Primary CD138 Research Articles

All‐trans retinoic acid can intensify the growth inhibition and differentiation induction effect of rosiglitazone on multiple myeloma cells

Activation of PPARgamma by its ligands has shown potential anti-neoplastic effects in solid tumors. In this study, we investigate the effects of rosiglitazone (RGZ) alone as well as in combination with all trans-retinoic acid (ATRA) on human myeloma cell lines and try to address its potential mechanism. U266, RPMI-8226 and primary myeloma cells from patients were treated with different concentrations of RGZ in the presence or absence of ATRA and various biological responses were studied by the methods of [3H] thymidine incorporation, MTT, cell cycle analysis, Annexin V-PI staining, Wright-Giemsa staining, CD49e expression assay, light chain protein detection, RT-PCR and caspase-3 activity assay. We report that exposure to RGZ induced proliferation inhibition and viability reduction in a dose-dependent manner in both U266 and RPMI-8226 cells. A similar exposure to RGZ also induced cell cycle arrest and cell apoptosis of myeloma cells. A combination of RGZ with ATRA enhanced the effects of RGZ and induced cell cycle arrest and apoptosis more profoundly in both cell lines. RGZ treated cells displayed morphological characteristics of cell differentiation, and more evident signs of differentiation were observed when combined with ATRA. These changes were confirmed by the detection of CD49e expression and light chain protein secretion. Similar cell apoptosis and differentiation were observed when primary CD138+ myeloma cells were treated with RGZ and ATRA. The mRNA expressions of FLIP, XIAP and survivin were detected in both cell lines and the levels decreased significantly after culture with RGZ. The addition of ATRA in culture medium made these changes more apparently. Caspase-3 activity was increased upon exposure to RGZ in both U266 and RPMI-8226 cells while combination of RGZ and ATRA brought out more effective activation of caspase-3. Similar apoptosis and cell differentiation induced by RGZ and ATRA can also be observed in primary CD138+ cells from myeloma patients. Concomitant RXRalpha activation by ATRA enhanced the inhibitory effects of RGZ on myeloma cell proliferation, cell cycle, apoptosis and differentiation. Combination of RGZ and ATRA may be a useful therapy for human multiple myeloma.

MicroRNAs 15a and 16–1 regulates tumor proliferation in multiple myeloma

e14640 Background: MicroRNAs (miRNAs) are non-coding RNAs that act as negative regulators of gene expression. They have been described to play roles in solid tumors and hematologic malignancies. However the role of miRNAs in multiple myeloma (MM) has not been yet fully described. Methods: We performed miRNA-profiling of primary bone marrow-derived CD138+ MM cells, compared to their normal cellular counterparts and validated data by qRT-PCR. In vitro and in vivo functional studies were performed on miRNA-15a- and -16–1-precursors-transfected MM cells. Effect of miRNA-15a and -16–1 on signaling cascades have been evaluated by western blot and immunofluorescence. NF-kB activity has been studied using the Active Motif TransAM kit. In vivo MM cell growth has been evaluated by either using an in vivo imaging model or bioluminescence. Angiogenesis has been studied both in vitro and in vivo using the chorioallantoic membrane model. Results: We identified a MM-specific miRNA signature characterized by down-expression of miRNA-15a, -16–1 and over-expression of miRNA-222/-221/-382/-181a/-181b (P<0.01). Data were confirmed by qRT-PCR on matched samples. Predicted targets for the decreased miRNAs in MM patients included pro-angiogenic cytokines, oncogenes, cell cycle regulators, NFkB activators. Conversely, predicted targets for the increased miRNAs included cell cycle inhibitors, suppressors of cytokine signaling, and pro-apoptotic factors. We demonstrated that miRNA-15a and - 16–1 regulate proliferation and growth of MM cells. Indeed, transfected cells showed decreased DNA synthesis; decreased cyclinD1/cyclinD3/CDC25a/pRb protein expression; phase G1 cell cycle arrest. Moreover, transfected cells showed inhibition of NFkB pathway as shown by reduced p65-/p50-/p52-NFkB activities; downregulation of p-p65/p50/p52 nuclear protein level; upregulation of cttosolic phospho-IkB; and inhibited translocation of p-p65 from the cytolplasm to the nucleus. Similarly, inhibition of MM cell growth was confirmed in vivo; and anti-angiogenic properties of miRNA-15a and -16–1 were demonstrated both in vitro and in vivo. Conclusions: These data indicate that miRNAs play a pivotal role in the biology of MM; providing the basis for the development of new miRNA-based targeted therapies in this disease. No significant financial relationships to disclose.

Endoplasmic reticulum stress is a target for therapy in Waldenstrom macroglobulinemia

AbstractWaldenstrom macroglobulinemia (WM) is an incurable low-grade lymphoma characterized by bone marrow (BM) involvement of IgM secreting lymphoplasmacytic cells. The induction of unfolded protein response (UPR) genes (“physiologic” UPR) enables cells to differentiate into professional secretory cells capable of production of high amounts of endoplasmic reticulum (ER)–processed proteins, such as immunoglobulins. Ultimately, the initially cytoprotective UPR triggers an apoptotic cascade if ER stress is not corrected, called proapoptotic/terminal UPR. We show that WM cells inherently express the physiologic UPR machinery compared with normal BM cells, and that increased ER stress leads to proapoptotic/terminal UPR in WM cells. We therefore examined tunicamycin, ER stress inducer, for potential antitumor effects in WM. Tunicamycin induced significant cytotoxicity, apoptosis and cell-cycle arrest, and inhibited DNA synthesis in WM cell lines and primary BM CD19+ cells from patients with WM with an inhibitory concentration (IC50) of 0.5 μg/mL to 1 μg/mL, but not in healthy donor cells. Importantly, coculture of WM cells in the context of the BM microenvironment did not inhibit tunicamycin-induced cytotoxicity. Finally, we demonstrate that ER stress inducer synergizes with other agents used in the treatment of WM. These preclinical studies provide a framework for further evaluation of ER stress inducing agents as therapeutic agents in WM.

