Germline Point Mutations Research Articles

The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis

MYCN oncogene amplification is frequently observed in aggressive childhood neuroblastoma. Using an unbiased large-scale mutagenesis screen in neuroblastoma-prone transgenic mice, we identify a single germline point mutation in the transcriptional corepressor Runx1t1, which abolishes MYCN-driven tumorigenesis. This loss-of-function mutation disrupts a highly conserved zinc finger domain within Runx1t1. Deletion of one Runx1t1 allele in an independent Runx1t1 knockout mouse model is also sufficient to prevent MYCN-driven neuroblastoma development, and reverse ganglia hyperplasia, a known pre-requisite for tumorigenesis. Silencing RUNX1T1 in human neuroblastoma cells decreases colony formation in vitro, and inhibits tumor growth in vivo. Moreover, RUNX1T1 knockdown inhibits the viability of PAX3-FOXO1 fusion-driven rhabdomyosarcoma and MYC-driven small cell lung cancer cells. Despite the role of Runx1t1 in MYCN-driven tumorigenesis neither gene directly regulates the other. We show RUNX1T1 forms part of a transcriptional LSD1-CoREST3-HDAC repressive complex recruited by HAND2 to enhancer regions to regulate chromatin accessibility and cell-fate pathway genes.

The Contribution of Mutation to Variation in Temperature-Dependent Sprint Speed in Zebrafish, Danio rerio.

AbstractThe contribution of new mutations to phenotypic variation and the consequences of this variation for individual fitness are fundamental concepts for understanding genetic variation and adaptation. Here, we investigated how mutation influenced variation in a complex trait in zebrafish, Danio rerio. Typical of many ecologically relevant traits in ectotherms, swimming speed in fish is temperature dependent, with evidence of adaptive evolution of thermal performance. We chemically induced novel germline point mutations in males and measured sprint speed in their sons at six temperatures (between 16°C and 34°C). Heterozygous mutational effects on speed were strongly positively correlated among temperatures, resulting in statistical support for only a single axis of mutational variation, reflecting temperature-independent variation in speed (faster-slower mode). These results suggest pleiotropic effects on speed across different temperatures; however, spurious correlations arise via linkage or heterogeneity in mutation number when mutations have consistent directional effects on each trait. Here, mutation did not change mean speed, indicating no directional bias in mutational effects. The results contribute to emerging evidence that mutations may predominantly have synergistic cross-environment effects, in contrast to conditionally neutral or antagonistic effects that underpin thermal adaptation. We discuss several aspects of experimental design that may affect resolution of mutations with nonsynergistic effects.

WP1066 induces cell death in a schwannomatosis patient–derived schwannoma cell line

Schwannomatosis is a rare genetic disorder that predisposes individuals to development of multiple schwannomas mainly in spinal and peripheral nerves and to debilitating chronic pain often unrelated to any schwannoma. Pathogenic variants of two genes, SMARCB1 and LZTR1, are causal in familial cases. However, many schwannomatosis patients lack mutations in these genes. Surgery is the standard treatment for schwannomas but leaves patients with increasing neurological deficits. Pain management is a daily struggle controlled by the use of multiple analgesic and anti-inflammatory drugs. There is a need for both nonsurgical treatment to manage tumor growth and nonaddictive, nonsedative pain control. Because standard clinical trials are exceedingly difficult for patients with rare disorders, precision medicine approaches offer the possibility of bespoke therapeutic regimens to control tumor growth. As a proof of principle, we obtained a bio-specimen of paraspinal schwannoma from a schwannomatosis patient with a germline point mutation in the SMARCB1/INI gene. We created an hTERT immortalized cell line and tested the ability of targeted small molecules with efficacy in neurofibromatosis type 2–related schwannomas to reduce cell viability and induce cell death. We identified WP1066, a STAT3 inhibitor, currently in phase 2 clinical trials for pediatric and adult brain tumors as a lead compound. It reduced cell viability and STAT-3 phosphorylation and induced expression of markers for both necroptosis and caspase-dependent cell death. The results demonstrate feasibility in creating patient-derived cell lines for use in precision medicine studies.

