Small-molecule inhibition of the METTL3/METTL14 complex suppresses neuroblastoma tumor growth and promotes differentiation.

The N6-methyladenosine (m6A) RNA modification is an important regulator of gene expression. m6A is deposited by a methyltransferase complex that includes methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14). High levels of METTL3/METTL14 drive the growth of many types of adult cancer, and METTL3/METTL14 inhibitors are emerging as new anticancer agents. However, little is known about the m6A epitranscriptome or the role of the METTL3/METTL14 complex in neuroblastoma, a common pediatric cancer. Here, we show that METTL3 knockdown or pharmacologic inhibition with the small molecule STM2457 leads to reduced neuroblastoma cell proliferation and increased differentiation. These changes in neuroblastoma phenotype are associated with decreased m6A deposition on transcripts involved in nervous system development and neuronal differentiation, with increased stability of target mRNAs. In preclinical studies, STM2457 treatment suppresses the growth of neuroblastoma tumors in vivo. Together, these results support the potential of METTL3/METTL14 complex inhibition as a therapeutic strategy against neuroblastoma.

5-Hydroxymethylcytosine Profiling of Cell-Free DNA Identifies Bivalent Genes That Are Prognostic of Survival in High-Risk Neuroblastoma.

PURPOSE: Neuroblastoma is the most common extracranial solid tumor in childhood. We previously showed that circulating cell-free DNA (cfDNA) and tumor biopsy derived 5-hydroxymethylcytosime (5-hmC) profiles identified patients with neuroblastoma who experienced subsequent relapse. Here, we hypothesized that 5-hmC modifications selectively enriched in cfDNA compared with tumor biopsy samples would identify epigenetic changes associated with aggressive tumor behavior and identify novel biomarkers of outcome in patients with high-risk neuroblastoma. METHODS: 5-hmC profiles from cfDNA (n = 64) and tumor biopsies (n = 48) were compared. Two neuroblastoma cell lines underwent chromatin immunoprecipitation followed by sequencing (ChIP-Seq) for H3K27me3, H3K4me3, and H3K27ac; kethoxal-associated single-stranded DNA sequencing; hmC-Seal for 5-hmC; and RNA-sequencing (RNA-Seq). Genes enriched for both H3K27me3 and H3K4me3 in the included cell lines were defined as bivalent. Using bivalent genes defined in vitro, a bivalent signature was established in three publicly available cohorts of patients with neuroblastoma through gene set variation analysis. Differences between tumors with high or low bivalent signatures were assessed by the Kaplan-Meier method and Cox proportional hazards models. RESULTS: In cfDNA compared with tumor biopsy derived 5-hmC profiles, we found increased 5-hmC deposition on Polycomb Repressive Complex 2 target genes, a finding previously described in the context of bivalent genes. We identified 313 genes that bore bivalent chromatin marks, were enriched for mediators of neuronal differentiation, and were transcriptionally repressed across a panel of heterogeneous neuroblastoma cell lines. In three distinct clinical cohorts, low bivalent signature was significantly and independently associated with worse clinical outcome in patients with high-risk neuroblastoma. CONCLUSION: Low expression of bivalent genes is a biomarker of worse outcome in patients with high-risk neuroblastoma.

Adrenergic and mesenchymal signatures are identifiable in cell-free DNA and correlate with metastatic disease burden in children with neuroblastoma.

BACKGROUND: Cell-free DNA (cfDNA) profiles of 5-hydroxymethylcytosine (5-hmC), an epigenetic marker of open chromatin and active gene expression, are correlated with metastatic disease burden in patients with neuroblastoma. Neuroblastoma tumors are comprised of adrenergic (ADRN) and mesenchymal (MES) cells, and the relative abundance of each in tumor biopsies has prognostic implications. We hypothesized that ADRN and MES-specific signatures could be quantified in cfDNA 5-hmC profiles and would augment the detection of metastatic burden in patients with neuroblastoma. METHODS: We previously performed an integrative analysis to identify ADRN and MES-specific genes (n = 373 and n = 159, respectively). Purified DNA from cell lines was serial diluted with healthy donor cfDNA. Using Gene Set Variation Analysis (GSVA), ADRN and MES signatures were optimized. We then quantified signature scores, and our prior neuroblastoma signature, in cfDNA from 84 samples from 46 high-risk patients including 21 patients with serial samples. RESULTS: Samples from patients with higher metastatic burden had increased GSVA scores for both ADRN and MES gene signatures (p < .001). While ADRN and MES signature scores tracked together in serially collected samples, we identified instances of patients with increases in either MES or ADRN score at relapse. CONCLUSIONS: While it is feasible to identify ADRN and MES signatures using 5-hmC profiles of cfDNA from neuroblastoma patients and correlate these signatures to metastatic burden, additional data are needed to determine the optimal strategies for clinical implementation. Prospective evaluation in larger cohorts is ongoing.

