Novel In Vivo Model of Multiple Myeloma Bone Disease Using ß-Tricalcium Phosphate Artificial Bone Grafts

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4200-4200
Author(s):  
Miroslav Koulnis ◽  
Homare Eda ◽  
Loredana Santo ◽  
Ka Tat Siu ◽  
Janani Ramachandran ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Growth ◽  
Bone Graft ◽  
Bone Disease ◽  
Tricalcium Phosphate ◽  
Research Funding ◽  
Bone Graft Substitute ◽  
Artificial Bone ◽  
Fetal Bone

Abstract Model systems to study Multiple Myeloma (MM) related bone disease exist but have a number of limitations. Disseminated MM models have variable cell homing and do not precisely recapitulate the human microenvironment interactions with myeloma cells. Severe combined immunodeficiency (SCID) mice engrafted with human fetal bone (SCID-hu) have been used by us, and are able to recapitulate the human bone marrow microenvironment. The fetal bone chips are however difficult to obtain, and vary in size and shape, complicating inter-sample comparison. Similarly, the poly-ε-caprolactone polymeric scaffold, previously used to seed murine or human stromal compartment, may not correctly reproduce bone destruction and inhibition of osteogenesis by MM as seen in patients, making this model difficult to test therapies targeting the MM niche. β-tricalcium phosphate (β-TCP) is a biocompatible and biodegradable bone graft substitute that is uniform in structure and easily available, and may be a viable alternative to overcome SCID-hu difficulties in modeling MM bone disease. Here, we utilized β-TCP bone graft substitute to develop a novel in vivo MM model where β-TCP permits the development of the bone microenvironment, supports MM development, and is technically feasible and highly reproducible. Using this model, we aim to better understand the biology of the niche in MM by genetically modifying its components and by testing new niche-targeting therapies. Our initial results show that osteogenesis takes place in the β-TCP bone graft, and the implant is supportive of MM tumor growth. Inter-scapular subcutaneous implantation of β-TCP alone, or co-implantation with human-derived stromal cell line HS27A in immunocompromised recipients resulted in the expression of osteogenic markers Runx2, alkaline phosphatase (ALP), Col1A1, and Osteocalcin (OCN), as well as a marker of bone resorption. Further, implants supported the growth of human-derived MM1.S and murine 5TGM1 cells, as visualized directly in vivo by serial luciferase bioluminescence imaging (BLI) and by immunohistochemistry. Modifying the niche compartment in Cre/iDTR animals with MM disease is an exciting novel strategy to understand which niche component in vivo may be targeted to suppress MM development. Mouse strains with promoter-specific Cre recombinase that induces the expression of the diphtheria toxin (DT) receptor (iDTR) can be utilized to selectively ablate a cell population of interest in vivo, via intraperitoneal DT injection. Here, we first utilized OCN-Cre/iDTR mice to test the deletion of mature osteoblasts in β-TCP artificial bone graft post-implantation. Our data show a dose-dependent reduction in osteoblastic markers OCN, ALP, Runx2, Sclerostin, Osteoprotegerin and RANKL. Importantly, DT ablation of osteoblasts in the β-TCP implant resulted in a significantly increased 5TGM1 tumor growth, as judged by BLI and tumor weight. Our data show that the mature osteocalcin-positive niche population is protective against MM disease. Ongoing studies of the β-TCP mouse model will address the relative contribution of various osteogenic populations to the course of MM development in vivo, and test the efficacy of novel MM drugs. Disclosures Raje: BMS: Consultancy; Amgen: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; Onyx: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Onyx: Consultancy; BMS: Consultancy; AstraZeneca: Research Funding; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Millenium: Consultancy; Eli Lilly: Research Funding; Novartis: Consultancy; Acetylon: Research Funding; Millenium: Consultancy; Novartis: Consultancy; Acetylon: Research Funding.

scholarly journals TBK1/Ikkε Inhibitor Amlx Blocks Multiple Myeloma Cell Growth in Vitro and In Vivo

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4504-4504
Author(s):  
Quanhong Sun ◽  
Peng Zhang ◽  
Juraj Adamik ◽  
Konstantinos Lontos ◽  
Valentina Marchica ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Growth ◽  
Cell Lines ◽  
Bone Disease ◽  
Research Funding ◽  
Ex Vivo ◽  
Dominant Negative ◽  
Induction Of Apoptosis

