Pathway In Multiple Myeloma Research Articles

Canonical and Non Canonical Activation of Hedgehog Pathway in Multiple Myeloma

To identify genes or Tumor-Associated Antigens (TAA) potentially involved in the transition from Monoclonal Gammopathy of Undetermined Significance (MGUS) to Multiple Myeloma (MM) and in the MM pathogenesis, we used SEREX and screened serum of MGUS and MM patients with a MM cDNA library. We identified 10 novel antigens and focused our attention on Oral-facial-digital syndrome 1 gene (Ofd1) due to a specific antibody response in MGUS (20.6%) as well as post-transplant MM patients (27.2%). Ofd1 is an embryonal antigen with an important role in the organogenesis and fetal survival; however, in the adult life its expression decreases significantly and is restricted to few tissues. Additionally it is a basal body/centrosome-related protein and a component of Tip60-HAT complex which is involved in cilia formation and functional regulation of Hedgehog (Hh)- and Wnt- pathways in mouse models. We first studied the expression of Ofd1 by western blotting analysis and observed that 11 MM cell lines and 4 primary MM patient cells express high levels compared with PBMCs. To define its role in MM pathogenesis, we specifically knocked-down Ofd1 using 100nM and 200nM of specific siRNA in MM1s cell line. Time-dependent but dose-independent inhibition of Ofd1 does not affect viability and proliferation of MM1s at 48h, as assayed by MTT and thymidine-uptake, respectively. Since mutations in proteins of cilia may block the Hh-signal transduction downstream to Smoothened (Smo) but upstream to Gli transcription factors, we next evaluated the effects of Ofd1 inhibition on the expression of Gli1 and Patched1 (Ptch1), hallmarks of Hh-activity. Western blotting analysis showed that specific knock-down of Ofd1 leads to down-regulation of Gli1 or Ptch1, and β-catenin. Therefore Ofd1 has a role in the functional regulation of Hh- and Wnt-pathway, and its overexpression might lead to constitutive activation of these tumorigenic pathways. These data strongly indicate the existence of an alternative and not canonical activation of Hh-pathway in MM. We also investigated if the canonical way is active in MM. To demonstrate that Hh-pathway is important in CD138+ MM patient cells, we used double staining with antibody anti-CD138 and anti-Sonic (Shh) to show that MM cells co-express Shh, the most common Hh-ligand, suggesting that MM cells might have autocrine and/or paracrine Hh-activity. We confirmed the expression of Gli1 and Ptch1, by western blotting, in 10 on 11 MM cell lines tested and using antibody anti-Gli1 we demonstrated, by immunofluorescence, predominant nuclear localization of Gli1 suggesting constitutive Hh-pathway activation. Treatment with 5μM of a specific Smo-inhibitor for 12–24 and 36 hours, induces down-regulation of Gli1 and Ptch1 in 50% MM cell lines. While down-regulation of Ptch1, a Gli-target gene, is an early event starting at 12 hours, down-regulation of Gli1 starts later at 36 hours. Therefore Gli1 is not the most important transducer of Hh-signaling in MM. In 50% MM cell lines, however, we didn't observe any down-regulation of Gli1 and Ptch1 after treatment with Smo-inhibitor; in these MM cell lines, the predominant nuclear localization of Gli1 is completely abrogated by treatment for 24 hours with 10μM of forskolin, a compound inhibiting the nuclear translocation of Gli1 via PKA. Taken together our data show canonical as well as alternative not canonical activation of Hh-pathway associated with overexpression of ciliary protein Ofd1. Further delineation of the role of primary cilia in regulating cellular response to Hh-signaling in MM will both define their role in disease pathogenesis and support novel therapies targeting Hh-signaling to improve patient outcome in MM.

Combined Treatment with the Mek Inhibitor PD0325901 and Arsenic Trioxide Has Potent Antitumor Activity in Vivo against Human Multiple Myeloma Xenograft Model.