Multiple myeloma cells undergo differentiation upon exposure to rosiglitazone and all-trans retinoic acid

Activation of PPARγ by its ligands has shown differentiating effects in solid tumors. However, few reports addressed its role in myeloma cells. Our study demonstrated that exposure to PPARγ ligand (rosiglitazone, RGZ) induced proliferation inhibition and cell cycle arrest in myeloma cells. A combination of RGZ with all-trans retinoic acid (ATRA) can enhance the growth inhibition effects of RGZ. Further study shows that RGZ-treated myeloma cells displayed morphological characteristics of cell differentiation, and more evident signs of differentiation were observed when RGZ was combined with ATRA. These changes were confirmed by the detection of CD49e expression and light chain protein secretion. Similar results were also observed when primary CD138+ cells were treated with RGZ and ATRA. Collectively, our study revealed that RGZ can induce cell differentiation in myeloma cells and concomitant treatment with ATRA can enhanced the effects of RGZ.

Survival Signals, Growth Cytokines, Immunosuppressive Factors and Their Cellular Perpetrators in the Myeloma Tumor Microenvironment.

Multiple myeloma (MM) is an incurable neoplasia of terminally differentiated plasma cells in the bone marrow. Essential interactions of MM cells with host bone marrow stromal cells (BMSC) induce growth factors essential for MM progression and pathogenesis, as well as induce an immunosuppressive environment that inhibits endogenous and therapeutically-induced immune responses against the MM cells. However, despite their importance, little is known about the identity of these BMSC cells or the molecular basis of their interaction with myeloma cells. A potential MM surface protein that could be involved in these interactions is CD28, based on its known pro-survival role in T cells. Clinical studies have shown that expression of CD28 in multiple myeloma highly correlates (p=0.006) with myeloma disease progression. Moreover, CD28+ MM cells invariably express the CD28 ligand CD86. A survival role for MM-CD28 might involve interactions with cellular partners that express the B7 (CD80/CD86) ligands. Potential candidates would include CD86+ myeloma cells themselves or B7+ dendritic cells (DC) that are known to be closely associated with myeloma cells in the patient bone marrow. When myeloma-myeloma interactions were disrupted by using the high affinity CD80/CD86 blocker CTLA4Ig (Abatacept®), increased sensitivity to arsenic trioxide (ATO) and melphalan (MEL) was observed in all the three MM cell lines U266, RPMI8226 and MM1S. For U266 viability was 93% in media alone, 84% with CTLA4Ig (100 μg/ml) alone, 86% with 2 μM ATO alone and was significantly reduced to 36% with CTLA4Ig + ATO. Similar drops in viability were observed with 25 μM MEL in combination with CTLA4Ig (33% as opposed to 71–74 % with CTLA4Ig or MEL alone). Our data suggests that this does not involve the downregulation of anti-apoptotic proteins Bcl-2, Bcl-xL or Mcl-1, commonly associated with drug resistance in myeloma. In the second part of the study, we demonstrate that myeloma cell lines or primary CD138+ myeloma cells can enhance via direct contact the ability of human monocyte derived immature DC to produce the immunosuppressive tryptophan depleting enzyme indoleamine 2,3 dioxygenase (IDO, as estimated by kynurenine (Kyn) (a tryptophan catabolite) levels in the supernatant) and also the pro-plasma cell survival cytokine IL-6. In co-cultures of IFNg treated immature DCs with either MM cell lines or with primary CD138+ myeloma cells from patient BM aspirates, the activity of IDO was enhanced ~ 2–8 fold (81 mM kyn with U266 and 20–43mM with primary cells) over that observed in control IFNg-treated DCs (9.7 mM Kyn). Western analysis also demonstrated increased IDO expression relative to IFNg activated DC controls. Blocking MM-CD28 with (Fab)2 fragments of anti-hCD28 mAb 9.3 downregulated IDO activity (9.3 mM) close to that of control, demonstrating the involvement of MM-CD28 in these interactions. We also demonstrated a significant up-regulation of the pro-myeloma survival cytokine IL-6 when immature DCs were co-cultured with CD28+ MM1S (90–300 pg/ml), a 4–9 fold increase over that of DC only control (25 – 35 pg/ml). This was further enhanced when immature DCs cultured with IL-10 (+ GM-CSF + IL-4) was used in co-cultures with MM-1S (800 – 1300 pg/ml), or with primary CD138+ myeloma cells from patient bone marrow aspirates (128–1142 pg/ml). In conclusion, our data demonstrates that blocking myeloma-CD28 - myeloma-CD86 “autocrine” interaction can enhance drug cytotoxicity, while interactions with DCs produce the essential growth cytokines IL-6 and immunosuppressive enzyme IDO with potential implications in MM survival and immune escape. Use of clinically approved agents (e.g. Abatacept®) to block myeloma-CD28 binding to its B7 ligands (increase chemotherapeutic efficacy), 1-MT to inhibit IDO and targeting DCs in the microenvironment to disrupt the tumor microenvironment could be viable therapeutic strategies for the future treatment of multiple myeloma.

