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Evaluation of cellular response to mTORC1 inhibition, BLI analysis of visceral metastases in PyMT-BO1 tumor-bearing mice receiving combination therapies, and αvβ3-NP-RAPA toxicity studies.

Breast cancer bone metastases are common and incurable. Tumoral integrin β3 (β3) expression is induced through interaction with the bone microenvironment. Although β3 is known to promote bone colonization, its functional role during therapy of established bone metastases is not known. We found increased numbers of β3+ tumor cells in murine bone metastases after docetaxel chemotherapy. β3+ tumor cells were present in 97% of post-neoadjuvant chemotherapy triple-negative breast cancer patient samples (n = 38). High tumoral β3 expression was associated with worse outcomes in both pre- and postchemotherapy triple-negative breast cancer groups. Genetic deletion of tumoral β3 had minimal effect in vitro, but significantly enhanced in vivo docetaxel activity, particularly in the bone. Rescue experiments confirmed that this effect required intact β3 signaling. Ultrastructural, transcriptomic, and functional analyses revealed an alternative metabolic response to chemotherapy in β3-expressing cells characterized by enhanced oxygen consumption, reactive oxygen species generation, and protein production. We identified mTORC1 as a candidate for therapeutic targeting of this β3-mediated, chemotherapy-induced metabolic response. mTORC1 inhibition in combination with docetaxel synergistically attenuated murine bone metastases. Furthermore, micelle nanoparticle delivery of mTORC1 inhibitor to cells expressing activated αvβ3 integrins enhanced docetaxel efficacy in bone metastases. Taken together, we show that β3 integrin induction by the bone microenvironment promotes resistance to chemotherapy through an altered metabolic response that can be defused by combination with αvβ3-targeted mTORC1 inhibitor nanotherapy. Our work demonstrates the importance of the metastatic microenvironment when designing treatments and presents new, bone-specific strategies for enhancing chemotherapeutic efficacy.

Additional details for methods and materials.

Background. DCIS consists of a molecularly heterogeneous group of premalignant lesions, with variable risk of invasive progression. Understanding biomarkers for invasive progression could help individualize treatment recommendations based upon tumor biology. As part of the NCI Human Tumor Atlas Network (HTAN), we conducted comprehensive genomic analyses on two large DCIS case-control cohorts. Methods. We performed smart3-seq and low-pass whole genome sequencing on two independent, retrospective, longitudinally sampled DCIS case-control cohorts. TBCRC 038 was a multicenter cohort diagnosed with DCIS between 1998 and 2016 at one of the Translational Breast Cancer Research sites; the RAHBT (Resource of Archival Human Breast Tissue) cohort included women identified through the St. Louis Breast Tissue Repository, and the Women’s Health Repository diagnosed between 1997 and 2001. We studied the spectrum of molecular changes present and sought genomic predictors of subsequent ipsilateral breast events (iBEs: DCIS recurrence or invasive progression) in both DCIS epithelium and stroma in formalin fixed paraffin embedded tissue. We generated de novo tumor and stroma-centric subtypes for DCIS that represents fundamental transcriptomic organization. Copy number analysis was performed using low-pass DNA sequencing. Non-negative matrix factorization (NMF) was applied to the RNA expression of all coding genes to identify clusters. A negative-binomial regression model was used to identify differentially expressed genes. Results. We analyzed 677 DCIS samples from 481 patients with 7.1 years median follow-up. In TBCRC samples, we identified three clusters via NMF in TBCRC referred to as ER low, quiescent, and ER high. The ER-low cluster had significantly higher levels of ERBB2 and lower levels of ESR1 compared to quiescent and ER-high clusters. Quiescent cluster lesions were less proliferative and less metabolically active than ER high and ER low subtypes. These findings were replicated in the RAHBT cohort. Focusing on the stromal component of DCIS from laser capture microdissection in RAHBT samples, we identified four distinct DCIS-associated stromal clusters. A “normal-like” stromal cluster with ECM organization and PI3K-AKT signaling; a “collagen-rich” stromal cluster; a “desmoplastic” stromal cluster with high fibroblast and total myeloid abundance, mostly associated with macrophages and myeloid dendritic cells (mDC); and an “immune-dense” stromal cluster. Further, we compared differentially expressed genes in patients with or without subsequent iBEs within 5 years of diagnosis. Hypothesizing that the resulting 812 DE genes (DESeq2) represent multiple routes to subsequent iBEs, we leveraged NMF to identify paths to progression. In both TBCRC and RAHBT cohorts, poor outcome groups exhibited increased ER, MYC signaling, and oxidative phosphorylation, supporting that these pathways are important for DCIS recurrence and progression. Conclusion. Comprehensive genomic profiling in two independent DCIS cohorts with longitudinal outcomes shows distinct DCIS stromal expression patterns and immune cell composition. RNA expression profiles reveal underlying tumor biology that is associated with later iBEs in both cohorts. These studies provide new insight into DCIS biology and will guide the design of diagnostic strategies to prevent invasive progression. Citation Format: Siri H Strand, Belén Rivero-Gutiérrez, Kathleen E Houlahan, Jose A Seoane, Lorraine M King, Tyler Risom, Lunden Simpson, Sujay Vennam, Aziz Khan, Timothy Hardman, Bryan E Harmon, Fergus J Couch, Kristalyn Gallagher, Mark Kilgore, Shi Wei, Angela DeMichele, Tari King, Priscilla F McAuliffe, Julie Nangia, Joanna Lee, Jennifer Tseng, Anna Maria Storniolo, Alastair Thompson, Gaorav Gupta, Robyn Burns, Deborah J Veis, Katherine DeSchryver, Chunfang Zhu, Magdalena Matusiak, Jason Wang, Shirley X Zhu, Jen Tappenden, Daisy Yi Ding, Dadong Zhang, Jingqin Luo, Shu Jiang, Sushama Varma, Cody Straub, Sucheta Srivastava, Christina Curtis, Rob Tibshirani, Robert Michael Angelo, Allison Hall, Kouros Owzar, Kornelia Polyak, Carlo Maley, Jeffrey R Marks, Graham A Colditz, E Shelley Hwang, Robert B West. The Breast PreCancer Atlas DCIS genomic signatures define biology and correlate with clinical outcomes: An analysis of TBCRC 038 and RAHBT cohorts [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS4-07.

Ductal carcinoma in situ; Tumor microenvironment; Whole genome sequencing Carcinoma ductal in situ; Microambiente tumoral; Secuenciación del genoma completo Carcinoma ductal in situ; Microambient tumoral; Seqüenciació del genoma complet Ductal carcinoma in situ (DCIS) is the most common precursor of invasive breast cancer (IBC), with variable propensity for progression. We perform multiscale, integrated molecular profiling of DCIS with clinical outcomes by analyzing 774 DCIS samples from 542 patients with 7.3 years median follow-up from the Translational Breast Cancer Research Consortium 038 study and the Resource of Archival Breast Tissue cohorts. We identify 812 genes associated with ipsilateral recurrence within 5 years from treatment and develop a classifier that predicts DCIS or IBC recurrence in both cohorts. Pathways associated with recurrence include proliferation, immune response, and metabolism. Distinct stromal expression patterns and immune cell compositions are identified. Our multiscale approach employed in situ methods to generate a spatially resolved atlas of breast precancers, where complementary modalities can be directly compared and correlated with conventional pathology findings, disease states, and clinical outcome. This publication is part of the HTAN (Human Tumor Atlas Network) Consortium paper package. A list of HTAN members is available at humantumoratlas.org/htan-authors/. R01 CA185138-01 (E.S.H.); U2C CA-17-035 Pre-Cancer Atlas (PCA) Research Centers (E.S.H., R.B.W., C.M., K.P., G.A.C., K.O.); UO1 CA214183 (J.R.M.); DOD BC132057 (E.S.H., C.M.); BCRF 19-074 (E.S.H.); BCRF 19-028 (G.A.C.); PRECISION CRUK Grand Challenge (E.S.H.); R01CA193694 (R.B.W., G.A.C.), BCRF PPI-18-006 (R.B.W.). AEI RYC2019- 026576-I, "LaCaixa" Foundation LCF/PR/PR17/51120011 (J.A.S.). S.H.S. was supported by the Lundbeck Foundation (R288-2018-35) and the Danish Cancer Society (R229-A13616). K.E.H. was supported by a CIHR Banting Postdoctoral Fellowship. TBCRC 038 was conducted by the TBCRC, which receives major funding support from The Breast Cancer Research Foundation and Susan G. Komen. Some results in this paper are based upon data generated by the TCGA Research Network.

