ZCCHC17 Modulates Neuronal RNA Splicing and Supports Cognitive Resilience in Alzheimer's Disease.

ZCCHC17 is a putative master regulator of synaptic gene dysfunction in Alzheimer's disease (AD), and ZCCHC17 protein declines early in AD brain tissue, before significant gliosis or neuronal loss. Here, we investigate the function of ZCCHC17 and its role in AD pathogenesis using data from human autopsy tissue (consisting of males and females) and female human cell lines. Co-immunoprecipitation (co-IP) of ZCCHC17 followed by mass spectrometry analysis in human iPSC-derived neurons reveals that ZCCHC17's binding partners are enriched for RNA-splicing proteins. ZCCHC17 knockdown results in widespread RNA-splicing changes that significantly overlap with splicing changes found in AD brain tissue, with synaptic genes commonly affected. ZCCHC17 expression correlates with cognitive resilience in AD patients, and we uncover an APOE4-dependent negative correlation of ZCCHC17 expression with tangle burden. Furthermore, a majority of ZCCHC17 interactors also co-IP with known tau interactors, and we find a significant overlap between alternatively spliced genes in ZCCHC17 knockdown and tau overexpression neurons. These results demonstrate ZCCHC17's role in neuronal RNA processing and its interaction with pathology and cognitive resilience in AD, and suggest that the maintenance of ZCCHC17 function may be a therapeutic strategy for preserving cognitive function in the setting of AD pathology.

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.

Characterizing relationships between T-cell inflammation and outcomes in patients with high-risk neuroblastoma according to mesenchymal and adrenergic signatures.

Recent insights have identified adrenergic (ADRN) and mesenchymal (MES) cell lineages as distinct biologic cell types and T-cell inflammation as a prognostic marker in neuroblastoma. We hypothesized that elucidating unique and overlapping aspects of these biologic features could serve as novel biomarkers for informing ongoing efforts to improve therapeutic approaches for children with high-risk neuroblastoma. We identified lineage-specific, single-stranded super-enhancers to define ADRN and MES specific genes. Publicly available RNA-seq of diagnostic tumor biopsies was used in Discovery and Validation cohorts. Each tumor was assigned a relative MES score and T-cell-inflamed (TCI) score. Survival was assessed using the Kaplan-Meier method, and differences were assessed by the log-rank test. Inflammation scores were correlated with MES scores and anticorrelated with MYCN-amplification in both cohorts. Among patients with high-risk, ADRN tumors, those with TCI tumors had superior overall survival to those with non-inflamed tumors. A similar, but non-significant, trend was observed in the Validation cohort. Conversely, there was no difference according to TCI status in the MES cohort in either the Discover or Validation Cohorts. High inflammation scores were correlated with improved survival in some patients with high-risk, ADRN but not MES neuroblastoma. Our findings bolster support for further developing T-cell based immunotherapy-based approaches for children with high-risk neuroblastoma of varying MES and ADRN expression.

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.

T-cell inflammation is prognostic of survival in patients with high-risk neuroblastoma enriched for an adrenergic signature.

Purpose: T-cell inflammation (TCI) has been shown to be a prognostic marker in neuroblastoma, a tumor comprised of cells that can exist in two epigenetic states, adrenergic (ADRN) and mesenchymal (MES). We hypothesized that elucidating unique and overlapping aspects of these biologic features could serve as novel biomarkers. Patients and Methods: We detected lineage-specific, single-stranded super-enhancers defining ADRN and MES specific genes. Publicly available neuroblastoma RNA-seq data from GSE49711 (Cohort 1) and TARGET (Cohort 2) were assigned MES, ADRN, and TCI scores. Tumors were characterized as MES (top 33%) or ADRN (bottom 33%), and TCI (top 67% TCI score) or non-inflamed (bottom 33% TCI score). Overall survival (OS) was assessed using the Kaplan-Meier method, and differences were assessed by the log-rank test. Results: We identified 159 MES genes and 373 ADRN genes. TCI scores were correlated with MES scores (R=0.56, p<0.001 and R=0.38, p<0.001) and anticorrelated with MYCN -amplification (R=-0.29, p<0.001 and -0.18, p=0.03) in both cohorts. Among Cohort 1 patients with high-risk, ADRN tumors (n=59), those with TCI tumors (n=22) had superior OS to those with non-inflammed tumors (n=37) (p=0.01), though this comparison did not reach significance in Cohort 2. TCI status was not associated with survival in patients with high-risk MES tumors in either cohort. Conclusions: High inflammation scores were correlated with improved survival in some high-risk patients with, ADRN but not MES neuroblastoma. These findings have implications for approaches to treating high-risk neuroblastoma.

ZCCHC17 modulates neuronal RNA splicing and supports cognitive resilience in Alzheimer's disease.

ZCCHC17 is a putative master regulator of synaptic gene dysfunction in Alzheimer's Disease (AD), and ZCCHC17 protein declines early in AD brain tissue, before significant gliosis or neuronal loss. Here, we investigate the function of ZCCHC17 and its role in AD pathogenesis. Co-immunoprecipitation of ZCCHC17 followed by mass spectrometry analysis in human iPSC-derived neurons reveals that ZCCHC17's binding partners are enriched for RNA splicing proteins. ZCCHC17 knockdown results in widespread RNA splicing changes that significantly overlap with splicing changes found in AD brain tissue, with synaptic genes commonly affected. ZCCHC17 expression correlates with cognitive resilience in AD patients, and we uncover an APOE4 dependent negative correlation of ZCCHC17 expression with tangle burden. Furthermore, a majority of ZCCHC17 interactors also co-IP with known tau interactors, and we find significant overlap between alternatively spliced genes in ZCCHC17 knockdown and tau overexpression neurons. These results demonstrate ZCCHC17's role in neuronal RNA processing and its interaction with pathology and cognitive resilience in AD, and suggest that maintenance of ZCCHC17 function may be a therapeutic strategy for preserving cognitive function in the setting of AD pathology.