Publications by authors named "Andrew C W Zannettino"

129 Publications

CKLF and IL1B transcript levels at diagnosis are predictive of relapse in children with pre-B-cell acute lymphoblastic leukaemia.

Br J Haematol 2021 Apr 23;193(1):171-175. Epub 2021 Feb 23.

Adelaide Medical School, Faculty of Health and Medical Science, University of Adelaide, Adelaide, SA, Australia.

Disease relapse is the greatest cause of treatment failure in paediatric B-cell acute lymphoblastic leukaemia (B-ALL). Current risk stratifications fail to capture all patients at risk of relapse. Herein, we used a machine-learning approach to identify B-ALL blast-secreted factors that are associated with poor survival outcomes. Using this approach, we identified a two-gene expression signature (CKLF and IL1B) that allowed identification of high-risk patients at diagnosis. This two-gene expression signature enhances the predictive value of current at diagnosis or end-of-induction risk stratification suggesting the model can be applied continuously to help guide implementation of risk-adapted therapies.
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http://dx.doi.org/10.1111/bjh.17161DOI Listing
April 2021

The mTORC1 complex in pre-osteoblasts regulates whole-body energy metabolism independently of osteocalcin.

Bone Res 2021 Feb 8;9(1):10. Epub 2021 Feb 8.

Adelaide Medical School, Faculty of Health and Medical Science, University of Adelaide, Adelaide, 5005, SA, Australia.

Overnutrition causes hyperactivation of mTORC1-dependent negative feedback loops leading to the downregulation of insulin signaling and development of insulin resistance. In osteoblasts (OBs), insulin signaling plays a crucial role in the control of systemic glucose homeostasis. We utilized mice with conditional deletion of Rptor to investigate how the loss of mTORC1 function in OB affects glucose metabolism under normal and overnutrition dietary states. Compared to the controls, chow-fed Rptor mice had substantially less fat mass and exhibited adipocyte hyperplasia. Remarkably, upon feeding with high-fat diet, mice with pre- and post-natal deletion of Rptor in OBs were protected from diet-induced obesity and exhibited improved glucose metabolism with lower fasting glucose and insulin levels, increased glucose tolerance and insulin sensitivity. This leanness and resistance to weight gain was not attributable to changes in food intake, physical activity or lipid absorption but instead was due to increased energy expenditure and greater whole-body substrate flexibility. RNA-seq revealed an increase in glycolysis and skeletal insulin signaling pathways, which correlated with the potentiation of insulin signaling and increased insulin-dependent glucose uptake in Rptor-knockout osteoblasts. Collectively, these findings point to a critical role for the mTORC1 complex in the skeletal regulation of whole-body glucose metabolism and the skeletal development of insulin resistance.
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http://dx.doi.org/10.1038/s41413-020-00123-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7868369PMC
February 2021

Macrophages in multiple myeloma: key roles and therapeutic strategies.

Cancer Metastasis Rev 2021 Mar 6;40(1):273-284. Epub 2021 Jan 6.

Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia.

Macrophages are a vital component of the tumour microenvironment and crucial mediators of tumour progression. In the last decade, significant strides have been made in understanding the crucial functional roles played by macrophages in the development of the plasma cell (PC) malignancy, multiple myeloma (MM). Whilst the interaction between MM PC and stromal cells within the bone marrow (BM) microenvironment has been extensively studied, we are only just starting to appreciate the multifaceted roles played by macrophages in disease progression. Accumulating evidence demonstrates that macrophage infiltration is associated with poor overall survival in MM. Indeed, macrophages influence numerous pathways critical for the initiation and progression of MM, including homing of malignant cells to BM, tumour cell growth and survival, drug resistance, angiogenesis and immune suppression. As such, therapeutic strategies aimed at targeting macrophages within the BM niche have promise in the clinical setting. This review will discuss the functions elicited by macrophages throughout different stages of MM and provide a comprehensive evaluation of potential macrophage-targeted therapies.
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http://dx.doi.org/10.1007/s10555-020-09943-1DOI Listing
March 2021

Tumour Dissemination in Multiple Myeloma Disease Progression and Relapse: A Potential Therapeutic Target in High-Risk Myeloma.

Cancers (Basel) 2020 Dec 4;12(12). Epub 2020 Dec 4.

Myeloma Research Laboratory, Faculty of Health and Medical Sciences, Adelaide Medical School, The University of Australia, Adelaide 5005, Australia.

Multiple myeloma (MM) is a plasma cell (PC) malignancy characterised by the presence of MM PCs at multiple sites throughout the bone marrow. Increased numbers of peripheral blood MM PCs are associated with rapid disease progression, shorter time to relapse and are a feature of advanced disease. In this review, the current understanding of the process of MM PC dissemination and the extrinsic and intrinsic factors potentially driving it are addressed through analysis of patient-derived MM PCs and MM cell lines as well as mouse models of homing and dissemination. In addition, we discuss how patient cytogenetic subgroups that present with highly disseminated disease, such as t(4;14), t(14;16) and t(14;20), suggest that intrinsic properties of MM PC influence their ability to disseminate. Finally, we discuss the possibility of using therapeutic targeting of tumour dissemination to slow disease progression and prevent overt relapse.
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http://dx.doi.org/10.3390/cancers12123643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761917PMC
December 2020

The Balance of Stromal BMP Signaling Mediated by GREM1 and ISLR Drives Colorectal Carcinogenesis.

Gastroenterology 2021 Mar 14;160(4):1224-1239.e30. Epub 2020 Nov 14.

Adelaide Medical School, University of Adelaide, Adelaide, Australia; South Australian Health and Medical Research Institute, Adelaide, Australia. Electronic address:

Background & Aims: Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored.

Methods: Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC.

Results: We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor β and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5 intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)-mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis.

Conclusions: Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver.
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http://dx.doi.org/10.1053/j.gastro.2020.11.011DOI Listing
March 2021

Expression of the chemokine receptor CCR1 promotes the dissemination of multiple myeloma plasma cells in vivo.

Haematologica 2020 11 5;Online ahead of print. Epub 2020 Nov 5.

Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, Australia; Precision Medicine Theme, South Australian Health and Medical Research Institute, Adelaide.

Multiple myeloma (MM) disease progression is dependent on the ability of MM plasma cells (PCs) to egress from the bone marrow (BM), enter the circulation and disseminate to distal BM sites. Expression of the chemokine CXCL12 by BM stromal cells is crucial for MM PC retention within the BM. However, the mechanisms which overcome CXCL12-mediated retention to enable dissemination are poorly understood. We have previously identified that treatment with the CCR1 ligand CCL3 inhibits the response to CXCL12 in MM cell lines, suggesting that CCL3/CCR1 signalling may enable egress of MM PC from the BM. Here, we demonstrated that CCR1 expression was an independent prognostic indicator in newly diagnosed MM patients. Furthermore, we showed that CCR1 is a crucial driver of dissemination in vivo, with CCR1 expression in the murine MM cell line 5TGM1 being associated with an increased incidence of bone and splenic disseminated tumours in C57BL/KaLwRij mice. Furthermore, we demonstrated that CCR1 knockout in the human myeloma cell line OPM2 resulted in a >95% reduction in circulating MM PC numbers and BM and splenic tumour dissemination following intratibial injection in NSG mice. Therapeutic targeting of CCR1 with the inhibitor CCX9588 significantly reduced OPM2 or RPMI-8226 dissemination in intratibial xenograft models. Collectively, our findings suggest a novel role for CCR1 as a critical driver of BM egress of MM PCs during tumour dissemination. Furthermore, these data suggest that CCR1 may represent a potential therapeutic target for the prevention of MM tumour dissemination.
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http://dx.doi.org/10.3324/haematol.2020.253526DOI Listing
November 2020

Characterization of the role of Samsn1 loss in multiple myeloma development.

FASEB Bioadv 2020 Sep 5;2(9):554-572. Epub 2020 Aug 5.

Myeloma Research Laboratory Adelaide Medical School Faculty of Health and Medical Sciences University of Adelaide Adelaide Australia.

The protein SAMSN1 was recently identified as a putative tumor suppressor in multiple myeloma, with re-expression of Samsn1 in the 5TGM1/KaLwRij murine model of myeloma leading to a near complete abrogation of intramedullary tumor growth. Here, we sought to clarify the mechanism underlying this finding. Intratibial administration of 5TGM1 myeloma cells into KaLwRij mice revealed that Samsn1 had no effect on primary tumor growth, but that its expression significantly inhibited the metastasis of these primary tumors. Notably, neither in vitro nor in vivo migration was affected by Samsn1 expression. Both knocking-out SAMSN1 in the RPMI-8226 and JJN3 human myeloma cell lines, and retrovirally expressing SAMSN1 in the LP-1 and OPM2 human myeloma cell lines had no effect on either cell proliferation or migration in vitro. Altering SAMSN1 expression in these human myeloma cells did not affect the capacity of the cells to establish either primary or metastatic intramedullary tumors when administered intratibially into immune deficient NSG mice. Unexpectedly, the tumor suppressive and anti-metastatic activity of Samsn1 in 5TGM1 cells were not evidenced following cell administration either intratibially or intravenously to NSG mice. Crucially, the growth of Samsn1-expressing 5TGM1 cells was limited in C57BL/6/Samsn1 mice but not in C57BL/6 Samsn1 mice. We conclude that the reported potent in vivo tumor suppressor activity of Samsn1 can be attributed, in large part, to graft-rejection from Samsn1 recipient mice. This has broad implications for the design and interpretation of experiments that utilize cancer cells and knockout mice that are mismatched for expression of specific proteins.
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http://dx.doi.org/10.1096/fba.2020-00027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7475304PMC
September 2020

Targeted Disruption of Bone Marrow Stromal Cell-Derived Gremlin1 Limits Multiple Myeloma Disease Progression In Vivo.

Cancers (Basel) 2020 Aug 3;12(8). Epub 2020 Aug 3.

Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA 5000, Australia.

In most instances, multiple myeloma (MM) plasma cells (PCs) are reliant on factors made by cells of the bone marrow (BM) stroma for their survival and growth. To date, the nature and cellular composition of the BM tumor microenvironment and the critical factors which drive tumor progression remain imprecisely defined. Our studies show that Gremlin1 (Grem1), a highly conserved protein, which is abundantly secreted by a subset of BM mesenchymal stromal cells, plays a critical role in MM disease development. Analysis of human and mouse BM stromal samples by quantitative PCR showed that / expression was significantly higher in the MM tumor-bearing cohorts compared to healthy controls ( < 0.05, Mann-Whitney test). Additionally, BM-stromal cells cultured with 5TGM1 MM PC line expressed significantly higher levels of , compared to stromal cells alone ( < 0.01, -test), suggesting that MM PCs promote increased expression in stromal cells. Furthermore, the proliferation of 5TGM1 MM PCs was found to be significantly increased when co-cultured with -overexpressing stromal cells ( < 0.01, -test). To examine the role of Grem1 in MM disease in vivo, we utilized the 5TGM1/KaLwRij mouse model of MM. Our studies showed that, compared to immunoglobulin G (IgG) control antibody-treated mice, mice treated with an anti-Grem1 neutralizing antibody had a decrease in MM tumor burden of up to 81.2% ( < 0.05, two-way ANOVA). The studies presented here demonstrate, for the first time, a novel positive feedback loop between MM PCs and BM stroma, and that inhibiting this vicious cycle with a neutralizing antibody can dramatically reduce tumor burden in a preclinical mouse model of MM.
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http://dx.doi.org/10.3390/cancers12082149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464474PMC
August 2020

LCRF-0006, a small molecule mimetic of the N-cadherin antagonist peptide ADH-1, synergistically increases multiple myeloma response to bortezomib.

FASEB Bioadv 2020 Jun 15;2(6):339-353. Epub 2020 Jun 15.

Myeloma Research Laboratory Adelaide Medical School Faculty of Health and Medical Sciences The University of Adelaide Adelaide Australia.