Mutations of the DNA repair gene PNKP in a patient with microcephaly, seizures, and developmental delay (MCSZ) presenting with a high-grade brain tumor

Polynucleotide Kinase-Phosphatase (PNKP) is a bifunctional enzyme that possesses both DNA 3′-phosphatase and DNA 5′-kinase activities, which are required for processing termini of single- and double-strand breaks generated by reactive oxygen species (ROS), ionizing radiation and topoisomerase I poisons. Even though PNKP is central to DNA repair, there have been no reports linking PNKP mutations in a Microcephaly, Seizures, and Developmental Delay (MSCZ) patient to cancer. Here, we characterized the biochemical significance of 2 germ-line point mutations in the PNKP gene of a 3-year old male with MSCZ who presented with a high-grade brain tumor (glioblastoma multiforme) within the cerebellum. Functional and biochemical studies demonstrated these PNKP mutations significantly diminished DNA kinase/phosphatase activities, altered its cellular distribution, caused defective repair of DNA single/double stranded breaks, and were associated with a higher propensity for oncogenic transformation. Our findings indicate that specific PNKP mutations may contribute to tumor initiation within susceptible cells in the CNS by limiting DNA damage repair and increasing rates of spontaneous mutations resulting in pediatric glioma associated driver mutations such as ATRX and TP53.

Fanci-FANCD2 Promotes Genome Stability and DNA Repair By Down-Regulating BLM Helicase Activity

Background: Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure, developmental defects, and higher risk of cancer. Mutations in FA genes have been detected commonly in a large swath of cancers. In the FA DNA repair pathway, DNA damage induces the mono-ubiquitination of the FANCI-FANCD2 (ID2) heterodimer and this regulation licenses the execution of downstream DNA damage signaling and repair steps. In response to replication stress, FANCD2 also prevents replication fork collapse during S phase. Bloom syndrome (BS) is also a genomic instability disease, characterized by growth abnormalities and cancer predisposition. The single BS protein, BLM helicase, participates in DNA repair by promoting DNA end resection and double Holliday junction dissolution. It has been shown that BLM is involved in restart of stalled replication fork. FA and BS have functional interactions. In tumor DNA sequencing of the Yale Precision Tumor board, we identified a somatic 6 amino acid deletion in FANCD2 in a head and neck tumor, while a germline point mutation was found on the other allele. We have identified a FANCD2-L822A mutant with defective BLM binding, which was used to further investigate the role of FANCD2-BLM interaction in genome stability and DNA repair.Methods: Highly purified proteins were used to investigate how ID2 affects helicase and DNA end resection activity of the BLM complex. HeLa, FANCD2-deficient, and FANCD2 corrected fibroblast cell lines were used to examine pRPA2 and RAD51 foci formation. We also used DNA fiber assay to detect end resection and isolation of proteins on nascent DNA (iPOND) assay to examine the RAD51 recruitment on replication fork.Results: A somatic 6 amino acid deletion (p819-824) in FANCD2 was identified in a head and neck tumor. FA-D2 mutant cells expressing the mutant cDNA demonstrated defects in FANCD2 mono-ubiquitination and DNA damage hypersensitivity. A FANCD2-L822A mutant with defective BLM binding was identified (Figure A, B). We found that Bloom helicase and its DNA end resection activity within BLM-DNA2-RPA were negatively regulated by the heterodimer ID2 (Figure C, D). Both DNA and BLM binding of the ID2 are required for the inhibitory function. The premature DNA end resection and HU sensitivity in FANCD2 deficient and mutant cells are rescued by BLM knockdown. By iPOND assay, we discovered that FANCD2 antagonizes BLM to promote RAD51 recruitment on HU-stalled replication fork.Conclusions: Our study suggests that the DNA end resection activity of BLM-DNA2 is tightly regulated by FANCD2 to ensure that the nuclease DNA2 normally resects the DNA intermediate needed for efficient DNA repair and RAD51 recruitment to protect replication forks. Our findings highlight that ID2-BLM interaction functions in DNA damage repair to maintain genome stability. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Impact of DNA Methylation Dynamics on the Mutation Rate During Human Germline Development

