Bioinformatic Data Mining for Candidate Drugs Affecting Risk of Bisphosphonate-Related Osteonecrosis of the Jaw (BRONJ) in Cancer Patients.

Background: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) leads to significant morbidity. Other coadministered drugs may modulate the risk for BRONJ. The present study aimed to leverage bioinformatic data mining to identify drugs that potentially modulate the risk of BRONJ in cancer. Methods: A GEO gene expression dataset of peripheral blood mononuclear cells related to BRONJ in multiple myeloma patients was downloaded, and differentially expressed genes (DEGs) in patients with BRONJ versus those without BRONJ were identified. A protein-protein interaction network of the DEGs was constructed using experimentally validated interactions in the STRING database. Overrepresented Gene Ontology (GO) molecular function terms and KEGG pathways in the network were analysed. Network topology was determined, and 'hub genes' with degree ≥2 in the network were identified. Known drug targets of the hub genes were mined from the 'drug gene interaction database' (DGIdb) and labelled as candidate drugs affecting the risk of BRONJ. Results: 751 annotated DEGs (log FC ≥ 1.5, p < 0.05) were obtained from the microarray gene expression dataset GSE7116. A PPI network with 633 nodes and 168 edges was constructed. Data mining for drugs interacting with 49 gene nodes was performed. 37 drug interactions were found for 9 of the hub genes including TBP, TAF1, PPP2CA, PRPF31, CASP8, UQCRB, ACTR2, CFLAR, and FAS. Interactions were found for several established and novel anticancer chemotherapeutic, kinase inhibitor, caspase inhibitor, antiangiogenic, and immunomodulatory agents. Aspirin, metformin, atrovastatin, thrombin, androgen and antiandrogen drugs, progesterone, Vitamin D, and Ginsengoside 20(S)-Protopanaxadiol were also documented. Conclusions: A bioinformatic data mining strategy identified several anticancer, immunomodulator, and other candidate drugs that may affect the risk of BRONJ in cancer patients.

Macrophage miR-149-5p induction is a key driver and therapeutic target for BRONJ.

Bisphosphonate-related (BP-related) osteonecrosis of the jaw (BRONJ) is one of the severe side effects of administration of BPs, such as zoledronic acid (ZA), which can disrupt the patient's quality of life. Although the direct target of skeletal vasculature and bone resorption activity by BPs has been phenomenally observed, the underlying mechanism in BRONJ remains largely elusive. Thus, it is urgently necessary to discover effective therapeutic targets based on the multifaceted underlying mechanisms in the development of BRONJ. Here, we determined the inhibitory role of ZA-treated macrophages on osteoclast differentiation and type H vessel formation during tooth extraction socket (TES) healing. Mechanistically, ZA activated the NF-κB signaling pathway and then induced p65 nuclear translocation in macrophages to promote miR-149-5p transcription, resulting in impaired osteoclast differentiation via directly binding to the Traf6 3'-UTR region. Moreover, we identified that miR-149-5p-loaded extracellular vesicles derived from ZA-treated bone marrow-derived macrophages could regulate biological functions of endothelial cells via the Rap1a/Rap1b/VEGFR2 pathway. Furthermore, local administration of chemically modified antagomiR-149-5p was proven to be therapeutically effective in BRONJ mice. In conclusion, our findings illuminate the dual effects of miR-149-5p on skeletal angiogenesis and bone remolding, suggesting it as a promising preventive and therapeutic target for BRONJ.

Analysis of the effect of zoledronic acid on gene differences in rat jaw.

