Systematic transcriptome profiling of hPSC-derived osteoblasts unveils CORIN's mastery in governing osteogenesis through CEBPD modulation.

The commitment of stem cells to differentiate into osteoblasts is a highly regulated and complex process that involves the coordination of extrinsic signals and intrinsic transcriptional machinery. While rodent osteoblastic differentiation has been extensively studied, research on human osteogenesis has been limited by cell sources and existing models. Here, we systematically dissect human pluripotent stem cell-derived osteoblasts to identify functional membrane proteins and their downstream transcriptional networks involved in human osteogenesis. Our results reveal an enrichment of type II transmembrane serine protease CORIN in humans but not rodent osteoblasts. Functional analyses demonstrated that CORIN depletion significantly impairs osteogenesis. Genome-wide chromatin immunoprecipitation enrichment and mechanistic studies show that p38 MAPK-mediated CCAAT enhancer binding protein delta (CEBPD) upregulation is required for CORIN-modulated osteogenesis. Contrastingly, the type I transmembrane heparan sulfate proteoglycan SDC1 enriched in mesenchymal stem cells exerts a negative regulatory effect on osteogenesis through a similar mechanism. Chromatin immunoprecipitation-seq, bulk and single-cell transcriptomes, and functional validations indicated that CEBPD plays a critical role in controlling osteogenesis. In summary, our findings uncover previously unrecognized CORIN-mediated CEBPD transcriptomic networks in driving human osteoblast lineage commitment.

Hereditary retinoblastoma iPSC model reveals aberrant spliceosome function driving bone malignancies.

The RB1 gene is frequently mutated in human cancers but its role in tumorigenesis remains incompletely defined. Using an induced pluripotent stem cell (iPSC) model of hereditary retinoblastoma (RB), we report that the spliceosome is an up-regulated target responding to oncogenic stress in RB1-mutant cells. By investigating transcriptomes and genome occupancies in RB iPSC­derived osteoblasts (OBs), we discover that both E2F3a, which mediates spliceosomal gene expression, and pRB, which antagonizes E2F3a, coregulate more than one-third of spliceosomal genes by cobinding to their promoters or enhancers. Pharmacological inhibition of the spliceosome in RB1-mutant cells leads to global intron retention, decreased cell proliferation, and impaired tumorigenesis. Tumor specimen studies and genome-wide TCGA (The Cancer Genome Atlas) expression profile analyses support the clinical relevance of pRB and E2F3a in modulating spliceosomal gene expression in multiple cancer types including osteosarcoma (OS). High levels of pRB/E2F3a­regulated spliceosomal genes are associated with poor OS patient survival. Collectively, these findings reveal an undiscovered connection between pRB, E2F3a, the spliceosome, and tumorigenesis, pointing to the spliceosomal machinery as a potentially widespread therapeutic vulnerability of pRB-deficient cancers.

Patient-derived iPSCs link elevated mitochondrial respiratory complex I function to osteosarcoma in Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive genetic disorder characterized by poikiloderma, small stature, skeletal anomalies, sparse brows/lashes, cataracts, and predisposition to cancer. Type 2 RTS patients with biallelic RECQL4 pathogenic variants have multiple skeletal anomalies and a significantly increased incidence of osteosarcoma. Here, we generated RTS patient-derived induced pluripotent stem cells (iPSCs) to dissect the pathological signaling leading to RTS patient-associated osteosarcoma. RTS iPSC-derived osteoblasts showed defective osteogenic differentiation and gain of in vitro tumorigenic ability. Transcriptome analysis of RTS osteoblasts validated decreased bone morphogenesis while revealing aberrantly upregulated mitochondrial respiratory complex I gene expression. RTS osteoblast metabolic assays demonstrated elevated mitochondrial respiratory complex I function, increased oxidative phosphorylation (OXPHOS), and increased ATP production. Inhibition of mitochondrial respiratory complex I activity by IACS-010759 selectively suppressed cellular respiration and cell proliferation of RTS osteoblasts. Furthermore, systems analysis of IACS-010759-induced changes in RTS osteoblasts revealed that chemical inhibition of mitochondrial respiratory complex I impaired cell proliferation, induced senescence, and decreased MAPK signaling and cell cycle associated genes, but increased H19 and ribosomal protein genes. In summary, our study suggests that mitochondrial respiratory complex I is a potential therapeutic target for RTS-associated osteosarcoma and provides future insights for clinical treatment strategies.

