AG-120, an Oral, Selective, First-in-Class, Potent Inhibitor of Mutant IDH1, Reduces Intracellular 2HG and Induces Cellular Differentiation in TF-1 R132H Cells and Primary Human IDH1 Mutant AML Patient Samples Treated Ex Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3734-3734 ◽  
Author(s):  
Erica Hansen ◽  
Cyril Quivoron ◽  
Kim Straley ◽  
René M Lemieux ◽  
Janeta Popovici-Muller ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cellular Differentiation ◽  
Ex Vivo ◽  
Day Treatment ◽  
Relative Proportion ◽  
Idh1 R132h ◽  
Idh Mutations ◽  
Recherche Médicale ◽  
Mutant Idh1 ◽  
Institut National Du Cancer

Abstract Point mutations in isocitrate dehydrogenase (IDH) define distinct subsets of acute myelogenous leukemia (AML). IDH is a metabolic enzyme that interconverts isocitrate and α-ketoglutarate (α-KG), but cancer-associated point mutations in IDH1 and IDH2 confer a neomorphic activity that allows reduction of α-KG to the oncometabolite R-2-hydroxyglutarate (2-HG). High levels of 2-HG have been shown to inhibit α-KG-dependent dioxygenases including histone and DNA demethylases, which play a key role in regulating the epigenetic state of cells, but the relationship between 2-HG and oncogenesis is not completely understood. Consistent with 2-HG promoting cancer via an effect on chromatin structure, patients harboring IDH mutations display a CpG island methylator phenotype (CIMP) and several studies have shown that overexpression of IDH mutant enzymes can induce histone and DNA hypermethylation as well as block cellular differentiation. In addition, mice engineered to express IDH1-R132H in hematopoietic tissue have increased early hematopoietic progenitors, splenomegaly, anemia, hypermethylated histones and altered DNA methylation patterns similar to those found in AML patients harboring IDH1/2 mutations.[i] Taken together, these data suggest that cancer-associated IDH mutations may induce a block in cellular differentiation to promote tumorigenesis. To investigate whether selective pharmacological inhibition of the mutant IDH1 enzyme could provide an effective way to lower intracellular 2-HG levels and restore normal differentiation, we treated TF-1 cells or primary human AML patient samples expressing mutant IDH1 with AG-120, an oral, selective, first-in-class, potent IDH1 mutant inhibitor currently in phase I clinical trials. Treatment with AG-120 decreased intracellular 2-HG levels, inhibited growth factor independent proliferation and restored erythropoietin (EPO)-induced differentiation in TF-1 IDH1-R132H cells. Similarly, pharmacological inhibition of mutant IDH1 enzyme with AG-120 in primary human blast cells cultured ex vivo provided an effective way to lower intracellular 2-HG levels and induced myeloid differentiation. Taken together, these data demonstrate that AG-120 is effective at lowering 2-HG levels and restoring cellular differentiation, and support further clinical development of this compound. Figure 1: Diagnosis and karyotypes of primary AML patient samples used in ex vivo studies Figure 1:. Diagnosis and karyotypes of primary AML patient samples used in ex vivo studies PB = peripheral blood, BM = bone marrow Figure 2: Percent 2-HG remaining relative to DMSO control after 6-day treatment with AG-120 in IDH1 R132H or IDH1 R132C patient samples Figure 2:. Percent 2-HG remaining relative to DMSO control after 6-day treatment with AG-120 in IDH1 R132H or IDH1 R132C patient samples or following 6 days of treatment with control (DMSO) or AG-120 (0.5, 1.0, and 5.0 μM) Figure 3: Relative proportion of cell types in human AML bone marrow samples untreated Figure 3:. Relative proportion of cell types in human AML bone marrow samples untreated [i] M. Sasaki et al., IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics. Nature 488(7413):656-9, 2012. Disclosures Hansen: Agios Pharmaceuticals: Employment, Stockholder Other. Quivoron:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la recherche contre le Cancer (ARC): Grant, Grant Other; AGIOS: Grant Other. Straley:Agios Pharmaceuticals: Employment, Stockholder Other. Lemieux:Agios Pharmaceuticals: Employment, Stockholder Other, US20130190249 (pending) Patents & Royalties. Popovici-Muller:Agios Pharmaceuticals: Employment, Stockholder Other. Fathi:Agios Pharmaceuticals: Advisory board participation Other. Gliser:Agios Pharmaceuticals: Employment, Stockholder Other. David:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la Recherche contre le Cancer (ARC): Grant, Grant Other; Association Laurette Fugain: Grant, Grant Other; AGIOS: Grant Other. Bernard:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Ligue Nationale contre le cancer (LNCC): Grant, Grant Other; AGIOS: Grant Other. Dorsch:Agios Pharmaceuticals: Employment, Stockholder Other. Yang:Agios Pharmaceuticals: Employment, Stockholder Other. Su:Agios Pharmaceuticals: Employment, Stockholder Other. Agresta:Agios Pharmaceuticals: Employment, Stockholder Other. de Botton:AGIOS: Grant Other. Penard-Lacronique:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la recherche contre le Cancer (ARC): Grant, Grant Other; AGIOS: Grant Other. Yen:Agios Pharmaceuticals: Employment, Stockholder Other.