DNA methylation is a dynamic epigenetic modification found in most eukaryotic genomes. It is known to lead to a high CpG to TpG mutation rate. However, the relationship between the methylation dynamics in germline development and the germline mutation rate remains unexplored. In this study, we used whole genome bisulfite sequencing (WGBS) data of cells at 13 stages of human germline development and rare variants from the 1000 Genome Project as proxies for germline mutations to investigate the correlation between dynamic methylation levels and germline mutation rates at different scales. At the single-site level, we found a significant correlation between methylation and the germline point mutation rate at CpG sites during germline developmental stages. Then we explored the mutability of methylation dynamics in all stages. Our results also showed a broad correlation between the regional methylation level and the rate of C > T mutation at CpG sites in all genomic regions, especially in intronic regions; a similar link was also seen at all chromosomal levels. Our findings indicate that the dynamic DNA methylome during human germline development has a broader mutational impact than is commonly assumed.

Germline heterozygous mutations in Nxf1 perturb RNA metabolism and trigger thrombocytopenia and lymphopenia in mice

In eukaryotic cells, messenger RNA (mRNA) molecules are exported from the nucleus to the cytoplasm, where they are translated. The highly conserved protein nuclear RNA export factor1 (Nxf1) is an important mediator of this process. Although studies in yeast and in human cell lines have shed light on the biochemical mechanisms of Nxf1 function, its contribution to mammalian physiology is less clear. Several groups have identified recurrent NXF1 mutations in chronic lymphocytic leukemia (CLL), placing it alongside several RNA-metabolism factors (including SF3B1, XPO, RPS15) whose dysregulation is thought to contribute to CLL pathogenesis. We report here an allelic series of germline point mutations in murine Nxf1. Mice heterozygous for these loss-of-function Nxf1 mutations exhibit thrombocytopenia and lymphopenia, together with milder hematological defects. This is primarily caused by cell-intrinsic defects in the survival of platelets and peripheral lymphocytes, which are sensitized to intrinsic apoptosis. In contrast, Nxf1 mutations have almost no effect on red blood cell homeostasis. Comparative transcriptome analysis of platelets, lymphocytes, and erythrocytes from Nxf1-mutant mice shows that, in response to impaired Nxf1 function, the cytoplasmic representation of transcripts encoding regulators of RNA metabolism is altered in a unique, lineage-specific way. Thus, blood cell lineages exhibit differential requirements for Nxf1-mediated global mRNA export.

Patient with confirmed LEOPARD syndrome developing multiple melanoma.

LEOPARD syndrome, also known as Gorlin syndrome II, cardiocutaneous syndrome, lentiginosis profusa syndrome, Moynahan syndrome, was more recently coined as Noonan syndrome with multiple lentigines (NSML), inside the RASopathies. Historically, the acronym LEOPARD refers to the presence of distinctive clinical features such as: lentigines (L), electrocardiographic/conduction abnormalities (E), ocular hypertelorism (O), pulmonary stenosis (P), genital abnormalities (A), retardation of growth (R), and sensorineural deafness (D). This condition is identified in 85% of patients with phenotype hallmarks caused by presence a germline point mutation in PTPN11 gene. Association of melanoma to NSML seems to be rare: to our knowledge, two patients so far were reported in the literature. We herein present a patient diagnosed with LEOPARD syndrome, in whom molecular investigation confirmed the presence of the c.1403C>T mutation in exon 12 of the PTPN11 gene, who developed four superficial spreading melanomas and three atypical lentiginous hyperplasias. Three of the melanomas were achromic or hypochromic, three were in situ, and one had a Breslow index under 0.5 mm. Dermoscopic examination showed some characteristic white structures in most of the lesions, which were a signature pattern and a key for the diagnosis.