Biallelic variants in CRIPT cause a Rothmund-Thomson-like syndrome with increased cellular senescence.

PURPOSE: Rothmund-Thomson syndrome (RTS) is characterized by poikiloderma, sparse hair, small stature, skeletal defects, cancer, and cataracts, resembling features of premature aging. RECQL4 and ANAPC1 are the 2 known disease genes associated with RTS in >70% of cases. We describe RTS-like features in 5 individuals with biallelic variants in CRIPT (OMIM 615789). METHODS: Two newly identified and 4 published individuals with CRIPT variants were systematically compared with those with RTS using clinical data, computational analysis of photographs, histologic analysis of skin, and cellular studies on fibroblasts. RESULTS: All CRIPT individuals fulfilled the diagnostic criteria for RTS and additionally had neurodevelopmental delay and seizures. Using computational gestalt analysis, CRIPT individuals showed greatest facial similarity with individuals with RTS. Skin biopsies revealed a high expression of senescence markers (p53/p16/p21) and the senescence-associated ß-galactosidase activity was elevated in CRIPT-deficient fibroblasts. RECQL4- and CRIPT-deficient fibroblasts showed an unremarkable mitotic progression and unremarkable number of mitotic errors and no or only mild sensitivity to genotoxic stress by ionizing radiation, mitomycin C, hydroxyurea, etoposide, and potassium bromate. CONCLUSION: CRIPT causes an RTS-like syndrome associated with neurodevelopmental delay and epilepsy. At the cellular level, RECQL4- and CRIPT-deficient cells display increased senescence, suggesting shared molecular mechanisms leading to the clinical phenotypes.

Mutations in ANAPC1, Encoding a Scaffold Subunit of the Anaphase-Promoting Complex, Cause Rothmund-Thomson Syndrome Type 1.

Rothmund-Thomson syndrome (RTS) is an autosomal-recessive disorder characterized by poikiloderma, sparse hair, short stature, and skeletal anomalies. Type 2 RTS, which is defined by the presence of bi-allelic mutations in RECQL4, is characterized by increased cancer susceptibility and skeletal anomalies, whereas the genetic basis of RTS type 1, which is associated with juvenile cataracts, is unknown. We studied ten individuals, from seven families, who had RTS type 1 and identified a deep intronic splicing mutation of the ANAPC1 gene, a component of the anaphase-promoting complex/cyclosome (APC/C), in all affected individuals, either in the homozygous state or in trans with another mutation. Fibroblast studies showed that the intronic mutation causes the activation of a 95 bp pseudoexon, leading to mRNAs with premature termination codons and nonsense-mediated decay, decreased ANAPC1 protein levels, and prolongation of interphase. Interestingly, mice that were heterozygous for a knockout mutation have an increased incidence of cataracts. Our results demonstrate that deficiency in the APC/C is a cause of RTS type 1 and suggest a possible link between the APC/C and RECQL4 helicase because both proteins are involved in DNA repair and replication.

Risk factors associated with delayed methotrexate clearance and increased toxicity in pediatric patients with osteosarcoma.

High-dose methotrexate (HD-MTX; 12 g/m2 ) is part of standard therapy for pediatric osteosarcoma (OS). Risk factors associated with MTX toxicity in children with OS are not well defined. We investigated the association between peak MTX levels (four-hour) and delayed MTX clearance or treatment toxicity. Information was retrieved from electronic medical records of 33 OS patients treated with HD-MTX at Texas Children's Hospital from 2008 to 2015. We found that the four-hour MTX level did not contribute to toxicity or delayed MTX clearance. We demonstrated that certain demographic characteristics are associated with delayed clearance and increased toxicity.

