Questions and answers in the management of children with medulloblastoma over the time. How did we get here? A systematic review.

Introduction: Treatment of children with medulloblastoma (MB) includes surgery, radiation therapy (RT) and chemotherapy (CT). Several treatment protocols and clinical trials have been developed over the time to maximize survival and minimize side effects. Methods: We performed a systematic literature search in May 2023 using PubMed. We selected all clinical trials articles and multicenter studies focusing on MB. We excluded studies focusing exclusively on infants, adults, supratentorial PNETs or refractory/relapsed tumors, studies involving different tumors or different types of PNETs without differentiating survival, studies including <10 cases of MB, solely retrospective studies and those without reference to outcome and/or side effects after a defined treatment. Results: 1. The main poor-prognosis factors are: metastatic disease, anaplasia, MYC amplification, age younger than 36 months and some molecular subgroups. The postoperative residual tumor size is controversial.2. MB is a collection of diseases.3. MB is a curable disease at diagnosis, but survival is scarce upon relapse.4. Children should be treated by experienced neurosurgeons and in advanced centers.5. RT is an essential treatment for MB. It should be administered craniospinal, early and without interruptions.6. Craniospinal RT dose could be lowered in some low-risk patients, but these reductions should be done with caution to avoid relapses.7. Irradiation of the tumor area instead of the entire posterior fossa is safe enough.8. Hyperfractionated RT is not superior to conventional RT9. Both photon and proton RT are effective.10. CT increases survival, especially in high-risk patients.11. There are multiple drugs effective in MB. The combination of different drugs is appropriate management.12. CT should be administered after RT.13. The specific benefit of concomitant CT to RT is unknown.14. Intensified CT with stem cell rescue has no benefit compared to standard CT regimens.15. The efficacy of intraventricular/intrathecal CT is controversial.16. We should start to think about incorporating targeted therapies in front-line treatment.17. Survivors of MB still have significant side effects. Conclusion: Survival rates of MB improved greatly from 1940-1970, but since then the improvement has been smaller. We should consider introducing targeted therapy as front-line therapy.

A synthetic mRNA cell reprogramming method using CYCLIN D1 promotes DNA repair, generating improved genetically stable human induced pluripotent stem cells.

A key challenge for clinical application of induced pluripotent stem cells (iPSC) to accurately model and treat human pathologies depends on developing a method to generate genetically stable cells to reduce long-term risks of cell transplant therapy. Here, we hypothesized that CYCLIN D1 repairs DNA by highly efficient homologous recombination (HR) during reprogramming to iPSC that reduces genetic instability and threat of neoplastic growth. We adopted a synthetic mRNA transfection method using clinically compatible conditions with CYCLIN D1 plus base factors (OCT3/4, SOX2, KLF4, LIN28) and compared with methods that use C-MYC. We demonstrate that CYCLIN D1 made iPSC have (a) lower multitelomeric signal, (b) reduced double-strand DNA breaks, (c) correct nuclear localization of RAD51 protein expression, and (d) reduced single-nucleotide polymorphism (SNP) changes per chromosome, compared with the classical reprogramming method using C-MYC. CYCLIN D1 iPSC have reduced teratoma Ki67 cell growth kinetics and derived neural stem cells successfully engraft in a hostile spinal cord injury (SCI) microenvironment with efficient survival, differentiation. We demonstrate that CYCLIN D1 promotes double-stranded DNA damage repair predominantly through HR during cell reprogramming to efficiently produce iPSC. CYCLIN D1 reduces general cell stress associated with significantly lower SIRT1 gene expression and can rescue Sirt1 null mouse cell reprogramming. In conclusion, we show synthetic mRNA transfection of CYCLIN D1 repairs DNA during reprogramming resulting in significantly improved genetically stable footprint in human iPSC, enabling a new cell reprogramming method for more accurate and reliable generation of human iPSC for disease modeling and future clinical applications.

Generation of mice with longer and better preserved telomeres in the absence of genetic manipulations.

