Ceftriaxone exerts antitumor effects in MYCN-driven retinoblastoma and neuroblastoma by targeting DDX3X for translation repression.

MYCN proto-oncogene, bHLH transcription factor (MYCN) amplification is associated with aggressive retinoblastoma (RB) and neuroblastoma (NB) cancer recurrence that is resistant to chemotherapies. Therefore, there is an urgent need to identify new therapeutic tools. This study aimed to evaluate the potential repurposing of ceftriaxone for the treatment of MYCN-amplified RB and NB, based on the clinical observations that the drug was serendipitously found to decrease the volume of the MYCN-driven RB subtype. Using patient-derived tumor organoids and tumor cell lines, we demonstrated that ceftriaxone is a potent and selective growth inhibitor targeting MYCN-driven RB and NB cells. Profiling of drug-induced transcriptomic changes, cell-cycle progression, and apoptotic death indicated cell-cycle arrest and death of drug-treated MYCN-amplified tumor cells. Drug target identification, using an affinity-based proteomic and molecular docking approach, and functional studies of the target proteins revealed that ceftriaxone targeted DEAD-box helicase 3 X-linked (DDX3X), thereby inhibiting translation in MYCN-amplified tumors but not in MYCN-nonamplified cells. The data suggest the feasibility of repurposing ceftriaxone as an anticancer drug and provide insights into the mechanism of drug action, highlighting DDX3X as a potential target for treating MYCN-driven tumors.

Sunitinib efficacy with minimal toxicity in patient-derived retinoblastoma organoids.

BACKGROUND: Recurrence of retinoblastoma (RB) following chemoreduction is common and is often managed with local (intra-arterial/intravitreal) chemotherapy. However, some tumors are resistant to even local administration of maximum feasible drug dosages, or effective tumor control and globe preservation may be achieved at the cost of vision loss due to drug-induced retinal toxicity. The aim of this study was to identify drugs with improved antitumor activity and more favorable retinal toxicity profiles via screening of potentially repurposable FDA-approved drugs in patient-derived tumor organoids. METHODS: Genomic profiling of five RB organoids and the corresponding parental tissues was performed. RB organoids were screened with 133 FDA-approved drugs, and candidate drugs were selected based on cytotoxicity and potency. RNA sequencing was conducted to generate a drug signature from RB organoids, and the effects of drugs on cell cycle progression and proliferative tumor cone restriction were examined. Drug toxicity was assessed with human embryonic stem cell-derived normal retinal organoids. The efficacy/toxicity profiles of candidate drugs were compared with those of drugs in clinical use. RESULTS: RB organoids maintained the genomic features of the parental tumors. Sunitinib was identified as highly cytotoxic against both classical RB1-deficient and novel MYCN-amplified RB organoids and inhibited proliferation while inducing differentiation in RB. Sunitinib was a more effective suppressor of proliferative tumor cones in RB organoids and had lower toxicity in normal retinal organoids than either melphalan or topotecan. CONCLUSION: The efficacy and retinal toxicity profiles of sunitinib suggest that it could potentially be repurposed for local chemotherapy of RB.

Germline RB1 Mutation in Retinoblastoma Patients: Detection Methods and Implication in Tumor Focality.

Purpose: The study aimed to generate a stepwise method to reduce the workload of full-scale RB1 sequencing for germline mutation screening in retinoblastoma (RB) patients. The implication of germline mutation in tumor focality was also determined in this study. Methods: A stepwise method was created on the basis of "hotspot" exons analyzed using data on germline RB1 mutation in the RB1-Leiden Open Variation Database and then tested for mutation screening in the blood DNA of 42 patients with RB. The method was compared with the clinical next-generation sequencing (NGS) panel in terms of sequencing outcomes. The germline RB1 mutation was examined in association with multifocality in RB. Results: Germline RB1 mutation was identified in 61% of all bilateral cases in the first step of the 3 stepwise method and in 78% and 89% for the two and three steps combined, respectively. NGS detected a mosaic variant of RB1 that was not detected by the first two steps and increased the sensitivity from 78% to 83%. Analysis of the relationship between mutation status and tumor focality indicated that multifocality in RB was dependent on germline RB1 mutation, confirming a higher tendency to have a germline RB1 mutation in patients with multifocal RB. Conclusions: A 3 stepwise method reduces the workload needed for sequencing of the RB1 for bilateral cases. NGS outweighs conventional sequencing in terms of the identification of germline mosaic variants. Multifocal tumors in RB may be used to presume germline mutation. Translational Relevance: The presence of "hotspot" exons of germline RB1 mutation in bilateral cases facilitates a mutation screening. However, when genetic testing is not available, multifocality in RB regardless of tumor laterality is predictive of germline RB1 mutation.