Abstract Multiple myeloma (MM) is the most frequent cancer to involve the skeleton and remains incurable for most patients, thus novel therapies are needed. MM bone disease is characterized by osteolytic lesions that contribute significantly to patient morbidity and mortality. We showed that TBK1 signaling is a novel pathway that increases osteoclast (OCL) formation in Paget's disease, an inflammatory bone disease. Therefore, we hypothesized that TBK1 plays a similar role in MM induction of OCL. We found that MM conditioned media (MM-CM) dose-dependently increased bone marrow monocyte (BMM) expression of activated TBK1 protein and enhanced RANKL-driven OCL formation. TBK1 knockdown by shRNA transduction into BMM significantly attenuated the ability of MM-CM to increase OCL differentiation without altering OCL differentiation in control media. We found that the TBK1/IKKε inhibitor Amlexanox (Amlx) blocked normal and MM-enhanced OCL formation. Importantly, TBK1 mRNA expression in CD138+ plasma cells (PC) isolated from MM or PC leukemia patients is significantly higher as compared to PC from Monoclonal Gammopathy of Undetermined Significance (MGUS) patients. Therefore, we tested whether targeting the TBK1/ IKKε signaling pathways would also affect MM cells. We found that Amlx strongly decreased the viability of several MM cell lines and primary MM cells via induction of apoptosis. Amlx treatment of MM cell lines also induced a G1/S blockade, decreased activated ERK1/2, and increased translation of the dominant-negative C/EBPb-LIP isoform in several MM cell lines. The positive-acting C/EBPb-LAP isoform was previously shown to be a critical transcription factor for MM viability. Importantly, Amlx also enhanced the effectiveness of the proteasome inhibitors bortezomib and carfilzomib to kill MM cells in culture. Further, Amlx sensitized MM1.S cells to the induction of apoptosis by the autophagic inhibitor Bafilomycin A. Amlx dose-dependently inhibited tumor growth in a syngeneic MM mouse model in which 5TGM1 MM cells expressing secreted GLuc were injected subcutaneously into immunocompetent C57Bl/KaLwRij. Tumor growth was assessed by measuring tumor volumes and by the levels of secreted GLuc in the blood. Further, OCL formation ex vivo from bone marrow monocytes obtained from AMLX-treated mice versus controls was decreased. Amlx did not affect the viability of primary BMM, bone marrow stromal cells (BMSC), or splenocytes. Further, Amlx treatment of primary BMSC from MM patients or normal donors decreased expression of TNFα, IL-6 and RANKL, thereby decreasing BMSC support of MM survival and OCL differentiation. Amlx pretreatment of BMSC and murine pre-osteoblast MC4 cells also decreased VCAM1 expression and reduced MM cell adhesion, another mechanism for Amlx reduction of bone microenvironmental MM support. These data suggest that targeting TBK1/IKKε signaling may decrease MM bone disease by slowing MM growth, directly and indirectly, and preventing MM-induced osteolysis. Disclosures Giuliani: Janssen Pharmaceutica: Other: Avisory Board, Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Takeda Pharmaceutical Co: Research Funding. Roodman:Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.

IN VIVO EVALUATION OF A NEW β-TRICALCIUM PHOSPHATE BONE SUBSTITUTE IN A RABBIT FEMUR DEFECT MODEL

2015 ◽  
Vol 27 (03) ◽  
pp. 1550028 ◽  
Author(s):  
Kam-Kong Chan ◽  
Chia-Hsien Chen ◽  
Lien-Chen Wu ◽  
Yi-Jie Kuo ◽  
Chun-Jen Liao ◽  
...  
Keyword(s):  
Bone Graft ◽  
Tricalcium Phosphate ◽  
Bone Substitute ◽  
Zealand White Rabbit ◽  
Bone Substitutes ◽  
Bone Graft Substitute ◽  
Defect Model ◽  
In Vivo Evaluation ◽  
Rabbit Femur