N-cadherin is a homophilic cell-cell adhesion molecule that plays a critical role in maintaining vascular stability and modulating endothelial barrier permeability. Pre-clinical studies have shown that the N-cadherin antagonist peptide, ADH-1, increases the permeability of tumor-associated vasculature thereby increasing anti-cancer drug delivery to tumors and enhancing tumor response. Small molecule library screens have identified a novel compound, LCRF-0006, that is a mimetic of the classical cadherin His-Ala-Val sequence-containing region of ADH-1. Here, we evaluated the vascular permeability-enhancing and anti-cancer properties of LCRF-0006 using in vitro vascular disruption and cell apoptosis assays, and a well-established pre-clinical model (C57BL/KaLwRij/5TGM1) of the hematological cancer multiple myeloma (MM). We found that LCRF-0006 disrupted endothelial cell junctions in a rapid, transient and reversible manner, and increased vascular permeability in vitro and at sites of MM tumor in vivo. Notably, LCRF-0006 synergistically increased the in vivo anti-MM tumor response to low-dose bortezomib, a frontline anti-MM agent, leading to regression of disease in 100% of mice. Moreover, LCRF-0006 and bortezomib synergistically induced 5TGM1 MM tumor cell apoptosis in vitro. Our findings demonstrate the potential clinical utility of LCRF-0006 to significantly increase bortezomib effectiveness and enhance the depth of tumor response in patients with MM.
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http://dx.doi.org/10.1096/fba.2019-00073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325588PMC
June 2020

P-Related Protein Accelerates Human Mesenchymal Stromal Cell Migration by Modulating VLA-5 Interactions with Fibronectin.

Cells 2020 04 29;9(5). Epub 2020 Apr 29.

Stem Cell Research, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9BQ, UK.

P-related protein (PZR), a Noonan and Leopard syndrome target, is a member of the transmembrane Immunoglobulin superfamily. Its cytoplasmic tail contains two immune-receptor tyrosine-based inhibitory motifs (ITIMs), implicated in adhesion-dependent signaling and regulating cell adhesion and motility. PZR promotes cell migration on the extracellular matrix (ECM) molecule, fibronectin, by interacting with SHP-2 (Src homology-2 domain-containing protein tyrosine phosphatase-2), a molecule essential for skeletal development and often mutated in Noonan and Leopard syndrome patients sharing overlapping musculoskeletal abnormalities and cardiac defects. To further explore the role of PZR, we assessed the expression of PZR and its ITIM-less isoform, PZRb, in human bone marrow mesenchymal stromal cells (hBM MSC), and its ability to facilitate adhesion to and spreading and migration on various ECM molecules. Furthermore, using siRNA knockdown, confocal microscopy, and immunoprecipitation assays, we assessed PZR and PZRb interactions with β1 integrins. PZR was the predominant isoform in hBM MSC. Migrating hBM MSCs interacted most effectively with fibronectin and required the association of PZR, but not PZRb, with the integrin, VLA-5(α5β1), leading to modulation of focal adhesion kinase phosphorylation and vinculin levels. This raises the possibility that dysregulation of PZR function may modify hBM MSC migratory behavior, potentially contributing to skeletal abnormalities.
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http://dx.doi.org/10.3390/cells9051100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290418PMC
April 2020

CMTM8 Is a Suppressor of Human Mesenchymal Stem Cell Osteogenic Differentiation and Promoter of Proliferation Via EGFR Signaling.

Stem Cells Dev 2020 07 4;29(13):823-834. Epub 2020 May 4.

South Australian Health and Medical Research Institute (SAHMRI), Adelaide, Australia.

Multipotent bone marrow-derived mesenchymal stem/stromal cells (BMSCs) exhibit a finite life span after ex vivo expansion leading to cellular senescence. Many factors can contribute to this. Recently, our group has identified for the first time expression of the chemokine-like factor superfamily 8 (CMTM8) gene in cultured human BMSCs. In this study, we examine the role of CMTM8 in BMSC proliferation, migration, and differentiation. Functional studies using siRNA-mediated knockdown of in human BMSCs resulted in decreased capacity to undergo proliferation and migration and an increased capacity for osteogenic differentiation in vitro. Furthermore, reduced levels led to a decrease in the epidermal growth factor receptor (EGFR) signaling pathway during BMSC proliferation and migration, respectively. Supportive studies using retroviral mediated enforced expression of in BMSC resulted in an increased capacity for proliferation and migration but a decreased osteogenic differentiation potential. Collectively, these data suggest that CMTM8 promotes BMSC proliferation and BMSC migration through the EGFR/ERK1/2 pathway. This study provides insight into novel regulatory mechanisms of human BMSC growth and cell fate determination.
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http://dx.doi.org/10.1089/scd.2020.0007DOI Listing
July 2020

Reciprocal signaling between mTORC1 and MNK2 controls cell growth and oncogenesis.

Cell Mol Life Sci 2021 Jan 13;78(1):249-270. Epub 2020 Mar 13.

Lifelong Health Theme, South Australian Health and Medical Research Institute, North Terrace, Adelaide, SA, 5000, Australia.

eIF4E plays key roles in protein synthesis and tumorigenesis. It is phosphorylated by the kinases MNK1 and MNK2. Binding of MNKs to eIF4G enhances their ability to phosphorylate eIF4E. Here, we show that mTORC1, a key regulator of mRNA translation and oncogenesis, directly phosphorylates MNK2 on Ser74. This suppresses MNK2 activity and impairs binding of MNK2 to eIF4G. These effects provide a novel mechanism by which mTORC1 signaling impairs the function of MNK2 and thereby decreases eIF4E phosphorylation. MNK2[S74A] knock-in cells show enhanced phosphorylation of eIF4E and S6K1 (i.e., increased mTORC1 signaling), enlarged cell size, and increased invasive and transformative capacities. MNK2[Ser74] phosphorylation was inversely correlated with disease progression in human prostate tumors. MNK inhibition exerted anti-proliferative effects in prostate cancer cells in vitro. These findings define a novel feedback loop whereby mTORC1 represses MNK2 activity and oncogenic signaling through eIF4E phosphorylation, allowing reciprocal regulation of these two oncogenic pathways.
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http://dx.doi.org/10.1007/s00018-020-03491-1DOI Listing
January 2021

Twist-1 is upregulated by NSD2 and contributes to tumour dissemination and an epithelial-mesenchymal transition-like gene expression signature in t(4;14)-positive multiple myeloma.

