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Mantle cell lymphoma (MCL) is a comparatively rare non-Hodgkin’s lymphoma characterised by overexpression of cyclin D1. Many patients present with or progress to advanced stage disease within 3 years. MCL is considered an incurable disease with median survival between 3 and 4 years. We have investigated the role(s) of CCN1 (CYR61) and cell cycle regulators in progressive MCL. We have used the human MCL cell lines REC1 G519 > JVM2 cells by RQ-PCR, depicting a decrease in CCN1 expression with disease progression. Investigation of CCN1 isoform expression by western blotting showed that whilst expression of full-length CCN1 was barely altered in the cell lines, expression of truncated forms (18–20 and 28–30 kDa) decreased with disease progression. We have then demonstrated that cyclin D1 and cyclin dependent kinase inhibitors (p21(CIP1)and p27(KIP1)) are also involved in disease progression. Cyclin D1 was highly expressed in REC1 cells (OD: 1.0), reduced to one fifth in G519 cells (OD: 0.2) and not detected by western blotting in JVM2 cells. p27(KIP1) followed a similar profile of expression as cyclin D1. Conversely, p21(CIP1) was absent in the REC1 cells and showed increasing expression in G519 and JVM2 cells. Subcellular localization detected p21(CIP1)/ p27(KIP1) primarily within the cytoplasm and absent from the nucleus, consistent with altered roles in treatment resistance. Dysregulation of the CCN1 truncated forms are associated with MCL progression. In conjunction with reduced expression of cyclin D1 and increased expression of p21, this molecular signature may depict aggressive disease and treatment resistance.

Chronic Myeloid Leukaemia (CML) is a myeloproliferative disorder characterized by the expression of the oncoprotein, Bcr-Abl kinase. CCN3 normally functions as a negative growth regulator, but it is downregulated in CML, the mechanism of which is not known. MicroRNAs (miRNAs) are small non-coding RNAs, which negatively regulate protein translation by binding to the complimentary sequences of the 3′ UTR of messenger RNAs. Deregulated miRNA expression has emerged as a hallmark of cancer. In CML, BCR-ABL upregulates oncogenic miRNAs and downregulates tumour suppressor miRNAs favouring leukaemic transformation. We report here that the downregulation of CCN3 in CML is mediated by BCR-ABL dependent miRNAs. Using the CML cell line K562, we profiled miRNAs, which are BCR-ABL dependent by transfecting K562 cells with anti-BCR-ABL siRNA. MiRNA expression levels were quantified using the Taqman Low Density miRNA array platform. From the miRNA target prediction databases we identified miRNAs that could potentially bind to CCN3 mRNA and reduce expression. Of these, miR-130a, miR-130b, miR-148a, miR-212 and miR-425-5p were significantly reduced on BCR-ABL knockdown, with both miR-130a and miR-130b decreasing the most within 24 h of siRNA treatment. Transfection of mature sequences of miR-130a and miR-130b individually into BCR-ABL negative HL60 cells resulted in a decrease of both CCN3 mRNA and protein. The reduction in CCN3 was greatest with overexpression of miR-130a whereas miR-130b overexpression resulted only in marginal repression of CCN3. This study shows that miRNAs modulate CCN3 expression. Deregulated miRNA expression initiated by BCR-ABL may be one mechanism of downregulating CCN3 whereby leukaemic cells evade negative growth regulation.