Osteoclasts are multinucleated cells with the unique ability to resorb bone matrix. Excessive production or activation of osteoclasts leads to skeletal pathologies that affect a significant portion of the population. Although therapies that effectively target osteoclasts have been developed, they are associated with sometimes severe side effects, and a fuller understanding of osteoclast biology may lead to more specific treatments. Along those lines, a rich body of work has defined essential signaling pathways required for osteoclast formation, function, and survival. Nonetheless, recent studies have cast new light on long-held views regarding the origin of these cells during development and homeostasis, their life span, and the cellular sources of factors that drive their production and activity during homeostasis and disease. In this review, we discuss these new findings in the context of existing work and highlight areas of ongoing and future investigation. Expected final online publication date for the

Breast cancer bone metastases are common and incurable. Tumoral integrin β3 (β3) expression is induced through interaction with the bone microenvironment. Although β3 is known to promote bone colonization, its functional role during therapy of established bone metastases is not known. We found increased numbers of β3+ tumor cells in murine bone metastases after docetaxel chemotherapy. β3+ tumor cells were present in 97% of post-neoadjuvant chemotherapy triple-negative breast cancer patient samples (n = 38). High tumoral β3 expression was associated with worse outcomes in both pre- and postchemotherapy triple-negative breast cancer groups. Genetic deletion of tumoral β3 had minimal effect in vitro, but significantly enhanced in vivo docetaxel activity, particularly in the bone. Rescue experiments confirmed that this effect required intact β3 signaling. Ultrastructural, transcriptomic, and functional analyses revealed an alternative metabolic response to chemotherapy in β3-expressing cells characterized by enhanced oxygen consumption, reactive oxygen species generation, and protein production. We identified mTORC1 as a candidate for therapeutic targeting of this β3-mediated, chemotherapy-induced metabolic response. mTORC1 inhibition in combination with docetaxel synergistically attenuated murine bone metastases. Furthermore, micelle nanoparticle delivery of mTORC1 inhibitor to cells expressing activated αvβ3 integrins enhanced docetaxel efficacy in bone metastases. Taken together, we show that β3 integrin induction by the bone microenvironment promotes resistance to chemotherapy through an altered metabolic response that can be defused by combination with αvβ3-targeted mTORC1 inhibitor nanotherapy. Our work demonstrates the importance of the metastatic microenvironment when designing treatments and presents new, bone-specific strategies for enhancing chemotherapeutic efficacy.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

The loss of estrogen (E(2)) initiates a rapid phase of bone loss leading to osteoporosis in one-half of postmenopausal women, but the mechanism is not fully understood. Here, we show for the first time how loss of E(2) activates low-grade inflammation to promote the acute phase of bone catabolic activity in ovariectomized (OVX) mice. E(2) regulates the abundance of dendritic cells (DCs) that express IL-7 and IL-15 by inducing the Fas ligand (FasL) and apoptosis of the DC. In the absence of E(2), DCs become long-lived, leading to increased IL-7 and IL-15. We find that IL-7 and IL-15 together, but not alone, induced antigen-independent production of IL-17A and TNFα in a subset of memory T cells (T(MEM)). OVX of mice with T-cell–specific ablation of IL15RA showed no IL-17A and TNFα expression, and no increase in bone resorption or bone loss, confirming the role of IL-15 in activating the T(MEM) and the need for inflammation. Our results provide a new mechanism by which E(2) regulates the immune system, and how menopause leads to osteoporosis. The low-grade inflammation is likely to cause or contribute to other comorbidities observed postmenopause. © 2020 American Society for Bone and Mineral Research.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused over 5 million deaths worldwide. Pneumonia and systemic inflammation contribute to its high mortality. Many viruses use heparan sulfate proteoglycans as coreceptors for viral entry, and heparanase (HPSE) is a known regulator of both viral entry and inflammatory cytokines. We evaluated the heparanase inhibitor Roneparstat, a modified heparin with minimum anticoagulant activity, in pathophysiology and therapy for COVID-19. We found that Roneparstat significantly decreased the infectivity of SARS-CoV-2, SARS-CoV-1, and retroviruses (human T-lymphotropic virus 1 [HTLV-1] and HIV-1)

NF-κB has been reported to both promote and inhibit bone formation. To further explore its role in osteolineage cells, we conditionally deleted IKKα, an upstream kinase required for non-canonical NF-κB activation, using Sp7/Osterix (Osx)-Cre. Surprisingly, we found no effect on either cancellous or cortical bone, even following mechanical loading. However, we noted that IKKα conditional knockout (cKO) mice began to lose body weight after 6 months of age with severe reductions in fat mass in geriatric animals. Low levels of recombination at the IKKα locus were detected in fat pads isolated from 15 month old cKO mice. To determine if these effects were mediated by unexpected deletion of IKKα in peripheral adipocytes, we looked for Osx-Cre-mediated recombination in fat using reporter mice, which showed increasing degrees of reporter activation in adipocytes with age up to 18 months. Since Osx-Cre-driven recombination in peripheral adipocytes increases over time, we conclude that loss of fat in aged cKO mice is most likely caused by progressive deficits of IKKα in adipocytes. To further explore the effect of IKKα loss on fat metabolism, we challenged mice with a high fat diet at 2 months of age, finding that cKO mice gained less weight and showed improved glucose metabolism, compared to littermate controls. Thus, Osx-Cre mediated recombination beyond bone, including within adipocytes, should be considered as a possible explanation for extraskeletal phenotypes, especially in aging and metabolic studies.

Tumor-infiltrating myeloid cells contribute to the development of the immunosuppressive tumor microenvironment. Myeloid cell expression of arginase 1 (ARG1) promotes a protumor phenotype by inhibiting T cell function and depleting extracellular l-arginine, but the mechanism underlying this expression, especially in breast cancer, is poorly understood. In breast cancer clinical samples and in our mouse models, we identified tumor-derived GM-CSF as the primary regulator of myeloid cell ARG1 expression and local immune suppression through a gene-KO screen of breast tumor cell-produced factors. The induction of myeloid cell ARG1 required GM-CSF and a low pH environment. GM-CSF signaling through STAT3 and p38 MAPK and acid signaling through cAMP were required to activate myeloid cell ARG1 expression in a STAT6-independent manner. Importantly, breast tumor cell-derived GM-CSF promoted tumor progression by inhibiting host antitumor immunity, driving a significant accumulation of ARG1-expressing myeloid cells compared with lung and melanoma tumors with minimal GM-CSF expression. Blockade of tumoral GM-CSF enhanced the efficacy of tumor-specific adoptive T cell therapy and immune checkpoint blockade. Taken together, we show that breast tumor cell-derived GM-CSF contributes to the development of the immunosuppressive breast cancer microenvironment by regulating myeloid cell ARG1 expression and can be targeted to enhance breast cancer immunotherapy.