Cancer Lett 2020 04 31;475:99-108. Epub 2020 Jan 31.

Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, Australia; Precision Medicine Theme, South Australian Health & Medical Research Institute (SAHMRI), Adelaide, Australia. Electronic address:

Approximately 15% of patients with multiple myeloma (MM) harbour the t(4;14) chromosomal translocation, leading to the overexpression of the histone methyltransferase NSD2. Patients with this translocation display increased tumour dissemination, accelerated disease progression and rapid relapse. Using publicly available gene expression profile data from NSD2 (n = 135) and NSD2 (n = 878) MM patients, we identified 39 epithelial-mesenchymal transition (EMT)-associated genes which are overexpressed in NSD2 MM plasma cells. In addition, our analyses identified Twist-1 as a key transcription factor upregulated in NSD2 MM patients and t(4;14)-positive cell lines. Overexpression and knockdown studies confirmed that Twist-1 is involved in driving the expression of EMT-associated genes in the human MM cell line KMS11 and promoted the migration of myeloma cell lines in vitro. Notably, Twist-1 overexpression in the mouse MM cell line 5TGM1 significantly increased tumour dissemination in an intratibial tumour model. These findings demonstrate that Twist-1, downstream of NSD2, contributes to the induction of an EMT-like signature in t(4;14)-positive MM and enhances the dissemination of MM plasma cells in vivo, which may, in part, explain the aggressive disease features associated with t(4;14)-positive MM.
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http://dx.doi.org/10.1016/j.canlet.2020.01.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829063PMC
April 2020

Fabrication of a Cartilage Patch by Fusing Hydrogel-Derived Cell Aggregates onto Electrospun Film.

Tissue Eng Part A 2020 08 19;26(15-16):863-871. Epub 2020 Mar 19.

School of Chemical Engineering and Advanced Materials, The University of Adelaide, Adelaide, Australia.

Irregular defects at sites of degenerative cartilage often accompany osteoarthritis (OA). The development of novel cell-/biomaterial-based cartilage tissue engineering methods to address these defects may provide a durable approach to hinder the development of OA. In this study, we fabricated a neocartilage patch by fusing cell aggregates onto a biodegradable nanofiber film for degenerative cartilage repair. Human mesenchymal stem/stromal cell (MSC) aggregates were prepared and induced for chondrogenesis in a thermosensitive hydrogel, poly (-isopropylacrylamide-co-acrylic acid (p(NIPAAm-AA)). Cell migration mediated the formation of cell aggregates in the thermosensitive hydrogel and led to a cell-dense hollow shell structure. The chondrocytes derived from MSC aggregates in the hydrogel were evidenced by the expression of chondrogenesis-related genes and extracellular matrices. They were fused onto an electrospun film by mechanical force and spatial confinement to generate a neo-cartilage patch. The fabricated neocartilage patches may be able to integrate into the irregular defects under compressive stresses and achieve cartilage regeneration . Impact statement The formation of human mesenchymal stem/stromal cells aggregates in thermosensitive hydrogels was mechanistically examined. These formed cell aggregates with enhanced chondrogenesis were bioengineered into a neocartilage patch for regeneration of superficial irregular cartilage defects.
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http://dx.doi.org/10.1089/ten.TEA.2019.0318DOI Listing
August 2020

GLIPR1 expression is reduced in multiple myeloma but is not a tumour suppressor in mice.

PLoS One 2020 29;15(1):e0228408. Epub 2020 Jan 29.

Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, Australia.

Multiple myeloma, a plasma cell malignancy, is a genetically heterogeneous disease and the genetic factors that contribute to its development and progression remain to be fully elucidated. The tumour suppressor gene GLIPR1 has previously been shown to be deleted in approximately 10% of myeloma patients, to inhibit the development of plasma cell tumours in ageing mice and to have reduced expression levels in the plasma cells of patients with light-chain amyloidosis, a myeloma-related malignancy. Therefore, we hypothesised that GLIPR1 may have tumour suppressor activity in multiple myeloma. In this study, we demonstrate that plasma cell expression of GLIPR1 is reduced in the majority of myeloma patients and Glipr1 expression is lost in the 5TGM1 murine myeloma cell line. However, overexpression of GLIPR1 in a human myeloma cell line did not affect cell proliferation in vitro. Similarly, re-expression of Glipr1 in 5TGM1 cells did not significantly reduce their in vitro proliferation or in vivo growth in C57BL/KaLwRij mice. In addition, using CRISPR-Cas9 genome editing, we generated C57BL/Glipr1-/- mice and showed that loss of Glipr1 in vivo did not affect normal haematopoiesis or the development of monoclonal plasma cell expansions in these mice up to one year of age. Taken together, our results suggest that GLIPR1 is unlikely to be a potent tumour suppressor in multiple myeloma. However, it remains possible that the down-regulation of GLIPR1 may cooperate with other genetic lesions to promote the development of myeloma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228408PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988976PMC
April 2020

Conditional knockout of ephrinB1 in osteogenic progenitors delays the process of endochondral ossification during fracture repair.

Bone 2020 03 19;132:115189. Epub 2019 Dec 19.

Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia; Cancer Theme, South Australian Health and Medical Research Institute, Adelaide, SA, Australia. Electronic address:

The Eph receptor tyrosine kinase ligand, ephrinB1 (EfnB1) is important for correct skeletal and cartilage development, however, the role of EfnB1 in fracture repair is unknown. This study investigated the role of EfnB1 during fracture repair where EfnB1 expression increased significantly at 1 and 2 weeks post fracture in C57Bl/6 wildtype mice, coinciding with the haematoma, soft callus formation/remodelling stages, respectively. To investigate the specific role of EfnB1 within the osteogenic lineage during fracture repair, male mice with a conditional deletion of EfnB1 in the osteogenic lineage (EfnB1), driven by the Osterix (Osx) promoter, and their male Osx:Cre counterparts were subject to a femoral fracture with internal fixation. Two weeks post fracture micro computed tomography (μCT) analysis revealed that EfnB1 mice displayed a significant decrease in bone volume relative to tissue volume within the fracture callus. This was attributed to an alteration in the distribution of osteoclasts within the fracture site, a significant elevation in cartilaginous tissue and reduction in the osteoprogenitor population and calcein labelled bone within the fracture site of EfnB1 mice. Supportive in vitro studies demonstrated that under osteogenic conditions, cultured EfnB1 stromal cells derived from the 2 week fracture site exhibited a reduced capacity to produce mineral and decreased expression of the osteogenic gene, Osterix, when compared to Osx:Cre controls. These findings suggest that the loss of EfnB1 delays the fracture repair process. The present study confirmed that EFNB1 activation in human BMSC, following stimulation with soluble-EphB2 resulted in de-phosphorylation of TAZ, demonstrating similarities in EfnB1 signalling between human and mouse stromal populations. Overall, the present study provides evidence that loss of EfnB1 in the osteo/chondrogenic lineages delays the soft callus formation/remodelling stages of the fracture repair process.
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http://dx.doi.org/10.1016/j.bone.2019.115189DOI Listing
March 2020

A niche-dependent myeloid transcriptome signature defines dormant myeloma cells.

Blood 2019 07 25;134(1):30-43. Epub 2019 Apr 25.

Division of Bone Biology, Garvan Institute of Medical Research, Sydney, NSW, Australia.

The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.
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http://dx.doi.org/10.1182/blood.2018880930DOI Listing
July 2019

Using genomics to better define high-risk MGUS/SMM patients.

Oncotarget 2018 Nov 27;9(93):36549-36550. Epub 2018 Nov 27.

Andrew C.W. Zannettino: Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, SA, Australia; Cancer Theme, South Australian Health and Medical Research Institute (SAHMRI), Adelaide, SA, Australia.

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http://dx.doi.org/10.18632/oncotarget.26390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290965PMC
November 2018

Loss of EfnB1 in the osteogenic lineage compromises their capacity to support hematopoietic stem/progenitor cell maintenance.

Exp Hematol 2019 01 13;69:43-53. Epub 2018 Oct 13.

Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, South Australia, Australia; Cancer Theme, South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia. Electronic address:

The bone marrow stromal microenvironment contributes to the maintenance and function of hematopoietic stem/progenitor cells (HSPCs). The Eph receptor tyrosine kinase family members have been implicated in bone homeostasis and stromal support of HSPCs. The present study examined the influence of EfnB1-expressing osteogenic lineage on HSPC function. Mice with conditional deletion of EfnB1 in the osteogenic lineage (EfnB1), driven by the Osterix promoter, exhibited a reduced prevalence of osteogenic progenitors and osteoblasts, correlating to lower numbers of HSPCs compared with Osx:Cre mice. Long-term culture-initiating cell (LTC-IC) assays confirmed that the loss of EfnB1 within bone cells hindered HSPC function, with a significant reduction in colony formation in EfnB1 mice compared with Osx:Cre mice. Human studies confirmed that activation of EPHB2 on CD34 HSPCs via EFNB1-Fc stimulation enhanced myeloid/erythroid colony formation, whereas functional blocking of either EPHB1 or EPHB2 inhibited the maintenance of LTC-ICs. Moreover, EFNB1 reverse signaling in human and mouse stromal cells was found to be required for the activation of the HSPC-promoting factor CXCL12. Collectively, the results of this study confirm that EfnB1 contributes to the stromal support of HSPC function and maintenance and may be an important factor in regulating the HSPC niche.
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http://dx.doi.org/10.1016/j.exphem.2018.10.004DOI Listing
January 2019

mTORC1 plays an important role in osteoblastic regulation of B-lymphopoiesis.

Sci Rep 2018 09 28;8(1):14501. Epub 2018 Sep 28.

Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, Australia.

Skeletal osteoblasts are important regulators of B-lymphopoiesis, serving as a rich source of factors such as CXCL12 and IL-7 which are crucial for B-cell development. Recent studies from our laboratory and others have shown that deletion of Rptor, a unique component of the mTORC1 nutrient-sensing complex, early in the osteoblast lineage development results in defective bone development in mice. In this study, we now demonstrate that mTORC1 signalling in pre-osteoblasts is required for normal B-lymphocyte development in mice. Targeted deletion of Rptor in osterix-expressing pre-osteoblasts (Rptor) leads to a significant reduction in the number of B-cells in the bone marrow, peripheral blood and spleen at 4 and 12 weeks of age. Rptor mice also exhibit a significant reduction in pre-B and immature B-cells in the BM, indicative of a block in B-cell development from the pro-B to pre-B cell stage. Circulating levels of IL-7 and CXCL12 are also significantly reduced in Rptor mice. Importantly, whilst Rptor-deficient osteoblasts are unable to support HSC differentiation to B-cells in co-culture, this can be rescued by the addition of exogenous IL-7 and CXCL12. Collectively, these findings demonstrate that mTORC1 plays an important role in extrinsic osteoblastic regulation of B-cell development.
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http://dx.doi.org/10.1038/s41598-018-32858-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162303PMC
September 2018

miRNA-376c-3p Mediates TWIST-1 Inhibition of Bone Marrow-Derived Stromal Cell Osteogenesis and Can Reduce Aberrant Bone Formation of TWIST-1 Haploinsufficient Calvarial Cells.

Stem Cells Dev 2018 12 23;27(23):1621-1633. Epub 2018 Oct 23.

1 Mesenchymal Stem Cell Laboratory, Faculty of Health and Medical Sciences, Adelaide Medical School, The University of Adelaide , Adelaide, Australia .