Despite recent advances in therapy, Multiple myeloma (MM) remains incurable because of the high resistance to apoptosis and both intrinsic and acquired drug resistance. Therefore, new therapeutic strategies are needed to improve patient outcome. We recently demonstrated that blockade of the MEK/ERK signaling module, using the small-molecule inhibitors PD184352 or PD0325901 (PD), strikingly enhances arsenic trioxide (ATO)-induced cytotoxicity in MM cells through a multiple modulation of apoptotic regulatory proteins, including p53 family proteins, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, several Bcl-2 family proteins and caspases, that depend on the functionality of the p53 pathway (Blood prebublished on line june 26, 2008). Furthermore, we also demonstrated that PD plus ATO treatment induces early tumor (volume approximately 200 mm3) regression, prolongs survival and is well tolerated in vivo in a human plasmacytoma xenograft model. The aim of this study was to investigate whether the combined treatment with PD and ATO is effective in animals with more advanced tumors; thus we used a murine model in which MM RPMI 8226 cells were injected subcutaneously into NOD-SCID mice and when the tumors reached approximately 1000mm3, mice were randomized (n=6/group) to receive vehicle or PD0325901 at 10 mg/kg administered by oral gavage or ATO (3.75 mg/kg) injected intraperitoneally or PD/ATO on a 5-days-a-week schedule for 3 consecutive weeks. Treatment of RPMI 8226 MM-advanced tumor-bearing mice with PD0325901 (10mg/kg) significantly reduced MM-tumor growth as compared to control (P <.01 Tukey-Kramer test), ATO (3.75 mg/kg) had minimal effect on the growth of tumors, which increased as in control mice. Importantly, when PD (10mg/kg) was combined with ATO (3.75mg/Kg), there was a significant reduction in tumor size and growth rate relative to untreated or PD treated mice (P<.001 for PD/ATO versus control, and P<.01 for PD/ATO versus PD Tukey-Kramer test). The combination of PD and ATO (3.75mg/Kg) significantly prolonged survival compared with treatment with either drug alone and was well tolerated in vivo because no differences in body weight and general appearance was noted in mice during the treatment. We next investigated the in vivo effects of the drug combination on proliferation and apoptosis; whole tumor-cell tissues and tumor lysates from mice treated for five days (n=2/group) were subjected to immunohistochemical staining and immunoblotting to assess in vivo phosphorylation of ERK, the proliferative antigen, Ki-67, and cleaved caspase-3. Tumor tissues from PD0325901 (10mg/kg) treatments resulted in profound p-ERK inhibition compared with tumor tissues from vehicle control or ATO-treated animals. In agreement with these data, a significant decrement in the number of Ki-67 positive plasma cells was noted in tumor sections from PD-treated mice relative to tumors from mice receiving either vehicle control or ATO (3.75mg/Kg) treatment alone thereby confirming the tumors growth retardation observed in PD-treated mice. Either PD (10mg/kg) or ATO (3.75mg/Kg) alone did not increase caspase activation compared with tumors from control cohorts. However, the combination PD/ATO dramatically activated caspase-3 in advanced tumors. Notably, consistent with our previous in vitro study demonstrating the involvement of the Bim pathway in MM PD/ATO-induced apoptosis, immunoblotting of MM tumors form PD plus ATO-treated mice showed an elevated ratio of proapoptotic Bim to antiapoptotic Mcl-1 compared with treatment with either drug alone. Collectively, our previous and present findings suggest that combining PD with ATO induces both cytostatic and cytotoxic responses in vivo, resulting in regression of early or advanced tumors, prolongs survival in vivo, and is well tolerated in vivo. In conclusion, our preclinical in vivo studies provide the framework for testing PD0325901 and ATO combination therapy in clinical trials aimed to improve patient outcome in MM.

Characterization of Human Multiple Myeloma Cells with High-Resolution Magic Angle Spinning Magnetic Resonance Spectroscopy

Advancements in the pathogenetic pathways in multiple myeloma have led to the identification of several primary and secondary genetic lesions and ultimately to a multiple myeloma genetic classification with prognostic implications. Although disregulation of cyclin activity has been recognized as a key event leading to the multiple myeloma phenotype, little is known about the metabolic consequences of this phenomenon. We have studied intact multiple myeloma cells by high resolution magnetic resonance spectroscopy to establish the metabolomic profiles of different native multiple myeloma cells as compared to other lymphoproliferative disorders.Multiple myeloma cells obtained from bone marrow aspirates (n =15), blood (n =3) or other biologic tissues (n =2) from 20 multiple myeloma patients and separated by density gradient centrifugation were evaluated and metabolic profiles were correlated with cytogenetic characteristics of the disease and patients clinical characteristics.Twelve patients were females (60%) with a median age of 65 years (range 50–82). Multiple myeloma monoclonal proteins were IgG (N=9), IgA (N=5) or BJ (N=6). Five of them (25%) had renal insufficiency. Nine patients (45%) had predominantly extramedullar diesase including four cases of plasma cell leukemia. IgH translocations were identified in 5 samples (25%), hyperploidy in 2 (10%), and other or no genetic lesions in 13 (65%), del13 was present in 9 samples (45%) and p53 alterations in 5 (25%).Bone marrow samples from thirteen patients with conventional multiple myeloma presented a relatively constant metabolic pattern with predominantly lipidic signals and a metilen to metil ratio ranging from 1.9 to 4.9 (median 2.9). No differences in this pattern were observed among subgroups of primary translocations or involvement of Rb and p53 genes. Four patients with plasma cell leukemia and three with predominant extranodal disease presented either non detectable lipid signals (N=3) or a higher metilen to metil ratio ranging from 2.8 to 3.9 (median 3.5). In fact, extranodal or leukemic disease was significantly associated to undetectable lipids (P < 0.031) or the composite variable undetectable lipids or metilen to metil ratio > 3 (P < 0.043). Furthermore, after a median follow-up of 18 months, absence of lipids in the metabolic profile was also associated to a shorter survival (median 0.45 years, 95%CI 0–1.03 versus 3 years, 95%CI 0.95–5.06, P < 0.022).These results suggest that metabolic profiles of different multiple myeloma genetic subtypes share common and reletively constant characteristics, while cells obtained from patients with plasma cell leukemia or predominantly extramedullar disease present a clearly distinct profile, probably reflecting the metabolic effect of clonal evolution at a genetic level.

The BCL9 Oncogene Promotes Tumor Progression by Conferring Enhanced Proliferative, Metastatic and Angiogenic Properties to Myeloma Cells