Although telomere length is genetically determined, mouse embryonic stem (ES) cells with telomeres of twice the normal size have been generated. Here, we use such ES cells with 'hyper-long' telomeres, which also express green fluorescent protein (GFP), to generate chimaeric mice containing cells with both hyper-long and normal telomeres. We show that chimaeric mice contain GFP-positive cells in all mouse tissues, display normal tissue histology and normal survival. Both hyper-long and normal telomeres shorten with age, but GFP-positive cells retain longer telomeres as mice age. Chimaeric mice with hyper-long telomeres also accumulate fewer cells with short telomeres and less DNA damage with age, and express lower levels of p53. In highly renewing compartments, such as the blood, cells with hyper-long telomeres are longitudinally maintained or enriched with age. We further show that wound-healing rates in the skin are increased in chimaeric mice. Our work demonstrates that mice with functional, longer and better preserved telomeres can be generated without the need for genetic manipulations, such as TERT overexpression.

ATRX driver mutation in a composite malignant pheochromocytoma.

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are tumors arising from the adrenal medulla and sympathetic/parasympathetic paraganglia, respectively. Approximately 40% of PCCs/PGLs are due to germline mutations in one of 16 susceptibility genes, and a further 30% are due to somatic alterations in 5 main genes. Recently, somatic ATRX mutations have been found in succinate dehydrogenase (SDH)-associated hereditary PCCs/PGLs. In the present study we applied whole-exome sequencing to the germline and tumor DNA of a patient with metastatic composite PCC and no alterations in known PCC/PGL susceptibility genes. A somatic loss-of-function mutation affecting ATRX was identified in tumor DNA. Transcriptional profiling analysis classified the tumor within cluster 2 of PCCs/PGLs (without SDH gene mutations) and identified downregulation of genes involved in neuronal development and homeostasis (NLGN4, CD99 and CSF2RA) as well as upregulation of Drosha, an important gene involved in miRNA and rRNA processing. CpG island methylator phenotype typical of SDH gene-mutated tumors was ruled out, and SNP array data revealed a unique profile of gains and losses. Finally, we demonstrated the presence of alternative lengthening of telomeres in the tumor, probably associated with the failure of ATRX functions. In conclusion, somatic variants affecting ATRX may play a driver role in sporadic PCC/PGL.

Localization-dependent and -independent roles of SLX4 in regulating telomeres.

SLX4, a scaffold for structure-specific DNA repair nucleases, is important for several types of DNA repair. Many repair proteins bind to sites of DNA damage, resulting in subnuclear "foci," but SLX4 forms foci in human cells even without DNA damage. Using several approaches, we show that most, but not all, SLX4 foci localize to telomeres in a range of human cell lines irrespective of the mechanisms used to maintain telomere length. The SLX1 Holliday-junction-processing enzyme is recruited to telomeres by SLX4, and SLX4, in turn, is recruited by a motif that binds to the shelterin subunit TRF2 directly. We also show that TRF2-dependent recruitment of SLX4 prevents telomere damage. Furthermore, SLX4 prevents telomere lengthening and fragility in a manner that appears to be independent of telomere association. These findings reveal that SLX4 plays multiple roles in regulating telomere homeostasis.

Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer.

A major goal in aging research is to improve health during aging. In the case of mice, genetic manipulations that shorten or lengthen telomeres result, respectively, in decreased or increased longevity. Based on this, we have tested the effects of a telomerase gene therapy in adult (1 year of age) and old (2 years of age) mice. Treatment of 1- and 2-year old mice with an adeno associated virus (AAV) of wide tropism expressing mouse TERT had remarkable beneficial effects on health and fitness, including insulin sensitivity, osteoporosis, neuromuscular coordination and several molecular biomarkers of aging. Importantly, telomerase-treated mice did not develop more cancer than their control littermates, suggesting that the known tumorigenic activity of telomerase is severely decreased when expressed in adult or old organisms using AAV vectors. Finally, telomerase-treated mice, both at 1-year and at 2-year of age, had an increase in median lifespan of 24 and 13%, respectively. These beneficial effects were not observed with a catalytically inactive TERT, demonstrating that they require telomerase activity. Together, these results constitute a proof-of-principle of a role of TERT in delaying physiological aging and extending longevity in normal mice through a telomerase-based treatment, and demonstrate the feasibility of anti-aging gene therapy.

Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites.