Key transcription factors, which activate or repress master gene regulators and signaling pathways, tightly regulate self-renewal and cell lineage differentiation of bone marrow-derived stromal cells (BMSC). Among these factors is the basic helix-loop-helix transcription factor Twist-related protein 1 (TWIST-1), which is important in BMSC self-renewal, life span, and differentiation. Another layer of gene regulation comes from microRNAs (miRNAs). miRNAs are short noncoding RNAs that interfere with translation of specific target mRNAs and thereby regulate diverse biological processes, including BMSC lineage commitment. However, little is known of how TWIST-1-regulated miRNAs control osteogenic commitment, and influence the fate of bone precursor cells. In this study, we have discovered a novel TWIST-1-regulated miRNA, miR-376c-3p. Reduced miR-376c-3p expression by a miR-376c-3p inhibitor or due to TWIST-1 haploinsufficiency promotes alkaline phosphatase (ALP) activity, mineral deposition, and expression of osteoblast-associated genes in BMSC and calvarial cells. Conversely, overexpression of miR-376c-3p using a miR-376c-3p mimic inhibited BMSC proliferation and the osteogenic potential of BMSC and TWIST-1 haploinsufficient calvarial cells. This was demonstrated by a decrease in insulin growth factor 1 receptor (IGF1R) levels, Akt signaling, ALP activity, mineral deposition, and expression of osteoblast-associated genes. Thus, miR-376c-3p reduces IGF1R/Akt signaling in BMSC and is one mechanism by which osteogenesis may be inhibited. Overall, we have identified miR-376c-3p as a TWIST-1-regulated miRNA, which plays an important role in the osteogenesis of bone precursor cells and can mediate TWIST-1 inhibition of osteogenesis. Furthermore, overexpression of miRNA-376c-3p in TWIST-1 haploinsufficient calvarial cells can decrease the aberrant osteogenesis of these cells, which contributes to increased calvarial bone volume and premature fusion of the coronal sutures.
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http://dx.doi.org/10.1089/scd.2018.0083DOI Listing
December 2018

The osteoprogenitor-specific loss of ephrinB1 results in an osteoporotic phenotype affecting the balance between bone formation and resorption.

Sci Rep 2018 08 24;8(1):12756. Epub 2018 Aug 24.

Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, 5005, SA, Australia.

The present study investigated the effects of conditional deletion of ephrinB1 in osteoprogenitor cells driven by the Osterix (Osx) promoter, on skeletal integrity in a murine model of ovariectomy-induced (OVX) osteoporosis. Histomorphometric and μCT analyses revealed that loss of ephrinB1 in sham Osx:cre-ephrinB1 mice caused a reduction in trabecular bone comparable to OVX Osx:Cre mice, which was associated with a significant reduction in bone formation rates and decrease in osteoblast numbers. Interestingly, these observations were not exacerbated in OVX Osx:cre-ephrinB1 mice. Furthermore, sham Osx:cre-ephrinB1 mice displayed significantly higher osteoclast numbers and circulating degraded collagen type 1 compared to OVX Osx:Cre mice. Confirmation studies found that cultured monocytes expressing EphB2 formed fewer TRAP multinucleated osteoclasts and exhibited lower resorption activity in the presence of soluble ephrinB1-Fc compared to IgG control. This inhibition of osteoclast formation and function induced by ephrinB1-Fc was reversed in the presence of an EphB2 chemical inhibitor. Collectively, these observations suggest that ephrinB1, expressed by osteoprogenitors, influences bone loss during the development of osteoporosis, by regulating both osteoblast and osteoclast formation and function, leading to a loss of skeletal integrity.
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http://dx.doi.org/10.1038/s41598-018-31190-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109077PMC
August 2018

Subclonal evolution in disease progression from MGUS/SMM to multiple myeloma is characterised by clonal stability.

Leukemia 2019 02 25;33(2):457-468. Epub 2018 Jul 25.

Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, SA, 5005, Australia.

Multiple myeloma (MM) is a largely incurable haematological malignancy defined by the clonal proliferation of malignant plasma cells (PCs) within the bone marrow. Clonal heterogeneity has recently been established as a feature in MM, however, the subclonal evolution associated with disease progression has not been described. Here, we performed whole-exome sequencing of serial samples from 10 patients, providing new insights into the progression from monoclonal gammopathy of undetermined significance (MGUS) and smouldering MM (SMM), to symptomatic MM. We confirm that intraclonal genetic heterogeneity is a common feature at diagnosis and that the driving events involved in disease progression are more subtle than previously reported. We reveal that MM evolution is mainly characterised by the phenomenon of clonal stability, where the transformed subclonal PC populations identified at MM are already present in the asymptomatic MGUS/SMM stages. Our findings highlight the possibility that PC extrinsic factors may play a role in subclonal evolution and MGUS/SMM to MM progression.
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http://dx.doi.org/10.1038/s41375-018-0206-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365384PMC
February 2019

Osteopontin in the pathophysiology of obesity: Is Opn a fat cell foe?

Obes Res Clin Pract 2018 May - Jun;12(3):249-250

Myeloma Research Laboratory, Faculty of Health and Medical Sciences, Adelaide Medical School, University of Adelaide and the Cancer Theme, South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia.

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http://dx.doi.org/10.1016/j.orcp.2018.06.004DOI Listing
April 2019

Tyrosine kinase receptor c-ros-oncogene 1 inhibition alleviates aberrant bone formation of TWIST-1 haploinsufficient calvarial cells from Saethre-Chotzen syndrome patients.

J Cell Physiol 2018 09 16;233(9):7320-7332. Epub 2018 Apr 16.

Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, South Australia, Australia.