RECQ DNA Helicases and Osteosarcoma.

The RECQ family of DNA helicases is a conserved group of enzymes that plays an important role in maintaining genomic stability. Humans possess five RECQ helicase genes, and mutations in three of them - BLM, WRN, and RECQL4 - are associated with the genetic disorders Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome (RTS), respectively. These syndromes share overlapping clinical features, and importantly they are all associated with an increased risk of cancer. Patients with RTS have the highest specific risk of developing osteosarcoma compared to all other cancer predisposition syndromes; therefore, RTS serves as a relevant model to study the pathogenesis and molecular genetics of osteosarcoma. The "tumor suppressor" function of the RECQ helicases continues to be an area of active investigation. This chapter will focus primarily on the known cellular functions of RECQL4 and how these may relate to tumorigenesis, as well as ongoing efforts to understand RECQL4's functions in vivo using animal models. Understanding the RECQ pathways will provide insight into avenues for novel cancer therapies in the future.

Generalized metabolic bone disease and fracture risk in Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder characterized by poikiloderma, small stature, sparse hair, skeletal abnormalities, increased risk of osteosarcoma, and decreased bone mass. To date, there has not been a comprehensive evaluation of the prevalence and extent of metabolic bone disease in RTS. Furthermore, the mechanisms that result in this phenotype are largely unknown. In this report, we provide a detailed evaluation of 29 individuals with RTS with respect to their metabolic bone status including bone mineral density, calcium kinetics studies, and markers of bone remodeling. We show that individuals with RTS have decreased areal bone mineral density. Additionally, we demonstrate that the presence of pathogenic variants in RECQL4 and low bone mineral density correlate with the history of increased risk of fractures. Using a RECQL4-deficient mouse model that recapitulates skeletal abnormalities seen in individuals with RTS, we demonstrate that generalized skeletal involvement is likely due to decreased osteogenesis. Our findings are clinically relevant as they may help in the risk stratification of patients with RTS and also in the identification of individuals who may benefit from additional surveillance and management of metabolic bone disease.

Generation of patient-derived tumor xenografts from percutaneous tumor biopsies in children with bone sarcomas.

One of the limitations of performing percutaneous biopsies in patients with bone sarcomas is the small amount of tumor that can be obtained for research purposes. Here, we describe our experience developing patient-derived tumor xenografts (PDXs) using percutaneous tumor biopsies in children with bone sarcomas. We generated 14 bone sarcoma PDXs from percutaneous tumor biopsies. We also developed eight bone sarcoma PDXs from surgical resection of primary bone tumors and pulmonary metastases. A multidisciplinary team approach was critical to establish an accurate diagnosis and to provide adequate tumor samples for PDX generation.

A novel prognostic model for osteosarcoma using circulating CXCL10 and FLT3LG.

