Supplementary Figure 1 from Crucial Roles for Protein Kinase C Isoforms in Tumor-Specific Killing by Apoptin

In the version of this article initially published, there was a mistake in the calculation of the nucleotide mutation rate per site per generation: 1 × 10 −9 mutations per site per generation was used, whereas 9.5 × 10 −9 was correct. This error affects the interpretation of population-size changes over time and their possible correspondence with known geological events, as shown in the original Fig. 4 and supporting discussion in the text, as well as details in the Supplementary Note. Neither the data themselves nor any other results are affected. Figure 4 has been revised accordingly. Images of the original and corrected figure panels are shown in the correction notice.

Podosomes are actin-based adhesions involved in migration of cells that have to cross tissue boundaries such as myeloid cells. The Wiskott Aldrich Syndrome Protein regulates de novo actin polymerization during podosome formation and it is cleaved by the protease calpain during podosome disassembly. The mechanisms that may induce the Wiskott Aldrich Syndrome Protein cleavage by calpain remain undetermined. We now report that in myeloid cells, tyrosine phosphorylation of the Wiskott Aldrich Syndrome Protein-tyrosine291 (Human)/tyrosine293 (mouse) not only enhances Wiskott Aldrich Syndrome Protein-mediated actin polymerization but also promotes its calpain-dependent degradation during podosome disassembly. We also show that activation of the Wiskott Aldrich Syndrome Protein leading to podosome formation occurs independently of tyrosine phosphorylation in spleen-derived dendritic cells. We conclude that tyrosine phosphorylation of the Wiskott Aldrich Syndrome Protein integrates dynamics of actin and cell adhesion proteins during podosome disassembly required for mobilization of myeloid cells during the immune response.

The response of the tumour microenvironment to anti-cancer drugs can influence treatment efficacy. Current drug-screening methodologies fail to distinguish and quantify simultaneously the concomitant effect of drugs on the tumour stroma and cancer cells. To overcome this limitation we have developed a fluorescence-based experimental model that employs mCherry-labelled stromal cells (e.g. bone marrow fibroblastic stromal cells) co-cultured in direct contact with enhanced green fluorescent protein-labelled tumour cell lines for accurate assessment of proliferation and viability in both cell compartments and adhesion of tumour cells. Additionally, we used fluorescence-based image analysis to determine morphological changes that correlate with cell function (e.g. morphology of the actin cytoskeleton and nuclearity of osteoclasts to predict their bone resorption activity). Using this platform we have revealed that dexamethasone induces HS5 fibroblast proliferation and contact with multiple myeloma cells via a process involving Src/c-Abl kinases. Osteoclasts also inhibited dexamethasone-induced apoptosis in myeloma cells while retaining their normal morphology and functionality in bone resorption. Myeloma resistance to dexamethasone mediated by HS5 cells and osteoclasts was reversed by treatment with the Src/c-Abl inhibitor dasatinib but not with bortezomib. This new experimental platform provides a more precise screening of new therapeutics for improved efficacy of tumour cell killing within the bone marrow microenvironment.

Downlo chicken anemia virus–derived protein apoptin induces apoptosis in a variety of human malignant and rmed cells but not in normal cells. However, the mechanisms through which apoptin achieves its sekilling effects are not well understood. We developed a lentiviral vector encoding a green fluorescent n–apoptin fusion gene (LV-GFP-AP) that can efficiently deliver apoptin into hematopoietic cells. Apopectively killed the human multiple myeloma cell lines MM1.R and MM1.S, and the leukemia cell lines HL60, U937, KG1, and NB4. In contrast, normal CD34 cells were not killed and maintained their differon potential in multilineage colony formation assays. In addition, dexamethasone-resistant MM1.R cells ound to be more susceptible to apoptin-induced cell death than the parental matched MM1.S cells. Death tibility correlated with increased phosphorylation and activation of the apoptin protein in MM1.R cells. sion array profiling identified differential kinase profiles between MM1.R and MM1.S cells. Among these s, protein kinase Cβ (PKCβ) was found by immunoprecipitation and in vitro kinase studies to be a cankinase responsible for apoptin phosphorylation. Indeed, shRNA knockdown or drug-mediated inhibition β significantly reduced apoptin phosphorylation. Furthermore, apoptin-mediated cell death proceeded h the upregulation of PKCβ, activation of caspase-9/3, cleavage of the PKCδ catalytic domain, and downtion of the MERTK and AKT kinases. Collectively, these results elucidate a novel pathway for apoptin actiregula vation involving PKCβ and PKCδ. Further, they highlight the potential of apoptin and its cellular regulators to purge bone marrow used in autologous transplantation for multiple myeloma. Cancer Res; 70(18); 7242–52. ©2010 AACR.

