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Type 1 diabetes mellitus (T1DM) patients have osteopenia and impaired fracture healing due to decreased osteoblast activity. Further, no adequate treatments are currently available that can restore impaired healing in T1DM; hence a significant need exists to investigate new therapeutics for treatment of orthopedic complications. Sclerostin (SOST), a WNT antagonist, negatively regulates bone formation, and SostAb is a potent bone anabolic agent. To determine whether SOST antibody (SostAb) treatment improves fracture healing in streptozotocin (STZ) induced T1DM mice, we administered SostAb twice weekly for up to 21days post-fracture, and examined bone quality and callus outcomes at 21days and 42days post-fracture (11 and 14weeks of age, respectively). Here we show that SostAb treatment improves bone parameters; these improvements persist after cessation of antibody treatment. Markers of osteoblast differentiation such as Runx2, collagen I, osteocalcin, and DMP1 were reduced, while an abundant number of SP7/osterix-positive early osteoblasts were observed on the bone surface of STZ calluses. These results suggest that STZ calluses have poor osteogenesis resulting from failure of osteoblasts to fully differentiate and produce mineralized matrix, which produces a less mineralized callus. SostAb treatment enhanced fracture healing in both normal and STZ groups, and in STZ+SostAb mice, also reversed the lower mineralization seen in STZ calluses. Micro-CT analysis of calluses revealed improved bone parameters with SostAb treatment, and the mineralized bone was comparable to Controls. Additionally, we found sclerostin levels to be elevated in STZ mice and β-catenin activity to be reduced. Consistent with its function as a WNT antagonist, SostAb treatment enhanced β-catenin activity, but also increased the levels of SOST in the callus and in circulation. Our results indicate that SostAb treatment rescues the impaired osteogenesis seen in the STZ induced T1DM fracture model by facilitating osteoblast differentiation and mineralization of bone.

NK cells are innate-like lymphocytes that eliminate virally infected and cancerous cells, but the mechanisms that control NK cell development and cytotoxicity are incompletely understood. We identified roles for sclerostin domain–containing-1 (Sostdc1) in NK cell development and function. Sostdc1-knockout (Sostdc1−/−) mice display a progressive accumulation of transitional NK cells (tNKs) (CD27+CD11b+) with age, indicating a partial developmental block. The NK cell Ly49 repertoire in Sostdc1−/− mice is also changed. Lower frequencies of Sostdc1−/− splenic tNKs express inhibitory Ly49G2 receptors, but higher frequencies express activating Ly49H and Ly49D receptors. However, the frequencies of Ly49I+, G2+, H+, and D+ populations were universally decreased at the most mature (CD27−CD11b+) stage. We hypothesized that the Ly49 repertoire in Sostdc1−/− mice would correlate with NK killing ability and observed that Sostdc1−/− NK cells are hyporesponsive against MHC class I–deficient cell targets in vitro and in vivo, despite higher CD107a surface levels and similar IFN-γ expression to controls. Consistent with Sostdc1’s known role in Wnt signaling regulation, Tcf7 and Lef1 levels were higher in Sostdc1−/− NK cells. Expression of the NK development gene Id2 was decreased in Sostdc1−/− immature NK and tNK cells, but Eomes and Tbx21 expression was unaffected. Reciprocal bone marrow transplant experiments showed that Sostdc1 regulates NK cell maturation and expression of Ly49 receptors in a cell-extrinsic fashion from both nonhematopoietic and hematopoietic sources. Taken together, these data support a role for Sostdc1 in the regulation of NK cell maturation and cytotoxicity, and identify potential NK cell niches.

SOST is a negative regulator of bone formation, and mutations in human SOST are responsible for sclerosteosis. In addition to high bone mass, sclerosteosis patients occasionally display hand defects, suggesting that SOST may function embryonically. Here we report that overexpression of SOST leads to loss of posterior structures of the zeugopod and autopod by perturbing anterior–posterior and proximal–distal signaling centers in the developing limb. Mutant mice that overexpress SOST in combination with Grem1 and Lrp6 mutations display more severe limb defects than single mutants alone, while Sost−/− significantly rescues the Lrp6−/− skeletal phenotype, signifying that SOST gain-of-function impairs limb patterning by inhibiting the WNT signaling through LRP5/6.