BACKGROUND: With the widespread use of bisphosphonates, there are more and more complications about bisphosphonates, bisphosphonate-related osteonecrosis of the jaw is one.In the past ten years, there have been many studies on the mechanism of bisphosphonate associated jaw necrosis. OBJECTIVE: To investigate the influence and analysis of zoledronic acid on gene differences in rat jaw. METHODS: Six Sprague-Dawley female rats were randomly divided into control group (n = 3) and experimental group (n = 3). The experimental group received zoledronic acid injection for 12 weeks (dose of 0.2 mg / kg, 3 times a week).Control groups were injected with normal saline for 12 weeks. All rats were subjected to left mandibular first molar extraction 12 weeks later.After 8 weeks of tooth extraction, all rats were sacrificed and the mandible was removed.RNA-seq was used to analyze differential gene changes in all mandibles. Bioinformatics analysis of differential genes. RESULTS: Compared with the two rat groups, there were 2,830 different genes, including 1,001 upregulated genes and 1,829 down-regulated genes. Gene Ontology analysis revealed that the upregulated genes were mainly associated with immune-related pathways. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that Hedgehog signaling pathway, Notch signaling pathway and Hippo signaling pathway were associated with upregulated genes. After the Gene Set Enrichment Analysis, the Gene Ontology analysis showed that 2559 / 6588 gene sets are upregulated in phenotype experimental group,and 342 gene sets with p <0.05. The Kyoto Encyclopedia of Genes and Genomes analysis revealed that 95 / 316 gene sets are upregulated in phenotype experimental group, and four gene sets(Notch pathway, other types of O-glycan biosynthesis, ovarian steridogenesis and Hippo pathway) with p <0.05. CONCLUSIONS: Changes in differential genes are mainly related to immune-related processes and pathways, and pathways related to bone metabolism. The up-regulation of some genes can promote the progress of Bisphosphonate-related osteonecrosis of the jaw.

[The role of semaphorin 4D in the mechanism of bisphosphonate-related osteonecrosis of the jaw in rats].

PURPOSE: To study the expression level of semaphorin 4D (Sema4D) in bisphosphonate-related osteonecrosis of the jaw (BRONJ) and to explore its possible role in the occurrence of BRONJ. METHODS: BRONJ-like rat model was established by intraperitoneal injection of zoledronic acid assisted with tooth extraction. The maxillary specimens were extracted for imaging and histological examination, and bone marrow mononuclear cells(BMMs) and bone marrow mesenchymal stem cells(BMSCs) of each group were obtained in vitro for co-culture. Trap staining and counting were performed on monocytes after osteoclast induction. RAW264.7 cells were induced by osteoclast orientation under bisphosphonates(BPs) environment, and Sema4D expression was detected. Similarly, MC3T3-E1 cells and BMSCs were induced to osteogenic orientation in vitro, and the expression level of osteogenic and osteoclastic related genes ALP, Runx2, and RANKL was detected under the intervention of BPs, Sema4D and Sema4D antibody. Statistical analysis of the data was performed using GraphPad Prism 8.0 software. RESULTS: BRONJ-like rat model was successfully constructed. Two weeks after tooth extraction, the healing of the tooth extraction wound in the experimental group was significantly limited, and the tooth extraction wound was exposed. H-E staining results showed that regeneration of new bone in the extraction socket of the experimental group was significantly restricted, dead bone was formed, and the healing of the soft tissue was limited. The results of trap staining showed that the number of osteoclasts in the experimental group was significantly less than that in the control group. Micro-CT results showed that bone mineral density and bone volume fraction in the extraction socket of the experimental group were significantly lower than those of the control group. Immunohistochemical results showed that compared with the control group, the expression level of Sema4D in the experimental group was significantly increased. In vitro studies showed that compared with the control group, the osteoclast induction of BMMs in the experimental group was significantly lower than that in the control group. BMSCs in the experimental group significantly reduced the induction of osteoclasts. Osteoclastic induction experiments revealed that bisphosphonates could effectively inhibit the formation of osteoclasts, and the expression of Sema4D was significantly reduced. Osteogenic induction experiment found that Sema4D significantly reduced the expression of Runx2 and RANKL genes in osteoblasts, while the expression of ALP gene decreased and the expression of RANKL up-regulated after adding Sema4D antibody. CONCLUSIONS: BPs can interfere with normal bone healing time by up-regulating the expression of Sema4D in tissues, leading to coupling disorder between osteoclasts and osteoblasts with inhibition of the maturation of osteoclasts, thereby inhibiting the growth of osteoblasts. Differentiation and expression of related osteogenic factors mediate the development of BRONJ.

Genome-wide Association Study Identified Chromosome 8 Locus Associated with Medication-Related Osteonecrosis of the Jaw.