Chronic Myeloid Leukaemia (CML) is characterized by expression of the constitutively active Bcr-Abl tyrosine kinase. We have shown previously that the negative growth regulator, CCN3, is down-regulated as a result of Bcr-Abl kinase activity and that CCN3 has a reciprocal relationship of expression with BCR-ABL. We now show that CCN3 confers growth regulation in CML cells by causing growth inhibition and regaining sensitivity to the induction of apoptosis. The mode of CCN3 induced growth regulation was investigated in K562 CML cells using gene transfection and treatment with recombinant CCN3. Both strategies showed CCN3 regulated CML cell growth by reducing colony formation capacity, increasing apoptosis and reducing ERK phosphorylation. K562 cells stably transfected to express CCN3 showed enhanced apoptosis in response to treatment with the tyrosine kinase inhibitor, imatinib. Whilst CCN3 expression was low or undetectable in CML stem cells, primary CD34+ CML progenitors were responsive to treatment with recombinant CCN3. This study shows that CCN3 is an important growth regulator in haematopoiesis, abrogation of CCN3 expression enhances BCR-ABL dependent leukaemogenesis. CCN3 restores growth regulation, regains sensitivity to the induction of apoptosis and enhances imatinib cell kill in CML cells. CCN3 may provide an additional therapeutic strategy in the management of CML.

We have adapted the CyQuant(R) assay to provide a simple, rapid, sensitive and highly reproducible method for measuring cell adhesion. The modified CyQuant(R) assay eliminates the requirement for labour intensive fluorescent labelling protocols prior to experimentation and has the sensitivity to measure small numbers (1000) of adherent cells.

FK506 binding protein-like (FKBPL) and its peptide derivatives exert potent anti-angiogenic activity in vitro and in vivo and control tumour growth in xenograft models, when administered exogenously. However, the role of endogenous FKBPL in angiogenesis is not well characterised. Here we investigated the molecular effects of the endogenous protein and its peptide derivative, AD-01, leading to their anti-migratory activity. Inhibition of secreted FKBPL using a blocking antibody or siRNA-mediated knockdown of FKBPL accelerated the migration of human microvascular endothelial cells (HMEC-1). Furthermore, MDA-MB-231 tumour cells stably overexpressing FKBPL inhibited tumour vascular development in vivo suggesting that FKBPL secreted from tumour cells could inhibit angiogenesis. Whilst FKBPL and AD-01 target CD44, the nature of this interaction is not known and here we have further interrogated this aspect. We have demonstrated that FKBPL and AD-01 bind to the CD44 receptor and inhibit tumour cell migration in a CD44 dependant manner; CD44 knockdown abrogated AD-01 binding as well as its anti-migratory activity. Interestingly, FKBPL overexpression and knockdown or treatment with AD-01, regulated CD44 expression, suggesting a co-regulatory pathway for these two proteins. Downstream of CD44, alterations in the actin cytoskeleton, indicated by intense cortical actin staining and a lack of cell spreading and communication were observed following treatment with AD-01, explaining the anti-migratory phenotype. Concomitantly, AD-01 inhibited Rac-1 activity, up-regulated RhoA and the actin binding proteins, profilin and vinculin. Thus the anti-angiogenic protein, FKBPL, and AD-01, offer a promising and alternative approach for targeting both CD44 positive tumours and vasculature networks. © 2013 Yakkundi et al.

CCN3, a tumour suppressor gene, is down-regulated as a result of BCR-ABL tyrosine kinase activity in Chronic Myeloid Leukaemia (CML). We have established a stable CCN3 expression model in the human K562 CML cell line and have further validated the role for CCN3 in the leukaemogenic process. K562 cells stably transfected with CCN3 (K562/CCN3; 2.25 × 10(6) copies per 50 ng cDNA) demonstrated over 50% reduction in cell growth in comparison to cells stably transfected with empty vector (K562/control; p = 0.005). K562/CCN3 cells had reduced colony formation capacity (reduced by 29.7%, p = 0.03) and reduced mitogenic signalling in comparison to K562/control cells (reduced by 29.5% (p = 0.002) and 37.4% (p = 0.017) for phosphorylation levels of ERK and AKT respectively). K562/CCN3 cells showed an accumulation of events within the subG(0) phase of the cell cycle and increased apoptosis was confirmed by a three-fold increase in annexin V binding (p 0.05). K562/CCN3 cells exposed to Imatinib (1 μM and 5 μM) showed an increase in events within the subG(0) phase of cell cycle over 96 h and mirrored the enhanced cell kill demonstrated by Annexin staining. Wild type K562 cells treated with recombinant human Ccn3 (10 nM) in combination with Imatinib (5 μM) also displayed enhanced cell kill (p = 0.008). K562/CCN3 cells displayed increased adhesion to matrigel™ (2.92 ± 0.52 fold increase compared to K562/control) which was commensurate with increased expression of the alpha 6 and beta 4 integrins (6.53 ± 0.47 and 1.94 ± 0.07 fold increase in gene expression respectively (n = 3, p 0.05)). CCN3 restores cellular growth regulatory properties that are absent in CML and sensitises CML cells to imatinib induced apoptosis. CCN3 may provide novel avenues for the development of alternate therapeutic strategies.

