Myeloma-bone marrow adipocyte axis in tumour survival and treatment response.

Multiple myeloma is an incurable cancer of the bone marrow that is dependent on its microenvironment, including bone marrow adipocytes (BMAds). Here, we discuss our findings that the reciprocal interaction of myeloma cells and BMAds, leads to myeloma cell survival and induces metabolic dysfunction and senescence-associated secretory phenotype in BMAds.

The CD38low natural killer cell line KHYG1 transiently expressing CD16F158V in combination with daratumumab targets multiple myeloma cells with minimal effector NK cell fratricide.

Multiple myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of the CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (MoAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells that mediate in vivo tumour immunosurveillance. NK cells also play an important role during MoAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their FcγRIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring variant of CD16 harbouring a single-point polymorphism (F158V). However, the contribution of NK cells to the efficacy of daratumumab remains debatable as clinical data clearly indicate the rapid depletion of CD38high peripheral blood NK cells in patients upon daratumumab administration. In contrast, CD38low peripheral blood NK cells have been shown to survive daratumumab mediated fratricide in vivo, while still retaining their potent anti-MM cytolytic effector functions ex vivo. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a "safe" mRNA electroporation-based approach on CD38low NK cells in combination with daratumumab could represent a novel therapeutic option for treatment of MM. In the present study, we investigate a NK cell line (KHYG-1), derived from a patient with aggressive NK cell leukemia, as a platform for generating CD38low NK cells expressing CD16F158V which can be administered as an "off-the-shelf" therapy to target both CD38high and CD38low tumour clones in patients receiving daratumumab.

Inhibiting the osteocyte-specific protein sclerostin increases bone mass and fracture resistance in multiple myeloma.

Multiple myeloma (MM) is a plasma cell cancer that develops in the skeleton causing profound bone destruction and fractures. The bone disease is mediated by increased osteoclastic bone resorption and suppressed bone formation. Bisphosphonates used for treatment inhibit bone resorption and prevent bone loss but fail to influence bone formation and do not replace lost bone, so patients continue to fracture. Stimulating bone formation to increase bone mass and fracture resistance is a priority; however, targeting tumor-derived modulators of bone formation has had limited success. Sclerostin is an osteocyte-specific Wnt antagonist that inhibits bone formation. We hypothesized that inhibiting sclerostin would prevent development of bone disease and increase resistance to fracture in MM. Sclerostin was expressed in osteocytes from bones from naive and myeloma-bearing mice. In contrast, sclerostin was not expressed by plasma cells from 630 patients with myeloma or 54 myeloma cell lines. Mice injected with 5TGM1-eGFP, 5T2MM, or MM1.S myeloma cells demonstrated significant bone loss, which was associated with a decrease in fracture resistance in the vertebrae. Treatment with anti-sclerostin antibody increased osteoblast numbers and bone formation rate but did not inhibit bone resorption or reduce tumor burden. Treatment with anti-sclerostin antibody prevented myeloma-induced bone loss, reduced osteolytic bone lesions, and increased fracture resistance. Treatment with anti-sclerostin antibody and zoledronic acid combined increased bone mass and fracture resistance when compared with treatment with zoledronic acid alone. This study defines a therapeutic strategy superior to the current standard of care that will reduce fractures for patients with MM.

The skeletal cell-derived molecule sclerostin drives bone marrow adipogenesis.