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but serious drug-related adverse event. To identify pharmacogenomic markers of MRONJ associated with bisphosphonate therapy, we conducted a genomewide association study (GWAS) meta-analysis followed by functional analysis of 5,008 individuals of European ancestry treated with bisphosphonates, which includes the largest number of MRONJ cases to date (444 cases and 4,564 controls). Discovery GWAS was performed in randomly selected 70% of the patients with cancer and replication GWAS was performed in the remaining 30% of the patients with cancer treated with intravenous bisphosphonates followed by meta-analysis of all 3,639 patients with cancer. GWAS was also performed in 1,369 patients with osteoporosis treated with oral bisphosphonates. The lead single-nucleotide polymorphism (SNP), rs2736308 on chromosome 8, was associated with an increased risk of MRONJ with an odds ratio (OR) of 2.71 and 95% confidence interval (CI) of 1.90-3.86 (P = 3.57*10-8 ) in the meta-analysis of patients with cancer. This SNP was validated in the MRONJ GWAS in patients with osteoporosis (OR: 2.82, 95% CI: 1.55-4.09, P = 6.84*10-4 ). The meta-analysis combining patients with cancer and patients with osteoporosis yielded the same lead SNP rs2736308 on chromosome 8 as the top SNP (OR: 2.74, 95% CI: 2.09-3.39, P = 9.65*10-11 ). This locus is associated with regulation of the BLK, CTSB, and FDFT1 genes, which had been associated with bone mineral density. FDFT1 encodes a membrane-associated enzyme, which is implicated in the bisphosphonate pathway. This study provides insights into the potential mechanism of MRONJ.

Medication-related osteonecrosis of the jaw (MRONJ) and eNOS Polymorphisms in multiple myeloma patients: a single center experience.

BACKGROUND: Multiple myeloma (MM) constitutes approximately 10% of hematological malignancies. Bisphosphonates have established themselves in solid organ metastasis and multiple myeloma lytic bone lesions by inhibiting osteoclast activation. Medication-related osteonecrosis of the jaw (MRONJ) emerges as an important complication. Investigating host-based factors, and developing personal risk factors gain importance in the development mechanism of MRONJ. We aimed to reveal the different genotype polymorphisms, and clinical effects of eNOS in patients with a diagnosis of MRONJ in MM patients. METHODS: Medical records and blood samples were collected from 60 MRONJ patients with MM and 60 healthy controls. Inclusion criteria was having an exposed maxillofacial bone for more than eight weeks, a history of bisphosphonates, and no history of radiation therapy for the jaws. eNOS G894T and intron 4 VNTR were calculated by polymerase chain reaction and/or restriction fragment length polymorphism. RESULTS: eNOS G894T and VNTR genotypes and alleles were compared statistically with the healthy control group. There was no significant difference between the two groups. In comparison between G894T and clinical parameters, aphthous stomatitis was more common in TT genotype, while DMFT > 3 was more common in TG-GG genotype (p = 0.035, 0.023). CONCLUSIONS: eNOS induces osteogenesis in bone metabolism, with its regulatory effects on bone remodeling and also NO induced angiogenesis takes place indirectly with its protective effect on endothelial functions. We see that these polymorphisms affecting the entire process of bone remodeling and angiogenesis, especially mucosal damage, which is the triggering factor of MRONJ pathology, have been revealed in the MM patient group. Considering the MRONJ initiating factors, it is necessary to emphasize the importance of our study results. It should be seen as an important step for new studies towards MRONJ and its treatment.

Immune-related expression profiles of bisphosphonates-related osteonecrosis of the jaw in multiple myeloma.