Natural killer (NK) cells are specialized lymphocytes with innate ability to eliminate virally infected and cancerous cells, but the mechanisms that control NK cell development and cytotoxicity are incompletely understood. We identified novel roles for sclerostin domain containing-1 (Sostdc1) in NK cell development and function. Sostdc1-knockout (KO) mice display a progressive accumulation of transitional NK cells (CD27+CD11b+, tNK) with age, indicating a partial developmental block. The Ly49 repertoire on NK cells in KO mice is also changed. Lower frequencies (%) of KO splenic tNKs express inhibitory Ly49G2, but higher % of activating Ly49H+ and D+ cells. However, the % of Ly49I+, G2+, H+ and D+ populations were universally decreased at the most mature (CD27− CD11b+, mNK) stage. We hypothesized that the Ly49 repertoire in Sostdc1-KO mice would correlate with NK killing ability. We observed that KO NK cells are hyporesponsive against MHC-I-deficient cell targets in vitro and in vivo, despite higher CD107a surface levels and similar IFN-gamma expression to controls. Further investigation is required to determine Sostdc1’s role in NK cell cytotoxicity and self/non-self recognition. Sostdc1 is expressed in the bone periosteum, and we discovered Sostdc1 expression in NK cells. Consistent with this, reciprocal BM transplant experiments showed that Sostdc1 regulates NK cells through cell-extrinsic and cell-intrinsic manners. Transcriptomic profiling should identify if Sostdc1’s known roles in Wnt and BMP signaling are active, and reveal novel effects of Sostdc1 on NK cells. Taken together, these data support a role for Sostdc1 in the regulation of NK cell maturation, and NK cell cytotoxicity, and identify potential NK cell niches.

Background: Bladder cancer is among the top ten most common cancers, with about ~380,000 new cases and ~150,000 deaths per year worldwide. Platinum-based combination chemotherapy is commonly used to treat advanced bladder cancer. It has been shown that only ~50% of the patients with advanced bladder cancer respond to platinum-based therapy. Methods: We have utilized a patient-derived bladder cancer xenograft (PDX) platform to characterize the molecular mechanisms that contribute to resistance of gemcitabine-cisplatin combination therapy in advanced bladder cancer. We have also identified key regulatory pathways in our PDX models that can be targeted to treat chemotherapy resistant bladder cancer using RNAseq analysis. Transcriptome profiling of P0 (passage 0) bladder cancer xenograft tumors from 4 PDX lines (2 gemcitabine-cisplatin resistant lines and 2 drug sensitive lines) was performed by RNA-Seq analysis, before and after a 21-day cisplatin/gemcitabine drug treatment regimen. Results: The RNA-seq data has indicated significant differences between the transcriptional profiles of drug-sensitive and drug-resistant tumors. PDXs retained morphology and shared 92-97% of genetic alterations of parental cancer cells. We identified 333 genes >2 fold up or down regulated in the drug resistant tumors compared to the drug sensitive tumors. Significantly up-regulated genes in drug resistant tumors analyzed include metabolic enzymes ALDH2, ALDH3A1, ALDH4A1 and ALDH7A1, transporter proteins ABCA1, SLC1A4, SLC2A5, SLC30A1, SLC39A6, SLC7A5 and SLC9A3, Notch ligand JAG2, Growth hormone receptor GHR and transmembrane protein GPNMB. Consistent with the change of cell surface proteins such as GHR and GPNMB, the MAPK and the PI3K-AKT pathways were upregulated when PDXs became resistant to cisplatin treatment. Additional changes in gene expression based on RNA-seq data before and after drug treatment were also found. Conclusion: Chemoresistance to gemcitabine and cisplatin is associated with altered expression of several cell surface proteins and upregulation of the downstream signaling pathways. Targeting these cell surface proteins can possibly be harnessed to overcome chemoresistance. GPNMB is a type I transmembrane protein that has previously been shown to be up-regulated in many metastatic cancers including breast cancer. Potentially targeting GPNMB with glembatumumab vedotin, an antibody-drug-conjugate has shown promising results in treating several cancers including breast cancer and osteosarcoma. Further studies will elucidate whether targeting GPNMB is an effective strategy for the treatment of chemotherapy resistant bladder cancer. This study received funding by a developmental grant from the UCDCC. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM number: LLNL-688318 Citation Format: Kelly A. Martin, Nicholas R. Hum, Aimy Sebastian, Deepa K. Murugesh, Chong-Xian Pan, Ai-Hong Ma, Ralph de Vere White, Gaby Loots. RNA sequencing of bladder cancer patient-derived xenograft models identifies genes associated with chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 97. doi:10.1158/1538-7445.AM2017-97