The bone marrow niche is a dynamic and complex microenvironment that can both regulate, and be regulated by the bone matrix. Within the bone marrow (BM), mesenchymal stromal cell (MSC) precursors reside in a multi-potent state and retain the capacity to differentiate down osteoblastic, adipogenic, or chondrogenic lineages in response to numerous biochemical cues. These signals can be altered in various pathological states including, but not limited to, osteoporotic-induced fracture, systemic adiposity, and the presence of bone-homing cancers. Herein we provide evidence that signals from the bone matrix (osteocytes) determine marrow adiposity by regulating adipogenesis in the bone marrow. Specifically, we found that physiologically relevant levels of Sclerostin (SOST), which is a Wnt-inhibitory molecule secreted from bone matrix-embedded osteocytes, can induce adipogenesis in 3T3-L1 cells, mouse ear- and BM-derived MSCs, and human BM-derived MSCs. We demonstrate that the mechanism of SOST induction of adipogenesis is through inhibition of Wnt signaling in pre-adipocytes. We also demonstrate that a decrease of sclerostin in vivo, via both genetic and pharmaceutical methods, significantly decreases bone marrow adipose tissue (BMAT) formation. Overall, this work demonstrates a direct role for SOST in regulating fate determination of BM-adipocyte progenitors. This provides a novel mechanism for which BMAT is governed by the local bone microenvironment, which may prove relevant in the pathogenesis of certain diseases involving marrow adipose. Importantly, with anti-sclerostin therapy at the forefront of osteoporosis treatment and a greater recognition of the role of BMAT in disease, these data are likely to have important clinical implications.

Exome sequencing reveals pathogenic mutations in 91 strains of mice with Mendelian disorders.

Spontaneously arising mouse mutations have served as the foundation for understanding gene function for more than 100 years. We have used exome sequencing in an effort to identify the causative mutations for 172 distinct, spontaneously arising mouse models of Mendelian disorders, including a broad range of clinically relevant phenotypes. To analyze the resulting data, we developed an analytics pipeline that is optimized for mouse exome data and a variation database that allows for reproducible, user-defined data mining as well as nomination of mutation candidates through knowledge-based integration of sample and variant data. Using these new tools, putative pathogenic mutations were identified for 91 (53%) of the strains in our study. Despite the increased power offered by potentially unlimited pedigrees and controlled breeding, about half of our exome cases remained unsolved. Using a combination of manual analyses of exome alignments and whole-genome sequencing, we provide evidence that a large fraction of unsolved exome cases have underlying structural mutations. This result directly informs efforts to investigate the similar proportion of apparently Mendelian human phenotypes that are recalcitrant to exome sequencing.

Discovery and characterization of spontaneous mouse models of craniofacial dysmorphology.

Craniofacial abnormalities are among the most common features of human genetic syndromes and disorders. The etiology of these conditions is often complex, influenced by both genetic context and the environment. Frequently, craniofacial abnormalities present as part of a syndrome with clear comorbid phenotypes, providing additional insight into mechanisms of the causative gene or pathway. The mouse has been a key tool in our understanding of the genetic mechanisms of craniofacial development and disease, and can provide excellent models for human craniofacial abnormalities. While powerful genetic engineering tools in the mouse have contributed significantly our understanding of craniofacial development and dysmorphology, forward genetic approaches provide an unbiased means to identify new genes and pathways. Moreover, spontaneous mutations can occur on any number of genetic backgrounds, potentially revealing critical genes that require a specific genetic context. Here we report discovery and phenotyping of 43 craniofacial mouse models, derived primarily from a screen for spontaneous mutations in production colonies at the Jackson Laboratory. We identify the causative gene for 33 lines, including novel genes in pathways not previously connected to craniofacial development, and novel alleles of known genes that present with unique phenotypes. Together with our detailed characterization, this work provides a valuable gene discovery resource for the craniofacial community, and a rich source of mouse models for further investigation.

Adipose, Bone, and Myeloma: Contributions from the Microenvironment.