Objective We have previously demonstrated that proteasome activity is higher in bone marrow from patients with chronic myeloid leukemia (CML) than normal controls. This study investigates whether there is any relationship between Bcr-Abl expression and proteasome activity. Materials and Methods Fluorogenic substrate assays and an activity-based probe were used to profile proteasome activity in CML cell-line models and the effect of the proteasome inhibitor BzLLLCOCHO on these cell-line models and primary CML cells was investigated. Results We have demonstrated that oncogenic transformation by BCR-ABL is associated with an increase in proteasome proteolytic activity. Furthermore, small interfering RNA targeted against BCR-ABL reduces proteasome activity. In addition, we have found that Bcr-Abl−positive cells are more sensitive than Bcr-Abl−negative cells to induction of apoptosis by the proteasome inhibitor BzLLLCOCHO, and that sequential addition of imatinib followed by BzLLLCOCHO has an additive effect on the induction of apoptosis in Bcr-Abl−positive cells. Finally, we demonstrate that cell lines that become resistant to imatinib remain sensitive to proteasome inhibition. Conclusion This is the first time that a direct relationship has been demonstrated between BCR-ABL transformation and the enzymatic activity of the proteasome. Our results suggest that the proteasome might provide a useful therapeutic target in CML, particularly in those patients who have developed resistance to conventional treatment.

Chronic Myeloid Leukaemia (CML) is characterized by expression of the constitutively active BCR-ABL tyrosine kinase. Previously, we identified down-regulation of the negative growth regulator, CCN3, as a result of BCR-ABL kinase activity. Reduced CCN3 expression is a prominent feature in both primary human CML cells and cell lines. We now show that CCN3 is growth inhibitory and enhances imatinib induced growth inhibition. To evaluate the biological consequence of CCN3 expression in CML, K562 cells were stably transfected with a construct containing CCN3 (pCMV82-23) and growth characteristics and activation of signaling pathways were compared to cells transfected with empty vector (control). CCN3 expression was undetected by Real-time PCR in control cells whilst pCMV82-23 cells expressed 2.25 × 106 copies per 50ng of cDNA. pCMV82-23 cells showed reduced colony formation capacity (p=0.003) and reduced cell growth over a period of five days (p=0.005). Investigation of cellular signaling showed CCN3 expression resulted in significant down-regulation of three major signaling pathways and demonstrated reduced phosphorylation of ERK (p=0.002), pAKT (p=0.017) and pSTAT5 (p=0.005) compared to control cells. Protein levels for total ERK, AKT and STAT5 were unaffected by CCN3 expression. Flow cytometry showed that sustained CCN3 expression resulted in an accumulation of cells within the subG0 stage of the cell cycle (11.4% ± 3 (p=0.040)). To determine if CCN3 expression could influence sensitivity to the BCR-ABL kinase inhibitor, imatinib, pCMV82-23 cells and control cells were treated with imatinib (5uM) for 48h. Control cells treated with imatinib showed moderate growth inihibition (19.6% ± 2.5) compared to untreated control. pCMV82-23 cells showed a significant increase in the magnitude of imatinib induced growth inhibition (63.3% ± 10.5 (p=0.043)). This was associated with an increased accumulation of cells in the subG0 area of the cell cycle, 34.6% ± 5 for pCMV82-23 cells compared to control cells (21.7% ± 8) in response to imatinib treatment (p=0.006). To then determine if these effects could be reproduced using recombinant CCN3 (rCCN3), K562 cells were treated with imatinib (5uM) alone or in combination with rCCN3 (10nM) for 48h. K562 cells treated with the combination of rCCN3 and imatinib showed enhanced growth inhibition (71.8% ± 7.9) compared to cells treated with imatinib alone (81.1% ± 9.2 (p=0.008)). Loss of CCN3 is consistent with properties associated with the CML phenotype. Sustained expression of CCN3 in K562 cells restores growth control and re-establishes induction of apoptosis. Both increased expression of CCN3 or addition of the recombinant protein provide additional benefit for imatinib induced growth inhibition thus providing a novel avenue for therapeutic intervention.

Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell, characterised by the constitutively active tyrosine kinase BCR-ABL. The current first-line therapy for CML is the tyrosine kinase inhibitor imatinib. Although imatinib induces durable responses, a number of patients develop resistance to this treatment, highlighting the need to identify new molecular targets in this disease. Proteasome inhibition has recently emerged as a novel anti-cancer therapy. There is evidence to suggest that the proteasome is a valid target in CML. We have previously reported that proteasome activity is higher in bone marrow from patients with CML than normal controls. Furthermore, we demonstrated using a cell line model that Bcr-Abl positive cells were more sensitive to induction of apoptosis by proteasome inhibition than Bcr-Abl negative cells. The present study investigates the relationship between Bcr-Abl expression and proteasome activity and the effect of proteasome inhibition on primary human CML cells. Conventional fluorogenic substrate assays for all three catalytic activities of the proteasome [chymotrypsin-like (CT-L), trypsin-like (T-L), post glutamyl peptide hydrolysing (PGPH)] and proteasome activesite label DansylAhx3L3VS were used to profile proteasome activity levels in ts-Bcr-Abl FDCP-Mix cells and mock transfected FDCP-Mix cells. Both methods confirmed that Bcr-Abl positive cells have higher levels of proteasome activity than Bcr-Abl negative cells (p ≤ 0.04; Figure 1a). Conversely, downregulation of BCR-ABL using si-RNA was associated with a significant decrease in proteasome activity (p < 0.05; Figure 1b). The ability of the proteasome inhibitor BzLLLCOCHO to induce apoptosis in primary human CML cells was evaluated using Mitosensor™ and Hoescht/Propidium Iodide staining. Treatment with BzLLLCOCHO (1μM), selectively induced apoptosis in primary CML cells compared to normal mononuclear cells (39 ± 9.6 % vs 18.1 ± 4.02 %, 72 hrs, p = 0.01). Drug combination experiments were performed with BzLLLCOCHO (1 μM) and imatinib (1 μM) in ts-Bcr-Abl FDCP-mix cells and primary CML cells. Using Calcusyn software to generate the median effect of Chou-Talalay, the sequential addition of imatinib followed by BzLLLCOCHO was found to synergistically enhance the induction of apoptosis in ts-Bcr-Abl FDCP-Mix cells and resulted in additive effects in primary CML cells (n=4). The effect of the compounds on Bcr-Abl kinase activity was assessed by immunoblotting for phosphorylated Crkl. No effect on Bcr-Abl activity was seen following treatment of ts-Bcr-Abl FDCP-Mix cells and primary CML cells with BzLLLCOCHO alone, however, the combination of BzLLLCOCHO and imatinib resulted in a greater reduction of Bcr- Abl activity (59.8 ± 9.9 %) than imatinib alone (34.1 ± 9.6 %). Finally, we investigated the effect of BzLLLCOCHO on two human CML cell lines which are resistant to imatinib (KCL22-r, LAMA84-r). Imatinib resistant cells were found to be equally as sensitive to induction of apoptosis by BzLLLCOCHO as their imatinib sensitive counterparts (KCL22-s 30.7 ± 5.7 % vs KCL22-r 32 ± 1.7 %; LAMA84-s 56.3 ± 3.2 % vs Lama84-r 57.2 ± 7.9 %; 72 hrs). The present findings suggest that higher proteasome activity in Bcr- Abl positive cells may render these cells more susceptible to induction of apoptosis by proteasome inhibition and provide a rational basis to examine the potential of proteasome inhibitors as a therapeutic target in CML, particularly in imatinib resistant disease. Figure 1a. Bcr-Abl+ cells contain significantly greater levels of proteasome activity than Bcr-Abl cells siRNA directed against BCR-ABL decreases proteasome activity. Figure 1a. Bcr-Abl+ cells contain significantly greater levels of proteasome activity than Bcr-Abl cells . / siRNA directed against BCR-ABL decreases proteasome activity.