Wnt signaling plays an important role in tissue development and maintenance during embryogenesis, cell differentiation, and stem cell growth. Several components of the Wnt signaling cascade have been shown to function as either tumor suppressor proteins or as oncogenes in multiple human cancers, underscoring the relevance of this pathway in oncogenesis. Deregulation of the canonical Wnt/b-catenin pathway has been implicated in numerous human epithelial malignancies as well as hematologic malignancies including multiple myeloma (MM), generating immense interest in these molecules as targets for cancer therapy. Activation of Wnt/b-catenin in cancer has been associated with mutations that enable b-catenin to escape degradation by the proteasome, thereby allowing its accumulation in the nucleus where it functions as a transcriptional regulator in conjunction with coactivators by constitutively activating target genes such as c-Myc and Cyclin D1. To date, however, no mutations in Wnt pathway have been documented in MM, suggesting that mechanisms other than gene mutation may contribute to Wnt pathway deregulation. BCL9, a key component of the Wnt pathway, is required for b-catenin transcriptional activity and resides on chromosome 1q21, a region frequently involved in secondary chromosomal aberrations associated with MM tumor progression. Here we provide evidence that dysregulation of BCL9 expression is a novel oncogenic mechanism of Wnt pathway activation in MM. Using in vitro and in vivo functional analyses, we demonstrate that BCL9 is a bonafide oncogene that is aberrantly expressed in MM and associated with survival. Using the TCF- specific luciferase reporter, we show that enforced expression of BCL9 in MM cells enhanced b-catenin mediated transcription by >12 fold, suggesting a possible role of BCL9 overexpression in the pathogenesis of MM. BCL9 enhanced proliferation (1.5 fold, P<0.02), migration (3.5 fold, P<0.0001) and the metastatic potential of MM cells. We also showed that BCL9 plays an important role in tumor progression by regulating Cyclin D1 and c-Myc mediated cell proliferation, CD44 mediated tumor metastasis, as well as VEGF mediated host angiogenesis. Importantly, BCL9 knockdown significantly increased the survival in a xenograft mouse model of human MM (P=0.001), associated with decreased tumor burden and host angiogenesis. In summary, we have demonstrated that BCL9 is a novel and potent oncogene of the Wnt pathway in MM, playing fundamental roles in tumor progression by regulating proliferation, migration, invasion, angiogenesis and the metastatic potential of tumor cells. The pleiotropic roles of BCL9 and its aberrant expression highlight its importance as an attractive and novel therapeutic target in the treatment of MM.

The effect of azacitidine on interleukin-6 signaling and nuclear factor- B activation and its in vitro and in vivo activity against multiple myeloma

Azacitidine is a DNA methyltransferase inhibitor and cytotoxic agent known to induce apoptosis of some cancer cells. This study evaluated the pre-clinical potential of azacitidine as a therapeutic agent for multiple myeloma. Dose responsiveness to azacitidine was determined utilizing a panel of genetically heterogenous human multiple myeloma cell lines. Azacitidine was also tested against primary multiple myeloma cells and in the 5T33MM murine model of systemic myelomatosis. Mechanistic studies included immunoblotting of key apoptosis signaling proteins, analysis of p16 gene methylation status, and characterization of both the interleukin-6 and nuclear factor-kappaB signaling pathways following azacitidine treatment. Human myeloma cell lines and primary multiple myeloma cells underwent apoptosis following exposure to clinically achievable concentrations of azacitidine (1 microM-20 microM). Similarly, azacitidine prolonged survival from 24.5 days to 32 days (p=0.001, log rank) in the 5T33MM model. At a mechanistic level azacitidine down-regulated two crucial cell survival pathways in multiple myeloma. First, it inhibited the elaboration of both interleukin-6 receptor-alpha and interleukin- 6 resulting in the reduced expression of both phospho-STAT3 and Bcl-xl. Secondly, azacitidine inhibited both nuclear factor-kappaB nuclear translocation and DNA binding in a manner independent of IkappaB. The kinetics of these azacitidine-induced responses was more consistent with protein synthesis inhibition than with either hypomethylation or another DNA-mediated effect. Azacitidine rapidly induces apoptosis of multiple myeloma cells, is effective in vivo against multiple myeloma and inhibits two crucial cell survival pathways in this disease. We conclude that azacitidine demonstrates novel and highly relevant anti-myeloma effects and warrants further evaluation in a clinical context.

Dual regulation of glucocorticoid-induced leucine zipper ( GILZ) by the glucocorticoid receptor and the PI3-kinase/AKT pathways in multiple myeloma

Glucocorticoids (GCs) are effective therapeutics commonly used in multiple myeloma (MM) treatment. Clarifying the pathway of GC-induced apoptosis is crucial to understanding the process of drug resistance and to the development of new targets for MM treatment. We have previously published results of a micro-array identifying glucocorticoid-induced leucine zipper ( GILZ) as GC-regulated gene in MM.1S cells. Consistent with those results, GCs increased GILZ in MM cell lines and patient samples. Reducing the levels of GILZ with siRNA decreased GC-induced cell death suggesting GILZ may mediate GC-killing. We conducted a screen to identify other pathways that affect GILZ regulation and report that inhibitors of PI3-kinase/AKT enhanced GILZ expression in MM cell lines and clinical samples. The combination of dexamethasone (Dex) and LY294002, wortmannin, triciribine, or AKT inhibitor VIII dramatically up regulated GILZ levels and enhanced apoptosis. Addition of interleukin-6 (IL-6) or insulin-like growth factor (IGF1), both which activate the PI3-kinase/AKT pathway and inhibit GC killing, blocked up regulation of GILZ by GC and PI3-kinase/AKT inhibitors. In summary, these results identify GILZ as a mediator of GC killing, indicate a role of PI3-kinase/AKT in controlling GILZ regulation and suggest that the combination of PI3-kinase/AKT inhibitors and GCs may be a beneficial MM treatment.

Targeting Akt and heat shock protein 90 produces synergistic multiple myeloma cell cytotoxicity in the bone marrow microenvironment.