Calcium phosphate ceramics, of a similar composition to that of mineral bone, and which possess the properties of bioactivity and osteoconductivity, have been widely used as substitutes for bone graft in orthopedic, plastic and craniofacial surgeries. A synthetic β-tricalcium phosphate, Osteocera™, a recently developed bone graft substitute, has been used in this study. To evaluate the affinity and efficacy of Osteocera™ as bone defect implant, we used a New Zealand white rabbit femur defect model to test the osteoconductivity of this new bone substitute. Alternative commercially available bone substitutes, Triosite® and ProOsteon500, were used as the control materials. These three bone substitutes show good biocompatibility, and no abnormal inflammation either infection was seen at the implantation sites. In the histological and histomorphometric images, newly formed bone grew into the peripheral pores in the bone substitutes. After six months implantation, the volume of bone formation was found to be 20.5 ± 5.2%, 29.8 ± 6.5% and 75.5 ± 4.9% of the potential total cavity offered by ProOsteon500, Triosite® and Osteocera™, respectively. The newly formed bone area within the femur defect section for Osteocera™ was significantly larger than ProOsteon500 and Triosite®. We concluded that Osteocera™ shows better bioresorbability, biocompatibility and osteoconductivity in the rabbit femur defect model.

Selective HDAC6 Inhibition Via ACY-1215, Either Alone or in Combination with Bortezomib, Restores Osteoblast Function and Suppresses Osteoclast Differentiation in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2908-2908
Author(s):  
Loredana Santo ◽  
Teru Hideshima ◽  
Andrew L. Kung ◽  
Jen-Chieh Tseng ◽  
David Tamang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Bone Formation ◽  
Board Of Directors ◽  
Bone Disease ◽  
Research Funding ◽  
Bone Homeostasis ◽  
Advisory Committees ◽  
Synergistic Cytotoxicity

Abstract Abstract 2908 Bone disease in multiple myeloma (MM) is due to the disruption of the delicate balance between osteoblast (OB)-mediated bone formation and osteoclast (OC)-mediated bone resorption. Agents that target both tumor cells and restore normal bone homeostasis can improve long-term disease control and prolong MM patient survival. It has been demonstrated that in vitro pan HDAC inhibitors accelerate OB maturation and suppress OC maturation, while bortezomib triggers OB activation and inhibits osteoclastogenesis. However it has recently been shown that vorinostat (SAHA), a non-selective HDAC inhibitor, causes bone loss in vivo by inhibiting immature OB. Here, we evaluated effects of a selective HDAC6 inhibitor ACY-1215 (Acetylon Pharmaceuticals, Inc), alone and in combination with bortezomib, on MM cell growth and related bone disease. ACY-1215 in combination with bortezomib has synergistic cytotoxicity due to simultaneous inhibition of the proteasome and aggresome pathways. We confirm the in vivo anti-MM activity of ACY-1215 in combination with bortezomib in two different xenograft mouse models: human MM injected subcutaneously; and luciferase-expressing human MM injected intravenously (disseminated MM model). Tumor growth was significantly delayed and overall host survival significantly prolonged in animals treated with combined therapy (34 vs 22 days, n=7, p<0.0011) in plasmacytoma model and (40 vs 17 days, n=12, p<0.0001) in disseminated model. Importantly, we show that ACY-1215 alone and in combination with bortezomib overcomes the proliferative effect of bone marrow stromal cells (BMSCs) and cytokines. MM cells stimulate OC formation and function, while inhibiting OB differentiation via both cell-to-cell contact and cytokine secretion. Therefore, osteoclastogenesis is an important therapeutic target in MM. In this context, we evaluated the effect of ACY-1215 (1μM) and bortezomib (2.5nM) on OCs generated from blood mononuclear cells stimulated with receptor activator of nuclear factor kappa B ligand (RANKL). ACY-1215 alone and in combination with bortezomib inhibited OC differentiation, evidenced by a decreased number of TRAP positive multinucleated cells and bone-resorbing activity. In addition, ACY-1215 (1μM) significantly decreased cell growth of mature OC in co-culture with MM cell lines. We next examined the effect of ACY-1215, alone and in combination with bortezomib, on downstream targets in RANKL/RANK signaling. ACY-1215 plus bortezomib inhibits transcription factors implicated in OC differentiation including p-ERK, p-AKT, c-FOS and NFATC1. Since there is decreased OB function and new bone formation in MM, we next assessed the effect of ACY-1215 on OB differentiation. ACY-1215, alone and in combination, enhanced OB differentiation, evidenced by increased alkaline phosphatase enzyme activity and alizarin red staining. In addition, we show increased mRNA expression of b-catenin, osteocalcin, Runx2 and Sp7 (OB differentiation markers) in immature OB triggered by ACY-1215. Finally, ACY-1215 was not toxic to PHA stimulated PBMCs, suggesting a favorable side effect profile and therapeutic index. Our studies therefore demonstrate that ACY-1215, alone and in combination with bortezomib, can inhibit osteoclastogenesis enhance osteoblastogenesis, and inhibit MM cell growth. Based upon these studies, ongoing clinical trials are examining the efficacy of ACY-1215 in relapsed MM and associated bone disease. Disclosures: Hideshima: Acetylon: Consultancy. Kung:Acetylon Pharmaceuticals, Inc.: Consultancy. Tamang:Acetylon Pharmaceuticals, Inc.: Employment. Yang:Acetylon Pharmaceuticals, Inc.: Employment. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment. Mazitschek:Acetylon Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees. Bradner:Acetylon Pharmaceuticals, Inc.: Consultancy. Anderson:Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon Pharmaceuticals, Inc.: founder; Merck: Membership on an entity's Board of Directors or advisory committees. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.