Objective: To investigate the immune cellular and genomic profiles of bisphosphonates-related osteonecrosis (BRONJ) of the jaw and excavate potential small molecule drugs. Methods and materials: The genomic profile of a multiple myeloma (MM) patient with BRONJ was downloaded from Gene Expression Omnibus (GEO). The 22 immune cell subsets infiltration in the patient were predicted by cell-type identification by estimating relative subsets of RNA transcripts. In addition, the differently expressed immune-related genes (DEMGs) of BRONJ were identified, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses for functional annotation. Then, potential drugs for BRONJ treatment were predicted by Connectivity Map (CMAP) based on DEGs. Results: High proportions of native CD4+T cells and M0 macrophages were observed while resting mast cells, NK cells, and eosinophils were downregulated in the BRONJ patient (P< 0.05). Resting dendritic cells and gamma delta T cells were positively correlated (r=0.93). Additionally, 36 DEMGs were screened from 336 DEGs in BRONJ expression profiles. GO enrichment analysis revealed that DEMGs were most associated with peptidyl-tyrosine modification, myeloid leukocyte migration, leukocyte chemotaxis and regulation of chemokine production(P<0.05). KEGG analysis indicated that DEMGs were mainly related to cytokine-cytokine receptor interaction, IL-17 signaling pathway and NF-Kappa B signaling pathway(P<0.05). Besides, 12 small molecule drugs were screened in MM patient with ONJ. Conclusion: The discovery of different composition of immune cell types and immune-related transcriptomes in BRONJ helps to explain the onset and development of MRONJ, which provides a novel target for BRONJ therapy.

SIRT1 Gene SNP rs932658 Is Associated With Medication-Related Osteonecrosis of the Jaw.

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but serious adverse drug reaction. Our previous whole-exome sequencing study found SIRT1 intronic region single-nucleotide polymorphism (SNP) rs7896005 to be associated with MRONJ in cancer patients treated with intravenous (iv) bisphosphonates (BPs). This study aimed to identify causal variants for this association. In silico analyses identified three SNPs (rs3758391, rs932658, and rs2394443) in the SIRT1 promoter region that are in high linkage disequilibrium (r2 > 0.8) with rs7896005. To validate the association between these SNPs and MRONJ, we genotyped these three SNPs on the germline DNA from 104 cancer patients of European ancestry treated with iv BPs (46 cases and 58 controls). Multivariable logistic regression analysis showed the minor alleles of these three SNPs were associated with lower odds for MRONJ. The odds ratios (95% confidence interval) and p values were 0.351 (0.164-0.751; p = 0.007) for rs3758391, 0.351 (0.164-0.751; p = 0.007) for rs932658, and 0.331 (0.157-0.697; p = 0.0036) for rs2394443, respectively. In the reporter gene assays, constructs containing rs932658 with variant allele A had higher luciferase activity than the reference allele, whereas constructs containing SNP rs3758391 and/or rs2394443 did not significantly affect activity. These results indicate that the promoter SNP rs932658 regulates the expression of SIRT1 and presumably lowers the risk of MRONJ by increasing SIRT1 expression. © 2020 American Society for Bone and Mineral Research (ASBMR).

Antimicrobial peptide gene expression in medication-related osteonecrosis of the jaw.

Bisphosphonates and denosumab are commonly used antiresorptive therapies in patients with bone metastasis and osteoporosis. Medication-related osteonecrosis of the jaw (MRONJ) is a serious side effect of these drugs, and infection has been recognized as a contributing factor. Current therapeutic options for MRONJ show limited effectiveness, therefore necessitating novel treatment strategies. Bisphosphonates have recently been reported to induce the expression of antimicrobial peptides (AMPs), an inherent component of the immune system. Therefore, the aim of the present study was to investigate and compare the influence of the anti-RANKL antibody denosumab and bisphosphonates on the gene expression of selected AMPs: human α-defensin-1, human α-defensin-3, human ß-defensin-1, and human ß-defensin-3. Bone specimens were collected from patients with MRONJ who had been treated with bisphosphonates (n = 6) or denosumab (n = 6), and from healthy subjects (n = 6) with no history of treatment with bone metabolism-influencing drugs. Reverse transcription-quantitative polymerase chain reaction was used to quantify the expression levels of selected AMPs. Samples from patients treated with denosumab showed significantly higher mRNA expression of human α-defensin-3 and human ß-defensin-3 than those from healthy subjects. This finding is similar to previously described upregulated expression of human defensins in patients with MRONJ after bisphosphonates treatment. This suggests that the elevated expression of defensins may be at least a part of the mechanism underlying the pathogenesis of osteonecrosis induced by antiresorptive therapies, which can serve as a new target for potential treatment of MRONJ.

Stress response in periodontal ligament stem cells may contribute to bisphosphonate­associated osteonecrosis of the jaw: A gene expression array analysis.