Synergistic Induction of Apoptosis in Multiple Myeloma Cells by Simultaneous Inhibition of the Raf/MEK/ERK and BCL-2 Pathways

The Raf/MEK/ERK pathway and the Bcl-2 family proteins are commonly overexpressed in hematologic malignancies, where they promote proliferation and survival of the neoplastic cells. We have previously demonstrated that selective MEK inhibitors (MEK-I) exert potent growth-inhibitory effects in preclinical models of both acute myeloid leukemia (AML) and multiple myeloma (MM) (Blood 2006, 108:254). More recently, we have obtained evidence that ABT-737, a Bcl-2/Bcl-xL (BH3 mimetic) inhibitor (kindly provided by Abbott Laboratories), shows potent in vitro growth-inhibitory and pro-apoptotic activity on MM cell lines and primary CD138+ bone marrow cells from MM patients, regardless of the disease status (Libotte F, ASH 2008). Moreover, in AML models, we and others have reported a highly synergistic pro-apoptotic interaction between inhibitors of the Raf/MEK/ERK pathway and of the Bcl-2 family (Blood 2002, Cancer Cell 2006, Ricciardi ASH 2008). Here, we analyzed the impact of the simultaneous inhibition of these two pathways on cell proliferation and apoptosis in MM cell lines. To this purpose, we exposed different MM cell lines to increasing concentrations of MEK-I and ABT-737, alone and in combination. While single compounds dose-dependently inhibited cell growth, as assessed by trypan blue exclusion counting, we observed that their combination synergistically enhanced this effect with combination indexes (CI), as measured by isobologram analysis (Chou–Talalay method), of 0.28 and 0.12 for KMS18 and KMS27 cells, respectively. We next analyzed the effects of combined MEK and Bcl-2/Bcl-xL inhibition on apoptosis induction. Both MEK-I and ABT-737 induced apoptosis in MM cells at high concentrations, as determined by sub-G1 DNA peak and Annexin V staining. When used at concentrations that induced minimal apoptosis as single agents (7.55% and 6.8% net apoptosis induction in KMS18 cells after 72 hours of exposure to MEK-I and ABT-737, respectively), the combination of MEK-I and ABT-737 was able to induce substantial apoptosis (more than 50% net apoptosis induction after 72 hours in both KMS18 and KMS27 cell lines). Such pro-apoptotic interaction was highly synergistic in nature, with CI, as defined using isobologram analysis, of 0.2 and 0.17 for KMS18 and KMS27 cells, respectively. Mitochondrial membrane depolarization was similarly enhanced by the combination of MEK-I and ABT-737. Conversely, in the MEK-I resistant MM cell line ARH-77, ABT-737 was still able to induce apoptosis (up to 40% of the cells) but its effect was not significantly potentiated by MEK inhibition. Preliminary results on primary CD138+ MM cells exposed to both inhibitors confirmed the higher cell growth inhibition induced by combining MEK-I and Bcl-2/Bcl-xL inhibition. In conclusion, we demonstrated a striking synergistic pro-apoptotic activity with combined inhibition of Raf/MEK/ERK and Bcl-2 signaling in MM cell lines. Simultaneous disruption of these two pathways warrants further investigation as novel therapeutic strategy for this disease.

Proteomic Analysis of Multiple Myeloma Identifies Upregulation of CRIK Protein, a Novel Protein Regulating Migration and Adhesion

PURPOSE: Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. In this study, we used a rapid and reproducible antibody-based protein microarray technique to screen the functional differences between malignant plasma cells in samples obtained from patients with MM compared to normal plasma cells (NPC) from the bone marrow of healthy volunteers.METHODS: We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM using high-throughput proteomic analysis with antibody-based protein microarray. Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. MM1.S was used in this study. Using immunohistochemistry and immunoblotting were confirmed. Lentivirus was used to knockdown CRIK. Gene expression datasets from the Mayo Clinic (accession number GSE 6477) and the UAMS (accession number GSE 2658) were obtained from the Gene Expression Omnibus for analysis. The Mayo dataset was generated using Affymetrix U133A platform whereas the UAMS dataset was generated using Affymetrix U133plus 2.0 platform.RESULTS: We identified four subgroups of MM using unsupervised clustering analysis. We confirmed overexpression of some of these proteins including CRIK and CDK4 using immunohistochemistry and immunoblotting. Many of these proteins are known to be deregulated in MM, indicating that this technique can accurately identify proteins that are over or under-expressed in MM in a high-throughput fashion. In addition, we identified novel proteins that are not previously known to be differentially expressed in MM, including the small GTPase member of the Rho family, CRIK protein. We then showed using knockdown of CRIK that this novel protein specifically regulates migration and adhesion in MM. There was no effect on survival of MM cells using the CRIK knockdown. Analyzing the GEP data of the 15 NPC, 46 monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM) and 101 MM samples from the Mayo Clinic, there was a significant increase in expression of CIT from NPC to MM. Among the 351 patients entered into TTII trials from UAMS, CIT expression was similar across the different TC class. Using a cut-off normalized expression level of 1.25 (a level above expression in NPC), MM with a high CIT expression (n=81) had a significantly shorter survival than the other patients.CONCLUSION: In this study, we show that MM cells express a high level of CRIK, and that inhibition of this protein leads to significant inhibition of adhesion and migration of MM cells. In addition, CRIK protein expression correlated with CIT gene expression, with high expression in MM samples compared to NPC. In addition, high CIT expression correlated with poor survival in patients with MM.