Abstract Abstract 5039 There is mounting evidence that malignant cells have an intrinsic ability to prevent apoptosis. In the present study we provide evidence that the ectopic expression of Apoptin can restore the failing apoptosis program in myeloma cells via protein kinase C b (PKCb) and overcome intrinsic or acquired resistance to cell death. Apoptin (VP3), a chicken anemia virus (CAV)-derived protein has been shown to possess tumor specific cytotoxicity; its expression induces apoptosis in human tumor and transformed cells but there is little or no cytotoxic effect in normal human cells or cell lines derived from different tissues including peripheral blood mononuclear cells, fibroblast and epithelial cells. Several studies have shown that the tumor specific killing of Apoptin correlates with its phosphorylation and its subcellular localization. In cancer cells, Apoptin is localized in the nucleus and is phosphorylated on threonine108 by an as yet unknown kinase, whereas in normal cells Apoptin is detected in the cytoplasm and is essentially unphosphorylated. We developed a lentiviral vector encoding a GFP-Apoptin fusion gene (LV-GFP-AP), which delivers the Apoptin gene efficiently to haematopoietic cells. Apoptin significantly and selectively killed a number of leukemia cell lines including K562, HL60, U937, KG1 and NB4. In particular, the dexamethasone resistant multiple myeloma cell line MM1.R and the dexamethasone sensitive cell line MM1.S were efficiently killed by Apoptin. In contrast normal CD34+ cells were not killed and maintained their differentiation potential in multilineage colony formation assays. In addition, we showed that the dexamethasone resistant MM1.R cells were considerably more susceptible to Apoptin induced cell death than the parental matched MM1.S cells. This correlated with increased phosphorylation and activation of the Apoptin protein in MM1.R cells. Expression profiling of MM1.R and MM1.S cells identified a number of differentially expressed kinases. PKCb was over-expressed 9 fold in MM1.R cells and we showed, by immunoprecipitation and in vivo kinase studies, that this kinase was responsible for Apoptin phosphorylation. Analysis of the Apoptin amino acid sequence for potential phosphorylation sites indicated seven putative phosphorylation sites corresponding to the PKC kinase consensus motifs (S/TXK/R or S/TXXK/R). These sites included Thr-108, which has been previously shown to be phosphorylated in tumor cells, but not in normal cells. In vitro studies showed that recombinant Apoptin protein was phosphorylated by recombinant GST-PKCb protein at the Thr-108 site. Addition of a PKCb specific inhibitor resulted in diminished Apoptin phosphorylation whilst an unrelated inhibitor had no such effect. Furthermore, shRNA knockdown or drug mediated inhibition of PKCb in vivo significantly reduced Apoptin phosphorylation. Finally, we found that Apoptin mediated cell death proceeded via the up-regulation of PKCb, activation of caspase-9/3, cleavage of the PKCd catalytic domain and down-regulation of MERTK and AKT protein kinases. Collectively these results demonstrate a novel pathway for Apoptin activation involving PKCb and PKCd. Our results show that Apoptin is able to effectively eliminate multiple myeloma cells which have become resistant to dexamethasone. In addition, this study has led to the identification of tumor specific cellular targets such as PKCb, whose modulation by shRNAs and small molecule drugs can induce strong anti-myeloma effects. Importantly, the evidence from our data suggests that protein kinase C inhibitors may have an important therapeutic role in plasma cell neoplasia. Disclosures: No relevant conflicts of interest to declare.