BACKGROUND: Osteosarcoma (OS) is the most common malignant pediatric bone tumor. The identification of novel biomarkers for early prognostication will facilitate risk-based stratification and therapy. This study investigated the significance of circulating cytokines/chemokines for predicting the prognosis at the initial diagnosis. METHODS: Luminex assays were used to measure cytokine/chemokine concentrations in blood samples from a discovery cohort of OS patients from Texas Children's Hospital (n = 37) and an independent validation cohort obtained from the Children's Oncology Group (n = 233). After the validation of the biomarkers, a multivariate model was constructed to stratify the patients into risk groups. RESULTS: The circulating concentrations of C-X-C motif chemokine ligand 10 (CXCL10), Fms-related tyrosine kinase 3 ligand (FLT3LG), interferon γ (IFNG), and C-C motif chemokine ligand 4 (CCL4) were significantly associated with overall survival in both cohorts. Among these candidates, CXCL10 and FLT3LG were independent of the existing prognostic factor, metastasis at diagnosis, and CCL4 further discriminated cancer cases from controls. CXCL10, FLT3LG, and the metastatic status at diagnosis were combined to develop a multivariate model that significantly stratified the patients into 4 distinct risk groups (P = 1.6 × 10-8 ). The survival analysis showed that the 5-year overall survival rates for the low-, intermediate-, high-, and very high-risk groups were 77%, 54%, 47%, and 10%, respectively, whereas the 5-year event-free survival rates were 64%, 47%, 27%, and 0%, respectively. Neither CXCL10 nor FLT3LG tumor expression was significantly associated with survival. CONCLUSIONS: High circulating levels of CXCL10 and FLT3LG predicted worse survival for patients with OS. Because both CXCL10 and FL3LG axes are potentially targetable, further study may lead to novel risk-based stratification and therapy for OS. Cancer 2017;144-154. © 2016 American Cancer Society.

RECQL4 helicase has oncogenic potential in sporadic breast cancers.

RECQL4 helicase is a molecular motor that unwinds DNA, a process essential during DNA replication and DNA repair. Germ-line mutations in RECQL4 cause type II Rothmund-Thomson syndrome (RTS), characterized by a premature ageing phenotype and cancer predisposition. RECQL4 is widely considered to be a tumour suppressor, although its role in human breast cancer is largely unknown. As the RECQL4 gene is localized to chromosome 8q24, a site frequently amplified in sporadic breast cancers, we hypothesized that it may play an oncogenic role in breast tumourigenesis. To address this, we analysed large cohorts for gene copy number changes (n = 1977), mRNA expression (n = 1977) and protein level (n = 1902). Breast cancer incidence was also explored in 58 patients with type II RTS. DNA replication dynamics and chemosensitivity was evaluated in RECQL4-depleted breast cancer cells in vitro. Amplification or gain in gene copy number (30.6%), high-level mRNA expression (51%) and high levels of protein (23%) significantly associated with aggressive tumour behaviour, including lymph node positivity, larger tumour size, HER2 overexpression, ER-negativity, triple-negative phenotypes and poor survival. RECQL4 depletion impaired the DNA replication rate and increased chemosensitivity in cultured breast cancer cells. Thus, although recognized as a 'safe guardian of the genome', our data provide compelling evidence that RECQL4 is tumour promoting in established breast cancers. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Extra-Anatomic Axillo-Mesenteric Reconstruction for Chronic Mesenteric Ischemia.

We present a case of extra-anatomic axillo-mesenteric reconstruction for chronic mesenteric ischemia. Endovascular access and retrograde bypass options were prohibited by severe aortoiliac occlusive disease. Standard antegrade bypass was impossible because of the presence of a thoracoabdominal aortic aneurysm. This unusual method of mesenteric reconstruction is a robust and viable option for patients with challenging anatomy and multiple comorbidities that preclude traditional endovascular and open surgical options.

Antitumor effects of chimeric receptor engineered human T cells directed to tumor stroma.

Cancer-associated fibroblasts (CAFs), the principle component of the tumor-associated stroma, form a highly protumorigenic and immunosuppressive microenvironment that mediates therapeutic resistance. Co-targeting CAFs in addition to cancer cells may therefore augment the antitumor response. Fibroblast activation protein-α (FAP), a type 2 dipeptidyl peptidase, is expressed on CAFs in a majority of solid tumors making it an attractive immunotherapeutic target. To target FAP-positive CAFs in the tumor-associated stroma, we genetically modified T cells to express a FAP-specific chimeric antigen receptor (CAR). The resulting FAP-specific T cells recognized and killed FAP-positive target cells as determined by proinflammatory cytokine release and target cell lysis. In an established A549 lung cancer model, adoptive transfer of FAP-specific T cells significantly reduced FAP-positive stromal cells, with a concomitant decrease in tumor growth. Combining these FAP-specific T cells with T cells that targeted the EphA2 antigen on the A549 cancer cells themselves significantly enhanced overall antitumor activity and conferred a survival advantage compared to either alone. Our study underscores the value of co-targeting both CAFs and cancer cells to increase the benefits of T-cell immunotherapy for solid tumors.