Adrenergic and mesenchymal signatures are identifiable in cell-free DNA and correlate with metastatic disease burden in children with neuroblastoma.

Background: Cell free DNA (cfDNA) profiles of 5-hydroxymethylcytosine (5-hmC), an epigenetic marker of open chromatin and active gene expression, are correlated with metastatic disease burden in patients with neuroblastoma. Neuroblastoma tumors are comprised of adrenergic (ADRN) and mesenchymal (MES) cells, and the relative abundance of each in tumor biopsies has prognostic implications. We hypothesized that ADRN and MES specific signatures could be quantified in cfDNA 5-hmC profiles and would augment the detection of metastatic burden in patients with neuroblastoma. Methods: We previously performed an integrative analysis to identify ADRN and MES specific genes (n=373 and n=159, respectively). Purified DNA from cell lines was serial diluted with healthy donor cfDNA. Using Gene Set Variation Analysis (GSVA), ADRN and MES signatures were optimized. We then quantified signature scores, and our prior neuroblastoma signature, in cfDNA from 84 samples from 46 high-risk patients including 21 patients with serial samples. Results: Samples from patients with higher metastatic burden had increased GSVA scores for both ADRN and MES gene signatures (p < 0.001). While ADRN and MES signature scores tracked together in serially collected samples, we identified instances of patients with increases in either MES or ADRN score at relapse. Conclusions: While it is feasible to identify ADRN and MES signatures using 5-hmC profiles of cfDNA from neuroblastoma patients and correlate these signatures to metastatic burden, additional data are needed to determine the optimal strategies for clinical implementation. Prospective evaluation in larger cohorts is ongoing.

5-hydroxymethylcytosine profiling of cell-free DNA identifies bivalent genes that are prognostic of survival in high-risk neuroblastoma.

Neuroblastoma is the most common extra-cranial solid tumor in childhood and epigenetic dysregulation is a key driver of this embryonal disease. In cell-free DNA from neuroblastoma patients with high-risk disease, we found increased 5-hydroxymethylcytosine (5-hmC) deposition on Polycomb Repressive Complex 2 (PRC2) target genes, a finding previously described in the context of bivalent genes. As bivalent genes, defined as genes bearing both activating (H3K4me3) and repressive (H3K27me3) chromatin modifications, have been shown to play an important role in development and cancer, we investigated the potential role of bivalent genes in maintaining a de-differentiated state in neuroblastoma and their potential use as a biomarker. We identified 313 genes that bore bivalent chromatin marks, were enriched for mediators of neuronal differentiation, and were transcriptionally repressed across a panel of heterogenous neuroblastoma cell lines. Through gene set variance analysis, we developed a clinically implementable bivalent signature. In three distinct clinical cohorts, low bivalent signature was significantly and independently associated with worse clinical outcome in high-risk neuroblastoma patients. Thus, low expression of bivalent genes is a biomarker of ultra-high-risk disease and may represent a therapeutic opportunity in neuroblastoma.

Persistence of Racial and Ethnic Disparities in Risk and Survival for Patients with Neuroblastoma over Two Decades.

BACKGROUND: Racial/ethnic survival disparities in neuroblastoma were first reported more than a decade ago. We sought to investigate if these disparities have persisted with current era therapy. METHODS: Two patient cohorts were identified in the International Neuroblastoma Risk Group Data Commons (INRGdc) (Cohort 1: diagnosed 2001-2009, n=4359; Cohort 2: diagnosed 2010-2019, n=4891). Chi-squared tests were used to assess the relationship between race/ethnicity and clinical and biologic features. Survival was estimated by the Kaplan-Meier method. Cox proportional hazards regression analyses were performed to investigate the association between racial/ethnic groups and prognostic markers. RESULTS: Significantly higher 5-year event-free survival (EFS) and overall survival (OS) were observed for Cohort 2 compared to Cohort 1 (P<0.001 and P<0.001, respectively). Compared to White patients, Black patients in both cohorts had a higher proportion of high-risk disease (Cohort 1: P<0.001; Cohort 2: P<0.001) and worse EFS (Cohort 1: P<0.001; Cohort 2 P<0.001) and OS (Cohort 1: P<0.001; Cohort 2: P<0.001). In Cohort 1, Native Americans also had a higher proportion of high-risk disease (P=0.03) and inferior EFS/OS. No significant survival disparities were observed for low- or intermediate-risk patients in either cohort or high-risk patients in Cohort 1. Hispanic patients with high-risk disease in Cohort 2 had significantly inferior OS (P=0.047). Significantly worse OS, but not EFS, (P=0.006 and P=0.02, respectively) was also observed among Black and Hispanic patients assigned to receive post-Consolidation dinutuximab on clinical trials (n=885). CONCLUSION: Racial/ethnic survival disparities have persisted over time and were observed among high-risk patients assigned to receive post-Consolidation dinutuximab.