scholarly journals Title: Genomic and Systemic Metabolism Differences Associated with Racial Disparities in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1601-1601
Author(s):  
Emily Gallagher ◽  
Alessandro Lagana ◽  
Jasmine Huang ◽  
David T. Melnekoff ◽  
Sundar Jagannath ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Multiple Myeloma ◽  
Tumor Growth ◽  
Racial Disparities ◽  
Research Funding ◽  
White Men ◽  
Mutational Signature ◽  
Prevalence Of Obesity

Abstract Background Racial disparities exist in the prevalence of multiple myeloma (MM) and monoclonal gammopathy of unknown significance (MGUS), with significantly higher prevalence in African American/ black (AA) compared to non-AA individuals. AA patients are also younger than non-AA at the time of diagnosis. We aimed to determine if differences in systemic metabolism and genetics might contribute to these racial disparities in MM. There are currently no approved anti-myeloma therapeutics that simultaneously reverse the metabolic drivers for MM tumor growth. Methods Studies were approved by the IRB and IACUC. We used a large health system electronic medical record database to characterize the metabolic phenotype of AA and white individuals with MM. Body mass index (BMI) was calculated from height and weight measures. BMI≥30kg/m 2 defined obesity. HbA1c ≥6.5% defined diabetes. Genomic analysis was performed by extracting mutational signatures from tumor samples of MM patients as previously described (PMID: 23945592), and comparing their prevalence between AA and white patients. We used an immunodeficient non-obese mouse model of type 2 diabetes (Rag1-/-/MKR) and controls (Rag1-/-) for in vivo MM1.S xenograft studies. ON123300, a dual ARK5/CDK4 kinase inhibitor was used for preclinical studies. Results Of our population of 3170 people, 2128 individuals were AA (21.7%) or white (78.3%). Females comprised 53.2% (n=246) of the AA population and 40.2% (n=669) of the white population. The highest prevalence of obesity was in AA women (46%) &gt; white men (41%) &gt; AA men (36%) &gt; white women (35%). The prevalence of diabetes was greater in AA men (37%) and women (34%) than white men (24%) and women (19%). BMI was a poor predictor of diabetes in the AA population with MM, where diabetes affected 1 in 5 AA individuals with normal BMI, but only 1 in 12 white individuals. Genomic characterization of AA patients, revealed a significant enrichment for the COSMIC SBS1 clock-like mutational signature in AA patients compared to white (p &lt; 0.05), which was confirmed after adjusting for age. The SBS1 signature correlates with the age of individuals and may represent a cell division clock. The male Rag1-/-/MKR mice had hyperglycemia and insulin resistance, and were not obese relative to control mice when fed a regular chow diet. The MM1.S tumor xenografts grew much more rapidly in the Rag1-/-/MKR mice compared with controls; 60 days post injection, tumors were approximately 5 times larger than controls and ex vivo analysis of protein lysates revealed increased activation of the insulin receptor (IR) / insulin-like growth factor 1 receptor (IGF-1R) and mTOR signaling pathway. Our previous studies (Perumal et al, CCR 2016) have characterized a role for ARK5(AMPK-related protein kinase 5), which is a major regulator of glutamine uptake in tumor cells that co-express ARK5 and MYC. Preclinical testing of MM cell lines and xenograft mouse models show a robust reduction of MM tumor growth as well as inhibition of IGF-1R-downstream signaling pathways, which contribute to tumor proliferation. Conclusions In our large diverse cohort of individuals with MM, we found significant racial disparities in the prevalence of obesity and type 2 diabetes, in addition to enrichment of a clock-like mutational signature in MM from AA individuals. Our findings suggest a signature of accelerated aging in MM from AA patients, which is concordant with younger age at diagnosis. In our mouse model, MM xenografts grew more rapidly in the diabetic mice and had more activation of mTOR signaling, a pathway known to regulate clock and aging gene signatures. ON123300 was able to reverse the metabolic drive as well as directly reduce tumor growth in vivo xenograft models. Disclosures Parekh: Foundation Medicine Inc: Consultancy; Amgen: Research Funding; PFIZER: Research Funding; CELGENE: Research Funding; Karyopharm Inv: Research Funding.