Osteomyelitis can result from the direct inoculation of pathogens into bone during injury or surgery, or from spread via the bloodstream, a condition called hematogenous osteomyelitis (HOM). HOM disproportionally affects children, and more than half of cases are caused by Staphylococcus (S.) aureus. Laboratory models of osteomyelitis mostly utilize direct injection of bacteria into the bone or the implantation of foreign material, and therefore do not directly interrogate the pathogenesis of pediatric hematogenous osteomyelitis. In this study, we inoculated mice intravenously and characterized resultant musculoskeletal infections using two strains isolated from adults (USA300-LAC and NRS384) and five new methicillin-resistant S. aureus isolates from pediatric osteomyelitis patients. All strains were capable of creating stable infections over five weeks, although the incidence varied. Micro-computed tomography (microCT) analysis demonstrated decreases in trabecular bone volume fraction but little effect on bone cortices. Histologic assessment revealed differences in the precise focus of musculoskeletal infection, with varying mixtures of bone-centered osteomyelitis and joint-centered septic arthritis. Whole genome sequencing of three new isolates demonstrated distinct strains, two within the USA300 lineage and one USA100 isolate. Interestingly, this USA100 isolate showed a distinct predilection for septic arthritis, compared to the other isolates tested, including NRS384 and LAC, which more frequently led to osteomyelitis or mixed bone and joint infections. Collectively, these data outline the feasibility of using pediatric osteomyelitis clinical isolates to study the pathogenesis of HOM in murine models and lay the groundwork for future studies investigating strain-dependent differences in musculoskeletal infection.

Articular cartilage has unique load-bearing properties but has minimal capacity for intrinsic repair. Here, we used three-dimensional weaving, additive manufacturing, and autologous mesenchymal stem cells to create a tissue-engineered, bicomponent implant to restore hip function in a canine hip osteoarthritis model. This resorbable implant was specifically designed to function mechanically from the time of repair and to biologically integrate with native tissues for long-term restoration. A massive osteochondral lesion was created in the hip of skeletally mature hounds and repaired with the implant or left empty (control). Longitudinal outcome measures over 6 months demonstrated that the implant dogs returned to normal preoperative values of pain and function. Anatomical structure and functional biomechanical properties were also restored in the implanted dogs. Control animals never returned to normal and exhibited structurally deficient repair. This study provides clinically relevant evidence that the bicomponent implant may be a potential therapy for moderate hip osteoarthritis.

Inhalant use disorder is a psychiatric condition characterized by repeated deliberate inhalation from among a broad range of household and industrial chemical products with the intention of producing psychoactive effects. In addition to acute intoxication, prolonged inhalation of fluorinated compounds can cause skeletal fluorosis (SF). We report a young woman referred for hypophosphatasemia and carrying a heterozygous ALPL gene variant (c.457TC, p.Trp153Arg) associated with hypophosphatasia, the heritable metabolic bone disease featuring impaired skeletal mineralization, who instead suffered from SF. Manifestations of her SF included recurrent articular pain, axial osteosclerosis, elevated bone mineral density, maxillary exostoses, and multifocal periarticular calcifications. SF was suspected when a long history was discovered of 'huffing' a computer cleaner containing 1,1-difluoroethane. Investigation revealed markedly elevated serum and urine levels of F

SUMMARYDuctal carcinoma in situ (DCIS) is the most common precursor of invasive breast cancer (IBC), with variable propensity for progression. We have performed the first multiscale, integrated profiling of DCIS with clinical outcomes by analyzing 677 DCIS samples from 481 patients with 7.1 years median follow-up from the Translational Breast Cancer Research Consortium (TBCRC) 038 study and the Resource of Archival Breast Tissue (RAHBT) cohorts. We identified 812 genes associated with ipsilateral recurrence within 5 years from treatment and developed a classifier that was predictive of DCIS or IBC recurrence in both cohorts. Pathways associated with recurrence include proliferation, immune response, and metabolism. Distinct stromal expression patterns and immune cell compositions were identified. Our multiscale approach employed in situ methods to generate a spatially resolved atlas of breast precancers, where complementary modalities can be directly compared and correlated with conventional pathology findings, disease states, and clinical outcome.HIGHLIGHTS⍰ Development of a new classifier for DCIS recurrence or progression⍰ Outcome associated pathways identified across multiple data types and compartments⍰ Four stroma-specific signatures identified⍰ CNAs characterize DCIS subgroups associated with high risk invasive cancers

Osteomyelitis can result from the direct inoculation of pathogens into bone during injury or surgery, or from spread via the bloodstream, a condition called hematogenous osteomyelitis (HOM). HOM disproportionally affects children, and more than half of cases are caused by Staphylococcus (S.) aureus. Laboratory models of osteomyelitis mostly utilize direct injection of bacteria into the bone or the implantation of foreign material, and therefore do not directly interrogate the pathogenesis of pediatric hematogenous osteomyelitis. In this study, we inoculated mice intravenously and characterized resultant musculoskeletal infections using two strains isolated from adults (USA300-LAC and NRS384) and five new methicillin-resistant S. aureus isolates from pediatric osteomyelitis patients. All strains were capable of creating stable infections over five weeks, although the incidence varied. Micro-computed tomography (microCT) analysis demonstrated decreases in trabecular bone volume fraction but little effect on bone cortices. Histologic assessment revealed differences in the precise focus of musculoskeletal infection, with varying mixtures of bone-centered osteomyelitis and joint-centered septic arthritis. Whole genome sequencing of three new isolates demonstrated distinct strains, two within the USA300 lineage and one USA100 isolate. Interestingly, the USA100 strain showed a distinct predilection for septic arthritis, compared to the USA300 strains, including NRS384 and LAC, which more frequently led to osteomyelitis or mixed bone and joint infections. Collectively, these data outline the feasibility of using pediatric osteomyelitis clinical isolates to study the pathogenesis of HOM in murine models and lay the groundwork for future studies investigating strain-dependent differences in musculoskeletal infection.ImportanceThe inflammation of bone tissue is called osteomyelitis, and more than half of cases are caused by an infection with the bacterium Staphylococcus aureus. In children, the most common route of infection is hematogenous, wherein bacteria seed the bone from the bloodstream without another known site of infection. Although these infections pose a significant health problem, they are understudied in the laboratory because of a dearth of robust animal models. In this study, we utilized several previously uncharacterized clinical isolates of S. aureus derived from children with bone infections to generate reproducible and stable musculoskeletal infection in mice with many features seen in human osteomyelitis, making them a valuable resource for future mechanistic and therapeutic studies.

Ductal carcinomain situ(DCIS) is a pre-invasive lesion that is thought to be a precursor to invasive breast cancer (IBC). To understand how the tumor microenvironment (TME) changes with transition to IBC, we used Multiplexed Ion Beam Imaging by time of flight (MIBI-TOF) and a 37-plex antibody staining panel to analyze 140 clinically annotated surgical resections covering the full spectrum of breast cancer progression. We compared normal, DCIS, and IBC tissues using machine learning tools for multiplexed cell segmentation, pixel-based clustering, and object morphometrics. Transition from DCIS to IBC was found to occur along a trajectory marked by coordinated shifts in location and function of myoepithelium, fibroblasts, and infiltrating immune cells in the surrounding stroma. Taken together, this comprehensive study within the HTAN Breast PreCancer Atlas offers insight into the etiologies of DCIS, its transition to IBC, and emphasizes the importance of the TME stroma in promoting these processes.