Loss of BCAT1 Expression is a Sensitive Marker for IDH-Mutant Diffuse Glioma

Neurosurgery ◽  
2018 ◽  
Vol 85 (3) ◽  
pp. 335-342
Author(s):  
Yen-Ying Chen ◽  
Hsiang-Ling Ho ◽  
Shih-Chieh Lin ◽  
Chih-Yi Hsu ◽  
Donald Ming-Tak Ho
Keyword(s):  
Dna Sequencing ◽  
Prognostic Significance ◽  
Surrogate Marker ◽  
Cost Effective ◽  
Mutation Rates ◽  
Diffuse Glioma ◽  
Idh Mutation ◽  
Idh1 R132h ◽  
Idh Mutations ◽  
Mutant Idh1

Abstract BACKGROUND IDH mutation is an important prognostic factor of diffuse astrocytomas. Although the majority of IDH mutations could be identified by immunohistochemical (IHC) stain for R132H-mutant IDH1, DNA sequencing would be required for IHC negative cases to determine their IDH mutation status. This approach is not cost-effective for tumors with low IDH mutation rates. OBJECTIVE To investigate whether BCAT1 could be used as a surrogate marker for IDH mutations, because BCAT1 is an enzyme related to IDH genes. METHODS A group of 120 anaplastic astrocytomas were immunostained for BCAT1, ATRX, and R132H-mutant IDH1. Staining results correlated with the results of DNA sequencing of IDH1/IDH2. RESULTS DNA sequencing showed IDH1/2 mutations in 50.8% of cases of which 73.8% had IDH1 R132H mutation. Several IDH1 noncodon 132 mutations, ie, G97D, S122N, G123E, I130K, and G131S, which had uncertain prognostic significance, were identified. IHC stain for R132H-mutant IDH1 identified 93.3% of IDH1 R132H mutations and 70.5% of all IDH mutations. BCAT1 loss was seen in 65.8% of cases, its sensitivity to identify IDH mutations was 96.7%. The sensitivity reached 100% for IDH1 codon 132 and IDH2 codon 172 mutations. CONCLUSION Positive BCAT1 stain could be used to exclude diffuse gliomas with IDH1 codon 132 and IDH2 codon 172 mutations. Selecting cases with negative BCAT1 and R132H-mutant IDH1 staining for DNA sequencing of IDH1/2 genes could improve the cost-effectiveness of detecting IDH mutations particularly in tumors with low IDH mutation rates, and confine the need of 1p/19q assay in IDH-mutant tumors.

