Genomic imprinting refers to the parent-of-origin specific monoallelic expression of a gene. Imprinted genes are often clustered in the genome and their expression is regulated by an imprinting centre (IC). ICs are regions of DNA that propagate the parental specific regulation of gene expression, which are usually characterized by differential DNA methylation, histone marks and the presence of non-coding RNAs. Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with the dysregulation of imprinted gene expression on human chromosome band 11p15.5. The 11p15.5 imprinted region has two imprinting centres, IC1 and IC2. IC1 is telomeric and regulates the imprinted expression of the genes H19 and IGF2. IC2 is ~700kb centromeric and is associated with a cluster of nine imprinted genes including CDKN1C, KCNQ1 and an imprinted non-coding RNA associated with IC2, KCNQ1OT1. Loss of differential DNA methylation at IC2 is seen in 50% of patients with BWS with loss of imprint of the non-coding RNA KCNQ1OT1 and associated with a decreased expression of the putative tumour suppressor CDKN1C. Patients with BWS also have a thousand-fold increased risk of pediatric cancer. The focus of this thesis involves investigation of dysregulation of imprinting in three groups of BWS patients. Firstly, I show that BWS patients with alveolar rhabdomyosarcoma have constitutional loss of methylation at IC2 and biallelic expression of KCNQ1OT1. Secondly, loss of methylation at IC2 has been previously associated with female monozygotic twins discordant for BWS. In male monozygotic twins with BWS, however, the molecular lesions reflect the molecular heterogeneity seen in BWS singletons. Thirdly, BWS patients associated with translocations and inversions that have breakpoints within the KCNQ1 gene near IC2 show regional gain of DNA methylation around the breakpoint and decreased expression of CDKN1C. Therefore, using a rare collection of BWS patients, I have attempted to determine the various roles of the imprinting centres IC1 and IC2 and their involvement in tumourigenesis, monozygotic twinning and structural chromosomal rearrangements causing BWS.

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Apoptosis is the mechanism by which complex multicellular organisms induce the programmed death of damaged cells, thus maintaining tissue homeostasis. One of the main hallmarks of cancer, apoptosis is tightly regulated by pro- and anti-apoptotic factors whose equilibrium will decide of the fate of the cell. Among these factors, the cellular inhibitor of apoptosis cIAP1 is a key regulator of nuclear factor-κB dependent signaling and of caspase-8 mediated apoptosis. cIAP1 expression is controlled primarily at the translational level through an internal ribosome entry site (IRES) that facilitates the recruitment of the ribosome to the translation initiation start independently of the 5’ cap. We have previously identified four putative IRES trans-acting factors (ITAFs) that bind specifically to the cIAP1 IRES, namely NF45, NF90, IGF2BP1 and RH1. My research project characterised NF45 as an ITAF that positively regulates the IRES-mediated translation of cIAP1 and of the Xlinked inhibitor of apoptosis, XIAP. This regulation is important for maintaining Survivin and Cyclin E protein levels and insuring proper cell division. Furthermore, I showed that IGF2BP1 is another ITAF that is overexpressed in rhabdomyosarcoma cancer (RMS) and positively regulates cIAP1 translation, thus leading to apoptotic resistance in these cells. Importantly, the use of Smac mimetics, chemical compounds that cause cIAP1 proteasomal degradation, induces TNFα-mediated apoptosis of RMS cells and leads to growth inhibition of RMS xenograft tumors as well as significantly improved survival. Finally, I show that certain modulators of innate immunity synergize with Smac mimetics to improve the killing of RMS cancer cells. Hence, cIAP1 translation regulation by NF45 and IGF2BP1 is highly important for maintaining proper functioning of the cell and dysregulation of these ITAFs can lead to carcinogenesis.

Over the past decade several converging lines of evidence have highlighted the importance of post-transcriptional events in skeletal muscle. This level of regulation is controlled by multi-functional RNA-binding proteins and trans-acting factors. In fact, several RNA-binding proteins are implicated in neuromuscular disorders including myotonic dystrophy type I, spinal muscular atrophy and amyotrophic lateral sclerosis. Therefore, it is necessary to examine the impact of RNA-binding proteins during skeletal muscle development and plasticity in order to understand the consequences linked to their misregulation in disease. Here, we focused on the RNA-binding protein Staufen1, which assumes multiple roles in both skeletal muscle and neurons. We previously demonstrated that Staufen1 is regulated during myogenic differentiation and that its expression is increased in denervated and in myotonic dystrophy type I skeletal muscles. The increased expression of Staufen1 initially appeared beneficial for DM1 since further elevating Staufen1 levels rescued key hallmarks of the disease. However, based on the multi-functional nature of Staufen1, we hypothesized that Staufen1 acts as a disease modifier in DM1. To test this, we investigated the roles of Staufen1 in skeletal muscle biology and their implications for disease. Our data demonstrated that Staufen1 is required during the early stages of muscle development, however its expression must remain low in postnatal skeletal muscle. Interestingly, the overexpression of Staufen1 impaired myogenesis through the regulation of c-myc translation. Since the function of c-myc in oncogenesis is well described, we investigated the role of Staufen1 in cancer biology. In particular, we determined novel functions of Staufen1 in rhabdomyosarcoma tumorigenesis, thus providing the first direct evidence for Staufen1’s involvement in cancer. Moreover, based on Staufen1’s role in myogenic differentiation and in myotonic dystrophy type I, we generated muscle-specific transgenic mice to examine the impact of sustained Staufen1 expression in postnatal skeletal muscle. Staufen1 transgenic mice developed a myopathy characterized by histological and functional abnormalities via atrogene induction and the regulation of PTEN mRNAs. In parallel, we further investigated Staufen1-regulated alternative splicing and our data demonstrated that Staufen1 regulates multiple alternative splicing events in normal and myotonic dystrophy type I skeletal muscles, both beneficial and detrimental for the pathology. Collectively, these findings uncover several novel functions of Staufen1 in skeletal muscle biology and highlight Staufen1’s role as a disease modifier in DM1.