Uptake and Distribution of Administered Bone Marrow Mesenchymal Stem Cell Extracellular Vesicles in Retina.

Cell replacement therapy using mesenchymal (MSC) and other stem cells has been evaluated for diabetic retinopathy and glaucoma. This approach has significant limitations, including few cells integrated, aberrant growth, and surgical complications. Mesenchymal Stem Cell Exosomes/Extracellular Vesicles (MSC EVs), which include exosomes and microvesicles, are an emerging alternative, promoting immunomodulation, repair, and regeneration by mediating MSC's paracrine effects. For the clinical translation of EV therapy, it is important to determine the cellular destination and time course of EV uptake in the retina following administration. Here, we tested the cellular fate of EVs using in vivo rat retinas, ex vivo retinal explant, and primary retinal cells. Intravitreally administered fluorescent EVs were rapidly cleared from the vitreous. Retinal ganglion cells (RGCs) had maximal EV fluorescence at 14 days post administration, and microglia at 7 days. Both in vivo and in the explant model, most EVs were no deeper than the inner nuclear layer. Retinal astrocytes, microglia, and mixed neurons in vitro endocytosed EVs in a dose-dependent manner. Thus, our results indicate that intravitreal EVs are suited for the treatment of retinal diseases affecting the inner retina. Modification of the EV surface should be considered for maintaining EVs in the vitreous for prolonged delivery.

Systematic review and meta-analysis of Roux-en-Y gastric bypass against laparoscopic sleeve gastrectomy for amelioration of NAFLD using four criteria.

Nonalcoholic fatty liver disease (NAFLD) prevalence is rising worldwide, as a direct consequence of the obesity epidemic. Bariatric surgery provides proven NAFLD amelioration, although questions remain regarding whether Roux-en-Y gastric bypass (RYGB) or laparoscopic sleeve gastrectomy (LSG) is more effective. To answer this question, we conducted a systematic review and meta-analysis exclusively comparing RYGB and LSG for amelioration of NAFLD using 4 separate criteria: alanine transaminase, aspartate transaminase, NAFLD activity score, and NAFLD fibrosis score. Our search included 1290 initial studies, which were narrowed to 20 final studies in the meta-analysis. Overall, both RYGB and LSG significantly improved alanine transaminase, aspartate transaminase, NAFLD activity score, and NAFLD fibrosis score postoperatively. Direct comparisons of RYGB to LSG in any of the 4 criteria failed to demonstrate superiority. Our findings corroborate the current literature showing that bariatric surgery significantly improves biochemical and histologic parameters in patients with NAFLD. The novel individual comparisons of 4 criteria failed to show superiority between RYGB and LSG in ameliorating NAFLD. Despite several limitations, our study can assist clinicians by supporting the notion that RYGB and LSG may be equally efficacious in ameliorating NAFLD.

Mesenchymal stem cell-derived extracellular vesicles and retinal ischemia-reperfusion.

Retinal ischemia is a major cause of vision loss and impairment and a common underlying mechanism associated with diseases such as glaucoma, diabetic retinopathy, and central retinal artery occlusion. The regenerative capacity of the diseased human retina is limited. Our previous studies have shown the neuroprotective effects of intravitreal injection of mesenchymal stem cells (MSC) and MSC-conditioned medium in retinal ischemia in rats. Based upon the hypothesis that the neuroprotective effects of MSCs and conditioned medium are largely mediated by extracellular vesicles (EVs), MSC derived EVs were tested in an in-vitro oxygen-glucose deprivation (OGD) model of retinal ischemia. Treatment of R28 retinal cells with MSC-derived EVs significantly reduced cell death and attenuated loss of cell proliferation. Mechanistic studies on the mode of EV endocytosis by retinal cells were performed in vitro. EV endocytosis was dose- and temperature-dependent, saturable, and occurred via cell surface heparin sulfate proteoglycans mediated by the caveolar endocytic pathway. The administration of MSC-EVs into the vitreous humor 24 h after retinal ischemia in a rat model significantly enhanced functional recovery, and decreased neuro-inflammation and apoptosis. EVs were taken up by retinal neurons, retinal ganglion cells, and microglia. They were present in the vitreous humor for four weeks after intravitreal administration, with saturable binding to vitreous humor components. Overall, this study highlights the potential of MSC-EV as biomaterials for neuroprotective and regenerative therapy in retinal disorders.