IDH1 Mutant Inhibitor Induces Cellular Differentiation and Offers a Combination Benefit With Ara-C In a Primary Human Idh1 Mutant AML Xenograft Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3946-3946 ◽  
Author(s):  
Katharine Yen ◽  
Rene Lemieux ◽  
Janeta Popovici-Muller ◽  
Yue Chen ◽  
Hua Yang ◽  
...  
Keyword(s):  
Cellular Differentiation ◽  
Low Dose ◽  
Ex Vivo ◽  
Point Mutations ◽  
Xenograft Model ◽  
Tumor Burden ◽  
Employment Equity ◽  
Isocitrate Dehydrogenase 1 ◽  
Idh Mutations ◽  
Equity Ownership

Abstract Somatic point mutations in isocitrate dehydrogenase 1/2 have a gain-of-function neomorphic activity that converts alpha-ketoglutarate to the oncometabolite, R (-)-2-hydroxyglutarate (2HG). Prospective studies of AML patients carrying IDH mutations have shown that intracellular concentrations of 2HG can range from 3-10 mM. This abnormal level of 2HG results in dysregulation of alpha-ketoglutarate dependent enzymes leading to alterations in the epigenetic state of hematopoietic progenitor/stem cells and functionally blocks their ability to fully differentiate. We have developed a potent and selective, orally available IDH1 mutant inhibitor AGI-14100, that is able to reduce intracellular 2HG concentrations to baseline levels found in wildtype cells. Ex vivo treatment of IDH1 mutant-containing primary human AML patient samples with AGI-14100 induced a proliferative burst followed by cellular differentiation as shown by flow cytometry and cytology. We next treated a primary human IDH1 (R132H)/FLT3-ITD mutant xenograft model with AGI-14100 either alone or in combination with Ara-c. In these studies, AGI-14100 alone significantly decreased tumor burden in the peripheral blood after 1 month of continuous BID treatment. In combination with a short-term, low-dose course of Ara-C, we also observed a decrease in the bone marrow tumor burden that was better than either treatment alone. Furthermore, this response was sustainable for >3 weeks even after dosing of both drugs had been terminated. Taken together, these data suggest that inhibition of mutant IDH1with AGI-14100 and low dose Ara-c could provide a combination benefit for patients with AML. Disclosures: Yen: Agios Pharmaceuticals: Employment, Equity Ownership. Lemieux:agios Pharmaceuticals: Employment, Equity Ownership. Popovici-Muller:agios Pharmaceuticals: Employment, Equity Ownership. Chen:agios Pharmaceuticals: Employment, Equity Ownership. Yang:Agios Pharmaceuticals: Employment, Equity Ownership. Straley:Agios Pharmaceuticals: Employment, Equity Ownership. Choe:agios Pharmaceuticals: Employment, Equity Ownership. Dorsch:agios Pharmaceuticals: Employment, Equity Ownership. Agresta:agios Pharmaceuticals: Employment, Equity Ownership. Schenkein:agios Pharmaceuticals: Employment, Equity Ownership. Biller:agios Pharmaceuticals: Employment, Equity Ownership. Su:agios Pharmaceuticals: Employment, Equity Ownership. Wang:Agios Pharmaceuticals: Employment, Equity Ownership.

BCL-2 Inhibition As a Synthetic Lethal Approach To Target Isocitrate Dehydrogenase Mutations In Acute Myeloid Leukemia Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 885-885 ◽  
Author(s):  
Steven M. Chan ◽  
Bruno C Medeiros ◽  
Ravi Majeti
Keyword(s):  
Cell Lines ◽  
Ex Vivo ◽  
Leukemic Cells ◽  
Synthetic Lethal ◽  
Nsg Mice ◽  
Idh Mutations ◽  
Selective Elimination ◽  
Shrna Library ◽  
Mutant Idh1