We hypothesized that targeting both Akt and heat shock protein (HSP) 90 would induce cytotoxic activity against multiple myeloma (MM) cells and target the bone marrow (BM) microenvironment to inhibit angiogenesis, osteoclast formation, as well as migration and adhesion of MM cells. MM cell lines were incubated with perifosine (5 and 10 micromol/L) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG; 50 and 100 nmol/L) alone and in combination. The combination of Akt inhibitor perifosine and HSP90 inhibitor 17-DMAG was synergistic in inducing MM cell cytotoxicity, evidenced by inhibition of DNA synthesis and induction of apoptosis. In addition, perifosine and 17-DMAG almost completely inhibited osteoclast formation: perifosine interfered with both early and late stages of osteoclast progenitor development, whereas 17-DMAG targeted only early stages. We next showed that combined therapy overcomes tumor growth and resistance induced by BM stromal cells and endothelial cells as well as the proliferative effect of exogenous interleukin-6, insulin-like growth factor-I, and vascular endothelial growth factor. Moreover, the combination also induced apoptosis and growth inhibition in endothelial cells and inhibited angiogenesis. Finally, we showed that the two agents prevented migration of MM cells toward stromal-derived factor-1 and vascular endothelial growth factor, which are present in the BM milieu, and also prevented adhesion of MM cells to fibronectin. This study provides the preclinical framework for treatment protocols targeting both the Akt and HSP pathways in MM.

Activation of B-Cell Maturation Antigen (BCMA) on Human Multiple Myeloma Cells by a Proliferation-Inducing Ligand (APRIL) Promotes Myeloma Cell Function in the Bone Marrow Microenvironment.

A proliferation-inducing ligand (APRIL), a close member of B-cell-activating factor (BAFF) belonging to the TNF superfamily that stimulates growth and survival of multiple myeloma (MM) cells, is mainly produced by MM patient bone marrow stromal cells (BMSCs) and osteoclasts in the BM microenvironment. Like BAFF, APRIL binds two receptors, B-cell maturation antigen (BCMA) and transmembrane activator and calcium modulator (TACI). Since BCMA has significantly higher affinity for APRIL than for BAFF (nM vs. mM range), APRIL/BCMA signaling may be more important than BAFF/BCMA in MM. Specifically, BCMA but not TACI is unequivocally expressed at mRNA level in the majority of CD138-expressing MM cell lines and patient MM cells. To date, it is unclear how APRIL signaling through BCMA contributes to BM microenvironment in support of MM cells. We here studied the functional sequelae of APRIL/BCMA pathway in MM cells, and further identified molecular mechanisms regulating these processes. Using flow cytometric analysis, cell surface BCMA expression was confirmed in the majority of MM cell lines. Microarray gene expression analysis showed significant expression of BCMA mRNA in CD138+ patient MM cells (n=105). Higher BCMA mRNA levels were observed in patient MM cells than in normal plasma cells (n=27) (p< 0.03). Importantly, CD138+ patient MM cells express BCMA protein on the cell surface (n=15), as confirmed by CD38+BCMA+ dual immunofluorescence staining followed by flow cytometric analysis and immunohistochemical (IHC) study. Next, H929 MM cell line that expresses only BCMA, but not TACI, and Daudi B cells transduced with huBCMA (huBCMA-Daudi) were stimulated with APRIL. These cells were subjected to mRNA extraction at different time points followed by microarray analysis to identify downstream transcriptional targets. Stimulation of these two cell lines by APRIL activated the NF-kB signaling in a dose-dependent manner. More than 6-fold increase in NF-kB activity was induced by APRIL (100 ng/ml) using a p65 enzyme-linked DNA/protein interaction assay. IL-6 and PI3K/AKT signaling cascades were concurrently induced. Additionally, APRIL upregulated chemotactic/osteoclast activating factors, i.e., MIP-1a and MIP1b, in a dose-dependent manner, which was further validated by specific ELISA. Multi-Analyte Profiles also confirmed that angiogenesis and adhesion/chemoattractant factors, i.e., IL-8, CXCL10, RANTES, MDC (ccl22), were significantly induced upon APRIL stimulation. Conversely, a soluble BCMA-Fc protein inhibited APRIL binding to BCMA and blocked secretion of APRIL-induced chemokines, indicating specific functional interaction of BCMA with APRIL. Importantly, APRIL similarly induced these signaling cascades in CD138+ patient MM cells. Taken together, our studies are the first to demonstrate cell surface BCMA on the majority of CD138+ patient MM cells. Furthermore, APRIL induced osteoclasts-, migration-, and angiogenesis-associated factors in MM cells. These studies therefore establish a role of BCMA activation in the BM microenvironment that provides a niche for MM disease progression, supporting novel therapeutics specifically targeting APRIL/BCMA pathway in MM.

Potentiation of Melphalan-Induced Cytotoxicity through Targeting of the Base Excision Repair Pathway in Multiple Myeloma.

Melphalan (M) is an active agent against multiple myeloma (MM). One of the obstacles with M treatment is the patient's ability to tolerate side effects such as mucositis and pancytopenia. This is especially true for those patients >70 years of age. We hypothesize that potentiation of M-induced cytotoxicity is possible in MM with agents that target, and therefore further imbalance, multiple DNA repair pathways. A key protein in the Base Excision Repair (BER) pathway, Apurinic/apyrimidinic endonuclease/ redox factor (APE1/Ref-1 or APE1) plays a major role in the repair of damage caused by chemotherapeutic agents including M and Temozolomide (TMZ), interacts with a number of transcription factors (HIF1-a, p53, AP1, NFkB, etc) to regulate their function through oxidation/reduction (redox) signaling, and is overexpressed in refractory/relapsed MM cells. Furthermore, a reduction in APE1 protein sensitizes MM cells to melphalan indicating that inhibition of this protein may have therapeutic potential in MM. In order to decipher the importance of APE1's redox and repair functions in MM cells' response to DNA damage via melphalan and TMZ, we have available to us small molecule APE1 inhibitors that affect only the repair activity or only the redox activity of APE1. The mechanism of action of MLP is primarily via monoadduct leading to DNA interstrand cross-link (ICL) formation which is processed by the Nucleotide Excision Repair (NER) pathway. MLP also causes N7methyl-G and N3methyl-A adduct formation which are repaired by the BER pathway. For these studies, we treated RPMI 8226 cells with several chemotherapeutic agents:1.M;2.TMZ, which creates primarily N7methyl-G and N3methyl-A adducts;3.Methoxyamine (MX), which has been shown to inhibit further processing by the BER pathway; and4.a small molecule which blocks the redox function of APE1. Our purpose was to overwhelm the DNA repair pathways by causing the accumulation of DNA repair intermediates and inducing apoptosis.M-induced cytotoxicity is enhanced by TMZ (CI=0.08), MX (CI=0.89), and E3330 (CI=0.06), and this effect was synergistic as determined by CalcuSyn software which generates median effect and combinational index (CI) values, with CI<1 indicative of synergy. Using MX to inhibit APE1 in combination with TMZ results in an increase in DNA damage and an increase in apoptosis in 8226 cells. Furthermore, the combination of the redox inhibitor + MX which blocks both functions of APE1 also results in an increase in apoptosis in the MM cells. Further studies include the addition of M to these combinations that are demonstrating an increase in efficacy in MM cells. These results indicate that using these DNA repair-targeted agents in addition to MLP may be a feasible way to increase the effect of the M on MM cells. The potential advantages to patients would be that they would be able to tolerate more treatments and that the combination treatments would be more effective than treatment with M alone. We anticipate that effective modulation of M and/or TMZ will overcome resistance without compromising efficacy and help to alleviate some of the side effects patients have to endure with melphalan treatment. This may be particularly advantageous to the more elderly patients.