In Vivo Imaging Model of Multiple Myeloma and Its Cellular Interaction with the Bone Marrow Microenvironment

BACKGROUND: Imaging animal models that offer serial measurement of systemic tumor progression, such as the GFP+ or bioluminescence MM model, have been limited to low resolution, gross measurements of tumor progression that are insufficient to detect individual cells, and their interaction with their microenvironment. Therefore, the need exists for development of sensitive, high resolution three-dimensional imaging methods that identify the dynamic changes that occur during tumor initiation and progression. We here show the use of in vivo fluorescence confocal microscopy to follow MM tumor initiation and progression at the cellular level using stably GFP-transfected MM1S cells in a xenograft model of MM.METHODS: 5 × 10 6 MM1S-GFP-Luc cells were injected into the tail veins of non-irradiated SCID/Beige male mice. MM cell growth in the marrow of the calvarial bone was analyzed using in vivo flow cytometry and fluorescence confocal microscopy, as previously described (Sipkins et al 2005). High-resolution images with unprecedented cellular detail were obtained through the intact mouse skull at depths of up to 250μm. To visualize the bone marrow vasculature the mice were injected with a blood pool marker (Angiosense 680 or 750) immediately before imaging, and to delineate the surface of calvarial bone, a fluorescent hydroxyapatite tag (Osteosense) was used. The validity of the imaging data was established by sacrificing select mice, and analyzing the previously imaged tissues by standard histologic and immunohistologic techniques. After MM tumors became established in the fourth week following injection, 1 mg/kg Bortezomib was administered twice weekly to a subset of the mice, these were imaged following treatment along with controls that were not treated. For all mice imaged, the number and areas of the skull where GFP+ MM cells were found were recorded. Confirmation of homing and tumor progression was also performed using CD138+ selected primary tumor cells.RESULTS: Using this model, we were able to detect and monitor individual GFP+MM cells within the bone marrow microenvironment. We demonstrate that MM.1 S cells and primary CD138+ cells exit the systemic circulation within one hour of injection, followed by specific rolling and adhesion to the vasculature of the bone marrow microenvironment. Within 4 days post after injection, the MM cells were fully engrafted along the bone marrow sinusoids, which were surrounded by bisphosphonate-rich bone structures including ostoeoblasts. Within the second week, loose clusters of a few cells began to form around the blood vessels. Growth and expansion appeared to be closely associated with the vasculature. Tumor growth dramatically increased in the third week following cell injection when areas of the parasagittal regions became completely involved with MM cells. In contrast, standard bioluminescence imaging performed concurrently detected tumor initiation only at 4 weeks post-injection, indicating that confocal microscopy is a much more sensitive technique in detecting early tumor proliferation. Imaging of bortezomib-treated mice demonstrated that tumor size and density was reduced in the skull, but even more dramatically the number of sites containing GFP+MM was greatly reduced.CONCLUSIONS: Our imaging model differs from other models due to its unprecedented resolution. Therefore it is particularly useful for following small numbers of tumor cells either early in disease progression or after therapeutic treatment. This model offers a more sensitive spatial and temporal live imaging of MM cells in the BM microenvironment and can be used to explore the dynamic interaction of MM with different structures and environments of the BM. We anticipate that this model will allow for a better understanding of the biologic effects of therapeutic agents on the growth of MM cells within the bone marrow niches.

Nocodazole Induces Myeloma Cell Death by Disrupting the Microtubular Network, Resulting in G2/M Arrest

Patients with relapsed multiple myeloma (MM) previously treated with bortezomib and lenalidomide often fail to respond to further therapies. To identify potential new treatment approaches for MM, we used Luminex technology to screen a library of 1,120 compounds provided by the Multiple Myeloma Research Foundation.By multiplex cytokine array, we identified benzimidazoles including the anthelmintics mebendazole, fenbendazole, albendazole, nocodazole and pyrvinium pamoate, as inhibiting the production of cytokines essential for MM cell growth and survival, such as IL-6 (inhibition rate 40–70%), MIP-1α (inhibition rate 65–75%), VEGF (inhibition rate 75%), and soluble IL-6R (inhibition rate 40–52%). Consequently, these anthelmintics demonstrated dose-dependent inhibition of myeloma cell (RPMI-8226, H929, U266 and MM1S) proliferation. The lead compound, nocodazole, caused nuclear fragmentation and caspase-8 activation in MM cell lines and primary CD138+ cells in dose- and time-dependent fashion (IC50: 30–60 nM). Importantly, growth and survival signals provided by bone marrow stromal cells in bone marrow co-cultures failed to protect MM cells from nocodazole-induced cell death. In the apoptotic cells, caspase-8 was more activated than caspase-9, suggesting that mitochondrial signaling is not a major apoptotic pathway. Cell cycle analysis indicated that G2/M cell cycle arrest reached a peak at 17 hr. Sub-G1 proportion was strongly increased after treatment for 24 hr in all tested cell lines. Electron microscope (EM) and nuclear staining studies consistently showed the accumulation of metaphase cells, and morphologic elongation at 7 hr, at which time G2/M arrest was obvious. Most of the elongated cells had only one nucleus, suggesting that they failed to progress to mitosis due to overall microtubular network disarray.We conclude that nocodazole exposure induced microtubular network disarray with cell elongation, and G2/M arrest with a late stage mitotic block resulting in cell death.Benzimidazoles including nocodazole, traditionally used as antihelmintic drugs, have shown antitumor activity against hepatocellular, lung and adrenocortical carcinoma, and melanoma. In our study, we identified the anthelmintic compound nocodazole as a new anti-myeloma agent. Nocodazole warrants further investigation for its anti-MM effects in vitro and in vivo.