Chronic Myeloid Leukemia (CML) is characterized by expression of the constitutively active BCR-ABL tyrosine kinase. Previously, we have identified downregulation of the negative growth regulator, CCN3, as a result of BCR-ABL kinase activity and detected reduced CCN3 expression in human CML cell lines and primary human CML cells. We now report a reciprocal relationship of BCR-ABL and CCN3 expression and the functional consequence of expressing CCN3 in BCR-ABL+ cells. Real-time PCR was used to examine the relationship between BCR-ABL and CCN3 expression in human K562 cells. Parental K562 cells showed high expression of BCR-ABL (4.68 x104 transcripts in 5 μL of cDNA) whilst CCN3 expression was not detected. Treatment with siRNA directed against BCR-ABL (0.5 μg per106 cells) for 72 hours resulted in a 3.7 fold decrease in BCR-ABL and 6.1 fold increase in CCN3 expression (mean Ct change 1.9 ± 0.2 and 2.6 ± 0.5 for BCR-ABL and CCN3 respectively, n=3, p=0.001). Similarly, K562 cells treated with imatinib (1 micromolar) for 96 hours showed a 5.9 fold decrease in BCR-ABL expression and a 4.2 fold increase in CCN3 expression (mean Ct change 2.5 ± 0.1 and 2.1± 0.2 for BCR-ABL and CCN3 respectively, n=3, p=0.001). To investigate CCN3 function, we expressed CCN3 in BCR-ABL+ cells using Nucleofector technology (Amaxa, GmbH). K562 cells were transfected with either the pCb6+ vector (Invitrogen,UK) or pCb6+ vector containing the CCN3 construct. Cells were analysed 24 hours post-transfection by flow cytometry and also after 7 days in methyl cellulose culture to determine clonogenicity. Cell cycle analysis was performed on 20,000 events using the winMDI software. CCN3 expression in BCR-ABL+ cells resulted in an accumulation of cells in the subG0 phase of the cell cycle (mean for subG0 9.9% ± 4.6 and 21.8% ± 0.7 for the pCb6+ vector alone and pCb6+ vector containing CCN3 construct respectively). CCN3 expression significantly increased the number of cells within the subG0 area of the cell cycle (n=3, p=0.027). In addition, CCN3 expression reduced the clonogenic capacity of BCR-ABL+ cells. K562 cells transfected with the pCb6+ vector containing CCN3 construct formed significantly fewer colonies on methyl cellulose in comparison to cells that had been transfected with the pCb6+ vector alone (n=3, p=0.027). This study demonstrates a reciprocal relationship between CCN3 and BCR-ABL expression. CCN3 is known to be a negative growth regulator and increased expression of CCN3 in BCR-ABL+ cells inhibits proliferation and decreases clonogenic potential. Thus CCN3 down-regulation mediated by BCR-ABL offers growth advantage to hematopoietic cells.