Silencing of the Long Noncoding RNA MYCNOS1 Suppresses Activity of MYCN-Amplified Retinoblastoma Without RB1 Mutation.

Purpose: MYCNOS (MYCN opposite strand) is co-amplified with MYCN in pediatric cancers, including retinoblastoma. MYCNOS encodes several RNA variants whose functions have not been elucidated in retinoblastoma. Thus, we attempted to identify MYCNOS variants in retinoblastoma and aimed to decipher the role of MYCNOS variant 1 (MYCNOS1) on the activity of MYCN-amplified retinoblastoma. Methods: The profiles of MYCNOS variants and MYCN status were determined in 17 retinoblastoma tissues, cell lines, retinas, and retinal organoids. A functional study of MYCNOS1 expression was conducted in patient-derived tumor cells and in retinoblastoma cell lines via short hairpin RNA-mediated gene silencing. We carried out MYCN expression, cell viability, cell cycle, apoptosis, soft agar colony formation, and transwell assays to examine the role of MYCNOS1 in MYCN and cell behaviors. We analyzed a transcriptome of MYCN-amplified retinoblastoma cells deficient for MYCNOS1 and, finally, tested the responses of these cells to chemotherapeutic agents. Results: Expression of MYCNOS1 was associated with the expression and copy number of MYCN. Knockdown of MYCNOS1 caused instability of the MYCN protein, leading to cell cycle arrest and impaired proliferation and chemotaxis-directed migration in MYCN-amplified retinoblastoma cells in which RB1 was intact. MYCNOS1 expression was associated with gene signatures of photoreceptor cells and epithelial-mesenchymal transition. MYCNOS1 silencing enhanced the response of retinoblastoma cells to topotecan but not carboplatin. Conclusions: MYCNOS1 supports progression of retinoblastoma. Inhibition of MYCNOS1 expression may be necessary to suppress MYCN activity when treating MYCN-amplified cancers without RB1 mutation.

Outcome of Repeated Use of Donor Site for Noncultured Epidermal Cellular Grafting in Stable Vitiligo: A Retrospective Study.

BACKGROUND: Noncultured epidermal suspension (NCES) is a surgical technique which employs cellular grafting onto depigmented lesions. However, scarring and dyschromia at the donor site often occurs. OBJECTIVE: To assess the outcome of reusing the same donor site in subsequent sessions of NCES procedures. METHODS: Electronic records of vitiligo patients who had undergone two sessions of NCES procedures were retrospectively reviewed. Information on the first and second NCES was retrieved for analyses. RESULTS: A total of 30 patients (female 19 and male 11) were included. The majority of patients had nonsegmental vitiligo (66.7%). The median donor-to-recipient ratios were 1 : 3 (1 : 1-1 : 20) for the first session and 1 : 3 (1 : 1-1 : 13.5) for the second session (p=0.661). The mean melanocyte count was 220.7 ± 65.5 cells/mm2 vs. 242.4 ± 55.3 cells/mm2 on the first and second sessions, respectively (p=0.440). The mean repigmentation rate was 84.2% (±21.1%) and 82.3 (±22.1%) for the first and second NCESs, respectively (p=0.645). The frequency of color mismatch and pigment loss were similar between both sessions (p=0.706 and p=1.000). CONCLUSIONS: Repeated use of donor sites in subsequent NCES sessions gave comparable repigmentation.

A three-dimensional organoid model recapitulates tumorigenic aspects and drug responses of advanced human retinoblastoma.

Persistent or recurrent retinoblastoma (RB) is associated with the presence of vitreous or/and subretinal seeds in advanced RB and represents a major cause of therapeutic failure. This necessitates the development of novel therapies and thus requires a model of advanced RB for testing candidate therapeutics. To this aim, we established and characterized a three-dimensional, self-organizing organoid model derived from chemotherapy-naïve tumors. The responses of organoids to drugs were determined and compared to relate organoid model to advanced RB, in terms of drug sensitivities. We found that organoids had histological features resembling retinal tumors and seeds and retained DNA copy-number alterations as well as gene and protein expression of the parental tissue. Cone signal circuitry (M/L+ cells) and glial tumor microenvironment (GFAP+ cells) were primarily present in organoids. Topotecan alone or the combined drug regimen of topotecan and melphalan effectively targeted proliferative tumor cones (RXRγ+ Ki67+) in organoids after 24-h drug exposure, blocking mitotic entry. In contrast, methotrexate showed the least efficacy against tumor cells. The drug responses of organoids were consistent with those of tumor cells in advanced disease. Patient-derived organoids enable the creation of a faithful model to use in examining novel therapeutics for RB.