Adrenocortical carcinoma characterized by gynecomastia: A case report.

.We present a 4-yr-old boy with adrenocortical carcinoma (ACC), diagnosed due to the appearance of gynecomastia as the presenting symptom. Six months prior to admission, an acute growth spurt along with the development of bilateral breast swelling was observed. He did not present any features of virilization, including enlargement of the testes, increase in testis volume, and penis size. Laboratory investigations showed gonadotropin-independent hypergonadism, with low LH/ FSH levels and elevated estradiol/testosterone levels. Abdominal computed tomography revealed a large heterogeneous mass adjacent to the right kidney and below the liver. Pathological investigations of the biopsy specimen demonstrated that the tumor was an ACC. Pre- and post-operative combination chemotherapy with mitotane was administered and surgical resection was carried out. Post-surgery, the elevated estradiol/testosterone concentrations reverted to within the reference range. Urinary steroid profile and tissue concentration analysis of estradiol and testosterone indicated the presence of estrogen in the ACC tissue. An investigation for TP53 gene aberrations revealed the presence of a germline point mutation in exon 4 (c.215C>G (p.Pro72Arg)). In ACC, the most common symptom is virilization, and feminization, characterized by gynecomastia, is very rare. However, a diagnostic possibility of ACC should be considered when we encounter patients who have developed gynecomastia without the influence of causative factors such as obesity or puberty, and do not present with the typical signs of virilization.

Congenital Adrenocortical Tumor in an Asymptomatic Neonate with Positive Newborn Screen for Congenital Adrenal Hyperplasia: A Case Report

Adrenocortical tumors (ACTs) are rare in childhood, particularly in the neonatal period. ACTs in children usually secrete hormones that can cause virilization, precocious puberty, or Cushing syndrome. However, in many cases the diagnosis is delayed because of the generally healthy appearance of the child and the lack of a palpable abdominal mass. ACTs can also be misdiagnosed as congenital adrenal hyperplasia (CAH) especially if 17-hydroxyprogesterone (17-OHP) is elevated. We describe a case of neonatal ACT diagnosed in an asymptomatic newborn with positive/elevated 17-OHP on newborn screening. On examination there were no signs of virilization, Cushing syndrome, hemihypertrophy, or hypertension, and there was no palpable abdominal mass. While awaiting confirmatory results of an elevated 17-OHP level, an abnormally low adrenocorticotropic hormone level was noted. Abdominal ultrasound, obtained to evaluate the adrenal glands, showed a right adrenal mass with peripheral calcification. MRI revealed a 3.6 x 3.5 x 3.4 cm heterogeneously enhancing ovoid mass in the right adrenal gland. Diagnosis of an ACT was also confirmed by laboratory data, and histopathology of the tumor. Surgical resection was safely performed with perioperative steroid replacement. Genetic analysis of CYP21A2 and CYP11B1 genes was negative. A germ-line point mutation in the TP53 region was discovered, suggesting Li-Fraumeni syndrome. ACTs should be considered in the differential diagnosis of neonates with elevated 17-OHP detected by newborn screening for CAH. Adrenal ultrasound is recommended in cases of significantly abnormal CAH screen results to rule out or treat adrenal neoplasms.

Abstract 3179: Germline deletion of FH in hereditary leiomyomatosis and renal cell carcinoma (HLRCC)