Cucurbitacin I (JSI-124) Has Potent Anti-Myeloma Effects Independent of Its Inhibition of JAK2-Induced STAT3 Activation.

Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. JSI-124 is a plant natural product identified previously as Cucurbitacin I, isolated from various plant families such as the Cucurbitaceae and Cruciferae and has been recently described as a specific inhibitor of Janus Kinase-2 (JAK-2)/signal transducer and activator of transcription-3 (STAT3). Based on the critical role of the IL-6/STAT3 pathway in MM we studied the effects of JSI-124 on different MM cells in vitro. Different human myeloma cell lines including MM1.S, IM9, OPM-2, RPMI-8226, ARH77 in addition to the murine 5TGM myeloma cell lines were incubated with increasing concentrations of JSI-124. The impact of JSI-124 on cell proliferation, cell cycle and induction of apoptosis was studied using [3H]-Thymidine incorporation, cell counting, flow cytometry and Annexin/PI staining and caspase 3, 8 and 9 activation. JSI-124 was able to inhibit proliferation and induce apoptosis in several MM cell lines, including MM1.S, IM9, OPM-2, RPMI-8226, ARH77, U266 and 5TGM in a dose- and time-dependent manner. JSI-124 lead to activation of caspase 3, 8 and 9 indicating involvement of both extrinsic and intrinsic apoptotic pathways. JSI-124-mediated induction of apoptosis was independent of JAK2/STAT3 inhibition as MM1.S and 5TGM cells, which lack constitutive STAT3 (Tyr705) activation were equally sensitive to JSI-124 compared to U266 cells which show a constitutive STAT3 Tyr705 phosphorylation. However, JSI-124 treatment was able to abrogate IL-6 and bone marrow stroma (BMSC)-induced STAT3 (Tyr705) activation in MM1.S cells. In addition we were able to observe JSI-124 dependent inhibition of constitutive STAT3 (Ser727) activation in MM cell lines. To further delineate the mechanism underlying its anti-myeloma effects we studied the impact of JSI-124 treatment on NF-κB, MAPK and PI3K pathways. Indeed, JSI-124 treatment resulted in inhibition of p-p65, p-MEK1,2 and p-Akt underscoring the effect of JSI-124 on STAT3-independent signaling. Our results indicate that JSI-124 is a powerful direct inhibitor of myeloma cells blocking constitutive and IL-6/BMSC-dependent STAT3 activation in addition to STAT3 independent signaling pathways. JSI-124 might therefore serve as a potent novel anti-myeloma agent targeting both myeloma cells and its bone marrow microenvironment. Further studies are warranted to evaluate the in vivo efficacy of JSI-124 and identify the STAT3 independent pathways contributing to myeloma cell growth and induction of apoptosis.

Induction of UBC9 and the Sumoylation Pathway in Multiple Myeloma Promotes Plasma Cell Growth and Correlates with Decreased Patient Survival.

The pursuit of rationale, targeted therapies relies on a detailed understanding of the mechanisms that subvert normal growth control and lead to development of Multiple Myeloma (MM). To further define the mechanistic steps that contribute to MM pathogenesis, we examined mRNA expression profiles of CD138+ plasma cells obtained from normal, Monoclonal Gammopathy of Unknown Significance (MGUS), and MM patient samples. Using genomic results in combination with molecular and cellular-based assays, we demonstrate a critical role for UBC9 and the sumoylation pathway in myeloma cell growth and survival. Notably, Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) demonstrated a ten-fold induction in UBC9; a gene that encodes the sole Sumo-conjugating enzyme in human cells. We demonstrated an elevation of UBC9 in both MM primary patient cells and in a number of patient-derived MM cell lines. In addition, a number of other sumoylation pathway components were induced in primary MM cells at the gene and protein level relative to normal plasma cells. We believe that induction of UBC9 is an early genetic event in the pathogenesis of MM since the induction was observed at the gene and protein level in plasma cells from patients with MGUS. Importantly, UBC9 induction was functionally significant since a different pattern of sumoylation was observed in total cell lysate from MM patient plasma cells relative to that of normal plasma cells. Furthermore, overexpresion of a mutant form of UBC9 deficient in Sumo-conjugating activity increased the sensitivity of plasma cells to apoptosis by chemotherapeutic agents and these cells were impaired in other essential functions that included cellular proliferation, DNA synthesis, resistance to apoptosis and adhesion to bone marrow stroma. Immunoblotting of MM patient cell lysates also demonstrated a similar induction of the UBC9 gene product (Ubc9) as well as induction of the Sumo ligases Nse2 and PIAS1. These studies identify UBC9 as a target upregulated early in the pathogenesis of MM and indicate a critical role for sumoylation in disrupting the controls that govern normal plasma cell growth. To further develop prognostically relevant molecular signatures and classifications of MM subtypes, we analyzed the survival outcome of patients that expressed induced levels of UBC9, as well as other sumoylation components, and demonstrate significantly reduced survival of such patients following current treatment modalities. The results provide evidence for critical role of UBC9 and sumoylation in MM pathogenesis. Furthermore, sumoylation pattern may serve as a therapeutic target in MM, help stratify clinical management and provide a framework for the identification of sumoylation pathway targets that govern MM cell growth and progression.