Natural killer (NK) cells are specialized lymphocytes with innate ability to eliminate virally infected and cancerous cells. NK cells stochastically express Ly49 activating and inhibitory surface receptors, through mechanisms that are not fully understood. Ly49 expression during NK cell development is regulated by a combination of external and internal factors, such as major histocompatibility complex class I (MHC-I) ligands and cytokines. Global deletion of sclerostin domain containing-1 (Sostdc1), a secreted protein, leads to altered bone homeostasis via dysregulation of Wnt and BMP signaling, which in turn, could result in altered immune cell development in the bone marrow (BM). Using Sostdc1-knockout (KO) mice, we have discovered a novel role for Sostdc1 in NK cell development. Flow cytometric analysis of the Ly49 repertoire in immature (CD27+CD11b-; iNK), transitional (CD27+CD11b+, tNK) and most mature (CD27-CD11b+, mNK) Sostdc1-KO NK cells demonstrated expression differences in Ly49G2, I, H, and D receptors. We also observed a progressive increase in tNK cell numbers and frequencies as the KOs aged from 12 weeks to 30 weeks, indicating a slowing or partial block in NK cell maturation with aging. Reciprocal BM chimera experiments showed that Sostdc1 regulates NK cells through cellintrinsic and cell-extrinsic manners. Consistent with this, Sostdc1 is expressed in the bone periosteum and we discovered Sostdc1 expression in CD45+ NK1.1-CD11b+ BM cells and iNK cells. Taken together, these data support a role for Sostdc1 in the regulation of NK cell differentiation and identify potential NK cell niches. Current studies are investigating whether Sostdc1-KO NK cells respond to stimulation and lyse MHC-I deficient target cells.

Investigation of the reciprocal effects of hematopoietic cell types and osteolineage cells on each other’s cell fates have clinical applications to improve bone fracture healing, prevent age-related bone loss and immune deficiencies. A significant knowledge gap exists as to the contribution of different osteolineage cells to the differentiation, proliferation, and long term survival of B cells in the BM. In global sclerostin knockout (SostKO) mice, we previously uncovered a cell-extrinsic requirement for sclerostin on B lymphocyte development. It is widely accepted that sclerostin is transcribed primarily in osteocytes (OCYs), but whether is it expressed and/or active in other bone progenitors has been controversial. In order to identify the Sost-expressing osteolineage cell type that is responsible for B cell support, we analyzed hematopoietic differentiation in mice lacking Sost in mesenchymal stem cells (MSCs) [Prx1-Cre], osteoblasts (OBs) [Col1a-Cre] and OCYs [Dmp1-Cre] through the use of conditional Sost-KO (cKO) mice. Loss of Sost expression in MSCs resulted in reduced BM cellularity, whereas this was unaffected by Sost-deficiency in OB and OCYs. Furthermore, MSC-specific depletion of Sost caused an accumulation of early B cell progenitors. In contrast, loss of Sost in OBs and OCYs resulted in a reduction of mature “recirculating” B cells in the BM. Remarkably, the percentage of long-term CD150+ CD48− HSCs was significantly reduced in MSC-cKO mice, but was not affected in OB- and OCY-cKOs. The data described herein suggest that Sost deficiency in MSCs, OBs, and OCYs differentially regulates hematopoietic stem cell retention, survival, maintenance and B cell differentiation in the bone microenvironment.

The role of apolipoprotein A-II (apoA-II) in high density lipoprotein (HDL) structure and metabolism has been studied previously in transgenic mice overexpressing either human or murine apoA-II. These studies have shown differences between these two groups of transgenic animals in the levels of very low density, low density, and high density lipoproteins, in the HDL particle size distribution, and in the relationship between apoA-II levels and lipoprotein levels. To determine whether these differences are due to the fact that human apoA-II is dimeric and murine apoA-II monomeric, we have examined the effects of monomeric human apoA-II (hA-IImon) in transgenic mice. Site-directed mutagenesis (Cys6→ Ser) was used to generate 15 transgenic founder lines of hA-IImon mice, that contained plasma hA-IImon concentrations over a 10-fold range (11 mg/dl to 185 mg/dl). The hA-IImon floated in the d ≤ 1.21 g/ml fraction and migrated as an apoA-II monomer by nonreducing SDS-polyacrylamide gel electrophoresis. HDL levels were not correlated with hA-IImon levels (r = −0.26); HDL particle size and size distribution, as well as very low density and low density lipoprotein levels and sizes, were unchanged compared to nontransgenic control mice. These results suggest that differences between mice overexpressing human dimeric apoA-II and those overexpressing murine apoA-II are the result of sequence differences between these two apoA-II molecules and are not solely due to the fact that human apoA-II exists as a dimer.