Researchers globally are working towards finding a cure for multiple myeloma (MM), a destructive blood cancer diagnosed yearly in ~750,000 people worldwide (Podar et al. in Expert Opin Emerg Drugs 14:99-127, 2009). Although MM targets multiple organ systems, it is the devastating skeletal destruction experienced by over 90 % of patients that often most severely impacts patient morbidity, pain, and quality of life. Preventing bone disease is therefore a priority in MM treatment, and understanding how and why myeloma cells target the bone marrow (BM) is fundamental to this process. This review focuses on a key area of MM research: the contributions of the bone microenvironment to disease origins, progression, and drug resistance. We describe some of the key cell types in the BM niche: osteoclasts, osteoblasts, osteocytes, adipocytes, and mesenchymal stem cells. We then focus on how these key cellular players are, or could be, regulating a range of disease-related processes spanning MM growth, drug resistance, and bone disease (including osteolysis, fracture, and hypercalcemia). We summarize the literature regarding MM-bone cell and MM-adipocyte relationships and subsequent phenotypic changes or adaptations in MM cells, with the aim of providing a deeper understanding of how myeloma cells grow in the skeleton to cause bone destruction. We identify avenues and therapies that intervene in these networks to stop tumor growth and/or induce bone regeneration. Overall, we aim to illustrate how novel therapeutic target molecules, proteins, and cellular mediators may offer new avenues to attack this disease while reviewing currently utilized therapies.

Kidney adysplasia and variable hydronephrosis, a new mutation affecting the odd-skipped related 1 gene in the mouse, causes variable defects in kidney development and hydronephrosis.

Many genes, including odd-skipped related 1 (Osr1), are involved in regulation of mammalian kidney development. We describe here a new recessive mutation (kidney adysplasia and variable hydronephrosis, kavh) in the mouse that leads to downregulation of Osr1 transcript, causing several kidney defects: agenesis, hypoplasia, and hydronephrosis with variable age of onset. The mutation is closely associated with a reciprocal translocation, T(12;17)4Rk, whose Chromosome 12 breakpoint is upstream from Osr1. The kavh/kavh mutant provides a model to study kidney development and test therapies for hydronephrosis.

Dsp rul: a spontaneous mouse mutation in desmoplakin as a model of Carvajal-Huerta syndrome.

Studies of spontaneous mutations in mice have provided valuable disease models and important insights into the mechanisms of human disease. Ruffled (rul) is a new autosomal recessive mutation causing abnormal hair coat in mice. The rul allele arose spontaneously in the RB156Bnr/EiJ inbred mouse strain. In addition to an abnormal coat texture, we found diffuse epidermal blistering, abnormal electrocardiograms (ECGs), and ventricular fibrosis in mutant animals. Using high-throughput sequencing (HTS) we found a frameshift mutation at 38,288,978bp of chromosome 13 in the desmoplakin gene (Dsp). The predicted mutant protein is truncated at the c-terminus and missing the majority of the plakin repeat domain. The phenotypes found in Dsp(rul) mice closely model a rare human disorder, Carvajal-Huerta syndrome. Carvajal-Huerta syndrome (CHS) is a rare cardiocutaneous disorder that presents in humans with wooly hair, palmoplantar keratoderma and ventricular cardiomyopathy. CHS results from an autosomal recessive mutation on the 3' end of desmoplakin (DSP) truncating the full length protein. The Dsp(rul) mouse provides a new model to investigate the pathogenesis of CHS, as well as the underlying basic biology of the adhesion molecules coded by the desmosomal genes.

A mutation in the nuclear pore complex gene Tmem48 causes gametogenesis defects in skeletal fusions with sterility (sks) mice.