Ductal carcinoma in situ (DCIS) is the most commonly diagnosed precursor of invasive breast cancer (IBC), with variable propensity for progression. We have performed the first multiscale, integrated profiling of DCIS with clinical outcomes to identify predictors of subsequent ipsilateral breast events. We analyzed 677 DCIS samples from 481 patients with 7.1 years median follow-up from two independent cohorts. We made observations on DNA, RNA, and protein expression, and generated a de novo clustering scheme for DCIS that represents the fundamental transcriptomic organization at this early stage of breast neoplasia. Distinct DCIS stromal expression patterns and immune cell compositions were identified. We also found RNA expression patterns that correlate with later events in both cohorts. Our multiscale approach employed in situ methods to generate a spatially resolved atlas of breast precancers, where complementary modalities can be directly compared and correlated with conventional pathology findings, disease states, and clinical outcome.

Breast cancer is a heterogeneous collection of diseases grouped by hormone receptor status or by expression of key subtype-determining genes. Breast cancer subtypes, in particular basal-like breast cancer and the luminal breast cancer subtypes, differ by hormonal receptor status, proliferation, genomic instability and mutational signatures, treatment response, and prognosis. The highly distinct signatures of basal-like and luminal breast cancer suggest that they may have different cells of origin within the breast duct. As part of the Washington University Human Tumor Atlas Network (WU-HTAN) program, we generated multi-omic data for 53 samples from 37 breast cancer tumors and 4 normal adjacent tissues. Genomic subtyping was applied to both bulk and single-nucleus RNA sequencing. Analysis of single-nucleus gene expression and chromatin accessibility in epithelial cells underscores similarities between basal-like breast cancer and luminal progenitor cells within the breast duct, and between luminal breast cancer and mature luminal ductal cells. This study links distinct breast cancer subtypes to normal breast cell populations and suggests distinct cells of origin for these cancer types. Citation Format: Michael D. Iglesia, Reyka G. Jayasinghe, Daniel Cui Zhou, Nadezhda V. Terekhanova, John Herndon, Alla Karpova, Siqi Chen, Nataly Naser Al Deen, Kazuhito Sato, Feng Chen, Deborah J. Veis, Ryan C. Fields, William E. Gillanders, Li Ding. Multi-omic characterization of transitional cell populations in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2936.

Immunostaining is the process of identifying proteins in tissue sections by incubating the sample with antibodies specific to the protein of interest, then visualizing the bound antibody using a chromogen (immunohistochemistry or IHC) or fluorescence (immunofluorescence or IF). Unlike in situ hybridization, which identifies gene transcripts in cells, immunostaining identifies the products themselves and provides information about their localization within cells (nuclear, cytoplasmic, or membrane) or extracellular matrix. This can be particularly important in the context of bone and cartilage because they contain many cell types as well as matrix components, each with distinct protein expression patterns. As the number of antibodies continues to grow, this technique has become vital for research laboratories studying the skeleton. Here, we describe a detailed protocol for antibody-based in situ analysis of bone and associated tissues, addressing specific issues associated with staining of hard and matrix-rich tissues.

Juvenile Paget's disease (JPD) became in 1974 the commonly used name for ultra-rare heritable occurrences of rapid bone remodeling throughout of the skeleton that present in infancy or early childhood as fractures and deformity hallmarked biochemically by marked elevation of serum alkaline phosphatase (ALP) activity (hyperphosphatasemia). Untreated, JPD can kill during childhood or young adult life. In 2002, we reported that homozygous deletion of the gene called tumor necrosis factor receptor superfamily, member 11B (TNFRSF11B) encoding osteoprotegerin (OPG) explained JPD in Navajos. Soon after, other bi-allelic loss-of-function TNFRSF11B defects were identified in JPD worldwide. OPG inhibits osteoclastogenesis and osteoclast activity by decoying receptor activator of nuclear factor κ-B (RANK) ligand (RANKL) away from its receptor RANK. Then, in 2014, we reported JPD in a Bolivian girl caused by a heterozygous activating duplication within TNFRSF11A encoding RANK. Herein, we identify mutation of a third gene underlying JPD. An infant girl began atraumatic fracturing of her lower extremity long-bones. Skull deformity and mild hearing loss followed. Our single investigation of the patient, when she was 15 years-of-age, showed generalized osteosclerosis and hyperostosis. DXA revealed a Z-score of +5.1 at her lumbar spine and T-score of +3.3 at her non-dominant wrist. Biochemical studies were consistent with positive mineral balance and several markers of bone turnover were elevated and included striking hyperphosphatasemia. Iliac crest histopathology was consistent with rapid skeletal remodeling. Measles virus transcripts, common in classic Paget's disease of bone, were not detected in circulating mononuclear cells. Then, reportedly, she responded to several months of alendronate therapy with less skeletal pain and correction of hyperphosphatasemia but had been lost to our follow-up. After we detected no defect in TNFRSF11A or B, trio exome sequencing revealed a de novo heterozygous missense mutation (c.926C>G; p.S309W) within SP7 encoding the osteoblast transcription factor osterix (specificity protein 7, transcription factor SP7). Thus, mutation of SP7 represents a third genetic cause of JPD.

Dynamic regulation of the mitochondrial network by mitofusins (MFNs) modulates energy production, cell survival, and many intracellular signaling events, including calcium handling. However, the relative importance of specific mitochondrial functions and their dependence on MFNs vary greatly among cell types. Osteoclasts have many mitochondria, and increased mitochondrial biogenesis and oxidative phosphorylation enhance bone resorption, but little is known about the mitochondrial network or MFNs in osteoclasts. Because expression of each MFN isoform increases with osteoclastogenesis, we conditionally deleted MFN1 and MFN2 (double conditional KO (dcKO)) in murine osteoclast precursors, finding that this increased bone mass in young female mice and abolished osteoclast precursor differentiation into mature osteoclasts in vitro. Defective osteoclastogenesis was reversed by overexpression of MFN2 but not MFN1; therefore, we generated mice lacking only MFN2 in osteoclasts. MFN2-deficient female mice had increased bone mass at 1 year and resistance to Receptor Activator of NF-κB Ligand (RANKL)-induced osteolysis at 8 weeks. To explore whether MFN-mediated tethering or mitophagy is important for osteoclastogenesis, we overexpressed MFN2 variants defective in either function in dcKO precursors and found that, although mitophagy was dispensable for differentiation, tethering was required. Because the master osteoclastogenic transcriptional regulator nuclear factor of activated T cells 1 (NFATc1) is calcium-regulated, we assessed calcium release from the endoplasmic reticulum and store-operated calcium entry and found that the latter was blunted in dcKO cells. Restored osteoclast differentiation by expression of intact MFN2 or the mitophagy-defective variant was associated with normalization of store-operated calcium entry and NFATc1 levels, indicating that MFN2 controls mitochondrion–endoplasmic reticulum tethering in osteoclasts.

