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.

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.

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.

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.

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.

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.

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.

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.

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.

Generation of an induced pluripotent stem cell line from an individual with a heterozygous RECQL4 mutation.

The DNA helicase RECQL4 is known for its roles in DNA replication and repair. RECQL4 mutations cause several genetic disorders including Rothmund-Thomson syndrome (RTS), characterized by developmental defects and predisposition to osteosarcoma. Here we reprogrammed fibroblasts with a heterozygous RECQL4 mutation (c.1878 + 32_1878 + 55del24) to induced pluripotent stem cells (iPSCs). These iPSCs are pluripotent and are able to be differentiated into all three germ layers, providing a novel tool to further interrogate the role of RECQL4 DNA helicase in vitro.

Osteosarcoma: Molecular Pathogenesis and iPSC Modeling.

Rare hereditary disorders provide unequivocal evidence of the importance of genes in human disease pathogenesis. Familial syndromes that predispose to osteosarcomagenesis are invaluable in understanding the underlying genetics of this malignancy. Recently, patient-derived induced pluripotent stem cells (iPSCs) have been successfully utilized to model Li-Fraumeni syndrome (LFS)-associated bone malignancy, demonstrating that iPSCs can serve as an in vitro disease model to elucidate osteosarcoma etiology. We provide here an overview of osteosarcoma predisposition syndromes and review recently established iPSC disease models for these familial syndromes. Merging molecular information gathered from these models with the current knowledge of osteosarcoma biology will help us to gain a deeper understanding of the pathological mechanisms underlying osteosarcomagenesis and will potentially aid in the development of future patient therapies.

Recommendations for Childhood Cancer Screening and Surveillance in DNA Repair Disorders.

DNA repair syndromes are heterogeneous disorders caused by pathogenic variants in genes encoding proteins key in DNA replication and/or the cellular response to DNA damage. The majority of these syndromes are inherited in an autosomal-recessive manner, but autosomal-dominant and X-linked recessive disorders also exist. The clinical features of patients with DNA repair syndromes are highly varied and dependent on the underlying genetic cause. Notably, all patients have elevated risks of syndrome-associated cancers, and many of these cancers present in childhood. Although it is clear that the risk of cancer is increased, there are limited data defining the true incidence of cancer and almost no evidence-based approaches to cancer surveillance in patients with DNA repair disorders. This article is the product of the October 2016 AACR Childhood Cancer Predisposition Workshop, which brought together experts from around the world to discuss and develop cancer surveillance guidelines for children with cancer-prone disorders. Herein, we focus on the more common of the rare DNA repair disorders: ataxia telangiectasia, Bloom syndrome, Fanconi anemia, dyskeratosis congenita, Nijmegen breakage syndrome, Rothmund-Thomson syndrome, and Xeroderma pigmentosum. Dedicated syndrome registries and a combination of basic science and clinical research have led to important insights into the underlying biology of these disorders. Given the rarity of these disorders, it is recommended that centralized centers of excellence be involved directly or through consultation in caring for patients with heritable DNA repair syndromes. Clin Cancer Res; 23(11); e23-e31. ©2017 AACRSee all articles in the online-only CCR Pediatric Oncology Series.

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.

Aging in Rothmund-Thomson syndrome and related RECQL4 genetic disorders.

Rothmund-Thomson Syndrome (RTS) is a rare autosomal recessive disease which manifests several clinical features of accelerated aging. These findings include atrophic skin and pigment changes, alopecia, osteopenia, cataracts, and an increased incidence of cancer for patients carrying RECQL4 germline mutations. Mutations in RECQL4 are responsible for the majority of cases of RTS. RECQL4 belongs to RECQ DNA helicase family which has been shown to participate in many aspects of DNA metabolism. In the past several years, accumulated evidence indicates that RECQL4 is important not only in cancer development but also in the aging process. In this review, based on recent research data, we summarize the common aging findings in RTS patients and propose possible mechanisms to explain the aging features in these patients.

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.

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.

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.

Human Epidermal Growth Factor Receptor 2 (HER2) -Specific Chimeric Antigen Receptor-Modified T Cells for the Immunotherapy of HER2-Positive Sarcoma.

PURPOSE: The outcome for patients with metastatic or recurrent sarcoma remains poor. Adoptive therapy with tumor-directed T cells is an attractive therapeutic option but has never been evaluated in sarcoma. PATIENTS AND METHODS: We conducted a phase I/II clinical study in which patients with recurrent/refractory human epidermal growth factor receptor 2 (HER2) -positive sarcoma received escalating doses (1 × 10(4)/m(2) to 1 × 10(8)/m(2)) of T cells expressing an HER2-specific chimeric antigen receptor with a CD28.ζ signaling domain (HER2-CAR T cells). RESULTS: We enrolled 19 patients with HER2-positive tumors (16 osteosarcomas, one Ewing sarcoma, one primitive neuroectodermal tumor, and one desmoplastic small round cell tumor). HER2-CAR T-cell infusions were well tolerated with no dose-limiting toxicity. At dose level 3 (1 × 10(5)/m(2)) and above, we detected HER2-CAR T cells 3 hours after infusion by quantitative polymerase chain reaction in 14 of 16 patients. HER2-CAR T cells persisted for at least 6 weeks in seven of the nine evaluable patients who received greater than 1 × 10(6)/m(2) HER2-CAR T cells (P = .005). HER2-CAR T cells were detected at tumor sites of two of two patients examined. Of 17 evaluable patients, four had stable disease for 12 weeks to 14 months. Three of these patients had their tumor removed, with one showing ≥ 90% necrosis. The median overall survival of all 19 infused patients was 10.3 months (range, 5.1 to 29.1 months). CONCLUSION: This first evaluation of the safety and efficacy of HER2-CAR T cells in patients with cancer shows the cells can persist for 6 weeks without evident toxicities, setting the stage for studies that combine HER2-CAR T cells with other immunomodulatory approaches to enhance their expansion and persistence.

RECQL4 Regulates p53 Function In Vivo During Skeletogenesis.

RECQ DNA helicases play critical roles in maintaining genomic stability, but their role in development has been less well studied. Rothmund-Thomson syndrome, RAPADILINO, and Baller-Gerold syndrome are rare genetic disorders caused by mutations in the RECQL4 gene. These patients have significant skeletal developmental abnormalities including radial ray, limb and craniofacial defects. To investigate the role of Recql4 in the developing skeletal system, we generated Recql4 conditional knockout mice targeting the skeletal lineage. Inactivation of Recql4 using the Prx1-Cre transgene led to limb abnormalities and craniosynostosis mimicking the major bone findings in human RECQL4 patients. These Prx1-Cre(+) ;Recql4(fl/fl) mice as well as Col2a1-Cre(+) ;Recql4(fl/fl) mice exhibited growth plate defects and an increased p53 response in affected tissues. Inactivation of Trp53 in these Recql4 mutants resulted in genetic rescue of the skeletal phenotypes, indicating an in vivo interaction between Recql4 and Trp53, and p53 activation as an underlying mechanism for the developmental bone abnormalities in RECQL4 disorders. Our findings show that RECQL4 is critical for skeletal development by modulating p53 activity in vivo.