Abstract Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a familial cancer syndrome characterized by germline mutations in the fumarate hydratase (FH) gene that encodes the enzyme responsible for conversion of fumarate to malate in the Kreb's Cycle. HLRCC is associated with the development of cutaneous and uterine leiomyomas, and a highly aggressive form of type 2 papillary kidney cancer. HLRCC renal tumors can be extremely aggressive with patients demonstrating locally advanced or disseminated disease even when associated with small tumors 1-2 cm in size. Therefore, accurate identification of the “at risk” individuals is very important. In our experience, FH point mutations are identified in 90% of individuals that are clinically diagnosed with HLRCC, while in the remainder, FH may be inactivated by deletion or other causes. Several studies have reported complete deletions of the FH gene and a few have estimated the extent of these deletions and identified the additional genes that would be lost in conjunction with FH. Interestingly, families with complete FH deletion have demonstrated cutaneous and uterine leiomyomas but no families have yet been reported with evidence of kidney cancer. A single family has been identified that demonstrates a partial deletion resulting in the loss of exon 1, and in this case kidney cancer has identified within the family. In this study, patients with suspected HLRCC hereditary kidney cancer but no germline point mutation were enrolled on an NCI-IRB approved protocol and an Agilent custom high-definition comparative genomic hybridization (CGH) array was used to identify FH gene deletions. The assay identified complete germline deletion of a single copy of FH within seven suspected HLRCC patients deletion ranging from 249kb to 4.74Mb that in all cases resulted in a minimum addition loss of the KMO, OPN3, CHML, and WDR6 genes. All results were confirmed by CLIA approved tests and a further five new patients were identified by direct CLIA testing, including a novel partial deletion. In contrast to previous reports, seven out of twelve (58.3%) patients/families in our cohort demonstrated a personal or family history of HLRCC associated Type 2 papillary kidney cancer. In conclusion, complete or partial deletion of the FH gene is an infrequent but important cause of HLRCC and has no protective effect on the incidence of HLRCC associated type 2 papillary kidney cancer. Citation Format: Cathy D. Vocke, Christopher J. Ricketts, Lindsay A. Middelton, Youfeng Yang, W. Marston Linehan. Germline deletion of FH in hereditary leiomyomatosis and renal cell carcinoma (HLRCC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3179.

Emerging Therapeutics for Radioiodide-Refractory Thyroid Cancer

Although uncommon, thyroid cancer constitutes the main endocrine neoplasia with an incidence rate that has been increasing steadily over the past decades. Recently, remarkable advances have occurred in understanding the biology of thyroid cancer. Novel germline and somatic point mutations as well as somatic chromosomal rearrangements associated with thyroid carcinogenesis have been discovered. Strikingly, acquired knowledge in the genetics of thyroid cancer has been translated into clinical practice, offering better diagnostic and prognostic accuracy and enabling the development of novel compounds for the treatment of advanced thyroid carcinomas. Even after 70 years, radioiodide therapy remains as the central treatment for advanced or metastatic differentiated thyroid cancer. However, the mechanisms leading to reduced radioiodide accumulation in the tumor cell remain partially understood. Radioiodide-refractory thyroid cancer metastasis constitutes a central problem in the management of thyroid cancer patients. In recent years, the antiangiogenic tyrosine kinase inhibitors sorafenib and lenvatinib have been approved for the treatment of advanced radioiodide-refractory thyroid carcinoma. Moreover, still on clinical phase of study, oncogene-specific and oncogene-activated signaling inhibitors have shown promising effects in recovering radioiodide accumulation in radioiodide-refractory thyroid cancer metastasis. Further clinical trials of these therapeutic agents may soon change the management of thyroid cancer. This review summarizes the latest advances in the understanding of the molecular basis of thyroid cancer, the mechanisms leading to reduced radioiodide accumulation in thyroid tumors and the results of clinical trials assessing emerging therapeutics for radioiodide-refractory thyroid carcinomas in the era of targeted therapies.

Diagnosis and Management of Hereditary Thyroid Cancer.