The Effect of Insulin-Like Growth Factor 1 on Waldenstrom Macroglobulinemia

Background: Waldenstrom's Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Insulin-like growth factor 1 (IGF1) is a polypeptide hormone that has been shown to have a proactive role in many cancer cell types, including multiple myeloma and solid tumor cells. We evaluated the role of IGF1 in WM.Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow-derived primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. The small molecule IGF-1R inhibitor II (Calbiochem) and the inhibitory antibody αIR3 (Calbiochem) were used. Cytotoxicity and DNA synthesis were measured by MTT assay and thymidine uptake assay, respectively. Cell signaling and apoptotic pathways were determined by Western Blot. Cell cycle and receptor analysis was obtained through flow cytometry. IGF1 levels were measured by ELISA. Receptor tyrosine kinase activity was evaluated in WM cell lines using the Luminex-microbeads-based assay.Results: We demonstrated that the IGF1 receptor (IGF1R) was expressed on WM primary patients and normal CD19+ control B cells, while IGF1 levels of serum and bone marrow samples in both patient and healthy donor samples were similar, suggesting a possible constitutive activation of the pathway downstream of IGF1R in WM and independent of receptor activity or cytokine levels. Surface IGF1R was expressed on BCWM1 cells, in serum-starved conditions, while, it was internalized with the addition of FBS that includes IGF-1. Finally, we also showed that IGF1 induces the activation of IGF1R as demonstrated by the induction of related tyrosine kinase activity. To demonstrate the protective effect of IGF1 on WM cells, IGF1 was added to serum starved BCWM1 cells, and we found it rescued nearly 100% of the cells from apoptosis in concentrations as low as 25ng/ml. The effect of IGF1R inhibitor on WM cells has been investigated and found that it blocked migration, induced cytotoxicity and decreased cell proliferation; without any effect on healthy donor peripheral blood mononuclear cells. Furthermore, the inhibitor decreased the tyrosine kinase activity of IGF1R, overcoming its initial activation in the presence of IGF1.Conclusion: IGF1 plays a complex role in WM cells with no evidence of constitutive activation of the receptor itself or higher levels of cytokine in WM marrow samples, indicating that activation of this pathway is downstream of IGF1R. Inhibition of IGF1R by a specific small molecule or antibody inhibitor led to a significant decrease in proliferation, survival, and migration in WM cells, but not in control mononuclear cells. These studies provide the framework to investigate the role of IGF1R inhibitors in clinical trials in WM.

The Tyrosine Kinase Src Regulates Adhesion and Survival in Waldenström Macroglobulinemia

BACKGROUND: Tyrosine phosphorylation is a central event in the regulation of a variety of biological processes such as cell proliferation, migration, adhesion, and survival. Waldenström Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy and characterized by widespread involvement of the bone marrow. We sought to determine whether the protein tyrosine kinase Src regulates adhesion and survival in WM.METHODS: The WM cell lines BCWM1, WM-WSU, and IgM secreting lymphoma cells lines MEC-1 and RL were used in these studies. Primary CD19+ WM cells were obtained from the bone marrow of patients after informed consent. AstraZeneca Biopharmaceuticals (London, England) provided the Src inhibitor AZD0503 (AZD). Cytotoxicity and DNA synthesis were measured using MTT assay and [3H]-thymidine uptake, respectively. Apoptosis was measured using flow cytometry with Apo 2.7 staining. Western blotting was performed to determine downstream signaling pathways. Migration was performed using the transwell migration assay.RESULTS: We demonstrated that pSrc is overexpressed in WM cells compared to control B cells. Similarly, phospho-Src protein expression was upregulated in WM cell lines, specifically BCWM.1 but not in WM-WSU. We then showed that pSrc regulates migration and adhesion in response to the chemokine SDF-1, as well as in vivo homing using in vivo flow cytometry. The use of the specific Src inhibitor AZD0530 led to significant inhibition of adhesion and migration in cell lines with pSrc activation, but not in those deficient of Src activation. Similarly, inhibition of Src activity led to significant inhibition of proliferation and survival through inhibition of STAT3, Akt, and ERK/MAPK pathways. The monoclonal antibody rituximab signals through Src kinase, and the combination of AZD0530 and rituximab was synergistic in vitro.CONCLUSION: Taken together, these studies delineate the role of Src kinase activity in WM and provide the framework for future clinical trials using Src inhibitors in combination with rituximab to improve the outcome of patients with WM.