Fracture healing involves rapid stem and progenitor cell migration, homing, and differentiation. SDF-1 (CXCL12) is considered a master regulator of CXCR4-positive stem and progenitor cell trafficking to sites of ischemic (hypoxic) injury and regulates their subsequent differentiation into mature reparative cells. In this study, we investigated the role of SDF-1/CXCR4 signaling in fracture healing where vascular disruption results in hypoxia and SDF-1 expression. Mice were injected with AMD3100, a CXCR4 antagonist, or vehicle twice daily until euthanasia with the intent to impair stem cell homing to the fracture site and/or their differentiation. Fracture healing was evaluated using micro-computed tomography, histology, quantitative PCR, and mechanical testing. AMD3100 administration resulted in a significantly reduced hyaline cartilage volume (day 14), callus volume (day 42) and mineralized bone volume (day 42) and reduced expression of genes associated with endochondral ossification including collagen Type 1 alpha 1, collagen Type 2 alpha 1, vascular endothelial growth factor, Annexin A5, nitric oxide synthase 2, and mechanistic target of rapamycin. Our data suggest that the SDF-1/CXCR4 signaling plays a central role in bone healing possibly by regulating the recruitment and/or differentiation of stem and progenitor cells.

We have explored the application of fetal hematopoietic stem cell (HSC) transplants for cellular replacement in a murine model of beta-thalassemia. Liver-derived HSCs from nonthalassemic syngeneic murine fetal donors were transplanted into nonirradiated neonatal beta-thalassemic recipients. Significant erythrocyte chimerism (9-27%) was demonstrated in the majority of recipients at 1 month and remained stable or increased (up to 55%) during long-term follow-up in almost all cases. Chimeras had improved phenotypes, as evidenced by decreased reticulocyte counts, increased mean erythrocyte deformability, and decreased iron deposits in comparison to controls. To investigate whether the high degree of peripheral blood chimerism was predominantly a feature of erythroid elements or was a general feature of all hematopoietic elements, chimeras were created using donor HSCs "tagged" with a DNA transgene. Whereas donor hemoglobin comprised > 30% of total hemoglobin, nucleated tagged nonerythroid donor cells comprised < 1% of peripheral blood elements. Explanations for the observed selective increase in erythroid chimerism include longer survival of normal donor red cells compared to that of thalassemic red cells and the effective maturation of the donor erythroid elements in the bone marrow in chimeric animals. The latter explanation bears consideration because it is consistent with the process of ineffective erythropoiesis, well documented to occur in thalassemia, in which the majority of thalassemic erythroid cells are destroyed during erythropoiesis prior to release from the bone marrow. Overall, these data demonstrate the potential for significant erythroid chimerism and suggest that fetal HSC transplantation may play a significant role in future treatment.

We have used cationic liposomes to facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. AAV plasmid DNA complexed with liposomes showed levels of expression several fold higher than those of complexes with standard plasmids. In addition, long-term expression (> 30 days) of the gene, unlike the transient expression demonstrated by typical liposome-mediated transfection with standard plasmids, was observed. Southern analysis of chromosomal DNA further substantiated the hypothesis that the long-term expression was due to the presence of the transgene in the AAV plasmid-transfected group and not in the standard plasmid-transfected group. AAV plasmid-liposome complexes induced levels of transgene expression comparable to those obtained by recombinant AAV transduction. Primary breast, ovarian, and lung tumor cells were transfectable with the AAV plasmid DNA-liposome complexes. Transfected primary and cultured tumor cells were able to express transgene product even after lethal irradiation. High-level gene expression was also observed in freshly isolated CD3+, CD4+, and CD8+ T cells from normal human peripheral blood. Transfection efficiency ranged from 10 to 50% as assessed by intracellular interleukin-2 levels in interleukin-2-transfected cells. The ability to express transgenes in primary tumor and lymphoid cells may be applied toward tumor vaccine studies and protocols which may eventually permit highly specific modulation of the cellular immune response in cancer and AIDS.

We have previously shown that cationic liposomes facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. To test the clinical feasibility of using genetically modified tumor vaccines for the treatment of breast and ovarian cancers, we have constructed an expression plasmid pMP6IL2 and investigated the use of liposome-mediated gene delivery into primary, uncultured human breast and ovarian tumor cells to produce interleukin 2 (IL-2)-secreting tumor cells. We have demonstrated significant levels of IL-2 expression in tumor cell lines and primary breast and ovarian tumor cells using this AAV-based expression plasmid complexed to cationic liposomes. Transfections with the non-AAV plasmid containing the identical expression cassette as the AAV plasmid induced IL-2 expression in the tumor cell line but failed to produce IL-2 in primary tumor cells. Significant levels of IL-2 were induced with the AAV plasmid regardless of liposome compositions used for transfection. The transfected breast cell line and primary tumor cells were able to express the transgene product for up to 28 days after lethal radiation. The transfection efficiency was comparable for both the tumor cell line and primary tumor cells and ranged from 20 to 50% for both cell types as assessed by intracellular IL-2 staining. Although the primary tumor cell preparations consist of mixed population of cells, at least 40% of the tumor cells expressed the transgene as assessed by immunostaining for IL-2. The ability to efficiently express transgenes in freshly isolated, nondividing tumor cells may potentiate active immunotherapy strategies for gene-based cancer treatment.