Thyroid cancers are largely divided into medullary (MTC) and non-medullary (NMTC) cancers , depending on the cell type of origin. Familial non-medullary thyroid cancer (FNMTC) comprises about 5-15% of NMTC and is a heterogeneous group of diseases, including both non-syndromic and syndromic forms. Non-syndromic FNMTC tends to manifest papillary thyroid carcinoma , usually multifocal and bilateral . Several high-penetrance genes for FNMTC have been identified, but they are often confined to a few or single families, and other susceptibility loci appear to play a small part, conferring only small increments in risk. Familial susceptibility is likely to be due to a combination of genetic and environmental influences. The current focus of research in FNMTC is to characterise the susceptibility genes and their role in carcinogenesis. FNMTC can also occur as a part of multitumour genetic syndromes such as familial adenomatous polyposis , Cowden's disease , Werner's syndrome and Carney complex . These tend to present at an early age and are multicentric and bilateral with distinct pathology. The clinical evaluation of these patients is similar to that for most patients with a thyroid nodule. Medullary thyroid cancer (MTC) arises from the parafollicular cells of the thyroid which release calcitonin. The familial form of MTC accounts for 20-25% of cases and presents as a part of the multiple endocrine neoplasia type 2 (MEN 2) syndromes or as a pure familial MTC (FMTC). They are caused by germline point mutations in the RET oncogene on chromosome 10q11.2. There is a clear genotype-phenotype correlation, and the aggressiveness of FMTC depends on the specific genetic mutation, which should determine the timing of surgery.

Loss of IL-4Rα-mediated PI3K signaling accelerates the progression of IgE/mast cell-mediated reactions.

Clinical and experimental evidence indicate that polymorphisms within the interleukin 4 (IL-4) receptor (IL-4R) chain are sufficient for altered strength of IL-4/IL-13 signaling, leading to an exaggerated allergic inflammatory response and increase susceptibility to allergic phenotypes. In the present study, we show that ablation of IL-4Rα-induced phosphatidylinositol 3-kinase (PI3K) activating signal by germline point mutation within the IL-4Rα motif (Y500F) did not alter susceptibility to IgE-mediated, food-induced experimental anaphylaxis. Moreover, diarrhea occurrence, antigen-specific IgE and intestinal mastocytosis were comparable between WT and IL-4Rα(Y500F) mice. However, mice unable to stimulate IL-4Rα-mediated PI3K signaling had accelerated disease progression. Notably, the accelerated anaphylactic response was associated with more rapid histamine-induced hypovolemia. Mechanistic in vitro and in vivo analyses revealed that endothelial IL-4Rα PI3K signaling negatively regulates the histamine-induced endothelial leak response. These results define an unanticipated role for IL-4Rα-mediated PI3K signaling in negative regulation of IgE-mediated anaphylactic reactions.

Abstract 18: Molecular characterization of Brazilian patients suspected for Lynch syndrome

Abstract Lynch syndrome (LS), former known as Hereditary Non Polyposis Colorectal cancer (HNPCC), accounts for 3-5% of all colorectal cancers (CRC) and is inherited in an autossomal dominant fashion. This syndrome is characterized by early CRC onset, high incidence of tumors in the ascending colon, excess of synchronous and metachronous tumors, and accelerated transition adenoma-carcinoma (2-3 years). Nowadays, LS is regarded of patients who carry deleterious germline mutations in one of the five Mismatch repair genes, such as MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), post-meiotic segregation increased 1 (PMS1) or post-meiotic segregation increased 2 (PMS2). In order to characterize Brazilian patients suspect for LS, we assessed the frequency of germline point mutations in MSH6, PMS1 and PMS2 in patients negative for point mutations in MLH1 and MSH2 by capillary sequencing. We also assessed chromosomal deletions in MLH1 and MSH2 and MSH6 generating a complete characterization of MMR genes. The analysis of the five MMR genes revealed 46 carriers of pathogenic mutations, including 25 in MSH2 (54%), 16 in MLH1 (35%), 4 in MSH6 (9%) and one in PMS2 (2%) gene In addition, from the 70 negative patients, we selected one mutation negative family that met the stringent Amsterdam criteria (AC) for whole exome sequencing using the SOLID platform. For this family, we selected 5 close blood relatives, including 3 affected and 2 unaffected individuals in order to facilitate the identification of new susceptibility genes with autosomal dominant pattern. The results revealed two candidate genes, c-KIT and WDR27 that segregated with the disease. Additional analysis are under evaluation. Citation Format: Felipe C. Silva, Giovana T. Torrezan, Márcia CP Figueiredo, Jose Roberto O. Ferreira, Érika M. Santos, Wilson T. Nakagawa, Samuel Aguiar-Junior, Benedito M. Rossi, Fabio O. Ferreira, Dirce M. Carraro. Molecular characterization of Brazilian patients suspected for Lynch syndrome. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 18. doi:10.1158/1538-7445.CANSUSC14-18

A neuron autonomous role for the familial dysautonomia gene ELP1 in sympathetic and sensory target tissue innervation.