scholarly journals Silicate/zinc-substituted strontium apatite coating improves the osteoinductive properties of β-tricalcium phosphate bone graft substitute

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Hironori Sugimoto ◽  
Yusuke Inagaki ◽  
Akira Furukawa ◽  
Tsutomu Kira ◽  
Sachiko Kawasaki ◽  
...  
Keyword(s):  
Bone Graft ◽  
Mrna Expression ◽  
Tricalcium Phosphate ◽  
Bone Graft Substitute ◽  
Allogeneic Bone ◽  
Qrt Pcr ◽  
Alp Activity ◽  
Apatite Coating

Abstract Background β-Tricalcium phosphate (β-TCP) is a popular synthetic bone graft substitute with excellent osteoconductive properties and bioabsorbability. However, its osteoinductive properties are inferior to those of autologous or allogeneic bone. Trace elements such as strontium (Sr), silica (Si), and zinc (Zn) have been reported to promote osteogenesis in materials. In this study, we aimed to determine whether a Si/Zn-substituted Sr apatite coating of β-TCP could enhance osteoinductive properties. Methods The apatite-coated β-TCP disks were prepared using nanoparticle suspensions of silicate-substituted Sr apatite (SrSiP) or silicate- and Zn-co-substituted Sr apatite (SrZnSiP). Bone marrow mesenchymal cells (BMSCs) from rat femur were cultured and subsequently seeded at a density of 1.0 × 106/cm2 onto apatite-coated and non-coated β-TCP disks. In vitro, the β-TCP disks were then placed in osteogenic medium, and lactate dehydrogenase (LDH) activity was measured from supernatants after culture for 2 days. Additionally, after culture for 14 days, the mRNA expression of genes encoding osteocalcin (OC), alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and vascular endothelial growth factor (VEGF) was evaluated by qRT-PCR. In vivo, the β-TCP disks were transplanted subcutaneously into rats that were sacrificed after 4 weeks. Then, the harvested disks were evaluated biochemically (ALP activity, OC content, mRNA expression of OC, ALP, BMP-2, and VEGF measured by qRT-PCR), radiologically, and histologically. Results Significantly higher mRNA expression of almost all evaluated osteogenic and angiogenic genes was observed in the SrZnSiP and SrSiP groups than in the non-coated group, with no significant cytotoxicity elicited by the apatite coating in vitro. Moreover, in vivo, the SrZnSiP and SrSiP groups showed significantly higher osteogenic and angiogenic gene expression and higher ALP activity and OC content than the non-coated group (P < 0.05). Radiological and histopathological findings revealed abundant bone formation in the apatite-coated group. Conclusions Our findings indicate that apatite coating of β-TCP improves osteoinductive properties without inducing significant cytotoxicity.

Proline-Rich Tyrosine Kinase (Pyk2) Promotes Tumor Progression In Multiple Myeloma Through Modulation Of Wnt/β-Catenin Signaling Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3094-3094
Author(s):  
Yu Zhang ◽  
Yong Zhang ◽  
Yuji Mishima ◽  
Michele Moschetta ◽  
Wenjing Zhang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Tumor Growth ◽  
Dna Synthesis ◽  
Cell Viability ◽  
Cyclin D1 ◽  
Research Funding ◽  
Advisory Board