Luteinizing Hormone Receptor Gene and Regulator of G-protein Signaling 2 Gene Expression Level and Association with Oocyte Maturity in In vitro Fertilization/Intracytoplasmic Sperm Injection Cycle.

AIMS: The aim is to study the relation and distribution in gene expression level of the luteinizing hormone receptor (LHR) gene and regulator of G-protein signaling 2 (RGS2) gene expression with oocyte maturation. SETTING AND DESIGN: This cross-sectional study was undertaken in an instruction-based tertiary care infertility unit, department of obstetrics and gynecology. MATERIALS AND METHODS: After controlled ovarian hyperstimulation, cumulus granulosa cells (CCs) from 59 oocytes among 18 women being treated by in vitro fertilization/intracytoplasmic sperm injection cycle technique from November 2015 to January 2016 were collected on the day of oocyte retrieval. Total RNA was extracted and converted to cDNA in individual oocytes. LHR and RGS2 gene levels were measured and analyzed using digital droplet polymerase chain reaction. STATISTICAL ANALYSIS: Gene expression level was analyzed using software STATA, version 14.0 (College Station, TX: StataCorp LP, USA). RESULTS: CCs were obtained from 59 cumulus-oocyte complexes (COC), 46 COC from metaphase II (CCMII), 13 COC from metaphase I, and GV oocyte (CCMI + GV). The RGS2 gene expression level, when compared with the housekeeping gene in CCMII and CCMI + GV, was 0.15 (0.05-0.52) and 0.08 (0.02-0.27), respectively. The LHR gene expression when compared with the housekeeping gene in CCMII and CCMI + GV did not differ and was quite in the same value that was 0.02 (0.00-0.11) and 0.02 (0.00-0.06), respectively. CONCLUSION: This study showed that LHR gene expression did not differ in between oocyte groups. Even though the median of RGS2 gene expression was more in the mature oocyte group, the result was inconclusive due to scattering and overlapping of gene expression data between oocyte groups.

Spectrum of germline RB1 mutations and clinical manifestations in retinoblastoma patients from Thailand.

Purpose: Retinoblastoma (RB) is a retinal tumor that most commonly occurs in children. Approximately 40% of RB patients carry germline mutations in the RB1 gene. RB survivors with germline mutations are at increased risk of passing on the disease to future offspring and of secondary cancer in adulthood. This highlights the importance of genetic testing in disease management and counseling. This study aimed to identify germline RB1 mutations and to correlate the mutations with clinical phenotypes of RB patients. Methods: Genomic DNA was extracted from peripheral blood mononuclear cells isolated from 52 RB patients (27 unilaterally and 25 bilaterally affected probands). Mutations in the RB1 gene, including the promoter and exons 1-27 with flanking intronic sequences, were identified by direct sequencing. The samples with negative test results were subjected to multiplex ligation-dependent probe amplification (MLPA) to detect any gross mutations. A correlation of germline RB1 mutations with tumor laterality or age at diagnosis was determined for RB patients. Age at diagnosis was examined in regard to genetic test results and the presence of extraocular tumor extension. Results: Germline RB1 mutations were detected in 60% (31/52) of patients. RB1 mutations were identified in 92% (22/25) of bilateral RB patients, and a high rate of germline RB1 mutations was found in unilateral RB cases (33% or 9/27). Whole gene and exon deletions were reported in five patients. Twenty-three distinct mutations as a result of base substitutions and small deletions were identified in 26 patients; seven mutations were novel. Nonsense and splicing mutations were commonly identified in RB patients. Furthermore, a synonymous mutation was detected in a patient with familial RB; affected mutation carriers in this family exhibited differences in disease severity. The types of germline RB1 mutations were not associated with age at diagnosis or laterality. In addition, patients with positive and negative test results for germline RB1 mutations were similar in age at diagnosis. The incidence of extraocular tumors was high in patients with heritable RB (83% or 5/6), particularly in unilateral cases (33% or 3/9); the mean age at diagnosis of these patients was not different from that of patients with intraocular tumors. Conclusions: This study provides a data set of an RB1 genotypic spectrum of germline mutations and clinical phenotypes and reports the distribution of disease-associated germline mutations in Thai RB patients who attended our center. Our data and the detection methods could assist in identifying a patient with heritable RB, establishing management plans, and informing proper counseling for patients and their families.