Gene expression alterations in periodontal ligament stem cells (PDLSCs) during bisphosphonate (BP) usage and the transcriptomic mechanism underlying BP­related osteonecrosis of the jaw have not been fully elucidated. In the present study, human PDLSCs were isolated from adults with no history of periodontal disease, and subsequently incubated and treated with zoledronate on days 3 and 5. Subsequently, PDLSCs from all timepoints were screened using an Affymetrix Gene Expression Array. Limma differential expression analysis was performed on a normalized gene expression matrix, followed by cluster analysis, pathway and network analyses. Overall, 906 genes (352 upregulated and 554 downregulated) exhibited differential expression levels between days 0 and 5, and these were termed slow­response genes. These slow­response genes were enriched in cellular stress response signaling pathways (upregulated genes), as well as proliferation­ and ossification­associated signaling pathways (downregulated genes). Furthermore, 168 (day 3 vs. 0) and 105 (day 5 vs. 3) genes were differentially expressed between adjacent timepoints. These genes were also enriched in stress response­ and proliferation­associated signaling pathways, but not in ossification­associated signaling pathways. Poly(ADP­ribose) polymerase 1 (PARP1) and CYLD lysine 63 deubiquitinase (CYLD) had the most protein­protein interaction partners among the slow­response genes and were connected with both stress­ (e.g. caspase­1) and ossification­associated genes [e.g. secreted phosphoprotein 1 and collagen type I α1 chain (COL1A1)]. BP treatment induced stress response­like transcriptional alterations in PDLSCs, followed by inhibition of proliferation and ossification. These alterations may contribute to the onset of jaw osteonecrosis. PARP1 and CYLD may be two key genes involved in this pathological procedure.

Pharmacogenomics in medication-related osteonecrosis of the jaw: a systematic literature review.

OBJECTIVE: Medication-related osteonecrosis of the jaw (ONJ) is an adverse, severe and debilitating effect, which although infrequent, affects patients with osteoporosis or neoplasm who take bisphosphonates, antiresorptive drugs, and/or antiangiogenic drugs. Its etiopathogenesis is unknown, although genetic causes have been postulated. MATERIALS AND METHODS: This review analyzed articles published to date that have studied genetic factors associated with ONJ. Fifteen case-control studies were included, published between 2008 and 2018. RESULTS: Five set out to determine genetic causes by means of genome-centered techniques, while ten do so by investigating gene-centered variants. Nine works found statistically significant associations between one or various single nucleotide polymorphisms (SNPs) and the appearance of ONJ. None of the studies coincided as to which genes present some association. CONCLUSIONS: The review observed the moderate impact of genetic factors on the appearance of ONJ. It also showed the heterogeneity of the studies that have investigated ONJ to date. In future studies, involving international and interhospital collaboration will be necessary to recruit sample sizes of sufficient size, elaborate adequate study designs, obtain clear results, and advance our understanding of ONJ and make it possible to single out individual patients at risk.

Identifying genetic variants underlying medication-induced osteonecrosis of the jaw in cancer and osteoporosis: a case control study.

BACKGROUND: Bisphosphonate-induced osteonecrosis of the jaw (BRONJ) presents with a typical pattern of jaw necrosis in patients who have been prescribed bisphosphonates (BPs) and other antiangiogenetic drugs to treat osteoporosis or bone-related complications of cancer. METHODS: This study divided 38 patients with BRONJ into two groups according to the prescribing causes: cancer (n = 13) and osteoporosis (n = 25), and underwent whole exome sequencing and compared them with normal controls (n = 90). To identify candidate genes and variants, we conducted three analyses: a traditional genetic model, gene-wise variant score burden, and rare-variant analysis methods. RESULTS: The stop-gain mutation (rs117889746) of the PZP gene in the BRONJ cancer group was significantly identified in the additive trend model analysis. In the cancer group, ARIDS, HEBP1, LTBP1, and PLVAP were identified as candidate genes. In the osteoporosis group, VEGFA, DFFA, and FAM193A genes showed a significant association. No significant genes were identified in the rare-variant analysis pipeline. Biologically accountable functions related to BRONJ occurrence-angiogenesis-related signaling (VEGFA and PLVAP genes), TGF-ß signaling (LTBP1 and PZP genes), heme toxicity (HEBP1) and osteoblast maturation (ARIDS)-were shown in candidate genes. CONCLUSION: This study showed that the candidate causative genes contributing to the development of BRONJ differ according to the BP dose and background disease.