Saethre-Chotzen syndrome (SCS), associated with TWIST-1 mutations, is characterized by premature fusion of cranial sutures. TWIST-1 haploinsufficiency, leads to alterations in suture mesenchyme cellular gene expression patterns, resulting in aberrant osteogenesis and craniosynostosis. We analyzed the expression of the TWIST-1 target, Tyrosine kinase receptor c-ros-oncogene 1 (C-ROS-1) in TWIST-1 haploinsufficient calvarial cells derived from SCS patients and calvaria of Twist-1 mutant mice and found it to be highly expressed when compared to TWIST-1 wild-type controls. Knock-down of C-ROS-1 expression in TWIST-1 haploinsufficient calvarial cells derived from SCS patients was associated with decreased capacity for osteogenic differentiation in vitro. Furthermore, treatment of human SCS calvarial cells with the tyrosine kinase chemical inhibitor, Crizotinib, resulted in reduced C-ROS-1 activity and the osteogenic potential of human SCS calvarial cells with minor effects on cell viability or proliferation. Cultured human SCS calvarial cells treated with Crizotinib exhibited a dose-dependent decrease in alkaline phosphatase activity and mineral deposition, with an associated decrease in expression levels of Runt-related transcription factor 2 and OSTEOPONTIN, with reduced PI3K/Akt signalling in vitro. Furthermore, Crizotinib treatment resulted in reduced BMP-2 mediated bone formation potential of whole Twist-1 mutant mouse calvaria organotypic cultures. Collectively, these results suggest that C-ROS-1 promotes osteogenic differentiation of TWIST-1 haploinsufficient calvarial osteogenic progenitor cells. Furthermore, the aberrant osteogenic potential of these cells is inhibited by the reduction of C-ROS-1. Therefore, targeting C-ROS-1 with a pharmacological agent, such as Crizotinib, may serve as a novel therapeutic strategy to alleviate craniosynostosis associated with aberrant TWIST-1 function.
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http://dx.doi.org/10.1002/jcp.26563DOI Listing
September 2018

Enhanced multi-lineage differentiation of human mesenchymal stem/stromal cells within poly(N-isopropylacrylamide-acrylic acid) microgel-formed three-dimensional constructs.

J Mater Chem B 2018 Mar 9;6(12):1799-1814. Epub 2018 Mar 9.

School of Chemical Engineering, The University of Adelaide, Adelaide, SA 5005, Australia.

Human mesenchymal stem/stromal cells (hMSCs) are a potential cell source of stem cell therapy for many serious diseases and hMSC spheroids have emerged to replace single cell suspensions for cell therapy. Three-dimensional (3D) scaffolds or hydrogels which can mimic properties of the extracellular matrix (ECM) have been widely explored for their application in tissue regeneration. However, there are considerably less studies on inducing differentiation of hMSC spheroids using 3D scaffolds or hydrogels. This study is the first to explore multi-lineage differentiation of a stem cell line and primary stem cells within poly(N-isopropylacrylamide) (p(NIPAAm))-based thermosensitive microgel-formed constructs. We first demonstrated that poly(N-isopropylacrylamide-co-acrylic acid) (p(NIPAAm-AA)) was not toxic to hMSCs and the microgel-formed constructs facilitated formation of uniform stem cell spheroids. Due to functional enhancement of cell spheroids, hMSCs within the 3D microgel-formed constructs were induced for multi-lineage differentiation as evidenced by significant up-regulation of messenger RNA (mRNA) expression of chondrogenic and osteogenic genes even in the absence of induction media on day 9. When induction media were in situ supplied on day 9, mRNA expression of chondrogenic, osteogenic and adipogenic genes within the microgel-formed constructs were significantly higher than that in the pellet and 2D cultures, respectively, on day 37. In addition, histological and immunofluorescent images also confirmed successful multi-lineage differentiation of hMSCs within the 3D microgel-formed constructs. Hence, the thermosensitive p(NIPAAm-AA) microgel can be potentially used in an in vitro model for cell differentiation or in vivo transplantation of pre-differentiated human mesenchymal stromal cells into patients for specific lineage differentiation.
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http://dx.doi.org/10.1039/c8tb00376aDOI Listing
March 2018

Pentosan polysulfate binds to STRO-1 mesenchymal progenitor cells, is internalized, and modifies gene expression: a novel approach of pre-programing stem cells for therapeutic application requiring their chondrogenesis.

Stem Cell Res Ther 2017 Dec 13;8(1):278. Epub 2017 Dec 13.

Proteobioactives Pty. Ltd., PO Box 174, Balgowlah, Sydney, New South Wales, 2093, Australia.

Background: The pharmaceutical agent pentosan polysulfate (PPS) is known to induce proliferation and chondrogenesis of mesenchymal progenitor cells (MPCs) in vitro and in vivo. However, the mechanism(s) of action of PPS in mediating these effects remains unresolved. In the present report we address this issue by investigating the binding and uptake of PPS by MPCs and monitoring gene expression and proteoglycan biosynthesis before and after the cells had been exposed to limited concentrations of PPS and then re-established in culture in the absence of the drug (MPC priming).

Methods: Immuno-selected STRO-1 mesenchymal progenitor stem cells (MPCs) were prepared from human bone marrow aspirates and established in culture. The kinetics of uptake, shedding, and internalization of PPS by MPCs was determined by monitoring the concentration-dependent loss of PPS media concentrations using an enzyme-linked immunosorbent assay (ELISA) and the uptake of fluorescein isothiocyanate (FITC)-labelled PPS by MPCs. The proliferation of MPCs, following pre-incubation and removal of PPS (priming), was assessed using the Wst-8 assay method, and proteoglycan synthesis was determined by the incorporation of SO into their sulphated glycosaminoglycans. The changes in expression of MPC-related cell surface antigens of non-primed and PPS-primed MPCs from three donors was determined using flow cytometry. RNA sequencing of RNA isolated from non-primed and PPS-primed MPCs from the same donors was undertaken to identify the genes altered by the PPS priming protocol.

Results: The kinetic studies indicated that, in culture, PPS rapidly binds to MPC surface receptors, followed by internalisation and localization within the nucleus of the cells. Following PPS-priming of MPCs and a further 48 h of culture, both cell proliferation and proteoglycan synthesis were enhanced. Reduced expression of MPC-related cell surface antigen expression was promoted by the PPS priming, and RNA sequencing analysis revealed changes in the expression of 42 genes.

Conclusion: This study has shown that priming of MPCs with low concentrations of PPS enhanced chondrogenesis and MPC proliferation by modifying their characteristic basal gene and protein expression. These findings offer a novel approach to re-programming mesenchymal stem cells for clinical indications which require the repair or regeneration of cartilaginous tissues such as in osteoarthritis and degenerative disc disease.
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http://dx.doi.org/10.1186/s13287-017-0723-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5729458PMC
December 2017

DNA Barcoding Reveals Habitual Clonal Dominance of Myeloma Plasma Cells in the Bone Marrow Microenvironment.