Skeletal fusions with sterility (sks) is an autosomal recessive mutation of mouse that results in male and female sterility because of defects in gametogenesis. The mutants also have skeletal malformations with fused vertebrae and ribs. We examined testicular phenotypes of sks/sks mice to investigate the defects in spermatogenesis. Histological and immunocytochemical analyses and expression analyses of the marker genes demonstrated that spermatogenesis is arrested at mid to late pachytene stage of meiotic prophase with defective synapsis of the homologous chromosomes. Next, we determined the precise chromosomal localization of the sks locus on a 0.3-Mb region of mouse chromosome 4 by linkage analysis. By sequencing the positional candidate genes in this region and whole exome sequencing, we found a GG to TT nucleotide substitution in exon 6 of the Tmem48 gene that encodes a putative transmembrane protein with six transmembrane domains. The nucleotide substitution causes aberrant splicing, which deletes exon 6 of the Tmem48 transcript. Specific expression of TMEM48 was observed in germ cells of males and females. Furthermore, the phenotypes of the sks mutant were completely rescued by the transgenesis of a genomic fragment containing the wild-type Tmem48 gene. These findings indicate that the Tmem48 mutation is responsible for the gametogenesis defects and skeletal malformations in the sks mice. The TMEM48 protein is a nuclear membrane protein comprising the nuclear pore complex; its exact function in the nuclear pore complex is still unknown. Our finding suggested that the nuclear pore complex plays an important role in mammalian gametogenesis and skeletal development.

Adipocytes and metabolism: Contributions to multiple myeloma.

Obesity contributes to many cancers, including breast cancer and multiple myeloma, two cancers that often colonize the bone marrow (BM). Obesity often causes metabolic disease, but at the cellular level, there is uncertainty regarding how these shifts affect cellular phenotypes. Evidence is building that different types of fuel affect tumor cell metabolism, mitochondrial function, and signaling pathways differently, but tumor cells are also flexible and adapt to less-than ideal metabolic conditions, suggesting that single-pronged attacks on tumor metabolism may not be efficacious enough to be effective clinically. In this review, we describe the newest research at the pre-clinical level on how tumor metabolic pathways and energy sources affect cancer cells, with a special focus on multiple myeloma (MM). We also describe the known forward-feedback loops between bone marrow adipocytes (BMAds) and local tumor cells that support tumor growth. We describe how metabolic targets and transcription factors related to fatty acid (FA) oxidation, FA biosynthesis, glycolysis, oxidative phosphorylation (OXPHOS), and other pathways hold great promise as new vulnerabilities in myeloma cells. Specifically, we describe the importance of the acetyl-CoA synthetase (ACSS) and the acyl-CoA synthetase long chain (ACSL) families, which are both involved in FA metabolism. We also describe new data on the importance of lactate metabolism and lactate transporters in supporting the growth of tumor cells in a hypoxic BM microenvironment. We highlight new data showing the dependency of myeloma cells on the mitochondrial pyruvate carrier (MPC), which transports pyruvate to the mitochondria to fuel the tricarboxylic acid (TCA) cycle and electron transport chain (ETC), boosting OXPHOS. Inhibiting the MPC affects myeloma cell mitochondrial metabolism and growth, and synergizes with proteosome inhibitors in killing myeloma cells. We also describe how metabolic signaling pathways intersect established survival and proliferation pathways; for example, the fatty acid binding proteins (FABPs) affect MYC signaling and support growth, survival, and metabolism of myeloma cells. Our goal is to review the current the field so that novel, metabolic-focused therapeutic interventions and treatments can be imagined, developed and tested to decrease the burden of MM and related cancers.

Inhibition of Acyl-CoA Synthetase Long Chain Isozymes Decreases Multiple Myeloma Cell Proliferation and Causes Mitochondrial Dysfunction.

Multiple myeloma (MM) is an incurable cancer of plasma cells with a 5-year survival rate of 59%. Dysregulation of fatty acid (FA) metabolism is associated with MM development and progression; however, the underlying mechanisms remain unclear. Acyl-CoA synthetase long-chain family members (ACSLs) convert free long-chain fatty acids into fatty acyl-CoA esters and play key roles in catabolic and anabolic fatty acid metabolism. The Cancer Dependency Map data suggested that ACSL3 and ACSL4 were among the top 25% Hallmark Fatty Acid Metabolism genes that support MM fitness. Here, we show that inhibition of ACSLs in human myeloma cell lines using the pharmacological inhibitor Triascin C (TriC) causes apoptosis and decreases proliferation in a dose- and time-dependent manner. RNA-seq of MM.1S cells treated with TriC for 24 h showed a significant enrichment in apoptosis, ferroptosis, and ER stress. Proteomics of MM.1S cells treated with TriC for 48 h revealed that mitochondrial dysfunction and oxidative phosphorylation were significantly enriched pathways of interest, consistent with our observations of decreased mitochondrial membrane potential and increased mitochondrial superoxide levels. Interestingly, MM.1S cells treated with TriC for 24 h also showed decreased mitochondrial ATP production rates and overall lower cellular respiration.