SUMMARY Ductal carcinoma in situ (DCIS) is a pre-invasive lesion that is thought to be a precursor to invasive breast cancer (IBC). To understand the changes in the tumor microenvironment (TME) accompanying transition to IBC, we used multiplexed ion beam imaging by time of flight (MIBI-TOF) and a 37-plex antibody staining panel to interrogate 79 clinically annotated surgical resections using machine learning tools for cell segmentation, pixel-based clustering, and object morphometrics. Comparison of normal breast with patient-matched DCIS and IBC revealed coordinated transitions between four TME states that were delineated based on the location and function of myoepithelium, fibroblasts, and immune cells. Surprisingly, myoepithelial disruption was more advanced in DCIS patients that did not develop IBC, suggesting this process could be protective against recurrence. Taken together, this HTAN Breast PreCancer Atlas study offers insight into drivers of IBC relapse and emphasizes the importance of the TME in regulating these processes., In brief A spatial imaging atlas of patient-matched ductal carcinoma in situ and invasive breast cancer depicts coordinated changes in the tumor microenviroment associated with invasive relapse, suggesting a potential protective role of myoepithelial disruption against invasive progression., Graphical Abstract

Alternative NF-κB signaling promotes osteoclastogenesis and pathological bone loss, but the effect of sex on phenotype has not been explored. We disrupted alternative NF-κB signaling by deletion of upstream kinase NF-κB-inducing kinase (NIK) or NF-κB subunit RelB and found that both NIK-deficient and RelB-deficient female mice possessed more than twofold higher trabecular bone mass compared to controls, whereas no differences were observed in males. In vitro, RelB-deficient precursors from female mice showed a more severe osteoclast (OC) differentiation defect than male, while WT had no sex bias. Next, we asked whether pharmacologic activation of alternative NF-κB by inhibitor of apoptosis (IAP) antagonist BV6 has sex-dependent effects on bone. Unlike male mice that lost bone, female mice on BV6 for 4 weeks showed no changes in either trabecular bone mass or OC number. Because estrogen generally suppresses NF-κB, we hypothesized that estrogen protects bone from BV6 effects in vivo. Thus, we performed ovariectomy or sham surgery in female mice, then treated with BV6 or vehicle for 4 weeks. Although ovariectomy caused bone loss, BV6 did not have any additional impact, suggesting that direct estrogen effects do not cause resistance to BV6 in vivo. The osteopenic effects of IAP antagonists in males may have implications for their use in cancer therapy. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Pediatric granulomatous arthritis (PGA) refers to two formerly separate entities: autosomal dominant Blau syndrome (BS) and its sporadic phenocopy early-onset sarcoidosis (EOS). In 2001 BS and in 2005 EOS became explained by heterozygous mutations within the gene that encodes nucleotide-binding oligomerization domain-containing protein 2 (NOD2), also called caspase recruitment domain-containing protein 15 (CARD15). NOD2 is a microbe sensor in leukocyte cytosol that activates and regulates inflammation. PGA is characterized by a triad of autoinflammatory problems (dermatitis, uveitis, and arthritis) in early childhood, which suggests the causal NOD2/CARD15 mutations are activating defects. Additional complications of PGA were recognized especially when NOD2 mutation analysis became generally available. However, in PGA, hypercalcemia is only briefly mentioned, and generalized osteosclerosis is not reported, although NOD2 regulates NF-κB signaling essential for osteoclastogenesis and osteoclast function. Herein, we report a 4-year-old girl with PGA uniquely complicated by severe 1,25(OH)2 D-mediated hypercalcemia, nephrocalcinosis, and compromised renal function together with radiological and histopathological features of osteopetrosis (OPT). The classic triad of PGA complications was absent, although joint pain and an antalgic gait accompanied wrist, knee, and ankle swelling and soft non-tender masses over her hands, knees, and feet. MRI revealed tenosynovitis in her hands and suprapatellar effusions. Synovial biopsy demonstrated reactive synovitis without granulomas. Spontaneous resolution of metaphyseal osteosclerosis occurred while biochemical markers indicated active bone turnover. Anti-inflammatory medications suppressed circulating 1,25(OH)2 D, corrected the hypercalcemia, and improved her renal function, joint pain and swelling, and gait. Mutation analysis excluded idiopathic infantile hypercalcemia, type 1, and known forms of OPT, and identified a heterozygous germline missense mutation in NOD2 common in PGA (c.1001G>A, p.Arg334Gln). Thus, radiological and histological findings of OPT and severe hypercalcemia from apparent extrarenal production of 1,25(OH)2 D can complicate NOD2-associated PGA. Although the skeletal findings seem inconsequential, treatment of the hypercalcemia is crucial to protect the kidneys. © 2018 American Society for Bone and Mineral Research.

Osteoclasts are mitochondria-rich cells, but the role of these energy-producing organelles in bone resorption is poorly defined. To this end, we conditionally deleted the mitochondria-inducing co-activator, PGC1β, in myeloid lineage cells to generate PGC1βLysM mice. In contrast to previous reports, PGC1β-deficient macrophages differentiate normally into osteoclasts albeit with impaired resorptive function due to cytoskeletal disorganization. Consequently, bone mass of PGC1βLysM mice is double that of wild type. Mitochondrial biogenesis and function are diminished in PGC1βLysM osteoclasts. All abnormalities are normalized by PGC1β transduction. Furthermore, OXPHOS inhibitors reproduce the phenotype of PGC1β deletion. PGC1β's organization of the osteoclast cytoskeleton is mediated by expression of GIT1, which also promotes mitochondrial biogenesis. Thus, osteoclast mitochondria regulate the cell's resorptive activity by promoting cytoskeletal organization. © 2018 American Society for Bone and Mineral Research.

Aberrant NF-κB signaling fuels tumor growth in multiple human cancer types including both hematologic and solid malignancies. Chronic elevated alternative NF-κB signaling can be modeled in transgenic mice upon activation of a conditional NF-κB-inducing kinase (NIK) allele lacking the regulatory TRAF3 binding domain (NT3). Here, we report that expression of NT3 in the mesenchymal lineage with Osterix (Osx/Sp7)-Cre or Fibroblast-Specific Protein 1 (FSP1)-Cre caused subcutaneous, soft tissue tumors. These tumors displayed significantly shorter latency and a greater multiple incidence rate in Fsp1-Cre;NT3 compared to Osx-Cre;NT3 mice, regardless of sex. Histological assessment revealed poorly differentiated solid tumors with some spindled patterns, as well as robust RelB immunostaining, confirming activation of alternative NF-κB. Even though NT3 expression also occurs in the osteolineage in Osx-Cre;NT3 mice, we observed no bony lesions. The staining profiles and pattern of Cre expression in the two lines pointed to a mesenchymal tumor origin. Immunohistochemistry revealed that these tumors stain strongly for alpha-smooth muscle actin (αSMA), although vimentin staining was uniform only in Osx-Cre;NT3 tumors. Negative CD45 and S100 immunostains precluded hematopoietic and melanocytic origins, respectively, while positive staining for cytokeratin 19 (CK19), typically associated with epithelia, was found in subpopulations of both tumors. Principal component, differential expression, and gene ontology analyses revealed that NT3 tumors are distinct from normal mesenchymal tissues and are enriched for NF-κB related biological processes. We conclude that constitutive activation of the alternative NF-κB pathway in the mesenchymal lineage drives spontaneous sarcoma and provides a novel mouse model for NF-κB related sarcomas.