Familial dysautonomia (FD) is characterized by severe and progressive sympathetic and sensory neuron loss caused by a highly conserved germline point mutation of the human ELP1/IKBKAP gene. Elp1 is a subunit of the hetero-hexameric transcriptional elongator complex, but how it functions in disease-vulnerable neurons is unknown. Conditional knockout mice were generated to characterize the role of Elp1 in migration, differentiation and survival of migratory neural crest (NC) progenitors that give rise to sympathetic and sensory neurons. Loss of Elp1 in NC progenitors did not impair their migration, proliferation or survival, but there was a significant impact on post-migratory sensory and sympathetic neuron survival and target tissue innervation. Ablation of Elp1 in post-migratory sympathetic neurons caused highly abnormal target tissue innervation that was correlated with abnormal neurite outgrowth/branching and abnormal cellular distribution of soluble tyrosinated α-tubulin in Elp1-deficient primary sympathetic and sensory neurons. These results indicate that neuron loss and physiologic impairment in FD is not a consequence of abnormal neuron progenitor migration, differentiation or survival. Rather, loss of Elp1 leads to neuron death as a consequence of failed target tissue innervation associated with impairments in cytoskeletal regulation.

Involvement of G-quadruplex structures in regulation of human RET gene expression by small molecules in human medullary thyroid carcinoma TT cells

The RET (rearranged during transfection) proto-oncogene encodes a receptor tyrosine kinase for members of the glial cell line-derived neurotrophic factor family of extracellular signaling molecules. The activating germline point mutations in the RET, which are known to induce oncogenic activation of RET tyrosine kinase, are associated with the development of medullary thyroid carcinoma (MTC) and pathogenesis of multiple endocrine neoplasia type 2 (MEN2). The polypurine/polypyrimidine tract in the proximal promoter region of the human RET gene (-51 to -33 relative to transcription start site) is essential for basal transcriptional activity of this gene. This tract consists of a guanine-rich sequence containing five runs of at least three contiguous guanines separated by one or more bases, conforming to a general motif capable of forming an intramolecular G-quadruplex. Here, we show that specific G-quadruplex structures formed in the RET promoter region act to repress the transcription of this gene, and transcription of this gene can be controlled by ligand-mediated G-quadruplex stabilization. In this study, NSC194598, a derivative of indeno[1,2,3-de]quinazoline, was found to be a novel G-quadruplex interactive agent that interfered with transcriptional activation of mutated RET gene in human medullary thyroid carcinoma TT cells. This compound significantly reduced endogenous RET protein levels and increased apoptosis in these cells. Our results provide further support for the idea that G-quadruplex structures may have a critical role in transcriptional regulation of the RET gene in vivo, providing insight into a novel strategy for transcriptional repression of this gene by small molecules.