Rap1 is a component of the shelterin complex at mammalian telomeres, but its in vivo role in telomere biology has remained largely unknown to date. Here we show that Rap1 deficiency is dispensable for telomere capping but leads to increased telomere recombination and fragility. We generated cells and mice deleted for Rap1; mice with Rap1 deletion in stratified epithelia were viable but had shorter telomeres and developed skin hyperpigmentation in adulthood. By performing chromatin immunoprecipitation coupled with ultrahigh-throughput sequencing, we found that Rap1 binds to both telomeres and to extratelomeric sites through the (TTAGGG)(2) consensus motif. Extratelomeric Rap1-binding sites were enriched at subtelomeric regions, in agreement with preferential deregulation of subtelomeric genes in Rap1-deficient cells. More than 70% of extratelomeric Rap1-binding sites were in the vicinity of genes, and 31% of the genes deregulated in Rap1-null cells contained Rap1-binding sites, suggesting a role for Rap1 in transcriptional control. These findings place a telomere protein at the interface between telomere function and transcriptional regulation.

TPP1 is required for TERT recruitment, telomere elongation during nuclear reprogramming, and normal skin development in mice.

The TPP1/ACD protein (hereafter TPP1) is a component of the shelterin complex at mammalian telomeres. Here we find that Tpp1-deficient mouse embryonic fibroblasts (MEFs) show increased chromosomal instability including sister chromatid fusions and chromosomes with multitelomeric signals related to telomere fragility. Tpp1 deletion decreases both TERT (the telomerase catalytic subunit) binding to telomeres in MEFs and telomerase function at chromosome ends in vivo. Abrogation of Tpp1 abolished net telomere elongation in the context of nuclear reprogramming of MEFs into induced pluripotent stem cells, whereas Tpp1 deletion in stratified epithelia of Tpp1(Delta/Delta)K5-Cre mice resulted in perinatal death, severe skin hyperpigmentation, and impaired hair follicle morphogenesis. p53 deficiency rescues skin hyperpigmentation and hair growth in these mice, indicating that p53 restricts proliferation of Tpp1-deficient cells. These results suggest a telomere-capping model where TPP1 protects telomere integrity and regulates telomerase recruitment to telomeres, thereby preventing early occurrence of degenerative pathologies.

ATR suppresses telomere fragility and recombination but is dispensable for elongation of short telomeres by telomerase.

Telomere shortening caused by incomplete DNA replication is balanced by telomerase-mediated telomere extension, with evidence indicating that the shortest telomeres are preferred substrates in primary cells. Critically short telomeres are detected by the cellular DNA damage response (DDR) system. In budding yeast, the important DDR kinase Tel1 (homologue of ATM [ataxia telangiectasia mutated]) is vital for telomerase recruitment to short telomeres, but mammalian ATM is dispensable for this function. We asked whether closely related ATR (ATM and Rad3 related) kinase, which is important for preventing replicative stress and chromosomal breakage at common fragile sites, might instead fulfill this role. The newly created ATR-deficient Seckel mouse strain was used to examine the function of ATR in telomerase recruitment and telomere function. Telomeres were recently found to resemble fragile sites, and we show in this study that ATR has an important role in the suppression of telomere fragility and recombination. We also find that wild-type ATR levels are important to protect short telomeres from chromosomal fusions but do not appear essential for telomerase recruitment to short telomeres in primary mouse embryonic fibroblasts from the ATR-deficient Seckel mouse model. These results reveal a previously unnoticed role for mammalian ATR in telomere protection and stability.

Zidovudine induces S-phase arrest and cell cycle gene expression changes in human cells.