Mitochondria are essential organelles that form highly complex, interconnected dynamic networks inside cells. The GTPase mitofusin 2 (MFN2) is a highly conserved outer mitochondrial membrane protein involved in the regulation of mitochondrial morphology, which can affect various metabolic and signaling functions. The role of mitochondria in bone formation remains unclear. Since MFN2 levels increase during osteoblast (OB) differentiation, we investigated the role of MFN2 in the osteolineage by crossing mice bearing floxed Mfn2 alleles with those bearing Prx-cre to generate cohorts of conditional knock out (cKO) animals. By ex vivo microCT, cKO female mice, but not males, display an increase in cortical thickness at 8, 18, and 30 weeks, compared to wild-type (WT) littermate controls. However, the cortical anabolic response to mechanical loading was not different between genotypes. To address how Mfn2 deficiency affects OB differentiation, bone marrow-derived mesenchymal stromal cells (MSCs) from both wild-type and cKO mice were cultured in osteogenic media with different levels of β-glycerophosphate. cKO MSCs show increased mineralization and expression of multiple markers of OB differentiation only at the lower dose. Interestingly, despite showing the expected mitochondrial rounding and fragmentation due to loss of MFN2, cKO MSCs have an increase in oxygen consumption during the first 7 days of OB differentiation. Thus, in the early phases of osteogenesis, MFN2 restrains oxygen consumption thereby limiting differentiation and cortical bone accrual during homeostasis in vivo.

The inflammation of bone tissue is called osteomyelitis, and most cases are caused by an infection with the bacterium Staphylococcus aureus. To date, the bone-building cells, osteoblasts, have been implicated in the progression of these infections, but not much is known about how the bone-resorbing cells, osteoclasts, participate. In this study, we show that S. aureus can infect osteoclasts and proliferate inside these cells, whereas bone-residing macrophages, immune cells related to osteoclasts, destroy the bacteria. These findings elucidate a unique role for osteoclasts to harbor bacteria during infection, providing a possible mechanism by which bacteria could evade destruction by the immune system., Osteomyelitis (OM), or inflammation of bone tissue, occurs most frequently as a result of bacterial infection and severely perturbs bone structure. OM is predominantly caused by Staphylococcus aureus, and even with proper treatment, OM has a high rate of recurrence and chronicity. While S. aureus has been shown to infect osteoblasts, it remains unclear whether osteoclasts (OCs) are also a target of intracellular infection. Here, we demonstrate the ability of S. aureus to intracellularly infect and divide within OCs. OCs were differentiated from bone marrow macrophages (BMMs) by exposure to receptor activator of nuclear factor kappa-B ligand (RANKL). By utilizing an intracellular survival assay and flow cytometry, we found that at 18 h postinfection the intracellular burden of S. aureus increased dramatically in cells with at least 2 days of RANKL exposure, while the bacterial burden decreased in BMMs. To further explore the signals downstream of RANKL, we manipulated factors controlling OC differentiation, NFATc1 and alternative NF-κB, and found that intracellular bacterial growth correlates with NFATc1 levels in RANKL-treated cells. Confocal and time-lapse microscopy in mature OCs showed a range of intracellular infection that correlated inversely with S. aureus-phagolysosome colocalization. The propensity of OCs to become infected, paired with their diminished bactericidal capacity compared to BMMs, could promote OM progression by allowing S. aureus to evade initial immune regulation and proliferate at the periphery of lesions where OCs are most abundant.

Studies from global loss-of-function mutants suggest that alternative NF-κB downstream of NF-κB inducing kinase (NIK) is a cell-intrinsic negative regulator of osteogenesis. However, the interpretation of the osteoblast and/or osteocyte contribution to the bone phenotype is complicated by simultaneous osteoclast defects in these models. Therefore, we turned to a transgenic mouse model to investigate the direct role of NIK in the osteolineage. Osx-Cre;NT3 animals (NT3-Cre +), which bear a constitutively active NIK allele (NT3) driven by Osx-Cre, were compared with their Cre-negative, Control (Ctrl) littermates. NT3-Cre + mice had elevated serum P1NP and CTX levels. Despite this high turnover state, µCT showed that constitutive activation of NIK resulted in a net increase in basal bone mass in both cortical and cancellous compartments. Furthermore, NT3-Cre + mice exhibited a greater anabolic response following mechanical loading compared with controls. We next performed RNA-Seq on nonloaded and loaded tibias to elucidate possible mechanisms underlying the increased bone anabolism seen in NT3-Cre + mice. Hierarchical clustering revealed two main transcriptional programs: one loading-responsive and the other NT3 transgene-driven. Gene ontology (GO) analysis indicated a distinct upregulation of receptor, kinase, and growth factor activities including Wnts, as well as a calcium-response signature in NT3-Cre + limbs. The promoters of these GO-term associated genes, including many known to be bone-anabolic, were highly enriched for multiple κB recognition elements (κB-RE) relative to the background frequency in the genome. The loading response in NT3-Cre + mice substantially overlapped (>90%) with Ctrl. Surprisingly, control animals had 10-fold more DEGs in response to loading. However, most top DEGs shared between genotypes had a high incidence of multiple κB-RE in their promoters. Therefore, both transcriptional programs (loading-responsive and NT3 transgene-driven) are modulated by NF-κB. Our studies uncover a previously unrecognized role for NF-κB in the promotion of both basal and mechanically stimulated bone formation. © 2019 American Society for Bone and Mineral Research.

Type I interferons (IFNs) are key mediators of the innate immune response. Although members of this family of cytokines signal through a single shared receptor, biochemical and functional variation exists in response to different IFN subtypes. While previous work has demonstrated that type I IFNs are essential to control infection by chikungunya virus (CHIKV), a globally emerging alphavirus, the contributions of individual IFN subtypes remain undefined. To address this question, we evaluated CHIKV pathogenesis in mice lacking IFN-β (IFN-β knockout [IFN-β-KO] mice or mice treated with an IFN-β-blocking antibody) or IFN-α (IFN regulatory factor 7 knockout [IRF7-KO] mice or mice treated with a pan-IFN-α-blocking antibody). Mice lacking either IFN-α or IFN-β developed severe clinical disease following infection with CHIKV, with a marked increase in foot swelling compared to wild-type mice. Virological analysis revealed that mice lacking IFN-α sustained elevated infection in the infected ankle and in distant tissues. In contrast, IFN-β-KO mice displayed minimal differences in viral burdens within the ankle or at distal sites and instead had an altered cellular immune response. Mice lacking IFN-β had increased neutrophil infiltration into musculoskeletal tissues, and depletion of neutrophils in IFN-β-KO but not IRF7-KO mice mitigated musculoskeletal disease caused by CHIKV. Our findings suggest disparate roles for the IFN subtypes during CHIKV infection, with IFN-α limiting early viral replication and dissemination and IFN-β modulating neutrophil-mediated inflammation. IMPORTANCE Type I interferons (IFNs) possess a range of biological activity and protect against a number of viruses, including alphaviruses. Despite signaling through a shared receptor, there are established biochemical and functional differences among the IFN subtypes. The significance of our research is in demonstrating that IFN-α and IFN-β both have protective roles during acute chikungunya virus (CHIKV) infection but do so by distinct mechanisms. IFN-α limits CHIKV replication and dissemination, whereas IFN-β protects from CHIKV pathogenesis by limiting inflammation mediated by neutrophils. Our findings support the premise that the IFN subtypes have distinct biological activities in the antiviral response.