RECQ DNA helicases and osteosarcoma.

The RECQ family of DNA helicases is a conserved group of enzymes that are important for maintaining genomic integrity. In humans, there are five RECQ helicase genes, and mutations in three of them-BLM, WRN, and RECQL4-are associated with the genetic disorders Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome (RTS), respectively. Importantly all three diseases are cancer predisposition syndromes. Patients with RTS are highly and uniquely susceptible to developing osteosarcoma; thus, RTS provides a good model to study the pathogenesis of osteosarcoma. The "tumor suppressor" role of RECQL4 and the other RECQ helicases is an area of active investigation. This chapter reviews what is currently known about the cellular functions of RECQL4 and how these may relate to tumorigenesis, as well as ongoing efforts to understand RECQL4's functions in vivo using animal models. Understanding the RECQ pathways may provide insight into avenues for novel cancer therapies in the future.

The adolescent and young adult with cancer: state of the art -- bone tumors.

Primary malignant bone tumors in the pediatric to young adult populations are relatively uncommon and account for about 6 % of all cancers in those less than 20 years old [1] and 3 % of all cancers in adolescents and young adults (AYA) within the age range of 15 to 29 years [2]. Osteosarcoma (OS) and Ewing's sarcoma (ES) comprise the majority of malignant bone tumors. The approach to treatment for both tumors consists of local control measures (surgery or radiation) as well as systemic therapy with high-dose chemotherapy. Despite earlier advances, there have been no substantial improvements in outcomes over the past several decades, particularly for patients with metastatic disease. This review summarizes the major advances in the treatment of OS and ES and the standard therapies available today, current active clinical trials, and areas of investigation into molecularly targeted therapies.

Cancer risk among RECQL4 heterozygotes.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive cancer-predisposition disorder characterized by the presence of a wide range of clinical features including poikiloderma, sparse hair, growth deficiency, cataracts, and skeletal abnormalities. Importantly, two-thirds of individuals with RTS have a significant risk of developing osteosarcoma due to the presence of biallelic pathogenic variants in RECQL4, a critical gene involved in DNA repair and replication. It is unknown whether individuals who are heterozygous for a RECQL4 pathogenic variant also have an increased risk of cancer. To address this question, we examined the largest international RTS registry and analyzed 123 RECQL4 heterozygous family members of RTS probands. Overall, the prevalence of cancer among RECQL4 heterozygous family members was 2.4% (3/123). We found that compared to the age-adjusted population estimate of 5.6% from the Surveillance, Epidemiology, and End Results program, the prevalence of cancer was not significantly different in this cohort of RECQL4 heterozygotes (Fisher's exact test, P = 0.2). Given that the biological parents of individuals with RTS are obligate heterozygotes and that siblings have a fifty-percent chance of being asymptomatic heterozygotes, these findings provide valuable information to help guide clinicians in counseling RTS family members regarding the likelihood of developing cancer.

Identification of a novel C16orf57 mutation in Athabaskan patients with Poikiloderma with Neutropenia.