Autophagy as a Target Pathway in Multiple Myeloma: A Forward Chemical Genetic Approach.

Autophagy is an evolutionarily conserved process by which cellular structures may be degraded to support ongoing biogenesis. Recent evidence has identified the induction of autophagy following growth factor pathway inhibition, rapid tumor expansion and proteasome inhibition. Thus, autophagy is a highly desirable pathway for the development of targeted therapeutics in cancer, in particular in tumors characterized by misfolded protein stress such as multiple myeloma (MM). Unfortunately, there are only several known classes of autophagy inhibitors and the emerging biology of autophagy has identified few validated targets. We therefore performed a forward chemical genetic study of autophagy using novel techniques in high-content imaging, high-throughput screening and multidimensional data analysis. Using LN229 glioblastoma cells stably transfected with an LC3-EGFP construct, we created a robust screening platform capable of quantifying the number, size and fluorescence intensity of autophagosomes. Assay validation was performed with the known autophagy inhibitor chloroquine, which induces a characteristic shift from diffuse to punctate fluorescence. Initially, we profiled a library of approximately 3,500 structurally diverse bioactive small molecules to leverage possible downstream mechanistic insights. Assay positives and controls were re-tested in the adherent H1299 adenocarcinoma cell line likewise stably expressing LC3-EGFP. Known autophagy inhibitors were enriched with this approach: nocodazole, bafilomycin A1, colchicine and monensin. Structure-activity relationship analysis of the primary screening data and 34 novel autophagy modulators revealed a compelling activity of bis-indolyl maleimides. We confirmed sub-micromolar inhibition of autophagy by three such compounds using a quantitative, radiolabeled protein degradation assay (tritiated tyrosine pulse-chase), LC3-II immunoblotting and quantitative measurement of autophagosomes across three cell lines. From these studies we selected a lead compound, K252a, for further characterization and tested twenty-two closely related analogues. Detailed SAR analysis has identified features of the molecule required for inhibition of autophagy and sites permissive for further functionalization in ongoing target identification studies. Using MM as a translational model system of misfolded protein stress, we found dose-dependent, selective cytotoxicity in MM of K252a compared to analogues lacking autophagy inhibitory activity. We report here the results of a forward chemical genetic study of autophagy which identifies and characterizes novel small molecule inhibitors of autophagy and establishes a rationale for clinical studies of autophagy inhibitors in the treatment of MM.

Delineation of Canonical and Non-Canonical NF-κB Pathways in Multiple Myeloma: Therapeutic Implications.

NF-κB pathway plays a crucial role in the pathogenesis in cancer cells including multiple myeloma (MM). The NF-κB complex is dimer in different combinations of Rel family proteins, including p65 (RelA), RelB, c-Rel, p50 (NF-κB1), and p52 (NF-κB2). Recent studies have revealed that NF-κB activity is mediated via two distinct pathways. In the canonical pathway, NF-kB is typically a heterodimer composed of p50 and p65 subunits. In the non-canonical pathway, NF-kB is typically a heterodimer composed of RelB and p100 subunits. We have shown anti-MM activities of IKKβ inhibitors (PS-1145, MLN120B); however, effects of these agents were modest. Our studies therefore suggest that baseline NF-kB activity in MM cells is not totally dependent on the canonical pathway, and that inhibition of only canonical NF-κB pathway may not be sufficient to block total NF-kB activity. In this study, we therefore hypothesized whether non-canonical inhibitors significantly enhanced NF-κB inhibition induced by canonical inhibitors in MM cells. We first examined baseline NF-κB activity using electrophoretic mobility shift assay (EMSA). NF-κB activity varied between cell lines; for example MM.1S, MM.1R and H929 cells have higher level of NF-κB activity than in RPMI8226, INA6 and OPM2 cells. To define the role of canonical and non-canonical pathway, we next examined protein expression of p50, p65 and p52 NF-κB in these cell lines: p65 was highly expressed in all MM cell lines; however, expression of p50 and 52 is variable. Surprisingly, no detectable or weak expression of p50 was observed in U266, RPMI8226, LR5, H929 and OPM2 cell lines, suggesting that baseline NF-kB activity in these cell lines is not maintained only by the canonical pathway. We then attempted to block non-canonical NF-κB pathway in MM cell lines. Specifically since IKKα and IKKβ are client proteins of heat shock protein (Hsp) 90, we examined whether 17AAG could inhibit expression and/or function of IKKα and IKKβ in MM cells. Importantly, both IKKα and IKKβ were significantly downregulated by 17AAG in MM cell lines. To determine whether downregulation of these IKK proteins by 17AAG was due to inhibition of transcription, we next performed real-time quantitative PCR and no significant inhibition of relative expression of IKKβ was observed by 17AAG treatment, suggesting that downregulation of these proteins was a post transcription event. We further examined whether 17AAG enhanced the effect of IKKβ inhibitor MLN120B on NF-κB activity. Although the inhibitory effect by either MLN120B or 17AAG alone on phosphorylation (p) of IκBα triggered by TNFα was marginal, combination treatment of MLN120B with 17AAG almost completely blocked IκBα, suggesting that this combination synergistically inhibit canonical NF-κB activity in MM cells. Importantly, the combination of MLN120B with 17AAG also significantly blocked baseline and TNFα-triggered NF-κB activity, assessed by EMSA, in MM cells. Finally, 17AAG augmented the growth inhibitory effect of MLN120B in the context of bone marrow stromal cells. Taken together, these results showed that baseline and TNFα-triggered NF-κB activities were completely blocked by this combination treatment, and provide the rationale for its clinical evaluation to induce maximum inhibition of NF-κB activity and improve patient outcome in MM.