Regulation of Histone Deacetylase in Waldenström Macroglobulinemia

Background: Waldenström's Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Histone deacetylase (HDAC) inhibitors represent promising new treatment strategy in B cell malignancies. We therefore investigated the in vitro effect of the novel hydroxamic acid derivative HDAC inhibitor LBH589 in WM.Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured by MTS assay and thymidine uptake assay. Cell signaling and apoptotic pathways were determined by Western Blot and immunofluorescence.Results: LBH589 induced a significant decrease of proliferation and triggered cytotoxicity in all cell lines tested and primary CD19+ WM cells (IC50 of 20–40nM), even in the presence of BMSC, IL-6 and IGF-1, which induce resistance to conventional therapies. Importantly, LBH589 did not induce cytotoxicity in healthy donor peripheral blood mononuclear cells. LBH589 induced both intrinsic and extrinsic apoptotic pathways, with caspase-9, caspase-8, caspase-3, and PARP cleavage in a dose-dependent manner. We also demonstrated significant upregulation of the proapoptotic transcription factor p53 and down-regulation of the anti-apoptotic proteins BclxL, Mcl-1 and c-myc. We then demonstrated that LBH589 induced apoptosis in WM cells in a caspase-independent manner through induction of autophagy, as shown by upregulation of LC3B and Rab7 expression. We further determined the mechanism of action of LBH589 in WM, investigating the effect of LBH589 on histone acetylation and NF-kB pathways. We found that LBH589 induced a dose-dependent increase in histone H3-H4 acetylation; and inhibited both canonical and non-canonical pathways of NF-κB, as shown by western blot and immunofluorescence. In addition, LBH589 augmented rituximab, fludarabine, bortezomib, and perifosine-induced cyotoxicity in WM cells.Conclusion: LBH589 has significant antitumor activity in WM in vitro, providing the framework for clinical trials evaluating LBH589 as a new therapeutic agent in patients with WM.

Dual PI3K and mTOR Inhibition in Waldenstrom Macroglobulinemia

Background. We have previously showed that PI3K/Akt is constitutively active in Waldenström Macroglobulinemia (WM) malignancies, mediating growth, survival, cell cycle regulation, and migration in primary tumor cells. Once activated, Akt phosphorylates downstream targets, including mammalian target of rapamycin (mTOR). Both PI3K/Akt and mTOR represent valid targets for antitumor therapeutic strategies. We therefore evaluated the antitumor activity of NVP-BEZ235 (Novartis, MA) in WM.Methods: WM cell lines (BCWM.1) and IgM secreting cell lines (MEK1, Namalwa) were used. Bone marrow primary CD19+ malignant cells and bone marrow stromal cells (BMSC) were obtained from WM patients. Cytotoxicity, DNA synthesis, and cell cycle were measured using MTT assay, [3H]-thymidine uptake, PI staining/flow cytometry, respectively. Effects of NVP-BEZ235 on cell signaling cascades were determined using immunoblotting and immunofluorescence. Adhesion on fibronectin has been evaluated in WM cells in the presence of NVP-BEZ235.Results: NVP-BEZ235 induced cytotoxicity and inhibited DNA synthesis with an IC50 of 20–25nM in BCWM.1 at 48 hours. Similar effects were demonstrated in all IgM secreting cell lines and in primary CD19+ WM cells, with an IC50 between 20nM and 50nM. No cytotoxicity was observed on peripheral blood mononuclear cells, indicating selective toxicity of the compound on the malignant lymphoplasmacytic clone. We observed that NVP-BEZ235 inhibited Akt (but not ERK phosphorylation) in a dose-dependent manner in BCWM.1 cells at 6 hours. Phosphorylation of GSK3α/β and ribosomal protein-S6, downstream target proteins of Akt, were also markedly inhibited. NVP-BEZ235-inhibited Akt phosphorylation was further confirmed by immunofluorescence. NVP-BEZ235 induced caspase-9, PARP cleavage and increased the release of Smac/DIABLO from the mitochondria to the cytosol, suggesting an induction of apoptosis in a caspase-dependent and –independent manner. We showed that NVP-BEZ235 inhibited adhesion of BCWM.1 cells to fibronectin in a dose-dependent fashion. Lastly, adherence to BMSCs did not confer protection to WM cells against NVP-BEZ235- induced cytotoxicity.Conclusions. These data indicate that NVP-BEZ235 has significant antitumor activity in WM, thus providing the framework for clinical trials in this disease.

CAL-101, a Selective Inhibitor of the p110δ Isoform of Phosphatidylinositol 3-Kinase, Effectively Induces Apoptosis in Primary Chronic Lymphocytic Leukemia Cells Providing a Novel Therapeutic Strategy for the Treatment of This Disease

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a pivotal role in cell proliferation and survival that underlies the biology of many cancers including CLL. Of the eight distinct mammalian isoforms of PI3K, it is the class IA PI3Ks (p110α, p110β and p110δ) that are responsible for Akt activation and cellular transformation. The p110α and p110β isoforms both have a ubiquitous tissue distribution in adults, whereas p110δ expression is restricted to cells of hematopoietic origin. Recent studies have established a dominant role of p110δ isoform in B-cell responses. Deletion or inactivation of p110δ ablates B-cell antigen receptor (BCR)-induced phosphorylation of Akt and impairs cell cycle progression. Furthermore, CD40-ligand (CD40L) dependent survival is compromised in the absence of p110δ activity. Considering the role of p110δ-mediated BCR and CD40L-CD40 signaling to the enhanced survival in normal B cells, we hypothesized that inhibition of this kinase will induce cytotoxicity in B-CLL cells. We first examined the protein expression of p110δ in primary tumor CD19+ B cells from CLL patients and show that 24/24 CLL consistently overexpressed p110δ. The expression levels of p110α and p110β however, varied more widely, and were often undetectable. Treatment of primary tumor cells with CAL-101, a novel selective p110δ inhibitor, at concentrations of 0.1–10μM resulted in significant cell killing (linear mixed model; p=0.0004). As an example, 5μM CAL-101 resulted in a median of 59.6% viable cells (n=18 CLL patient samples). CAL-101 induced cytotoxicity was accompanied by PARP and caspase 3 cleavage. Previous published studies have demonstrated that CD40L-CD40 signaling promotes activation of CLL cells (as measured by up-regulation of CD40 and CD86) and also protection from spontaneous apoptosis ex vivo. Treatment of CLL cells in the presence of CAL-101 diminished the activation markers CD40 and CD86 induced by CD40L. In addition, an increase in CLL cell viability induced by CD40L was reversed by CAL-101 treatment. Contrasting with this, diminishment of apoptosis with IL-4 was not observed. Given the common finding of innate and cellular immune effects induced by therapies utilized in CLL, we next assessed the effect of CAL-101 on normal NK cells and T cells. Treatment of NK cells and T cells in vitro from healthy volunteers had no effect on cell viability. The lack of cytotoxic effect on normal NK cells and T cells was also assessed in vivo from a completed phase I trial of healthy volunteers that serves as a forerunner to the phase 1 clinical trial in CLL and related lymphoproliferative diseases currently ongoing. Here, treatment of normal human volunteers with CAL-101 for seven days achieved peak plasma concentrations up to 5μM without changes in general hematology or subpopulations of NK cells and T cells. Overall, our results identify the p110δ isoform as a potential therapeutic target in CLL where selective cytotoxicity is observed as compared to normal immune effector cells and the very important CD40-CD40L survival pathway is disrupted. Together, these in vitro and in vivo data provide sound validation for the ongoing Phase 1 clinical trial for the treatment of patients with CLL and related lymphoid malignancies.

