Disruption of the complex processes underlying central nervous system development leads to a broad spectrum of brain malformations and neurodevelopmental disorders, often with a genetic cause. Here, we report bi-allelic pathogenic variants in fibronectin type III and SPRY domain-containing 1-like (FSD1L), encoding a protein of unknown function, in eleven individuals, including five fetuses from six unrelated families. The phenotype ranges from severe hydrocephalus, corpus callosum agenesis, and absent pyramid decussation to a neurodevelopmental syndrome characterized by severe intellectual disability, spastic tetraparesis, reduced vision, and epilepsy, associated with corpus callosum agenesis/hypoplasia, mild ventricular dilation, optic nerve hypoplasia, and white matter reduction. This phenotype closely resembles that observed in L1 syndrome, caused by pathogenic variants in L1CAM, encoding a neural adhesion molecule. The knockdown of Fsd1l in mouse embryos recapitulated the ventricular dilation observed in affected fetuses. Immunohistochemical studies in human control fetuses revealed that FSD1L localized to neurons with commissural fate and projection neurons during human development. Induced pluripotent stem cell (iPSC)-derived neural progenitor cells from affected individuals failed to differentiate into premature neurons and to properly form neurospheres while undergoing increased cell death. In neural progenitors, FSD1L localized with microtubules of the mitotic spindle during M phase and to the transition zone and along the axoneme of the primary cilium during interphase. In line with this, fibroblasts from affected individuals exhibited marked alterations of the mitotic spindle and reduced ciliogenesis and ciliary length compared to control cells. Our findings define FSD1L as a microtubule-associated protein implicated in neuronal differentiation, axon guidance, and fasciculation.

Amyotrophic lateral sclerosis (ALS) is characterised by the aggregation of TDP-43 and mutant FUS in the cytoplasm of affected motor neurons. Accumulation of DNA damage is emerging as a novel correlative trait of ALS. We recently showed that formation of TDP-43 and FUS cytoplasmic inclusions (CIs) lead to DNA damage accumulation through dysregulation of the DNA damage response (DDR). However, the multiple molecular mechanisms contributing to DNA damage accumulation in affected motor neurons in ALS have not been fully elucidated. In recent years, chemical inhibition of the serine/threonine kinase CHK1 was shown to lead to accumulation of DNA breaks as well as increased apoptosis, in differentiated cortical neurons. Notably, CHK1 has been involved in DNA double-strand break repair in non-dividing cells, by acting through the histone chaperone ASF1A. In this article, we show that cells bearing FUS and TDP-43 CIs show downregulation of the protein levels of CHK1 and ASF1A. We observe CHK1 protein downregulation in neuronal cell lines, as well as in patient-derived motor neurons progenitors and in the spinal cord of a FUS-ALS mouse model. Restoration of the nuclear levels of CHK1 and ASF1A via transient overexpression, is sufficient to reduce DNA damage signal accumulation and rescues DDR defects. Importantly, we show that the ubiquitin-proteasome pathway is responsible for CHK1 degradation in cells bearing FUS CI, since its inhibition restores CHK1 and ASF1A protein levels. Our study demonstrates that proteasomal-dependent CHK1 and ASF1A downregulation contributes to accumulation of DNA damage in cells affected by ALS-linked protein aggregates.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, with chronic metabolic disorders increasing risk and severity. Prolonged exposure to altered metabolism changes specific metabolite levels, impacting epigenetic landscape contributing neoplastic lesion acquisition. This study examines the interplay between metabolism and epigenetics in dysmetabolic-driven PDAC tumorigenesis, exploiting LSL-KrasG12D;PDX-1-Cre mice (KC mice) exposed to high-fat diet (HFD) and KRAS-mutated human pancreatic ductal epithelial (HPDE) cells. Untargeted metabolomics of HFD-fed KC pancreata reveals altered free fatty acid and elevated S-adenosyl methionine levels during tumorigenesis. Targeted metabolomics shows increased succinate alongside reduced α-ketoglutarate levels. This imbalance suggests an epigenetic derangement, targeting DNA methylation. In KRAS-mutated HPDE cells exposed to altered metabolism, the DNA demethylation complex of ten-to-eleven-translocation methylcytosine 1 and thymine DNA glycosylase (TDG) is disrupted, leading to iterative cytosine modification and apurinic/apyrimidinic (AP) site accumulation. Succinate directly binds TDG at arginine 275, hyperactivating it and increasing AP site formation. This alteration combined with the methylation-prone metabolic environment, impairs the base excision repair pathway by hypermethylating and downmodulating DNA ligases LIG1 and LIG3. This predisposes to genomic instability and pancreatic preneoplastic lesion development. These findings uncover a metabolic-epigenetic axis in dysmetabolic PDAC, highlighting how metabolite-driven epigenetic changes compromise DNA repair and drive tumorigenesis.