Adipose-derived stem cells prevent the onset of bisphosphonate-related osteonecrosis of the jaw through transforming growth factor ß-1-mediated gingival wound healing.

BACKGROUND: Due to its complex pathogenesis and low clinical cure rate, bisphosphonate-related osteonecrosis of the jaw (BRONJ) poses a substantial challenge for oral and maxillofacial surgeons. Therefore, the treatment of BRONJ should focus on prevention. In clinical studies, primary wound closure can significantly reduce the incidence of BRONJ. Whether local stem cell transplantation can promote primary gingival healing in patients with a medication history and prevent BRONJ has not been reported. METHODS: In this study, animals were divided into a healthy group (non-drug treatment), a BP group, a hydroxyapatite (HA) group, and an adipose-derived stem cell (ADSC) group. All groups except the healthy group were treated with BPs and immunosuppressive drugs once per week for 8 weeks, simulating clinical use for the treatment of cancer patients with bone metastasis, to induce BRONJ-like animals. After the sixth drug treatment, the bilateral premolars were extracted in all groups. In contrast to the healthy and BP groups, the extraction sockets in the HA and ADSC groups were filled with HA or HA + ADSCs simultaneously post extraction to observe the preventive effect of ADSCs on the occurrence of BRONJ. At 2 and 8 weeks post extraction, animals from all groups were sacrificed. RESULTS: At 8 weeks post transplantation, ADSCs prevented the occurrence of BRONJ, mainly through accelerating healing of the gingival epithelium at 2 weeks post extraction. We also found that ADSCs could upregulate the expression of transforming growth factor ß1 (TGF-ß1) and fibronectin in tissue from animals with a medication history by accelerating gingival healing of the extraction socket. A rescue assay further demonstrated that TGF-ß1 and fibronectin expression decreased in TGF-ß1-deficient ADSC-treated animals, which partially abolished the preventive effect of ADSCs on the onset of BRONJ. CONCLUSION: ADSCs prevent the onset of BRONJ, mainly by upregulating the expression of TGF-ß1 and fibronectin to promote primary gingival healing, ultimately leading to bone regeneration in the tooth extraction socket. Our new findings provide a novel stem cell treatment for the prevention of BRONJ.

Pharmacogenomics of osteonecrosis of the jaw.

Osteonecrosis of the jaw (ONJ) is a rare but serious drug induced adverse event, mainly associated with the use of antiresorptive medications, such as intravenous (IV) bisphosphonates (BPs) in cancer patients. In this review, we evaluated all the pharmacogenomic association studies for ONJ published up to December 2018. To date, two SNPs (CYP2C8 rs1934951 and RBMS3 rs17024608) were identified to be associated with ONJ by two genome-wide association studies (GWAS). However, all six subsequent candidate gene studies failed to replicate these results. In addition, six discovery candidate gene studies tried to identify the genetic markers in several genes associated with bone remodeling, bone mineral density, or osteoporosis. After evaluating the results of these 6 studies, none of the SNPs was significantly associated with ONJ. Recently, two whole-exome sequencing (WES) analysis (including one from our group) were performed to identify variants associated with ONJ. So far, only our study successfully replicated discovery result indicating SIRT1 SNP rs7896005 to be associated with ONJ. However, this SNP also did not reach genome-wide significance. The major limitations of these studies include lack of replication phases and limited sample sizes. Even though some studies had larger sample sizes, they recruited healthy individuals as controls, not subjects treated with BPs. We conclude that a GWAS with a larger sample size followed by replication phase will be needed to fully investigate the pharmacogenomic markers of ONJ.

Altered microRNA expression profile in the peripheral lymphoid compartment of multiple myeloma patients with bisphosphonate-induced osteonecrosis of the jaw.