Obesity and myeloma: Clinical and mechanistic contributions to disease progression.

Obesity and obesogenic behaviors are positively associated with both monoclonal gammopathy of unknown significance (MGUS) and multiple myeloma (MM). As the only known modifiable risk factor, this association has emerged as a new potential target for MM prevention, but little is known about the mechanistic relationship of body weight with MM progression. Here we summarize epidemiological correlations between weight, body composition, and the various stages of myeloma disease progression and treatments, as well as the current understanding of the molecular contributions of obesity-induced changes in myeloma cell phenotype and signaling. Finally, we outline groundwork for the future characterization of the relationship between body weight patterns, the bone marrow microenvironment, and MM pathogenesis in animal models, which have the potential to impact our understanding of disease pathogenesis and inform MM prevention messages.

Targeting the fatty acid binding proteins disrupts multiple myeloma cell cycle progression and MYC signaling.

Multiple myeloma is an incurable plasma cell malignancy with only a 53% 5-year survival rate. There is a critical need to find new multiple myeloma vulnerabilities and therapeutic avenues. Herein, we identified and explored a novel multiple myeloma target: the fatty acid binding protein (FABP) family. In our work, myeloma cells were treated with FABP inhibitors (BMS3094013 and SBFI-26) and examined in vivo and in vitro for cell cycle state, proliferation, apoptosis, mitochondrial membrane potential, cellular metabolism (oxygen consumption rates and fatty acid oxidation), and DNA methylation properties. Myeloma cell responses to BMS309403, SBFI-26, or both, were also assessed with RNA sequencing (RNA-Seq) and proteomic analysis, and confirmed with western blotting and qRT-PCR. Myeloma cell dependency on FABPs was assessed using the Cancer Dependency Map (DepMap). Finally, MM patient datasets (CoMMpass and GEO) were mined for FABP expression correlations with clinical outcomes. We found that myeloma cells treated with FABPi or with FABP5 knockout (generated via CRISPR/Cas9 editing) exhibited diminished proliferation, increased apoptosis, and metabolic changes in vitro. FABPi had mixed results in vivo, in two pre-clinical MM mouse models, suggesting optimization of in vivo delivery, dosing, or type of FABP inhibitors will be needed before clinical applicability. FABPi negatively impacted mitochondrial respiration and reduced expression of MYC and other key signaling pathways in MM cells in vitro. Clinical data demonstrated worse overall and progression-free survival in patients with high FABP5 expression in tumor cells. Overall, this study establishes the FABP family as a potentially new target in multiple myeloma. In MM cells, FABPs have a multitude of actions and cellular roles that result in the support of myeloma progression. Further research into the FABP family in MM is warrented, especially into the effective translation of targeting these in vivo.