Abstract Background Proline-rich tyrosine kinase (Pyk2) is a non-receptor tyrosine kinase which belongs to the focal adhesion kinase (FAK) family. It is known to facilitate TNF-α induced EMT process in solid tumors, but this has not been investigated in the field of hematologic malignancies. We therefore dissected the role of PyK2 in multiple myeloma (MM) by looking at its ability to modulate MM biology both in vitro and in vivo. Methods Lentiviral packaged small hairpin RNA (shRNA), overexpression plasmid, related scramble probe and empty vector were introduced into MM1.S (GFP+/Luc+) cell line, to generate stable Pyk2 K.D. (#A2 and #A4), Pyk2 K.I., and control cells, respectively. The efficiency of knock-down or knock-in was validated by qPCR and immunoblotting. Cell viability and cell proliferation were detected by using CellTiter-Glo® luminescent assay and thymidine uptake, respectively. Gain- and loss-of fucntion studies were also performed on MM cells in presence of primary bone marrow stromal cells isolated from MM patients (MM-BMSCs). Adhesion of Pyk2 stable cells to fibronectin was measured by using a ECM cell adhesion assay kit. The synergistic effects of Pyk2 with Bortezomib was determined through calculating the DNA synthesis of Pyk2 K.D. cells treated with Bortezomib (2.5-5µM), using Calcusyn software and Chou-Talalay method. Pyk2 K.D. stable cells were intravenously injected into SCID-Biege mice to generate xenograft model. In vivo tumor growth was observed by Bioluminescent Imaging. Pyk2 -dependent-modulation of Wnt/β-catenin pathway signaling was indentified by using immunobloting. Results Knockdown of Pyk2 in MM cells significantly repressed cell viability and proliferation, as well as their adhesive ability to BMSCs, compared to scramble control cells. Moreover, Pyk2 knockdown induced de-adhesion of MM cells from BMSCs thus inducing chemosensitivity of tumor cells to Bortezomib. We next corroborated our findings by studying Pyk2 knock-in MM cells, and showed that stably upregulated Pyk2 expression promoted MM cell growth as measured by either ATP quantitation or DNA synthesis. Upregulation of Pyk2 expression also stablized the adhesion of MM cells to BMSCs, leading to a drug-resistance of MM cells to Bortezomib, compared with vector control cells. Pyk2 related tumor growth was further validated by establishing a xenograft mouse model. By using bioluminescence imaging, we found a significantly lower tumor burden in mice injected with Pyk2 K.D. cells, compared to mice controls (injected with scramble cells). We next dissected the effect of Pyk2 in modulation of cellular signaling in MM cells by using immunoblotting, and demonstrated that Pyk2 played an important role in regulating β-catenin signaling. Indeed, knockdown of Pyk2 induced GSK3β-phosphorylation, leading to increased β-catening-phosphorylation, thus resulting in β-catenin degradation and inhibited translocation to nucleus. Importantly, Pyk2 K.D. cells presented with reduced expression of c-myc and cyclin D1 at protein level. Conversely, Pyk2 overexpression enhanced β-catenin expression together with c-myc and cyclin D1 up-regulation, thus confirming the role of Pyk2 in modulating Wnt/β-catenin signaling activity in MM. Conclusion These findings indicate that Pyk2 exhibits pro-oncogenic properties in MM through modulation of Wnt/β-catenin signaling. Therefore, Pyk2 represents a novel therapeutic target in MM. Disclosures: Ghobrial: Sanofi: Research Funding; Noxxon: Research Funding; BMS: Advisory board, Advisory board Other, Research Funding; Onyx: Advisoryboard Other.

scholarly journals Silicate/zinc-substituted strontium apatite coating improves the osteoinductive properties of β-tricalcium phosphate bone graft substitute

2021 ◽  
Author(s):  
Hironori Sugimoto ◽  
Yusuke Inagaki ◽  
Akira Furukawa ◽  
Tsutomu Kira ◽  
Sachiko Kawasaki ◽  
...  
Keyword(s):  
Bone Graft ◽  
Mrna Expression ◽  
Tricalcium Phosphate ◽  
Bone Graft Substitute ◽  
Allogeneic Bone ◽  
Qrt Pcr ◽  
Alp Activity ◽  
Apatite Coating