Bisphosphonates are formidable inhibitors of osteoclast-mediated bone resorption employed for therapy of multiple myeloma (MM) subjects with osteolytic lesions. Osteonecrosis of the jaw (ONJ) is an uncommon drug-induced adverse event of these agents. MicroRNAs (miRNAs) are a group of small, noncoding RNAs nucleotides, which are essential post-transcriptional controllers of gene expression. They have a central role in the normal bone development. The goal of our study was to investigate 18 miRNAs, whose targets were previously validated and described in MM subjects without ONJ, in peripheral lymphocytes of MM subjects with bisphosphonate-induced ONJ. Utilizing reverse transcription quantitative polymerase chain reaction, we evaluated miRNAs in five healthy subjects and in five MM patients with ONJ. Our experimental data revealed that a diverse miRNA signature for ONJ subjects emerged with respect to control subjects. Using the filter for in silico analysis, among the 18 miRNAs, we recognized 14 dysregulated miRNAs. All these miRNAs were significantly over-expressed in patients vs controls (MIR-16-1, MIR-21, MIR-23A, MIR-28, MIR-101-1, MIR-124-1, MIR-129, MIR-139, MIR-145, MIR-149, MIR-202, MIR-221, MIR-424, MIR-520). Among them, six were strongly upregulated (fourfold upregulated and more). These miRNAs target numerous pathways and genes implicated in calcium ion binding, bone resorption, mineralization of bone matrix, and differentiation and maintenance of bone tissue. A modified microRNA expression profile after zoledronate therapy could participate to the onset of ONJ. Targeting these miRNAs could provide a new opportunity for the prevention or treatment of ONJ.

Bisphosphonate Modulation of the Gene Expression of Different Markers Involved in Osteoblast Physiology: Possible Implications in Bisphosphonate-Related Osteonecrosis of the Jaw.

The aim of the present study was to elucidate the role of osteoblasts in bisphosphonates-related osteonecrosis of the jaw (BRONJ). The specific objective was to evaluate the effect on osteoblasts of two nitrogen-containing BPs (zoledronate and alendronate) and one non-nitrogen-containing BP (clodronate) by analyzing modulations in their expression of genes essential for osteoblast physiology. Real-time polymerase chain reaction (RT-PCR) was used to study the effects of zoledronate, alendronate, and clodronate at doses of 10-5, 10-7, or 10-9 M on the expression of Runx-2, OSX, ALP, OSC, OPG, RANKL, Col-I, BMP-2, BMP-7, TGF-ß1, VEGF, TGF-ßR1, TGF-ßR2, and TGF-ßR3 by primary human osteoblasts (HOBs) and MG-63 osteosarcoma cells. Expression of these markers was found to be dose-dependent, with no substantive differences between these cell lines. In general, results demonstrated a significant increase in TFG-ß1, TGF-ßR1, TGF-ßR2, TGF-ßR3, and VEGF expressions and a significant reduction in RUNX-2, Col-1, OSX, OSC, BMP-2, BMP-7, ALP, and RANKL expressions, while OPG expression varied according to the dose and cell line. The results of this in vitro study of HOBS and MG-63 cell lines indicate that low BP doses can significantly affect the expression of genes essential for osteoblast growth and differentiation and of genes involved in regulating osteoblast-osteoclast interaction, possibly by increasing TGF-ß1 production. These findings suggest that osteoblasts may play an important role in BRONJ development, without ruling out other factors.

Zoledronate suppresses VEGF­induced capillary tube formation and inhibits expression of interferon­induced transmembrane protein­1 in human umbilical vein endothelial cells.

Interferon­induced transmembrane protein 1 (IFITM1) is a member of the interferon­induced transmembrane protein family and has recently been identified as a novel protein participant in angiogenesis. Zoledronate (ZON), a nitrogen­containing bisphosphonate, is widely used in the treatment of osteoporosis and to prevent bone metastases of certain cancer types. However, the association between ZON and IFITM1 has remained elusive. The present study investigated the effect of ZON on the expression of IFITM1 during vascular endothelial growth factor (VEGF)­induced capillary tube formation in human umbilical vein endothelial cells. It was observed that cell proliferation and VEGF­induced tube formation were significantly inhibited by treatment with 10 µM ZON. The expression of IFITM1 increased during VEGF­induced tube formation. However, the VEGF­induced increase in IFITM1 expression exhibited a dose­ and time­dependent decrease with ZON treatment at the mRNA and protein level. Furthermore, matrix metalloproteinase­9 activation was markedly decreased by ZON treatment. These results suggest that induction of IFITM1 expression may be involved in the anti­angiogenic activity of ZON.