Multiple myeloma is a type of blood cancer for which only a few treatments are available. Currently, only about half the patients with multiple myeloma survive for five years after diagnosis. Because obesity is a risk factor for multiple myeloma, researchers have been studying how fat cells or fatty acids affect multiple myeloma tumor cells to identify new treatment targets. Fatty acid binding proteins (FABPs) are one promising target. The FABPs shuttle fatty acids and help cells communicate. Previous studies linked FABPs to some types of cancer, including another blood cancer called leukemia, and cancers of the prostate and breast. A recent study showed that patients with multiple myeloma, who have high levels of FABP5 in their tumors, have worse outcomes than patients with lower levels. But, so far, no one has studied the effects of inhibiting FABPs in multiple myeloma tumor cells or animals with multiple myeloma. Farrell et al. show that blocking or eliminating FABPs kills myeloma tumor cells and slows their growth in a dish (in vitro) and in some laboratory mice. In the experiments, the researchers treated myeloma cells with drugs that inhibit FABPs or genetically engineered myeloma cells to lack FABPs. They also show that blocking FABPs reduces the activity of a protein called MYC, which promotes tumor cell survival in many types of cancer. It also changed the metabolism of the tumor cell. Finally, the team examined data collected from several sets of patients with multiple myeloma and found that patients with high FABP levels have more aggressive cancer. The experiments lay the groundwork for more studies to determine if drugs or other therapies targeting FABPs could treat multiple myeloma. More research is needed to determine why inhibiting FABPs worked in some mice with multiple myeloma but not others, and whether FABP inhibitors might work better if combined with other cancer therapies. There were no signs that the drugs were toxic in mice, but more studies must prove they are safe and effective before testing the drugs in humans with multiple myeloma. Designing better or more potent FABP-blocking drugs may also lead to better animal study results.

Development and characterization of three cell culture systems to investigate the relationship between primary bone marrow adipocytes and myeloma cells.

The unique properties of the bone marrow (BM) allow for migration and proliferation of multiple myeloma (MM) cells while also providing the perfect environment for development of quiescent, drug-resistant MM cell clones. BM adipocytes (BMAds) have recently been identified as important contributors to systemic adipokine levels, bone strength, hematopoiesis, and progression of metastatic and primary BM cancers, such as MM. Recent studies in myeloma suggest that BMAds can be reprogrammed by tumor cells to contribute to myeloma-induced bone disease, and, reciprocally, BMAds support MM cells in vitro. Importantly, most data investigating BMAds have been generated using adipocytes generated by differentiating BM-derived mesenchymal stromal cells (BMSCs) into adipocytes in vitro using adipogenic media, due to the extreme technical challenges associated with isolating and culturing primary adipocytes. However, if studies could be performed with primary adipocytes, then they likely will recapitulate in vivo biology better than BMSC-derived adipocytes, as the differentiation process is artificial and differs from in vivo differentiation, and progenitor cell(s) of the primary BMAd (pBMAds) may not be the same as the BMSCs precursors used for adipogenic differentiation in vitro. Therefore, we developed and refined three methods for culturing pBMAds: two-dimensional (2D) coverslips, 2D transwells, and three-dimensional (3D) silk scaffolds, all of which can be cultured alone or with MM cells to investigate bidirectional tumor-host signaling. To develop an in vitro model with a tissue-like structure to mimic the BM microenvironment, we developed the first 3D, tissue engineered model utilizing pBMAds derived from human BM. We found that pBMAds, which are extremely fragile, can be isolated and stably cultured in 2D for 10 days and in 3D for up to 4 week in vitro. To investigate the relationship between pBMAds and myeloma, MM cells can be added to investigate physical relationships through confocal imaging and soluble signaling molecules via mass spectrometry. In summary, we developed three in vitro cell culture systems to study pBMAds and myeloma cells, which could be adapted to investigate many diseases and biological processes involving the BM, including other bone-homing tumor types.

Witch Nails (Krt90whnl): A spontaneous mouse mutation affecting nail growth and development.