Poikiloderma with Neutropenia (PN), Clericuzio-Type (OMIM #604173) is characterized by poikiloderma, chronic neutropenia, recurrent sinopulmonary infections, bronchiectasis, and nail dystrophy. First described by Clericuzio in 1991 in 14 patients of Navajo descent, it has since also been described in non-Navajo patients. C16orf57 has recently been identified as a causative gene in PN. The purpose of our study was to describe a spectrum of C16orf57 mutations in a cohort of PN patients including five patients of Athabaskan (Navajo and Apache) ancestry. Eleven patients from eight kindreds were enrolled in an IRB-approved study at Baylor College of Medicine. Five patients were of Athabaskan ancestry. PCR amplification and sequencing of the entire coding region of the C16orf57 gene was performed on genomic DNA. We identified biallelic C16orf57 mutations in all 11 PN patients in our cohort. The seven new deleterious mutations consisted of deletion (2), nonsense (3), and splice site (2) mutations. The patients of Athabaskan ancestry all had a common deletion mutation (c.496delA) which was not found in the six non-Athabaskan patients. Mutations in the C16orf57 gene have been identified thus far in all patients studied with a clinical diagnosis of PN. We have identified seven new mutations in C16orf57 in PN patients. One of these is present in all patients of Athabaskan descent, suggesting that c.496delA represents the PN-causative mutation in this subpopulation.

Engineering Oncolytic Vaccinia Virus to redirect Macrophages to Tumor Cells.

Oncolytic virotherapy has been tested in numerous early phase clinical studies. However, the antitumor activity of oncolytic viruses thus far has been limited. Numerous strategies are being explored to enhance their antitumor activity by activating the adaptive arm of the immune system. We reasoned that it might also be possible to engineer oncolytic viruses to redirect tumor-associated macrophages to tumor cells for therapeutic benefit. We engineered an oncolytic vaccinia virus (VV) to disrupt the CD47/SIRPα interaction by expressing a chimeric molecule that consists of the ectodomain of SIRPα and the Fc domain of IgG4 (SIRPα-Fc-VV). SIRPα-Fc-VV readily replicated in tumor cells and redirected M1 as well as M2 macrophages to tumor cells in vitro. In contrast, control VVs that either encoded YFP (YFP-VV) or SIRPα (SIRPα-VV) did not. In vivo, SIRPα-Fc-VV had greater antitumor activity than YFP-VV and SIRPα-VV in an immune competent osteosarcoma model resulting in a significant survival advantage. Pretreatment with cytoxan further augmented the antitumor activity of SIRPα-Fc-VV. Thus, arming oncolytic viruses with SIRPα-Fc may present a promising strategy to enhance their antitumor activity for the virotherapy of solid tumors.

Sensitivity of RECQL4-deficient fibroblasts from Rothmund-Thomson syndrome patients to genotoxic agents.

RECQ helicase protein-like 4 (RECQL4) is a member of the human RECQ family of DNA helicases. Two-thirds of patients with Rothmund-Thomson syndrome (RTS) carry biallelic inactivating mutations in the RECQL4 gene. RTS is an autosomal recessive disorder characterized by poikiloderma, sparse hair, small stature, skeletal abnormalities, cataracts, and an increased risk of cancer. Mutations in two other RECQ helicases, BLM and WRN, are responsible for the cancer predisposition conditions Bloom and Werner syndromes, respectively. Previous studies have shown that BLM and WRN-deficient cells demonstrate increased sensitivity to hydroxyurea (HU), camptothecin (CPT), and 4-nitroquinoline 1-oxide (4NQO). Little is known about the sensitivity of RECQL4-deficient cells to these and other genotoxic agents. The purpose of this study was to determine if RTS cells display any distinct cellular phenotypes in response to DNA damaging agents or replication blocks that could provide insight into the molecular function of the RECQL4 protein. Our results show that primary fibroblasts from RTS patients carrying two deleterious RECQL4 mutations, compared to wild type (WT) fibroblasts, have increased sensitivity to HU, CPT, and doxorubicin (DOX), modest sensitivity to other DNA damaging agents including ultraviolet (UV) irradiation, ionizing radiation (IR), and cisplatin (CDDP), and relative resistance to 4NQO. The RECQ family of DNA helicases has been implicated in the regulation of DNA replication, recombination, and repair. Because HU, CPT, and DOX exert their effects primarily during S phase, these results support a greater role for the RECQL4 protein in DNA replication as opposed to repair of exogenous damage.