Cytokine storm syndrome (CSS) is a life-threatening condition characterized by excessive activation of T cells and uncontrolled inflammation, mostly described in patients with familial hemophagocytic lymphohistiocytosis and certain systemic auto-inflammatory diseases, such as systemic juvenile idiopathic arthritis (sJIA). Defects in T cell cytotoxicity as a mechanism for uncontrolled inflammation following viral infections fail to represent the whole spectrum of CSS. Evidence implicates dysregulated innate immune responses, especially activation of monocytes and macrophages, in patients with CSS. However, the direct contribution of monocytes/macrophages to CSS development and the signaling pathways involved in their activation have not been formally investigated. We find that depletion of monocytes/macrophages during early stages of CSS development, by clodronate-liposomes or neutralizing anti-CSF1 antibody, reduces mortality and inflammatory cytokine levels in two CSS mouse models, one dependent on T cells and the second induced by repeated TLR9 stimulation. We further demonstrate that activation of Plcγ2 in myeloid cells controls CSS development by driving macrophage pro-inflammatory responses. Intriguingly, the Plcγ2 downstream effector Tmem178, a negative modulator of calcium levels, acts in a negative feedback loop to restrain inflammatory cytokine production. Genetic deletion of Tmem178 leads to pro-inflammatory macrophage polarization in vitro and more severe CSS in vivo. Importantly, Tmem178 levels are reduced in macrophages from mice with CSS and after exposure to plasma from sJIA patients with active disease. Our data identify a novel Plcγ2/Tmem178 axis as a modulator of inflammatory cytokine production by monocytes/macrophages. We also find that loss of Tmem178 accentuates the pro-inflammatory responses in CSS.

Osteolytic bone lesions and hypercalcemia are common, serious complications in adult T cell leukemia/lymphoma (ATL), an aggressive T cell malignancy associated with human T cell leukemia virus type 1 (HTLV-1) infection. The HTLV-1 viral oncogene HBZ has been implicated in ATL tumorigenesis and bone loss. In this study, we evaluated the role of HBZ on ATL-associated bone destruction using HTLV-1 infection and disease progression mouse models. Humanized mice infected with HTLV-1 developed lymphoproliferative disease and continuous, progressive osteolytic bone lesions. HTLV-1 lacking HBZ displayed only modest delays to lymphoproliferative disease but significantly decreased disease-associated bone loss compared with HTLV-1-infected mice. Gene expression array of acute ATL patient samples demonstrated increased expression of RANKL, a critical regulator of osteoclasts. We found that HBZ regulated RANKL in a c-Fos-dependent manner. Treatment of HTLV-1-infected humanized mice with denosumab, a monoclonal antibody against human RANKL, alleviated bone loss. Using patient-derived xenografts from primary human ATL cells to induce lymphoproliferative disease, we also observed profound tumor-induced bone destruction and increased c-Fos and RANKL gene expression. Together, these data show the critical role of HBZ in driving ATL-associated bone loss through RANKL and identify denosumab as a potential treatment to prevent bone complications in ATL patients.

Skeletal fluorosis (SF) is endemic primarily in regions with fluoride (F)-contaminated well water, but can reflect other types of chronic F exposure. Calcium (Ca) and vitamin D (D) deficiency can exacerbate SF. A 51-year-old man with years of musculoskeletal pain and opiate use was hypocalcemic with secondary hyperparathyroidism upon manifesting recurrent long bone fractures. He smoked cigarettes, drank large amounts of cola beverage, and consumed little dietary Ca. Then, after 5 months of Ca and D(3) supplementation, serum 25(OH)D was 21 ng/mL (Nl, 30–100), corrected serum Ca had normalized from 7.8 to 9.4 mg/dL (Nl, 8.5–10.1), alkaline phosphatase (ALP) had decreased from 1080 to 539 U/L (Nl, 46–116), yet parathyroid hormone (PTH) had increased from 133 to 327 pg/mL (Nl, 8.7–77.1). Radiographs revealed generalized osteosclerosis and a cystic osteopenic area in the left femoral neck and intertrochanteric region. DXA BMD Z-scores were +7.4 and +0.4 at the lumbar spine and “1/3” radius, respectively. Bone scintigraphy showed increased uptake in two ribs, periarticular areas, and proximal left femur at the site of a subsequent atraumatic fracture. Elevated serum collagen type I C-telopeptide 2513 pg/mL (Nl, 87–345) and osteocalcin >300 ng/mL (Nl, 9–38) indicated rapid bone turnover. Negative studies included hepatitis C Ab, prostate-specific antigen, serum and urine electrophoresis, and Ion Torrent mutation analysis for dense or high-turnover skeletal diseases. After discovering markedly elevated F concentrations in his plasma [4.84 mg/L (Nl, 0.02–0.08)] and spot urine [42.6 mg/L (Nl, 0.2–3.2)], a two-year history emerged of “huffing” computer cleaner containing difluoroethane. Non-decalcified histology of a subsequent right femur fracture showed increased osteoblasts and osteoclasts and excessive osteoid. A 24-hour urine collection contained 27 mg/L F (Nl, 0.2–3.2) and < 2 mg/dL Ca. Then, 19 months after “huffing” cessation and improved Ca and D(3) intake, yet with persisting bone pain, serum PTH was normal (52 pg/mL) and serum ALP and urine F had decreased to 248 U/L and 3.3 mg/L, respectively. Our experience combined with 15 publications in PubMed concerning unusual causes of non-endemic SF where the F source became known (19 cases in all) revealed: 11 instances from high consumption of black tea and/or F-containing toothpaste, 1 due to geophagia of F-rich soil, and 7 due to “recreational” inhalation of F-containing vapors. Circulating PTH measured in 13 was substantially elevated in 2 (including ours) and mildly increased in 2. Their SF severity, including bone turnover rate, seemed to reflect cumulative F exposure, renal function, and Ca and D status. Several factors appeared to condition our patient’s skeletal disease: i) direct anabolic effects of toxic amounts of F on his skeleton, ii) secondary hyperparathyroidism from degradation-resistant fluorapatite bone crystals and low dietary Ca, and iii) impaired mineralization of excessive osteoid due to hypocalcemia.

We have previously demonstrated that expression of the integrin β3 (β3) subunit is consistently increased in breast cancer (BC) bone metastases across clinical samples and preclinical models. Bone metastases frequently exhibit resistance to chemotherapy. Based on evidence that integrin β3 can function as a promoter of tumor survival and metastasis, we asked whether increased β3 signaling in bone metastases promotes chemoresistance. In established murine bone metastases, the proportion of integrin β3+ tumor cells was increased after docetaxel administration. Genetic deletion of β3 in either MMTV-PyMT-derived BC cells (BO1-FL-GFP, modeling luminal B disease) or 4T1 BC cells (4T1-FL-GFP, modeling triple-negative disease) yielded bone metastases with increased sensitivity to docetaxel treatment in vivo. Retroviral rescue of β3 expression in β3-/- BO1-FL-GFP cells by a signaling-competent human integrin β3 construct (hβ3) restored relative docetaxel chemoresistance in bone metastases. By contrast, expression of a signaling-deficient mutant β3 (Δβ3) failed to restore chemoresistance, suggesting that signaling through β3 is critical to its capacity to promote docetaxel chemoresistance in bone-residing BC cells. Mechanistically, RNAseq analysis of docetaxel response in β3-/- and hβ3-rescued cells revealed β3-mediated enrichment in transcriptional pathways associated with oxidative phosphorylation and metabolism. Direct imaging of mitochondria by super-resolution microscopy and extracellular flux analysis of oxygen consumption rate further confirmed an alternative metabolic response to docetaxel in resistant, β3-expressing tumor cells. mTORC1 plays an important role in mitochondrial biogenesis and cellular metabolism, and can be activated downstream of integrin signaling in certain contexts. Based on transcriptomic data identifying enhanced mTORC1 activity as a possible mediator of β3-dependent metabolic changes, we asked whether mTOR inhibition could restore chemosensitivity in BC bone metastases. In mice bearing chemoresistant, β3-WT metastases, combination of the mTORC1 inhibitor rapamycin with docetaxel yielded synergistic, site-specific attenuation of bone tumor burden. Taken together, our data 1) establish integrin β3 as a mediator of chemoresistance in breast cancer bone metastases, 2) demonstrate a mechanistic link between integrin β3 expression and an alternative metabolic response to docetaxel in resistant cells, and 3) suggest mTORC1 inhibition as a candidate for rational combination with chemotherapy to interrupt treatment resistance in breast cancer bone metastases. Citation Format: Gregory C. Fox, Michael H. Ross, Xinming Su, Yalin Xu, Alison Esser, Elizabeth Cordell, Elizabeth Wilson, Barbara Muz, Ha Dang, Christopher A. Maher, Abdel Kareem Azab, Deborah Veis, Samuel Achilefu, Gregory M. Lanza, Katherine N. Weilbaecher. Integrin beta-3 signaling links chemoresistance and mitochondrial metabolism in breast cancer bone metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2613.