Numerous single gene mutations identified in humans and mice result in nail deformities with many similarities between the species. A spontaneous, autosomal, recessive mutation called witch nails (whnl) is described here where the distal nail matrix and nail bed undergo degenerative changes resulting in formation of an abnormal nail plate causing mice to develop long, curved nails. This mutation arose spontaneously in a colony of MRL/MpJ-Faslpr/J at The Jackson Laboratory. Homozygous mutant mice are recognizable by 8 weeks of age by their long, curved nails. The whnl mutation, mapped on Chromosome 15, is due to a 7-bp insertion identified in the 3' region of exon 9 in the Krt90 gene (formerly Riken cDNA 4732456N10Rik), and is predicted to result in a frameshift that changes serine 476 to arginine and subsequently introduces 36 novel amino acids into the protein before a premature stop codon (p. Ser476ArgfsTer36). By immunohistochemistry the normal KRT90 protein is expressed in the nail matrix and nail bed in control mice where lesions are located in mutant mice. Immunoreactivity toward equine KRT124, the ortholog of mouse KRT90, is restricted to the hoof lamellae (equine hoof wall and lamellae are homologous to the mouse nail plate and nail bed) and the mouse nail bed. Equine laminitis lesions are similar to those observed in this mutant mouse suggesting that the latter may be a useful model for hoof and nail diseases. This first spontaneous mouse mutation affecting the novel Krt90 gene provides new insight into the normal regulation of the molecular pathways of nail development.

Molecular characterization of an allelic series of mutations in the mouse Nox3 gene.

The inner ear consists of the cochlea (the organ of hearing) and the vestibular system (the organs of balance). Within the vestibular system, linear acceleration and gravity are detected by the saccule and utricle. Resting above the neurosensory epithelia of these organs are otoconia, minute proteinaceous and crystalline (calcite) inertial masses that shift under the physical forces imparted by linear movements and gravity. It is the transduction and sensation of these movements and their integration with vision and proprioceptive inputs that contribute to the sensation of balance. It has been proposed that a reactive oxygen species- (ROS-) generating NADPH oxidase comprising the gene products of the Nox3, Noxo1, and Cyba genes plays a critical and constructive role in the process of inner-ear development, specifically, the deposition of otoconia. Inactivation in mouse of any of the NADPH oxidase components encoded by the Nox3, Noxo1, or Cyba gene results in the complete congenital absence of otoconia and profound vestibular dysfunction. Here we describe our use of PCR, reverse transcription-PCR (RT-PCR), and rapid amplification of cDNA ends (RACE) with traditional and high-throughput (HTP) sequencing technologies to extend and complete the molecular characterization of an allelic series of seven mutations in the Nox3 gene. Collectively, the mutation spectrum includes an endogenous retrovirus insertion, two missense mutations, a splice donor mutation, a splice acceptor mutation, premature translational termination, and a small duplication. Together, these alleles provide tools to investigate the mechanisms of otoconial deposition over development, throughout aging, and in various disease states.

Bone Marrow Adipocytes: A Link between Obesity and Bone Cancer.

Cancers that grow in the bone marrow are for most patients scary, painful, and incurable. These cancers are especially hard to treat due to the supportive microenvironment provided by the bone marrow niche in which they reside. New therapies designed to target tumor cells have extended the life expectancy for these patients, but better therapies are needed and new ideas for how to target these cancers are crucial. This need has led researchers to interrogate whether bone marrow adipocytes (BMAds), which increase in number and size during aging and in obesity, contribute to cancer initiation or progression within the bone marrow. Across the globe, the consensus in the field is a unified "yes". However, how to target these adipocytes or the factors they produce and how BMAds interact with different tumor cells are open research questions. Herein, we review this research field, with the goal of accelerating research in the network of laboratories working in this area and attracting bright scientists with new perspectives and ideas to the field in order to bring about better therapies for patients with bone cancers.

Sclerostin-Neutralizing Antibody Treatment Rescues Negative Effects of Rosiglitazone on Mouse Bone Parameters.