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Rhabdomyosarcoma (RMS) is a type of soft tissue sarcoma that mainly occurs in children. RMS can be divided into two subtypes embryonal (ERMS) and alveolar (ARMS). The ARMS subtype can be especially aggressive when a balanced chromosomal translocation is present. This translocation results in the expression of a PAX3/7-FOXO1 fusion protein, an oncogenic transcription factor. PAX3-FOXO1 positive RMS has an especially bad prognosis and survival rate. In the cell painting assay relevant organelles are stained and morphological features are extracted on a cellular level. Based on these features, morphological profiles of each cell type and a similarity score can be calculated. The morphological profiles of ERMS cell lines RD and RD18 as well as the ARMS cell lines RH30 and CW9019 were obtained. RD and RD18 are most similar to each other followed by RH30. CW9019 has a very different profile from the other cell lines. Since ERMS is associated with a better survival rate a drug that reprograms the phenotype from ARMS to a more ERMS-like phenotype might sensitize the cells to the standard treatment of RMS. ARMS patients might then benefit from a combination therapy of such a drug and the standard treatment. To find such a drug a drug screen was conducted. Drugs were selected for the screen that either target fusion-protein stability or overexpressed targets of the fusion protein. Phenotypic reference compounds were included to get a first idea of the mechanisms and involved organelles of the screened drugs. In total, 30 compounds and 9 phenotypic reference compounds were screened for changing morphological profiles of RMS cell lines with the cell painting assay. 15 compounds were identified that change the phenotype of the ARMS cell line RH30. In addition, 7 of these compounds shift the phenotype of RH30 towards an ERMS-like phenotype. If that morphological resemblance of RH30 cells to ERMS cells translates into a change of a more ERMS-like behavior and sensitizes the cells for the standard treatment of RMS remains to be investigated. The reprogramming hits showed high similarity with phenotypic reference compounds that increase nucleus size which might suggest changed behavior of the fusion protein. Especially, Bosutinib, Midostaurin and Alisertib are promising new compounds for ARMS treatment. They shifted the ARMS phenotype towards an ERMS-like phenotype in the drug screen. This shift is likely a result of the interaction with PLK1 or Aurora-kinase A that are shown to have an influence on fusion-protein stability.

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No description available.

Rhabdomyosarcomas (RMS) are the most common type of soft tissue neoplasm in children. They typically arise from primitive skeletal muscle and are usually observed in either the head and neck or the genitourinary region. We report a rare case of an embryonal rhabdomyosarcoma lung malignancy and a formulated histopathological process to differentiate these lesions from its main differentials. We aim to bring attention to this case in an effort to assist physicians in making the correct diagnosis, should they be presented with a similar case. Here we present a case of a 56-year-old female with a past medical history of COPD and a 30-pack-year smoking history who was referred to a Regional Cancer Center clinic due to newly diagnosed lung cancer. The patient presented with anorexia, weight loss, cough, dyspnea for two months, and a 30-pound weight loss over the last month. CTA of the chest on 7/26/2021 revealed a 14 cm confluent mass extending throughout the mediastinum and the left hilum with associated narrowing of the left-sided pulmonary arteries and bronchi consistent with neoplasia. Several cavitary nodules in the left lung are present, consistent with metastatic disease. CT-guided biopsy on 8/2/2021 revealed a high-grade neoplasm with neuroendocrine features, and frequent mitotic figures, and tumor necrosis. Immunohistochemical staining was positive for synaptophysin, and CD56. Negative stains include cytokeratin TTF-1, AE1/AE3, CAM 5.2, CK 34 beta E12, CK5/6, CK7, CK20, p40, mart 1, SOX10 and CD45. The patient was then diagnosed with pulmonary embryonal rhabdomyosarcoma. In this case report the patient was diagnosed with pulmonary embryonal rhabdomyosarcoma, which characteristically has cells that show variable degrees of skeletal muscle differentiation with spindled morphology and differentiated rhabdomyoblasts. Desmin, myoD1, and myogenin are the key immunohistochemical stains that should be utilized to confirm the suspected diagnosis of a RMS. The staining pattern varies between different RMS subtypes, as the pulmonary subtype staining pattern is more focal compared to the others which tend to stain diffusely. CD56 staining can also be used to identify an alveolar RMS, however, is nonspecific. In this patient's case, CD56 was positive but the FISH analysis confirmed embryonal RMS as the final diagnosis. Previous studies have indicated that RMS can metastasize to the lung, thus this strain can prove to be a useful tool in rare cases such as this one, where the etiology of the cancer is unclear but has progressed to the lung. This unique case report highlights the diagnostic approach and aims to provide a differential diagnosis for a pulmonary embryonal rhabdomyosarcoma as well as an effective workup. Future research into the origin of the pulmonary embryonal rhabdomyosarcoma is indicated to provide comprehensive treatment for the patient and further understand the pathophysiology of the disease discussed.