SIRT1/HERC4 Locus Associated With Bisphosphonate-Induced Osteonecrosis of the Jaw: An Exome-Wide Association Analysis.

Osteonecrosis of the jaw (ONJ) is a rare, but serious drug side effect, mainly associated with the use of intravenous (iv) bisphosphonates (BPs). The purpose of this study was to identify genetic variants associated with ONJ in patients of European ancestry treated with iv BPs using whole-exome sequencing (WES). The WES phase 1 included 44 multiple myeloma patients (22 ONJ cases and 22 controls) and WES phase 2 included 17 ONJ patients with solid tumors. Multivariable logistic regression analysis was performed to estimate the odds ratios (ORs) and 95% confidence intervals (CI), adjusting for age, sex, and principal components for ancestry. Meta-analysis of WES phase 1 and 2 was performed to estimate the combined ORs. In silico analyses were then performed to identify expression quantitative loci (eQTL) single-nucleotide polymorphisms (SNPs) that are in high linkage disequilibrium (LD) with the top SNPs. The associations of the potentially functional SNPs were replicated and validated in an independent case-control study of 48 patients of European ancestry treated with iv BPs (19 ONJ cases and 29 controls). The top SNPs in the exome-wide association meta-analysis were two SNPs on chromosome 10: SIRT1 SNP rs7896005 and HERC4 SNP rs3758392 with identical OR of 0.07 (0.01-0.46; p = 3.83 × 10-5 ). In the in silico functional analyses, two promoter region SNPs (rs7894483 and rs3758391) were identified to be in high LD with the index SNPs and are eQTLs for SIRT1 gene in whole blood in the GTEx database. The ORs were 0.30 (0.10-0.88), 0.26 (0.12-0.55), and 0.26 (0.12-0.55) for the WES top SNP rs7896005 and two promoter SNPs rs7894483 and rs3758391, respectively, in the replication sample. In summary, we identified the SIRT1/HERC4 locus on chromosome 10 to be associated with iv BP-induced ONJ and two promoter SNPs that might be the potential genetic markers for this association. © 2017 The Authors.Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Identifying a combined biomarker for bisphosphonate-related osteonecrosis of the jaw.

BACKGROUND: For this study, the aim was to identify combined biomarkers associated with bisphosphonate-related osteonecrosis of the jaw (BRONJ). MATERIALS AND METHODS: Microarray data for GSE7116 were downloaded from the Gene Expression Omnibus database, which contains 26 samples, including without ONJ, and 5 healthy volunteers. The combined biomarkers were identified using principal component analysis, and the pathway enrichment analyses were performed using the DAVID online tool. RESULTS: Two hundred differently expressed genes between groups were detected according to the significances. From functional annotation, Y-box binding protein 1 and heterogeneous nuclear ribonucleoprotein C were found to be included in the most significant 10 pathways. Ten combined gene sets were identified that were effective in classifying multiple myeloma (MM) with ONJ and MM without ONJ. CONCLUSION: Identifying combined gene expression profiles is expected to contribute to more personalized management of BRONJ and to improve existing therapies, and it will be helpful in finding new therapies by identifying more predictive biomarkers.

Genetic factors related with early onset of osteonecrosis of the jaw in patients with multiple myeloma under zoledronic acid therapy.

Specific genetic polymorphisms (SNPs) have been correlated with the development of bisphosphonate-related osteonecrosis of the jaw (BRONJ) in small series. We screened 140 myeloma patients (36 patients with and 104 without BRONJ) for the presence of previously identified SNPs in PPARG and CYP2C8 genes. All the patients received exclusively zolendronic acid (ZA) therapy and were followed prospectively for BRONJ. SNPs in both genes were associated with a higher risk of development of early BRONJ, occurring within less than 2 years of ZA therapy (59% vs. 16%, p = .022 for PPARG and 29% vs. 7%, p = .07 for CYP2C8) and a shorter time to develop BRONJ (59% versus 12%, p = .011 for PPARG and 29% versus 0% at 2 years, p = .037 for CYP2C8), independently of indices of poor oral hygiene. Thus, although preliminary, our data indicate that the presence of SNPs in PPARG and CYP2C8 genes may be associated with increased risk of early BRONJ.