Supplementary Figure S8 shows migration assays of MYC-CaP cells treated with lactate or LDHA inhibitor FX11.

Excel file containing 19 supplementary tables

R-loops are three-stranded RNA-DNA hybrid structures that play important regulatory roles, but excessive or deregulated R-loops formation can trigger DNA damage and genome instability. Digestion of R-loops is mainly relying on the action of two specialized ribonucleases: RNaseH1 and RNaseH2. RNaseH2 is the main enzyme carrying out the removal of misincorporated rNMPs during DNA replication or repair, through the Ribonucleotide Excision Repair (RER) pathway. We have recently shown that the human RNA helicase DDX3X possessed RNaseH2-like activity, being able to substitute RNaseH2 in reconstituted RER reactions. Here, using synthetic R-loop mimicking substrates, we could show that human DDX3X alone was able to both displace and degrade the ssRNA strand hybridized to DNA. Moreover, DDX3X was found to physically interact with human RNaseH2. Such interaction suppressed the nuclease and helicase activities of DDX3X, but stimulated severalfold the catalytic activity of the trimeric RNaseH2, but not of RNaseH1. Finally, silencing of DDX3X in human cells caused accumulation of RNA-DNA hybrids and phosphorylated RPA foci. These results support a role of DDX3X as a scaffolding protein and auxiliary factor for RNaseH2 during R-loop degradation.

Supplementary Figure S1 shows the systemic effects of HFD.

Supplementary Figure S7 shows the output of mouse-based deconvolution models ImmuCC and mMCP.

Supplementary Figure S6 includes scRNA-seq data showing that Luminal (high Ly6d) cells in MYC-transformed DLP display glycolytic features.

Histone deacetylases (HDACs) are crucial regulators of gene expression, DNA synthesis, and cellular processes, making them essential targets in cancer research. HDAC6, specifically, influences protein stability and chromatin dynamics. Despite HDAC6’s potential therapeutic value, its exact role in gene regulation and chromatin remodeling needs further clarification. This study examines how HDAC6 inactivation influences lysine acetyltransferase P300 stabilization and subsequent effects on chromatin structure and function in cancer cells. We employed the HDAC6 inhibitor ITF3756, siRNA, or CRISPR/Cas9 gene editing to inactivate HDAC6 in different epigenomic backgrounds. Constantly, this inactivation led to significant changes in chromatin accessibility, particularly increased acetylation of histone H3 lysines 9, 14, and 27 (ATAC-seq and H3K27Ac ChIP-seq analysis). Transcriptomics, proteomics, and gene ontology analysis revealed gene changes in cell proliferation, adhesion, migration, and apoptosis. Significantly, HDAC6 inactivation altered P300 ubiquitination, stabilizing P300 and leading to downregulating genes critical for cancer cell survival. Our study highlights the substantial impact of HDAC6 inactivation on the chromatin landscape of cancer cells and suggests a role for P300 in contributing to the anticancer effects. The stabilization of P300 with HDAC6 inhibition proposes a potential shift in therapeutic focus from HDAC6 itself to its interaction with P300. This finding opens new avenues for developing targeted cancer therapies, improving our understanding of epigenetic mechanisms in cancer cells.

Supplementary Figure S10 shows the output of human-based deconvolution model QuanTIseq.