Ret Oncogene and Thyroid Carcinoma

Thyroid cancer is a malignant neoplasm that originates from follicular or parafollicular thyroid cells and is categorized as papillary (PTC), follicular (FTC), anaplastic (ATC) or medullary thyroid carcinoma (MTC). The alteration of the Rearranged during trasfection (RET) (proto-oncogene, a gene coding for a tyrosine-kinase receptor involved in the control of cell differentiation and proliferation, has been found to cause PTC and MTC. In particular, RET/PTC rearrangements and RET point mutations are related to PTC and MTC, respectively. Although RET/PTC rearrangements have been identified in both spontaneous and radiation-induced PTC, they occur more frequently in radiation-associated tumors. RET/PTC rearrangements have also been reported in follicular adenomas. Although controversial, correlations between RET/PTC rearrangements, especially RET/PTC3, and a more aggressive phenotype and a more advanced stage have been identified. Germline point mutations in the RET proto-oncogene are associated with nearly all cases of hereditary MTC, and a strict correlation between genotype and phenotype has been demonstrated. A large spectrum of RET point mutations has been reported to date. Somatic RET mutations, almost all of which are point mutations, have been described in approximately 40-50% of sporadic MTC cases. Although RET somatic mutations have been found in different codons, the M918T mutation in exon 16 is the most frequent. Detecting germline RET point mutations is a useful tool for identifying subjects who are clinically unaffected but who will develop the disease soon or late in their life, thus allowing presymptomatic treatment. From a clinical point of view, detecting RET somatic mutations in tumoral tissue is able to predict a poor outcome of the disease. In recent years, several drugs capable of inhibiting RET activity have been demonstrated to play an important role in the treatment of radiorefractory advanced thyroid carcinomas.

A germline point mutation in Runx1 uncouples its role in definitive hematopoiesis from differentiation

Definitive hematopoiesis requires the master hematopoietic transcription factor Runx1, which is a frequent target of leukemia-related chromosomal translocations. Several of the translocation-generated fusion proteins retain the DNA binding activity of Runx1, but lose subnuclear targeting and associated transactivation potential. Complete loss of these functions in vivo resembles Runx1 ablation, which causes embryonic lethality. We developed a knock-in mouse that expresses full-length Runx1 with a mutation in the subnuclear targeting cofactor interaction domain, Runx1(HTY350-352AAA). Mutant mice survive to adulthood, and hematopoietic stem cell emergence appears to be unaltered. However, defects are observed in multiple differentiated hematopoietic lineages at stages where Runx1 is known to play key roles. Thus, a germline mutation in Runx1 reveals uncoupling of its functions during developmental hematopoiesis from subsequent differentiation across multiple hematopoietic lineages in the adult. These findings indicate that subnuclear targeting and cofactor interactions with Runx1 are important in many compartments throughout hematopoietic differentiation.

Abstract 745: Role of G-quadruplex structures in the human RET promoter region in the regulation of this gene.

Abstract The medullary thyroid carcinoma (MTC) is a tumor type characterized by germline point mutations of the RET (REarranged during Transfection) proto-oncogene. Since these mutations are known to induce oncogenic activation of RET tyrosine kinase, various therapeutic strategies interfering with the oncogenic function of RET protein have been developed and tested for treating RET-associated cancers in several preclinical and clinical studies. Our previous study has demonstrated that the G-rich strand in the polypurine/polypyrimidine (pPu/pPy) region of the RET proximal promoter is capable of forming intramolecular G-quadruplex structures in vitro in the presence of K+, which can be further stabilized by a G-quadruplex interactive agents. Thus, the detailed mechanism by which G-quadruplex structures regulate RET transcription has been studied using mutation analysis and promoter reporter assays together with a detailed analysis of transcription factor binding to the RET promoter in vivo. To this end, we have explored the Flp-In cell system using homologous recombination, which allows integration of a single copy of a wild type or G-quadruplex-mutant RET promoter–luciferase construct into a specific genomic recombination locus (FRT site) in a host Flp-In the human embryonic kidney 293 (HEK293) cell line. Thus, differences in reporter expression levels from isolated isogenic cell clones can be ascribed more confidently to promoter-specific effects, rather than enhancer/silencer artifacts due to variations in integration sites. Using these isogenic cell lines, we are able to provide strong evidence for G-quadruplex-dependent transcriptional repression of the RET gene in vivo. Citation Format: Daekyu Sun, Yoon-Joo Shin. Role of G-quadruplex structures in the human RET promoter region in the regulation of this gene. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 745. doi:10.1158/1538-7445.AM2013-745