Abstract Mutations in isocitrate dehydrogenase (IDH) 1 and 2 occur in about 15% of acute myeloid leukemia (AML) patients. Studies of paired samples at diagnosis and relapse have demonstrated that IDH mutations, in contrast to FLT3 or RAS mutations, are stable during disease evolution. This finding indicates that a small population of cells harboring IDH mutations can persist in remission and eventually contribute to relapse. Therapeutic strategies that eradicate this IDH-mutated population have the potential to result in long-term remission. Small molecule inhibitors specific for the mutant IDH enzymes have recently been developed (Wang et al Science 2013), but it is not known if they are effective in targeting primary AML cells including the leukemic stem cell (LSC) compartment in vivo. We sought an alternative approach to target IDH-mutated cells based on the concept of non-oncogene addiction which refers to the increased dependence on a subset of non-mutated genes for survival in response to activation of a specific oncogene. This dependency can be exploited therapeutically by inhibiting the activity of these non-oncogenes resulting in selective elimination of malignant cells, a phenomenon known as synthetic lethality (SL). In an effort to identity SL targets against IDH mutations, we performed a pooled lentiviral RNA interference (RNAi) screen to search for genes that, when inhibited, led to the selective elimination of mutant IDH1 expressing cells. Our lentiviral short hairpin RNA (shRNA) library consisted of 27,500 unique shRNAs targeting 5,043 human genes of relevance to cancer biology. Each shRNA was tagged with a unique barcode sequence which permitted downstream identification by sequencing. The lentiviral shRNA library was transduced into a human AML cell line engineered to express mutant IDH1 (R132H) under the control of a doxycycline-inducible promoter. Following transduction, the cells were cultured in doxycycline to deplete shRNAs that were synthetic lethal to mutant IDH1. The relative abundance of each shRNA was subsequently determined by high-throughput sequencing of the barcode. Using a stringent algorithm designed to minimize false positive hits, the prosurvival gene BCL-2 was identified as one of the top SL hits. We confirmed that RNAi-mediated knockdown of BCL-2 expression was selectively lethal to mutant IDH expressing AML cell lines and further demonstrated that exposure to a cell-permeable form of (R)-2-hydroxyglutarate, the oncometabolite produced by mutant IDH, was sufficient to induce BCL-2 dependence. Similarly, pharmacologic inhibition of BCL-2 with ABT-199, a novel orally bioavailable and highly specific inhibitor of BCL-2 (Souers et al Nature Medicine 2013), was significantly more toxic to mutant IDH expressing AML cell lines than isogenic cell lines with wildtype (WT) IDH. We next investigated the impact of IDH mutation status on ABT-199 sensitivity of primary AML cells and found that FACS-purified blasts with IDH mutations were 10-fold more sensitive to ABT-199 than blasts with WT IDH in ex vivo culture conditions. Normal cord blood hematopoietic stem and progenitor cells were highly resistant to ABT-199 treatment ex vivo suggestive of a wide therapeutic index. To demonstrate in vivo activity, we treated immunodeficient NOD/SCID/IL2Rγ-null (NSG) mice engrafted with primary human IDH-mutated leukemic cells with either ABT-199 at a dose of 100 mg/kg/day or vehicle control for 7 consecutive days by oral administration. Bone marrow engraftment analysis before and after treatment showed a 10 to 20-fold reduction in leukemic burden in ABT-199 treated mice, whereas no difference was seen in vehicle-treated mice. Importantly, bone marrow cells collected from ABT-199 treated mice failed to engraft in secondary transplant recipients indicative of a loss of LSC activity. In separate experiments, lentiviral transduction of BCL-2 shRNA vectors into IDH-mutated primary AML cells to knockdown BCL-2 expression impaired their engraftment in NSG mice, further validating the detrimental effect of BCL-2 inhibition on IDH-mutated LSCs. In summary, our results indicate that IDH mutations increase BCL-2 dependence in leukemic cells including LSCs and identify a subgroup of patients that is likely to respond to pharmacologic BCL-2 inhibition. Our data provide the preclinical rationale for investigating the use ABT-199 in this patient subgroup in clinical trials. Disclosures: No relevant conflicts of interest to declare.

In Vitro, In Silico and Ex Vivo Studies of Dihydroartemisinin Derivatives as Antitubercular Agents

2019 ◽  
Vol 19 (8) ◽  
pp. 633-644 ◽  
Author(s):  
Komal Kalani ◽  
Sarfaraz Alam ◽  
Vinita Chaturvedi ◽  
Shyam Singh ◽  
Feroz Khan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Silico ◽  
Ex Vivo ◽  
Virulent Strain ◽  
Infection Model ◽  
Mouse Bone Marrow ◽  
Significant Activity ◽  
Macrophage Infection ◽  
In Silico Studies