Abstract Background: β-Tricalcium phosphate (β-TCP) is a popular synthetic bone graft substitute with excellent osteoconductive properties and bioabsorbability. However, its osteoinductive properties are inferior to those of autologous or allogeneic bone. Trace elements such as strontium (Sr), silica (Si), and zinc (Zn) have been reported to promote osteogenesis in materials. In this study, we aimed to determine whether a Si/Zn-substituted Sr apatite coating of β-TCP could enhance osteoinductive properties.Methods: The apatite-coated β-TCP disks were prepared using nanoparticle suspensions of silicate-substituted Sr apatite (SrSiP) or silicate- and Zn-co-substituted Sr apatite (SrZnSiP).Bone marrow mesenchymal cells (BMSCs) from rat femur were cultured and subsequently seeded at a density of 1.0 × 106/cm2 onto apatite-coated and non-coated β-TCP disks.In vitro, the β-TCP disks were then placed in osteogenic medium, and lactate dehydrogenase (LDH) activity was measured from supernatants after culture for 2 days. Additionally, after culture for 14 days, the mRNA expression of genes encoding osteocalcin (OC), alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and vascular endothelial growth factor (VEGF) was evaluated by qRT-PCR. In vivo, the β-TCP disks were transplanted subcutaneously into rats that were sacrificed after 4 weeks. Then, the harvested disks were evaluated biochemically (ALP activity, OC content, mRNA expression of OC, ALP, BMP-2, and VEGF measured by qRT-PCR), radiologically, and histologically.Results: Significantly higher mRNA expression of almost all evaluated osteogenic and angiogenic genes was observed in the SrZnSiP and SrSiP groups than in the non-coated group, with no significant cytotoxicity elicited by the apatite coating in vitro. Moreover, in vivo, the SrZnSiP and SrSiP groups showed significantly higher osteogenic and angiogenic gene expression and higher ALP activity and OC content than the non-coated group (P < 0.05). Radiological and histopathological findings revealed abundant bone formation in the apatite-coated group.Conclusions: Our findings indicate that apatite coating of β-TCP improves osteoinductive properties without inducing significant cytotoxicity.

scholarly journals Antibiotic loaded β-tricalcium phosphate/calcium sulfate for antimicrobial potency, prevention and killing efficacy of Pseudomonas aeruginosa and Staphylococcus aureus biofilms

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nan Jiang ◽  
Devendra H. Dusane ◽  
Jacob R. Brooks ◽  
Craig P. Delury ◽  
Sean S. Aiken ◽  
...  
Keyword(s):  
Tricalcium Phosphate ◽  
Calcium Sulfate ◽  
Bone Graft Substitute ◽  
In Vitro Assays ◽  
In Vivo Experiments ◽  
Orthopaedic Infections ◽  
Clinical Investigations ◽  
Antimicrobial Potency

AbstractThis study investigated the efficacy of a biphasic synthetic β-tricalcium phosphate/calcium sulfate (β-TCP/CS) bone graft substitute for compatibility with vancomycin (V) in combination with tobramycin (T) or gentamicin (G) evidenced by the duration of potency and the prevention and killing efficacies of P. aeruginosa (PAO1) and S. aureus (SAP231) biofilms in in vitro assays. Antibiotic loaded β-TCP/CS beads were compared with antibiotic loaded beads formed from a well characterized synthetic calcium sulfate (CS) bone void filler. β-TCP/CS antibiotic loaded showed antimicrobial potency against PAO1 in a repeated Kirby-Bauer like zone of inhibition assay for 6 days compared to 8 days for CS. However, both bead types showed potency against SAP231 for 40 days. Both formulations loaded with V + T completely prevented biofilm formation (CFU below detection limits) for the 3 days of the experiment with daily fresh inoculum challenges (P < 0.001). In addition, both antibiotic loaded materials and antibiotic combinations significantly reduced the bioburden of pre-grown biofilms by between 3 and 5 logs (P < 0.001) with V + G performing slightly better against PAO1 than V + T. Our data, combined with previous data on osteogenesis suggest that antibiotic loaded β-TCP/CS may have potential to stimulate osteogenesis through acting as a scaffold as well as simultaneously protecting against biofilm infection. Future in vivo experiments and clinical investigations are warranted to more comprehensively evaluate the use of β-TCP/CS in the management of orthopaedic infections.

scholarly journals Highly Differentiated Anti-CD47 Antibody, AO-176, Potently Inhibits Hematologic Malignancies Alone and in Combination

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1844-1844
Author(s):  
John Richards ◽  
Myriam N Bouchlaka ◽  
Robyn J Puro ◽  
Ben J Capoccia ◽  
Ronald R Hiebsch ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Combination Therapy ◽  
Tumor Growth ◽  
Growth Inhibition ◽  
Tumor Cells ◽  
Tumor Growth Inhibition ◽  
Employment Equity ◽  
Equity Ownership

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

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