PKC412, a Small Molecule Tyrosine Kinase Inhibitor, Regulates Survival and Proliferation Pathways in Waldenström Macroglobulinemia

Background: Waldenström's Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Protein kinase C (PKC) is a multigene family that that plays an important role in the regulation of cell growth and tumor progression. PKC412, a derivate of staurosporine, is a competitive inhibitor of ATP binding to PKC subtypes. This PKC inhibitor was used to examine its role in WM.Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured by MTT assay and thymidine uptake assay. Cell signaling of HSP response, VEGF signaling and AKT pathways were determined by Western Blot analysis. Cell cycle and apoptosis analysis were obtained through flow cytometry.Results: We showed that PKC412 induced cytotoxicity in WM cell lines tested (IC50 of 1uM) with no cytotoxic effect on healthy donor peripheral blood mononuclear cells at concentrations as high as 5uM. Cell proliferation and apoptosis analysis showed similar results, in a time independent manner. A G2/M arrest was observed upon cell cycle analysis, suggesting a possible mechanism for apoptosis. Similarly, the Akt pathway was investigated and was found to show a decrease in its activity in a dose dependent manner. Finally, PKC412 regulated VEGF levels in WM samples indicating an anti-angiogenic activity.Conclusion: PKC412 has a significant effect on the survival and proliferation of WM cells, including regulation of angiogenesis. These studies provide the framework for clinical evaluation of this novel PKC inhibitor in Waldenström Macroglobulinemia.

Epigenetic silencing of the interferon regulatory factor ICSBP/IRF8 in human multiple myeloma

Multiple myeloma (MM) is presently an incurable malignant plasma cell tumor. The objective of this study was to investigate expression of the interferon regulatory factor family (IRF1-9) and the potential role of DNA methylation in silencing IRF genes in MM cell lines and purified MM cells from patients. Using a panel of 13 human MM cell lines and purified CD138+ cells from nine MM patients, expression of IRF genes was investigated by quantitative reverse transcriptase polymerase chain reaction and Western blot. DNA methylation of the interferon consensus sequence-binding protein (ICSBP/IRF8) gene was measured using pyrosequencing, and the effect of promoter methylation on expression was analyzed by in vitro methylation of a cloned ICSBP/IRF8 promoter, and treatment of MM cells with 5-aza-2'-deoxycytidine (DAC). Eight of thirteen of the MM cell lines were found to lack ICSBP/IRF8 expression, associated with hypermethylation of the CpG island in the ICSBP/IRF8 promoter. We also found that ICSBP/IRF8 was significantly underexpressed in primary MM cells, whereas the ICSBP/IRF8 promoter was methylated in only one of nine of primary purified CD138+ MM samples. DAC-mediated demethylation restored endogenous ICSBP/IRF8 expression, whereas in vitro methylation silenced the promoter. Expression of the ICSBP/IRF8 gene is silenced in a majority of MM cell lines and primary CD138+ MM cells. DNA methylation of the ICSBP/IRF8 gene is a frequent event in MM cell lines, but silencing is also observed in the absence of methylation. These results suggest that silencing of ICSBP/IRF8 expression, by DNA methylation or other epigenetic mechanisms, may be associated with the malignant phenotype of MM.

Interruption of the Ras/MEK/ERK signaling cascade enhances Chk1 inhibitor–induced DNA damage in vitro and in vivo in human multiple myeloma cells

AbstractThe role of the Ras/MEK/ERK pathway was examined in relation to DNA damage in human multiple myeloma (MM) cells exposed to Chk1 inhibitors in vitro and in vivo. Exposure of various MM cells to marginally toxic concentrations of the Chk1 inhibitors UCN-01 or Chk1i modestly induced DNA damage, accompanied by Ras and ERK1/2 activation. Interruption of these events by pharmacologic (eg, the farnesyltransferase inhibitor R115777 or the MEK1/2 inhibitor PD184352) or genetic (eg, transfection with dominant-negative Ras or MEK1 shRNA) means induced pronounced DNA damage, reflected by increased γH2A.X expression/foci formation and by comet assay. Increased DNA damage preceded extensive apoptosis. Notably, similar phenomena were observed in primary CD138+ MM cells. Enforced MEK1/2 activation by B-Raf transfection prevented R115777 but not PD184352 from inactivating ERK1/2 and promoting Chk1 inhibitor–induced γH2A.X expression. Finally, coadministration of R115777 diminished UCN-01–mediated ERK1/2 activation and markedly potentiated γH2A.X expression in a MM xenograft model, associated with a striking increase in tumor cell apoptosis and growth suppression. Such findings suggest that Ras/MEK/ERK activation opposes whereas its inhibition dramatically promotes Chk1 antagonist–mediated DNA damage. Together, these findings identify a novel mechanism by which agents targeting the Ras/MEK/ERK pathway potentiate Chk1 inhibitor lethality in MM.