Introduction: As a part of our drug discovery program for anti-tubercular agents, dihydroartemisinin (DHA-1) was screened against Mtb H37Rv, which showed moderate anti-tubercular activity (>25.0 µg/mL). These results prompted us to carry out the chemical transformation of DHA-1 into various derivatives and study their antitubercular potential. Materials and Methods: DHA-1 was semi-synthetically converted into four new acyl derivatives (DHA-1A – DHA-1D) and in-vitro evaluated for their anti-tubercular potential against Mycobacterium tuberculosis H37Rv virulent strain. The derivatives, DHA-1C (12-O-(4-nitro) benzoyl; MIC 12.5 µg/mL) and DHA-1D (12-O-chloro acetyl; MIC 3.12µg/mL) showed significant activity against the pathogen. Results: In silico studies of the most active derivative (DHA-1D) showed interaction with ARG448 inhibiting the mycobacterium enzymes. Additionally, it showed no cytotoxicity towards the Vero C1008 cells and Mouse bone marrow derived macrophages. Conclusion: DHA-1D killed 62% intracellular M. tuberculosis in Mouse bone marrow macrophage infection model. To the best of our knowledge, this is the first-ever report on the antitubercular potential of dihydroartemisinin and its derivatives. Since dihydroartemisinin is widely used as an antimalarial drug; these results may be of great help in anti-tubercular drug development from a very common, inexpensive, and non-toxic natural product.

130 Burn Injury Reduces Bone Marrow Mesenchymal Stem Cells and Sensitizes Their Adrenergic Receptor Subgroups in a Murine Model

2021 ◽  
Vol 42 (Supplement_1) ◽  
pp. S87-S88
Author(s):  
Kuzhali Muthumalaiappan ◽  
Maria Camargo Johnson ◽  
Julia Walczak ◽  
Vimal Subramaniam ◽  
Anthony J Baldea ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Adrenergic Receptor ◽  
Burn Injury ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Traumatic Injury ◽  
Hematopoietic Cell ◽  
The Right

Abstract Introduction Previous burn and traumatic injury studies have established that adrenergic signaling is increased after burn injury and may lead to an impairment of hematopoietic cell development in the bone marrow (BM). Nonetheless, mesenchymal stem cells (MSCs), which have gained momentum in regenerative medicine also play a predominant role in the BM niche. Understanding the propensity of the adrenergic receptor (AR) response by MSCs can be utilized for devising targeted therapies. However, the traditional plastic adherence procedure using ex vivo culture of BM cells for several weeks may skew the actual characteristics of MSCs. Our current study focused on isolating MSCs from freshly obtained BM in a murine scald burn model with a goal to characterize the expression pattern of native AR subgroups present on BM MSCs as compared to sham mice. Methods Eight, two-month-old adult female mice were subjected to a 15% total body 3rd degree burn or sham burn. The mice were sacrificed 7 days later. Femurs were removed and total bone marrow cells were flushed out. Multi parametric flow cytometry was used to gate for cells negative for hematopoietic cell markers (CD45, CD11B) and positive for MSC markers (CD105, CD106, SSEA, Ly6A) and AR subgroups (α1, α2, β1, β2, β3). We measured the number of BM MSCs, quantified the subtypes of ARs present on MSCs, and compared the ratio of AR antibody binding per total MSC population. Results Overall the frequency of MSCs per million total BM cells decreased by 48% post-burn injury with165,300 ± 194 in sham versus 110,000 ± 30 in burn displayed as bar graph in Panel A. Over 90% of MSCs consistently express β2 AR and only 10% express α2 AR subgroup in both scald and sham burn. Presence of other subgroups ranged from 50% to 80% of MSCs as seen in histograms to the right of dotted line in Panel B. Our AR propensity score based on AR mean fluorescence intensity adjusted to total number of MSCs present was increased by 2.8-fold for α1, 2.5-fold for β1, 1.6-fold for β3, and 1.3-fold for β2 AR subgroups (Panel C). These findings indicate burn injury not only decreases the frequency of BM MSCs but also increases the affinity of certain AR subgroups present on MSCs. Since BM MSCs are the major source of cytokines, chemokines and growth factors; detailed studies on AR mediated signaling in BM MSCs is warranted. Conclusions Polarization of AR signaling in BM MSCs by burn-induced catecholamines may have broader implications for comorbidities such as bone resorption and muscle wasting observed in human patients post burn trauma.