Antiretroviral therapy for the human immunodeficiency virus-1 (HIV-1) typically includes two nucleoside reverse transcriptase inhibitors (NRTIs). 3'-Azido-3'-deoxythymidine (AZT, Zidovudine) plus 2'-deoxy-3'-thiacytidine (3TC, Lamivudine) is a combination that is used frequently. The NRTIs are mutagenic nucleoside analogs that become incorporated into DNA and terminate replication. We therefore hypothesized that exposure to this class of drug may alter cell cycle parameters. We used flow cytometry to examine the cell cycle in human epithelioid carcinoma (HeLa) cells exposed to AZT and 3TC alone, as well as a series of AZT/3TC dose combinations: (A) 125.0 microM AZT/12.5 microM 3TC; (B) 250.0 microM AZT/25.0 microM 3TC; and (C) 500 microM AZT/50 microM 3TC. At 24 h, at all doses, there was a good cell viability (>/=68%), and incorporation of AZT into nuclear DNA. Using flow cytometry, a dose-related increase in the percentage of cells in S phase, from 9.5% with no drug, to 36.0% with dose C, was observed in cells exposed for 24 h (P = 0.001, ANOVA). A concomitant decrease in the percentage of cells in G(1) phase, from 82.6% with no drug to 58.5% with dose C, was observed in cells exposed for 24 h (P = 0.017, ANOVA). A similar S phase arrest was seen in cells exposed to 125, 250 and 500 microM AZT alone, but there was no S phase alteration with 50 microM 3TC alone, suggesting that AZT is responsible for the accumulation of cells in S phase. To elucidate the accumulation of cells in S phase and explore the cell cycle gene expression changes induced by AZT and 3TC, we used c-DNA microarray, Cell Cycle Super Array and real-time PCR. There was a strong upregulation of the DNA damage-inducible transcript 3 (DDIT3 or GADD153) in NRTI-exposed cells. In addition, AZT induced an upregulation of cyclin D1 accompanied by a downregulation of the cyclin D1-associated inhibitors P18 and P57, and the G(1)-S check point gene P21, the net effect of which would be to foster a cell progression into S phase. Cyclin A2 was down-regulated in cells exposed to AZT, suggesting a block in S-G(2)-M progression that would also be consistent with the accumulation of cells in S phase. Overall, the study demonstrates that AZT, but not 3TC, causes an arrest of cells in S phase with a consistent alteration in the expression of several cell cycle genes.

Porphyrin derivatives for telomere binding and telomerase inhibition.

The capacity of G-quadruplex ligands to stabilize four-stranded DNA makes them able to inhibit telomerase, which is involved in tumour cell proliferation. A series of cationic metalloporphyrin derivatives was prepared by making variations on a meso-tetrakis(4-N-methyl-pyridiniumyl)porphyrin skeleton (TMPyP). The DNA binding properties of nickel(II) and manganese(III) porphyrins were studied by surface plasmon resonance, and the capacity of the nickel porphyrins to inhibit telomerase was tested in a TRAP assay. The nature of the metal influences the kinetics (the process is faster for Ni than for Mn) and the mode of interaction (stacking or external binding). The chemical alterations did not lead to increased telomerase inhibition. The best selectivity for G-quadruplex DNA was observed for Mn-TMPyP, which has a tenfold preference for quadruplex over duplex.

Desmopressin inhibits lung and lymph node metastasis in a mouse mammary carcinoma model of surgical manipulation.

BACKGROUND AND OBJECTIVES: Desmopressin (DDAVP) is a synthetic derivative of vasopressin with hemostatic and fibrinolytic properties that has been used during surgery in patients with bleeding disorders. Our aim was to investigate the effect of DDAVP on lung and lymph node metastatic cell colonization using a preclinical mouse mammary carcinoma model of subcutaneous tumor manipulation and surgical excision. METHODS: Female BALB/c mice bearing the highly aggressive F3II mammary carcinoma were subjected to repeated manipulations of primary tumors (0.5 kg/cm(2) during 2 min), followed (or not) by surgical excision. DDAVP was administered intravenously 30 min before and 24 h after each manipulation or surgery, at a dose of 2 microg/kg. At the end of the experiment, mice were sacrificed and necropsied. RESULTS: Tumor manipulation induced dissemination to the axillary nodes and increased up to 6-fold the number of metastatic lung nodules. Perioperative treatment with DDAVP dramatically reduced regional metastasis. The incidence of lymph node involvement in manipulated animals was 12% with DDAVP and 87% without treatment (P < 0.02). Histopathological analysis of axillary nodes from DDAVP-treated animals showed sinusal histiocytosis and no evidence of cancer cells. Metastatic lung nodules were also reduced about 65% in animals treated with DDAVP (P = 0.026). CONCLUSIONS: Our results suggest a potential clinical application of DDAVP in the management of breast cancer, as well as other aggressive solid tumors. DDAVP may be useful to reduce the risk of metastatic cell colonization both during and after surgical manipulation.