Screening of Homozygous Deletions Identifies Key Deregulated Genes and Pathways in Multiple Myeloma.

Homozygous deletions (HD) are important in cancer cell lines and have a non-random distribution in the genome. We have determined the frequency and distribution of HD, along with the genes affected, in 84 presenting multiple myeloma cases using the Affymetrix 500K SNP GeneChip mapping arrays. Initially we took a highly sensitive approach identifying regions with a copy number (CN) <1.0 in at least 4 adjacent SNPs. The analysis was done both manually, in dChip, and using an in-house algorithm. To reduce the complexity of the data generated we removed deletions that only occurred in one sample and further filtered the data using U133 Plus 2.0 expression array analysis. Genes passed expression filtering criteria by having a lower expression than the median expression of all samples. HD in plasma cells occur during IgH and IgL chain rearrangements and were used as internal controls to validate our approach. We found that regions of homozygous deletion were not randomly distributed. At the CN<1.0 level, we identified 1761 regions containing 6650 genes. After expression filtering 129 genes were identified (with an additional 116 genes which did not have expression array probesets) which fell in chromosomal regions 1p, 6q, 8p, 11q, 12p, 13q, 14q, 16q, 20q, and 22q, which are also the regions that most frequently have loss of heterozygosity (LOH). The distribution of deletions was even between different myeloma molecular groups. We then applied a more stringent copy number cut-off of 0.65 and identified 18/84 cases with HD (21%, median size 18 kb) and most cases had only one homozygous deletion. In this analysis the number of genomic regions falls to 29 spread over 10 chromosomes, including 241 genes of which only 20 genes survived expression filtering. Of these regions 3 are in known fragile sites and only 1p, 11q, 13q, and 22q showed HD containing genes with a decreased expression. Although deletions were seen on chromosome 13, none involved the RB1 locus. Several microRNA were also noted to be deleted on chromosomes 6, 13, and 22 indicating that these may also be important in myeloma and are being explored further. The NF-kB pathway inhibitors BIRC2 and BIRC3, on 11q, were homozygously deleted in 4 cases as were the neighbouring loci ANGPTL5 and TMEM123. Interestingly, the 11q region was the exception to the rule that HD occur only in regions of LOH. The most frequent regions of homozygous deletion involved CDKN2C (p18) and FAF1 on 1p32.3, which were deleted in 6 cases (CN<1.0). In addition we have demonstrated that deletion of this region is an important prognostic marker (median survival 19 months vs 33 months). We and others have suggested that p18 is the important event at this locus but in this study when stringent criteria (CN<0.65) were applied more frequent deletions were found at the FAF1 locus. To define the critical lesion we carried out mutation and methylation analysis on both these genes and did not find any abnormalities at p18 and are using this approach to analyse FAF1. FAF1 is potentially relevant to myeloma as it is a regulator of NF-kB which also induces apoptosis via the FAS pathway. Loss of FAF1 could enable cells to tolerate DNA damage better than when it is intact. We have attempted to differentiate these two roles further through correlation with downstream deregulated genes using global expression arrays.

Blockage of TRAF6 Signaling Inhibits Osteoclast Formation and Tumor Cell through Blocking the NF-kB and JNK Pathways in Multiple Myeloma.

We have recently shown that silencing of tumor necrosis factor receptor-associated factor 6 (TRAF6) with a C-terminal siRNA inhibits proliferation and increases apoptosis of multiple myeloma (MM) tumor cells. In addition, TRAF6 ubiquitin ligase is also essential for receptor activator of nuclear factor kappa B ligand (RANKL) signaling and osteoclast differentiation. Based on TRAF6, CD40, and RANKL sequences and the TRAF6 interaction domain with CD40 or RANKL resides between residues 333 to 508, we cloned a sequence representing a 167 amino acid sequence from this domain in order to produce a TRAF6 dominant negative fragment (TRAF6dn) from the NIH gene bank (U78798) into the PCRII-TOPO vector. Subsequently, we re-cloned this fragment into an expression vector (pLenti6.2-hTRAF6dn). Expression of the TRAF6 dominant negative peptide was confirmed by Western blot analysis. We used human MM monocytes isolated by anti-CD14 micro-bead affinity column from MM patients' peripheral blood (PB) or bone marrow (BM). In order to quantify osteoclast formation, the cells were fixed and stained for tartrate resistant acid phosphatase following seven days of culture. The BM and PB CD14+ cells were cultured on slide-culture dishes at a density of 2 × 105 cells per well. The cells infected with the pLenti6.2-hTRAF6dn or with the control vector, pLenti6.2/GW/EmGFP, were treated with 50ng/ml RANKL and 10ng/ml mCSF at the beginning of the culture period, and these factors were added again during a medium change after three days of incubation. We found that the TRAF6dn vector significantly inhibited osteoclast cell formation of CD14+ cells induced by RANKL and mCSF in a concentration dependent fashion compared with the control group. Furthermore, we examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. The results showed that total endogenous c-Jun is reduced after TRAF6dn blocks TRAF6 signaling whereas cells infected with the control vector showed no changes in c-Jun. We further examined the effects of TRAF6dn on MM cell growth and apoptosis. Both tumor cells from fresh MM BM and the U266 and MM1s cell lines showed decreased cell proliferation and increased apoptosis in the presence of the TRAF6dn vector at 72 hours whereas the control vector had no effect on MM tumor cell growth or apoptosis. Furthermore, the TRAF6dn vector led to marked decreases in phospho-NF-kB protein levels compared to the control vector. Thus, we have demonstrated that inhibition of TRAF6 with a dominant negative construct both inhibits MM cell growth as well as osteoclast formation, and also reduces NF-kB activation and c-Jun levels which likely results in decreased activation of TRE/AP-1 elements. These studies suggest that the inhibition of TRAF6 may be an excellent therapeutic target for multiple myeloma since its inhibition results in suppression of tumor growth as well as osteoclast formation.