scholarly journals Outcome of alloanergized haploidentical bone marrow transplantation after ex vivo costimulatory blockade: results of 2 phase 1 studies

Blood ◽  
2008 ◽  
Vol 112 (6) ◽  
pp. 2232-2241 ◽  
Author(s):  
Jeff K. Davies ◽  
John G. Gribben ◽  
Lisa L. Brennan ◽  
Dongin Yuk ◽  
Lee M. Nadler ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
T Cell ◽  
Marrow Transplantation ◽  
Viral Infections ◽  
Ex Vivo ◽  
Chronic Gvhd ◽  
Costimulatory Blockade ◽  
Donor T Cells

AbstractWe report the outcomes of 24 patients with high-risk hematologic malignancies or bone marrow failure (BMF) who received haploidentical bone marrow transplantation (BMT) after ex vivo induction of alloantigen-specific anergy in donor T cells by allostimulation in the presence of costimulatory blockade. Ninety-five percent of evaluable patients engrafted and achieved full donor chimerism. Despite receiving a median T-cell dose of 29 ×106/kg, only 5 of 21 evaluable patients developed grade C (n = 4) or D (n = 1) acute graft-versus-host disease (GVHD), with only one attributable death. Twelve patients died from treatment-related mortality (TRM). Patients reconstituted T-cell subsets and immunoglobulin levels rapidly with evidence of in vivo expansion of pathogen-specific T cells in the early posttransplantation period. Five patients reactivated cytomegalovirus (CMV), only one of whom required extended antiviral treatment. No deaths were attributable to CMV or other viral infections. Only 1 of 12 evaluable patients developed chronic GVHD. Eight patients survive disease-free with normal performance scores (median follow-up, 7 years). Thus, despite significant early TRM, ex vivo alloanergization can support administration of large numbers of haploidentical donor T cells, resulting in rapid immune reconstitution with very few viral infections. Surviving patients have excellent performance status and a low rate of chronic GVHD.

scholarly journals GPR109A mediates the effects of hippuric acid on regulating osteoclastogenesis and bone resorption in mice

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Jin-Ran Chen ◽  
Haijun Zhao ◽  
Umesh D. Wankhade ◽  
Sree V. Chintapalli ◽  
Can Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Mass ◽  
Hippuric Acid ◽  
Ex Vivo ◽  
Marrow Cell ◽  
Osteoclast Differentiation ◽  
Bone Homeostasis ◽  
Wild Type ◽  
Osteoclast Precursors

AbstractThe G protein-coupled receptor 109 A (GPR109A) is robustly expressed in osteoclastic precursor macrophages. Previous studies suggested that GPR109A mediates effects of diet-derived phenolic acids such as hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA) on promoting bone formation. However, the role of GPR109A in metabolic bone homeostasis and osteoclast differentiation has not been investigated. Using densitometric, bone histologic and molecular signaling analytic methods, we uncovered that bone mass and strength were significantly higher in tibia and spine of standard rodent diet weaned 4-week-old and 6-month-old GPR109A gene deletion (GPR109A−/−) mice, compared to their wild type controls. Osteoclast numbers in bone and in ex vivo bone marrow cell cultures were significantly decreased in GPR109A−/− mice compared to wild type controls. In accordance with these data, CTX-1 in bone marrow plasma and gene expression of bone resorption markers (TNFα, TRAP, Cathepsin K) were significantly decreased in GPR109A−/− mice, while on the other hand, P1NP was increased in serum from both male and female GPR109A−/− mice compared to their respective controls. GPR109A deletion led to suppressed Wnt/β-catenin signaling in osteoclast precursors to inhibit osteoclast differentiation and activity. Indeed, HA and 3-3-PPA substantially inhibited RANKL-induced GPR109A expression and Wnt/β-catenin signaling in osteoclast precursors and osteoclast differentiation. Resultantly, HA significantly inhibited bone resorption and increased bone mass in wild type mice, but had no additional effects on bone in GPR109A−/− mice compared with their respective untreated control mice. These results suggest an important role for GPR109A during osteoclast differentiation and bone resorption mediating effects of HA and 3-3-PPA on inhibiting bone resorption during skeletal development.

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