute to the drug resistance of acute myeloid leukaemia (AML). CAM-DR (cell adhesion-mediated drug resistance) is a reversible, de novo drug resistance induced by adhesion of tumour cell lines to fibronectin (FN). Adhesion was demonstrated to directly regulate the apoptotic machinery. And it was observed in previous studies that high levels of X-linked inhibitor of apoptosis protein (XIAP) were related to resistance to chemotherapeutics in many cancer cell lines. However, whether XIAP is relevant to CAM-DR of AML cells is unknown. In this report, we demonstrated that the mRNA and protein levels of XIAP were increased by 96.15% and 120.92%, respectively in U937 cells cocultured with FN as compared with controls. Antisense oligonucleotides targeting XIAP down-regulated the expression of XIAP and sensitized U937 cells to daunorubicin. In addition, we investigated the signalling pathway involved in the upregulation of XIAP. The levels of phosphorylated Akt (Ser473) were elevated in U937/FN cells and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 suppressed XIAP expression and restored the chemosensitivity to daunorubicin. Our findings suggested that adhesion-dependent activation of the PI3K/Akt/XIAP pathway may be one of the factors involved in the CAM-DR of U937 cells. Targeting this pathway may be a useful approach to improve the therapeutic responsiveness of leukaemia cells.

First clinical experience with simvastatin to overcome drug resistance in refractory multiple myeloma

In vitro statins overcome cell adhesion-mediated drug resistance at non-toxic concentrations that are achievable in humans by standard dose simvastatin. A pilot phase-II trial was initiated to determine feasibility and antimyeloma efficacy. In six myeloma patients refractory to two cycles of bortezomib or bendamustine simvastatin was concomitantly administered during further two cycles. The therapy was well tolerated without grade 3/4 toxicity. Intrapatient (cycles I/II vs. III/IV) and interpatient comparison (vs. ten patients without simvastatin) showed reduction of drug resistance by inhibition of HMG-CoA-reductase. In summary, this is the first phase II experience to study antimyeloma activity of statins in humans.

Wnt3/RhoA/ROCK signaling pathway is involved in adhesion-mediated drug resistance of multiple myeloma in an autocrine mechanism

Adhesion of myeloma cells to bone marrow stromal cells is now considered to play a critical role in chemoresistance. However, little is known about the molecular mechanism governing cell adhesion-mediated drug resistance (CAM-DR) of myeloma cells. In this study, we focused our interests on the implication of the Wnt signal in CAM-DR. We first screened the expression of Wnt family in myeloma cell lines and found that Wnt3 was overexpressed in all the myeloma cells examined. KMS-5 and ARH77, which highly expressed Wnt3 protein, tightly adhered to human bone marrow stromal cells, and accumulation of beta-catenin and GTP-bounded RhoA was observed in these myeloma cell lines. Conversely, RPMI8226 and MM1S, which modestly expressed Wnt3 protein, rather weakly adhered to human bone marrow stromal. We then examined the relevance of Wnt3 expression to adhesive property to stromal cells and to CAM-DR of myeloma cells. KMS-5 and ARH-77 exhibited apparent CAM-DR against doxorubicin. This CAM-DR was significantly reduced by anti-integrin beta(1) antibody, anti-integrin alpha(6) antibody and a Wnt-receptor competitor, secreted Frizzled-related protein-1, and Rho kinase inhibitor Y27632, but not by the specific inhibitor of canonical signaling (Dickkopf-1), indicating that Wnt-mediated CAM-DR that is dependent on integrin alpha(6)/beta(1) (VLA-6)-mediated attachment to stromal cells is induced by the Wnt/RhoA/Rho kinase pathway signal. This CAM-DR was also significantly reduced by Wnt3 small interfering RNA transfer to KMS-5. These results indicate that Wnt3 contributes to VLA-6-mediated CAM-DR via the Wnt/RhoA/ROCK pathway of myeloma cells in an autocrine manner. Thus, the Wnt3 signaling pathway could be a promising molecular target to overcome CAM-DR of myeloma cells.

Exploratory study of novel leukemia bone marrow stromal cell adhesion mediated drug resistance model

To construct cell adhesion mediated drug resistance (CAM-DR) model based on Acute lymphocyte leukemia bone marrow stromal cells(BMSC) for further studying drug resistance of leukemia. Firstly, we adhesively cultured Jurkat cell strain of human leukaemia lymphocyte with the matrix cell radiated by (60)Co to construct the model of CAM-DR, then evaluated the model in morph and construction by scanning electron microscope. The IC50 of DNR on Jurkat cell was examen by MTT and the concentration and distribution of DNR in the cell was detected by flow cytometry. When the Jurkat cells were cultured with BMSC for 24 h, we found that Jurkat cells had adhesioned the bone marrow stromal cell layer by parapodium and some of them had nichd the mesh consisted by the confluence of BMSC. At the point of 48 h, some Jurkat cells had migrated to underlayer of BMSC, and Jurkat cells were nichd the mesh of BMSC like nidi. The accumulation and distribution of DNR in the Jurkat cells were not affected in the model, but the reaction of Jurkat cells to DNR were significantly inhibited. The model of CAM-DR based on Acute lymphocyte leukemia bone marrow stromal cells was successfully built.