Obesity, a growing pandemic, is a risk factor for many cancers and causes increased bone marrow adipose tissue (BMAT). in vitro studies and obese animal models suggest that BMAT contributes to cancer progression, but there is a lack of preclinical models to directly test BMAT's role in cancer. Overactivation of peroxisome-proliferator-activated receptor-γ (PPARγ) can skew bone formation and resorption rates, resulting in increased BMAT and trabecular bone loss. Thiazolidinediones (eg, rosiglitazone) are anti-diabetic therapies that promote adipogenesis through PPARγ activation. We investigated if rosiglitazone increases BMAT in an immunocompromised model, commonly used in cancer research, and if these effects could be reversed by co-administering a bone anabolic agent (sclerostin-neutralizing antibody [Scl-Ab]), which has been shown to inhibit adipogenesis, using DXA, µCT, OsO4 µCT, and dynamic histomorphometry. Four weeks of rosiglitazone in female SCID Beige mice (cohort 1) significantly decreased trabecular bone volume (BV/TV) by about one-half, through increased osteoclast and suppressed osteoblast activity, and significantly increased BMAT. In cohort 2, mice were administered rosiglitazone ± Scl-Ab for 4 weeks, and then rosiglitazone was discontinued and Scl-Ab or vehicle were continued for 6 weeks. Scl-Ab significantly increased bone parameters (eg, BV/TV, N.Ob/B.Pm, and MS/BS) in both groups. Scl-Ab also overcame many negative effects of rosiglitazone (eg, effects on trabecular bone parameters, increased mineralization lag time [MLT], and decreased bone formation rate [BFR]). Interestingly, Scl-Ab significantly decreased rosiglitazone-induced BMAT in the femur, mostly due to a reduction in adipocyte size, but had a much weaker effect on tibial BMAT. These data suggest targeting sclerostin can prevent rosiglitazone-induced bone loss and reduce BM adiposity, in some, but not all BMAT locations. Collectively, our data demonstrate that rosiglitazone increases BMAT in SCID Beige mice, but concomitant changes in bone may confound its use to specifically determine BMAT's role in tumor models. © 2020 American Society for Bone and Mineral Research (ASBMR).

Sclerostin antibody increases trabecular bone and bone mechanical properties by increasing osteoblast activity damaged by whole-body irradiation in mice.

Irradiation therapy causes bone deterioration and increased risk for skeletal-related events. Irradiation interferes with trabecular architecture through increased osteoclastic activity, decreased osteoblastic activity, and increased adipocyte expansion in the bone marrow (BM), which further compounds bone-related disease. Neutralizing antibodies to sclerostin (Scl-Ab) increase bone mass and strength by increasing bone formation and reducing bone resorption. We hypothesized that treatment with Scl-Ab would attenuate the adverse effects of irradiation by increasing bone volume and decreasing BM adipose tissue (BMAT), resulting in better quality bone. In this study, 12-week-old female C57BL/6J mice were exposed to 6 Gy whole-body irradiation or were non-irradiated, then administered Scl-Ab (25 mg/kg) or vehicle weekly for 5 weeks. Femoral µCT analysis confirmed that the overall effect of IR significantly decreased trabecular bone volume/total volume (Tb.BV/TV) (2-way ANOVA, p < 0.0001) with a -43.8% loss in Tb.BV/TV in the IR control group. Scl-Ab independently increased Tb.BV/TV by 3.07-fold in non-irradiated and 3.6-fold in irradiated mice (2-way ANOVA, p < 0.0001). Irradiation did not affect cortical parameters, although Scl-Ab increased cortical thickness and area significantly in both irradiated and non-irradiated mice (2-way ANOVA, p < 0.0001). Femoral mechanical testing confirmed Scl-Ab significantly increased bending rigidity and ultimate moment independently of irradiation (2-way ANOVA, p < 0.0001). Static and dynamic histomorphometry of the femoral metaphysis revealed osteoblast vigor, not number, was significantly increased in the irradiated mice treated with Scl-Ab. Systemic alterations were assessed through serum lipidomic analysis, which showed that Scl-Ab normalized lipid profiles in the irradiated group. This data supports the theory of sclerostin as a novel contributor to the regulation of osteoblast activity after irradiation. Overall, our data support the hypothesis that Scl-Ab ameliorates the deleterious effects of whole-body irradiation on bone and adipose tissue in a mouse model. Our findings suggest that future research into localized and systemic therapies after irradiation exposure is warranted.