Neoplasia 2017 Dec 5;19(12):972-981. Epub 2017 Nov 5.

Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, South Australia, 5000, Australia; South Australian Health and Medical Research Institute, Adelaide, South Australia, 5000, Australia.

Multiple myeloma (MM) is a hematological malignancy resulting from the uncontrolled proliferation of antibody-producing plasma cells in the bone marrow. At diagnosis, independent plasma cell tumors are found throughout the skeleton. The recirculation of mutant plasma cells from the initial lesion and their recolonization of distant marrow sites are thought to occur by a process similar to solid tumor metastasis. However, the efficiency of this bone marrow homing process and the proportion of disseminated cells that actively divide and contribute to new tumor growth in MM are both unknown. We used the C57BL/KaLwRij mouse model of myeloma, lentiviral-mediated DNA barcoding of 5TGM1 myeloma cells, and next-generation sequencing to investigate the relative efficiency of plasma cell migration to, and growth within, the bone marrow. This approach revealed three major findings: firstly, establishment of metastasis within the bone marrow was extremely inefficient, with approximately 0.01% of circulating myeloma cells becoming resident long term in the bone marrow of each long bone; secondly, the individual cells of each metastasis exhibited marked differences in their proliferative fates, with the majority of final tumor burden within a bone being attributable to the progeny of between 1 and 8 cells; and, thirdly, the proliferative fate of individual clonal plasma cells differed at each bone marrow site in which the cells "landed." These findings suggest that individual myeloma plasma cells are subjected to vastly different selection pressures within the bone marrow microenvironment, highlighting the importance of niche-driven factors, which determine the disease course and outcome.
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http://dx.doi.org/10.1016/j.neo.2017.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678743PMC
December 2017

Mesenchymal progenitor cells primed with pentosan polysulfate promote lumbar intervertebral disc regeneration in an ovine model of microdiscectomy.

Spine J 2018 Mar 18;18(3):491-506. Epub 2017 Oct 18.

Department of Surgery, Monash University, 246 Clayton Rd, Clayton, VIC 3168, Australia; Department of Neurosurgery, Monash Medical Centre, 246 Clayton Rd, Clayton, VIC 3168, Australia; The Ritchie Centre, Hudson Institute of Medical Research, Monash University, 246 Clayton Rd, Clayton, VIC 3168, Australia.

Background Context: Neural compression associated with lumbar disc herniation is usually managed surgically by microdiscectomy. However, 10%-20% of patients re-present with debilitating back pain, and approximately 15% require further surgery.

Purpose: Using an ovine model of microdiscectomy, the present study investigated the relative potential of pentosan polysulfate-primed mesenchymal progenitor cells (pMPCs) or MPC alone implanted into the lesion site to facilitate disc recovery.

Study Design: An ovine model of lumbar microdiscectomy was used to compare the relative outcomes of administering MPCs or pMPCs to the injury site postsurgery.

Methods: At baseline 3T magnetic resonance imaging (MRI) of 18 adult ewes was undertaken followed by annular microdiscectomy at two lumbar disc levels. Sheep were randomized into three groups (n=6). The injured controls received no further treatment. Defects of the treated groups were implanted with a collagen sponge and MPC (5×10 cells) or pMPC (5×10 cells). After 6 months, 3T MRI and standard radiography were performed. Spinal columns were dissected, individual lumbar discs were sectioned horizontally, and nucleus pulposus (NP) and annulus fibrosus (AF) regions were assessed morphologically and histologically. The NP and AF tissues were dissected into six regions and analyzed biochemically for their proteoglycans (PGs), collagen, and DNA content.

Results: Both the MPC- and pMPC-injected groups exhibited less reduction in disc height (p<.05) and lower Pfirrmann grades (p≤.001) compared with the untreated injury controls, but morphologic scores for the pMPC-injected discs were lower (p<.05). The PG content of the AF injury site region (AF1) of pMPC discs was higher than MPC and injury control AF1 (p<.05). At the AF1 and contralateral AF2 regions, the DNA content of pMPC discs was significantly lower than injured control discs and MPC-injected discs. Histologic and birefringent microscopy revealed increased structural organization and reduced degeneration in pMPC discs compared with MPC and the injured controls.

Conclusions: In an ovine model 6 months after administration of pMPCs to the injury site disc PG content and matrix organization were improved relative to controls, suggesting pMPCs' potential as a postsurgical adjunct for limiting progression of disc degeneration after microdiscectomy.
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http://dx.doi.org/10.1016/j.spinee.2017.10.008DOI Listing
March 2018

HIF-2α Promotes Dissemination of Plasma Cells in Multiple Myeloma by Regulating CXCL12/CXCR4 and CCR1.

Cancer Res 2017 10 30;77(20):5452-5463. Epub 2017 Aug 30.

Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, Australia.

Disease progression and relapse in multiple myeloma is dependent on the ability of the multiple myeloma plasma cells (PC) to reenter the circulation and disseminate throughout the bone marrow. Increased bone marrow hypoxia is associated with increased recirculation of multiple myeloma PCs. Accordingly, we hypothesized that during chronic hypoxia, activation of HIF-2α may overcome the bone marrow retention signal provided by stromal-derived CXCL12, thereby enabling dissemination of multiple myeloma PCs. Here we demonstrate that HIF-2α upregulates multiple myeloma PC CXCL12 expression, decreasing migration toward CXCL12 and reducing adhesion to mesenchymal stromal cells We also found that HIF-2α strongly induced expression of the chemokine receptor CCR1 in multiple myeloma PCs. CCR1 activation potently induces multiple myeloma PC migration toward CCL3 while abrogating the multiple myeloma PC migratory response to CXCL12. In addition, increased CCR1 expression by multiple myeloma PCs conferred poor prognosis in newly diagnosed multiple myeloma patients and was associated with an increase in circulating multiple myeloma PCs in these patients. Taken together, our results suggest a role for hypoxia-mediated CCR1 upregulation in driving the egress of multiple myeloma PCs from the bone marrow. Targeting CCR1 may represent a novel strategy to prevent dissemination and overt relapse in multiple myeloma. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-0115DOI Listing
October 2017