Genes Regulating the Sensitivity of Solid Tumor Cell Lines to Cytotoxic Agents: A Literature Review

In order to review gene alterations associated with drug responses in vitro to identify candidate genes for predictive chemosensitivity testing, we selected from literature genes fulfilling at least one of the following criteria for the definition of 'in vitro chemosensitivity associated gene': (i) alterations of the gene can be identified in human solid tumor cell lines exhibiting drug-induced resistance; (ii) transfection of the gene induces drug resistance; (iii) down-regulation of the gene increases the drug sensitivity. We then performed Medline searches for papers on the association between gene alterations of the selected genes and chemosensitivity of cancer cell lines, using the name of the gene as a keyword. A total of 80 genes were identified, which were categorized according to the protein encoded by them as follows: transporters (n = 15), drug targets (n = 8), target-associated proteins (n = 7), intracellular detoxifiers (n = 7), DNA repair proteins (n = 10), DNA damage recognition proteins (n = 2), cell cycle regulators (n = 6), mitogenic and survival signal regulators (n = 7), transcription factors (n = 4), cell adhesion-mediated drug resistance protein (n = 1), and apoptosis regulators (n = 13). The association between the gene alterations and chemosensitivity of cancer cell lines was evaluated in 50 studies for 35 genes. The genes for which the association above was shown in two or more studies were those encoding the major vault protein, thymidylate synthetase, glutathione S-transferase pi, metallothionein, tumor suppressor p53, and bcl-2. We conclude that a total of 80 in vitro chemosensitivity associated genes identified in the literature are potential candidates for clinical predictive chemosensitivity testing.

β1 integrin mediated adhesion increases Bim protein degradation and contributes to drug resistance in leukaemia cells

It has been shown that the tumour microenvironment confers resistance to chemotherapy. Specifically, it was previously reported that adhesion of haematopoietic tumour cells to fibronectin (FN) via beta1 integrins confers a multi-drug resistance phenotype commonly referred to as cell adhesion mediated drug resistance. The present study showed that the pro-apoptotic BCL-2 family member Bim was reduced when leukaemia cells were adherent to FN via beta1 integrins. beta1 integrin-mediated regulation of Bim in K562 cells was demonstrated to be partly a result of increased proteasomal-mediated degradation of Bim protein levels, and proteasome inhibitors prevent Bim degradation. Increased degradation of Bim was not related to activation of the mitogen-activated protein kinase pathway, as adhesion of K562 cells caused a reduction in phospho-extracellular signal-related kinase (ERK)1/2 levels. In addition, pharmacological inhibition of MAP/ERK (MEK) with PD98059 did not increase Bim levels. Reducing Bim levels by short hairpin RNA targeting inhibited imatinib and mitoxantrone-induced cell death. These results showed that beta1 integrin-mediated adhesion regulates Bim degradation and may contribute to the minimal residual disease associated with many haematopoietic malignancies. Together our data indicate that disrupting beta1 integrin-mediated regulation of Bim degradation may increase the efficacy of drugs, including imatinib, used to treat haematopoietic malignancies.

Combination Therapy of Anti-Cancer Drug with FNIII14 Peptide of Fibronectin Effectively Overcome Cell Adhesion Mediated-Drug Resistance of Acute Myelogenous Leukemia.

We have previously reported that in acute myelogenous leukemia (AML), drug resistance is induced by the attachment of α4β1-integrin (very late antigen (VLA)-4) on leukemic cells (AML cells) to fibronectin (FN) on bone marrow (BM) stromal cells, and this interaction is crucial in BM minimal residual disease (MRD) and prognosis after chemotherapy (Matsunaga T et al, Nature Med 2003, 9, 1158–1165). We also reported a successful overcome of drug resistance in human AML cells transplanted mice with the use of anti-VLA-4 antibody. However, a drawback of progressive multifocal leukoencephalopathy by anti-VLA-4 antibody administration was reported in multiple sclerosis and Chron's disease patients. Therefore, in this study, we examined whether FNIII14, a 22-mer peptide derived from FN which was reportedly potent to block the interaction between FN and VLA-4, VLA-5 or αvβ3-integrin (Fukai F et al. Clin Cancer Res 2002, 8, 2455–2462), can overcome cell adhesion mediated drug resistance (CAM-DR) induced by VLA-4/FN interaction in AML cells. By in vitro culturing system, FNIII14 dose dependently inhibited adhesion of two AML cell lines (U937 cells, HL-60 cells) and fresh AML cells from patients to FN, and significantly circumvented CAM-DR of these AML cells to cytosine arabinoside (Ara C). The mechanism for this circumvention of CAM-DR involved conformational inactivation of β1-integrin as revealed by FACS through inside out inhibitory signal from complex FNIII14/its 50kDa receptor resulting in down regulation of FAK/Akt/Bcl-2 signal which is responsible for VLA-4/FN induced CAM-DR. In a mouse model of MRD, a 100% survival rate was achieved by combining FNIII14 and Ara C, in contrast to a slight prolongation of survival by Ara C alone. These results indicate that combination treatment of anticancer drugs and FNIII14 is the potent modality for eradication of MRD in BM, and should improve the prognosis of AML.