The HMG-CoA inhibitor, simvastatin, triggers in vitro anti-tumour effect and decreases IgM secretion in Waldenstrom macroglobulinaemia.

Waldenstrom macroglobulinaemia (WM) is an incurable lymphoplasmacytic lymphoma with secretion of serum monoclonal immunoglobulin M (IgM). We previously showed that patients receiving cholesterol-lowering statins, had the lowest IgM value in a large cohort of patients with WM. Simvastatin, a 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor, induced inhibition of proliferation, cytotoxic effect and apoptosis in IgM secreting cell lines as well as in primary CD19(+) WM cells. Interestingly, those effects were reversed by addition of mevalonate and geranylgeranylpyrophosphate, demonstrating that simvastatin inhibited cell growth, survival and IgM secretion on BCWM.1 WM cells by inhibition of geranylgeranylated proteins. Furthermore, simvastatin overcame tumour cell growth induced by co-culture of WM cells with bone-marrow stromal cells. Simvastatin also decreased IgM secretion by BCWM.1 cells at an early time-point that had not affected cell survival. Simvastatin-induced cytotoxicity was preceded by a decrease in Akt (protein kinase B, PKB) and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathways at 18 h. In addition, simvastatin induced an increase in stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) MAPK followed by caspase-8, -9, -3 and poly(ADP-ribose) polymerase (PARP) cleavages at 18 h, leading to apoptosis. Furthermore, simvastatin enhanced the cytotoxicity induced by bortezomib, fludarabine and dexamethasone. Our studies therefore support our earlier observation of statin-mediated anti-WM activity and provide the framework for future clinical trials testing simvastatin in WM.

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.

Mechanisms Regulating CXCR4 Expression in Myeloma Cells.

The chemokine receptor CXCR4 plays roles in the homing of myeloma cells to the bone marrow (BM) and in the progression of the disease. However, the regulation of CXCR4 expression in myeloma cells is poorly defined. This study investigated the mechanisms regulating CXCR4 expression in myeloma cells. RPMI8226 and U266 myeloma cells strongly expressed CXCR4 on the cell surface, whereas ARH77 myeloma cells expressed minimal CXCR4 on the cell surface, as determined by flow cytometry using three different monoclonal antibodies to CXCR4. However, Western blot analysis, flow cytometry after permeabilization, and immunofluorescence staining reveled that ARH77 cells have abundant CXCR4 in the cytoplasm, in amounts similar to those in RPMI8226 and U266 cells. The cell surface expression of CXCR4 in primary CD138+ cells obtained from the BM of multiple myeloma patients differed among different specimens. Similar to myeloma cell lines, the primary myeloma cells that expressed minimal CXCR4 on the cell surface had abundant CXCR4 in the cytoplasm. The transmigration of the cells induced by stromal cell-derived factor-1 (SDF-1) was correlated with the cell surface expression of CXCR4, indicating that only the CXCR4 on the cell surface is functional. These findings suggest that myeloma cells have their own intrinsic mechanisms for regulating CXCR4 expression on the cell surface. In all three myeloma cell lines and in some primary BM CD138+ cells, dexamethasone (Dex) enhanced CXCR4 expression both in the cytoplasm and on the cell surface while downregulating SDF-1; this led to enhanced cell migration in response to SDF-1. Cell surface CXCR4 expression was more prominent in annexin V-positive apoptotic cells. VEGF and proinflammatory cytokines, including TNF-α and TGF-β1, also upregulated the cell surface expression of CXCR4 in RPMI8226 and some primary BM CD138+ cells, but not in U266 and ARH77 cells. Myeloma cells, including all the three cell lines and some primary BM CD138+ cells, incubated under hypoxic conditions (1% O2) exhibited upregulation of CXCR4 both in the cytoplasm and on the cell surface. Again, surface CXCR4 expression was stronger in apoptotic cells than in non-apoptotic cells, suggesting that the upregulation of CXCR4 is a counter-regulatory phenomenon for stimuli causing cell damage. As proven previously for other cell types, hypoxia induced the accumulation of HIF-1α in myeloma cells. Topotecan, which inhibits HIF-1α, attenuated the hypoxia-induced upregulation of CXCR4, whereas the proteasome inhibitor bortezomib slightly enhanced it. Dex, VEGF, TNF-α, and TGF-β1 all induced the accumulation of HIF-1α in RPMI8226 and some primary BM CD138+ cells. In addition, the effects of topotecan and bortezomib under hypoxic conditions were observed in the change of CXCR4 expression mediated by Dex and cytokines. These results indicate that complex intrinsic and extrinsic mechanisms regulate CXCR4 expression in myeloma cells and suggest that HIF-1α is a common regulatory molecule, at least in the extrinsic mechanisms.