Abstract The chicken anemia virus–derived protein apoptin induces apoptosis in a variety of human malignant and transformed cells but not in normal cells. However, the mechanisms through which apoptin achieves its selective killing effects are not well understood. We developed a lentiviral vector encoding a green fluorescent protein–apoptin fusion gene (LV-GFP-AP) that can efficiently deliver apoptin into hematopoietic cells. Apoptin selectively killed the human multiple myeloma cell lines MM1.R and MM1.S, and the leukemia cell lines K562, HL60, U937, KG1, and NB4. In contrast, normal CD34+ cells were not killed and maintained their differentiation potential in multilineage colony formation assays. In addition, dexamethasone-resistant MM1.R cells were found to be more susceptible to apoptin-induced cell death than the parental matched MM1.S cells. Death susceptibility correlated with increased phosphorylation and activation of the apoptin protein in MM1.R cells. Expression array profiling identified differential kinase profiles between MM1.R and MM1.S cells. Among these kinases, protein kinase Cβ (PKCβ) was found by immunoprecipitation and in vitro kinase studies to be a candidate kinase responsible for apoptin phosphorylation. Indeed, shRNA knockdown or drug-mediated inhibition of PKCβ significantly reduced apoptin phosphorylation. Furthermore, apoptin-mediated cell death proceeded through the upregulation of PKCβ, activation of caspase-9/3, cleavage of the PKCδ catalytic domain, and downregulation of the MERTK and AKT kinases. Collectively, these results elucidate a novel pathway for apoptin activation involving PKCβ and PKCδ. Further, they highlight the potential of apoptin and its cellular regulators to purge bone marrow used in autologous transplantation for multiple myeloma. Cancer Res; 70(18); 7242–52. ©2010 AACR.

Abstract Abstract 982 Mounting evidence on the role of tumour microenvironment in supporting the growth and survival of multiple myeloma (MM) and other tumour cells makes testing of potential drug treatments in vitro in this setting almost obligatory. It is becoming evident that effective therapeutic approaches against MM must target not only MM cell viability but also the pro-survival support of the tumour cellular and non-cellular stroma. Recently developed strategies recreate the myeloma tumour microenvironment in vitro allowing for detection of myeloma cell proliferation or distribution in bone marrow compartments using cell imaging. However, there is a need for high troughput self-contained co-culture technology to facilitate differentiation in the behaviour of myeloma plasma cells from the accessory cells in the tumour microenvironment. Herein, we validate a high throughput in vitro co-culture experimental platform to analyse and measure simultaneously the effect of therapeutic agents on MM cells and the tumour stroma in co-culture. We have generated eGFP-MM cell lines (eGFP-MM1.S, eGFP-MM1.R, eGFP-U266) by lentiviral infection and clonal selection by limiting dilution and validated that eGFP-expressing cells maintain the properties of the parental cell lines. The same methodology was used to generate eGFP-K562 (myeloid malignancies), eGFP- PC3, eGFP-DU145 (both prostate cancer cell lines) and eGFP-HT29 (colon cancer cell line). We show that growth of eGFP-expressing cells can be estimated using fluorimetry (lexcitation 395/475; l emission 509) or image-based analysis in the presence of other BM cells in co-culture. The use of eGFP-MM cells also allows flow cytometry analysis of cell cycle profile and apoptosis with no significant cellular contamination from co-cultured cells such as fibroblasts, osteoclasts or stromal cells derived from bone marrow aspirates of MM patients. Additionally, we have generated and validated mCherry-expressing HS5 fibroblast cell lines as HS5 cells are commonly used to study the support by fibroblasts of proliferation and viability of MM cells. Proliferation of mCherry-HS5 cells can be evaluated using fluorimetry (l excitation 584; l emission 607) or image-based analysis in the presence of various tumour cells of haematological origin and solid tumours. The disadvantage of the MTT assay is that it is impossible to distinguish the signal of specific cell types in co-culture whereas the use of fluorescent cell lines allows us to discriminate these signals. We found that proliferation of stromal cells is differentially stimulated by different tumour cells and drug treatments. Additionally, the spatial organisation of stromal cells is also specifically altered depending on the type of tumour. These results suggest that tumour cells generate distinct signals that can affect stromal cells specifically resulting in explicit therapeutic requirements to prevent the support of the tumour microenvironment. We also found that when MM cells were seeded at low densitiy, stromal cells promote MM cell proliferation and resistance to therapeutic agents. However, when MM cells were plated at high densities and can easily enter exponential growth, tumour cells can grow independently of the presence of the stroma. However both at high and low densities, stromal cells abrogated either partially or completely the pro-apoptotic effect of therapeutic drugs. The use of eGFP-MM and mCherry HS5 cells enables application of laboratory techniques to effectively distinguish and screen the effects of drugs on the biology of myeloma cells and stromal cells in a co-culture setting that reproduces more accurately some key aspects of the tumour microenvironment. We propose the use of this experimental platform to evaluate therapeutic drugs against MM as well as other malignancies. Disclosures: No relevant conflicts of interest to declare.