Inhibition of lymphocyte function associated antigen 1 by LFA878 induces apoptosis in multiple myeloma cells and is associated with downregulation of the focal adhesion kinase/phosphatidylinositol 3 kinase/Akt pathway

Multiple myeloma (MM) is still an incurable disease and adhesion of MM cells to bone marrow stromal cells is one of the hallmarks of the disease. Lymphocyte function associated antigen 1 (LFA-1) is an adhesion molecule that mediates lymphocyte adhesion, but its role in MM is only poorly understood. The aim of the presented study was to improve knowledge on LFA-1 and associated pathways in MM for the development of molecular targeted therapies. We demonstrate that LFA-1 is expressed in U266, RPMI-8226, OPM-2, and NCI-H929 MM cell lines and in primary cells of eight tested patients. The LFA-1 inhibitor LFA878 induces apoptosis in all four cell lines as revealed by annexin V staining and caspase 3 cleavage. Apoptosis is not hampered by adhesion to stromal cells. Additionally, the soluble ligand, intracellular adhesion molecule 1 (ICAM-1), which is increased in the serum of MM patients, does not protect from melphalan-induced apoptosis. Western blots demonstrate downregulation of FAK, PI3-K, and Akt upon LFA878 treatment. Additionally, sequential inhibition of the pathway by simultaneous application of Src family kinase or PI3-K inhibitors significantly increases LFA878 induced apoptosis. We conclude that LFA-1/FAK/PI3-K/Akt is a survival pathway in MM and that targeted inhibition may provide new therapeutic options.

CD117 (c-kit) is aberrantly expressed in a subset of MGUS and multiple myeloma with unexpectedly good prognosis

CD117 (c-kit) was evaluated on normal plasma cells (PC) ( n = 10), PC of individuals with monoclonal gammopathy of undetermined significance (MGUS, n = 12), malignant PC from patients with multiple myeloma (MM) either at diagnosis ( n = 83) or relapse ( n = 38) and on 23 human myeloma cell lines (HMCL). Whereas CD117 is never expressed in normal PC, it is expressed in 50% of MGUS ( p = 0.015). Furthermore, 33% of MM at diagnosis do express CD117, as opposed to 8% of those in relapse ( p = 0.003). Finally, CD117 was never found in HMCL. CD117 expression was associated with a better prognosis: overall survival was 93% at 4 years in CD117+ MM versus 64% in CD117− MM ( p = 0.05). Conversely, lack of CD117, but also high beta-2 microglobulin, t(4;14) and CD221 (IGF-1R) expression were associated with a poorer prognosis. Multivariate analysis revealed that CD117− patients were those with CD221 and t(4;14) and had the poorest prognosis. In conclusion, CD117 (c-kit) is aberrantly expressed on a subset of MGUS and MM with a more indolent presentation and is functionally antinomic to CD221 (IGF-1R). CD117 expression could be related to a specific oncogenic pathway in MM.

Targeting the Phosphatidylinositol 3-Kinase Pathway in Multiple Myeloma

Multiple myeloma is a plasma cell neoplasm with a median survival of 3 to 5 years. Recent advances have improved patient outlook, but the disease remains incurable. Therefore, continued efforts to develop new therapies that target aberrant signaling pathways are needed. The phosphatidylinositol 3-kinase pathway regulates apoptosis, cell cycle regulation, and tumor proliferation. This pathway is constitutively activated in multiple myeloma and its inhibition induces apoptosis. Advances in understanding the signaling cascades mediating proliferation and survival of multiple myeloma cells have markedly improved the treatment of this disease. In this article, we review the role of the phosphatidylinositol 3-kinase/Akt pathway in the pathogenesis of multiple myeloma and the potential therapeutic implications of targeting this pathway in the treatment of multiple myeloma.

Epigenetic dysregulation of the death-associated protein kinase/p14/HDM2/p53/Apaf-1 apoptosis pathway in multiple myeloma

Aim: To study the role of gene promoter hypermethylation of the putative tumour suppressor genes involved in the death-associated protein (DAP) kinase/p14/HDM2/p53/Apaf-1 apoptosis pathway in multiple myeloma (MM). Method: DNAs...

Targeting Signaling Pathways in Multiple Myeloma

Multiple myeloma (MM) is an incurable malignancy of terminally differentiated B-cells accounting for approximately 1 to 2% of all human cancers. The development of MM is believed to be a multistep transformation process that leads to progressive accumulation of genetic alterations. The interaction between MM and bone marrow (BM) microenviroment triggers multiple proliferative and antiapoptotic signaling pathways. Here we discuss the current understanding of signaling pathways, and their potential implication in targeted therapies in MM.