Radiotherapy is a commonly used conditioning regimen for bone marrow transplantation (BMT). Cytotoxicity limits the use of this life-saving therapy, but the underlying mechanisms remain poorly defined. Here, we use the syngeneic mouse BMT model to test the hypothesis that lethal radiation damages tissues, thereby unleashing signals that indiscriminately activate the inflammasome pathways in host and transplanted cells. We find that a clinically relevant high dose of radiation causes severe damage to bones and the spleen through mechanisms involving the NLRP3 and AIM2 inflammasomes but not the NLRC4 inflammasome. Downstream, we demonstrate that gasdermin D (GSDMD), the common effector of the inflammasomes, is also activated by radiation. Remarkably, protection against the injury induced by deadly ionizing radiation occurs only when NLRP3, AIM2, or GSDMD is lost simultaneously in both the donor and host cell compartments. Thus, this study reveals a continuum of the actions of lethal radiation relayed by the inflammasome-GSDMD axis, initially affecting recipient cells and ultimately harming transplanted cells as they grow in the severely injured and toxic environment. This study also suggests that therapeutic targeting of inflammasome-GSDMD signaling has the potential to prevent the collateral effects of intense radiation regimens., Radiotherapy is a commonly used conditioning regimen for bone marrow transplantation; cytotoxicity limits the use of this life-saving therapy, but the underlying mechanisms remain poorly defined. This study reveals a continuum of the effects of radiation, relayed by the inflammasome–gasdermin D axis, initially affecting host cells and, ultimately, harming transplanted cells.

The SIBLINGs are a subfamily of the secreted calcium-binding phosphoproteins and comprise five small integrin-binding ligand N-linked glycoproteins [dentin matrix protein-1 (DMP1), secreted phosphoprotein-1 (SPP1) also called osteopontin (OPN), integrin-binding sialoprotein (IBSP) also called bone sialoprotein (BPS), matrix extracellular phosphoglycoprotein (MEPE), and dentin sialophosphoprotein (DSPP)]. Each SIBLING has at least one “acidic, serine- and aspartic acid-rich motif’ (ASARM) and multiple Ser-x-Glu/pSer sequences that when phosphorylated promote binding of the protein to hydroxyapatite for regulation of biomineralization. Mendelian disorders from loss-of-function mutation(s) of the genes that encode the SIBLINGs thus far involve DSPP causing various autosomal dominant dysplasias of dentin but without skeletal disease, and DMP1 causing autosomal recessive hypophosphatemic rickets, type 1 (ARHR1). No diseases have been reported from gain-of-function mutation(s) of DSPP or DMP1 or from alterations of SPP1, IBSP, or MEPE. Herein, we describe severe hypophosphatemic osteosclerosis and hyperostosis associated with skeletal deformity, short stature, enthesopathy, tooth loss, and high circulating FGF23 levels in a middle-aged man and young woman from an endogamous family living in southern India. Both shared novel homozygous mutations within two genes that encode a SIBLING protein: stop-gain (“non-sense”) DMP1 (c.556G>T, p.Glu186Ter) and missense SPP1 (c.769C>T, p.Leu266Phe). The man alone also carried novel heterozygous missense variants within two additional genes that condition mineral homeostasis and are the basis for autosomal recessive disorders: CYP27B1 underlying vitamin D dependent rickets, type 1, and ABCC6 underlying both generalized arterial calcification of infancy, type 2 and pseudoxanthoma elasticum (PXE). By immunochemistry, his bone contained high amounts of OPN, particularly striking surrounding osteocytes. We review how our patients’ disorder may represent the first digenic SIBLING protein osteopathy.

Unresolved inflammation is harmful to any tissues in the organism. Bone in particular is vulnerable to inflammatory assaults because its integrity depends on the activity of osteoclasts, which arise from myeloid precursors. Osteoclasts are responsible for bone resorption in normal and disease conditions. Increased osteolysis is a common feature of inflammatory disorders and a risk factor for bone fractures. Thus, bone is impacted negatively not only by local and systemic inflammatory mediators, but also directly, by alterations affecting myelopoiesis and lineage allocations. Such perturbations are characteristics of dysregulated inflammasomes, which are key regulators of innate immunity. In this review, we discuss the role of inflammasomes in bone diseases caused by sterile or non-sterile inflammation.

Background: NF-κB essential modulator (NEMO), encoded by IKBKG, is necessary for activation of the ubiquitous transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Animal studies suggest NEMO is required for NF-κB mediated bone homeostasis, but this has not been thoroughly studied in humans. IKBKG loss-of-function mutation causes incontinentia pigmenti (IP), a rare X-linked disease featuring linear hypopigmentation, alopecia, hypodontia, and immunodeficiency. Single case reports describe osteopetrosis (OPT) in boys carrying hypomorphic IKBKG mutations. Method: We studied the bone phenotype in women with IP with evaluation of radiographs of the spine and non-dominant arm and leg; lumbar spine and femoral neck aBMD using DXA; μ-CT and histomorphometry of trans-iliac crest biopsy specimens; bone turnover markers; and cellular phenotype in bone marrow skeletal (stromal) stem cells (BM-MSCs) in a cross-sectional, age-, sex-, and BMI-matched case-control study. X-chromosome inactivation was measured in blood leucocytes and BM-MSCs using a PCR method with methylation of HpaII sites. NF-κB activity was quantitated in BM-MSCs using a luciferase NF-κB reporter assay. Results: Seven Caucasian women with IP (age: 24–67 years and BMI: 20.0–35.2 kg/m 2 ) and IKBKG mutation (del exon 4–10 (n = 4); c.460C>T (n = 3)) were compared to matched controls. The IKBKG mutation carriers had extremely skewed X-inactivation (>90:10%) in blood, but not in BM-MSCs. NF-κB activity was lower in BM-MSCs from IKBKG mutation carriers (n = 5) compared to controls (3094 ± 679 vs. 5422 ± 1038/μg protein, p < 0.01). However, no differences were identified on skeletal radiographics, aBMD, μ-architecture of the iliac crest, or bone turnover markers. The IKBKG mutation carriers had a 1.7–fold greater extent of eroded surfaces relative to osteoid surfaces (p < 0.01), and a 2.0–fold greater proportion of arrested reversal surface relative to active reversal surface (p < 0.01). Conclusion: Unlike mutation-positive males, the IKBKG mutation-positive women did not manifest OPT.