Supplementary Figure S9 shows GSEA_Hallmark results in human PCa

Supplementary references for Materials and Methods

Abstract Background: Histone deacetylases (HDACs) play a pivotal role in gene regulation, DNA synthesis, and cellular metabolism, significantly influencing cancer development. Class IIb histone deacetylase 6 (HDAC6) is crucial in maintaining protein stability and regulating chromatin dynamics, with notable implications in cancer and immune responses. Despite this, the specific effects of HDAC6 inactivation on gene regulation and chromatin remodeling remain unclear due to the subtle phenotype changes following its genetic deactivation. This study delves into how inactivating HDAC6 affects the stabilization of lysine acetyltransferase P300 and the subsequent impacts on chromatin structure and function in cancer cells. Methods & Results: Using the HDAC6 inhibitor ITF3756 and CRISPR/Cas9 gene editing, we deactivated HDAC6 in various cancer cell lines. We observed profound changes in chromatin accessibility, particularly in the acetylation of histone H3 lysines 9, 14, and 27, which accumulated into introns and distal intergenic regions as determined through ATAC-seq and H3K27Ac ChIP-seq analyses. Transcriptomics, proteomics, and gene ontology analysis revealed alterations in gene function linked to cell proliferation, adhesion, migration, and apoptosis. A notable finding was the modification of P300 ubiquitination post HDAC6 inactivation, which increased P300 expression and activity, resulting in the downregulation of genes essential for cellular proliferation and survival. Conclusions: This research underscores the significant effect of HDAC6 inactivation on the chromatin landscape in cancer cells, shedding light on the crucial role of P300 in the HDAC6-mediated anticancer response. Specifically, the role of P300 stabilization in the effectiveness of an HDAC6 inhibitor suggests for a potential shift in focus from HDAC6 to its interaction with P300. This insight could contribute to developing more precise cancer treatments by targeting this specific pathway, thereby enhancing our knowledge of cancer cell behavior and offering new therapeutic perspectives.

Supplementary Figure S3 shows a graphical representation of key metabolic pathways altered by MYC alone or by obesogenic HFD in WT and MYC-transformed DLP.

Abstract Introduction: Histone deacetylases (HDACs)are crucial in regulating gene expression, DNA synthesis, and metabolic processes, including cancer, in various cellular environments. The unique structural and functional attributes of class IIb HDAC6 position it as a key player in influencing protein stability and chromatin dynamics. While its role in cancer progression and immunomodulation is well-recognized, the in-depth mechanisms following HDAC6 inactivation, particularly its influence on chromatin remodeling and gene regulation, are not thoroughly understood. This knowledge gap represents a critical area of investigation, given the potential of HDAC6 as a therapeutic target in oncology. This study aims to elucidate HDAC6 inactivation-dependent epigenetic effects of, exploring the possibility to exploit these latter for therapeutic purposes in cancer. This aim includes an examination of the stabilization of the lysine acetyltransferase P300 and its implications for chromatin structure and function. Methods & Results: In this study, ITF3756 and CRISPR/Cas9 were employed to inactivate HDAC6 across a variety of cancer cell lines (Jurkat, MDA 231, HCC1806, 4T1, and B16F10 melanoma). ATAC-seq and H3K27Ac ChIP-seq were used to investigate changes in chromatin accessibility, revealing significant alterations, particularly in introns and distal intergenic regions. Gene Ontology (GO) analysis further supported this alteration in the chromatin landscape, which indicated a differential expression of genes involved in critical cellular processes like cell proliferation, adhesion, migration, and apoptosis. A notable outcome of this study was the stabilization of P300 following ITF3756 treatment, an effect that was paralleled by changes in the expression of critical genes, including AKT1, ITGB3, GAS6, SOX9, NF1, TGFB2, and CASP7. These genes are integral to cellular survival mechanisms, and their modulation emphasizes the role of HDAC6 in cancer cell physiology. Conclusions: This research shows that the chemical or genetic inactivation of HDAC6 impacts cancer cell proliferation and chromatin architecture, underscoring the potential of HDAC6 as a target in cancer therapeutics.. A critical finding of this study is the stabilization of P300, which offers new perspectives on HDAC6's functionality and its role as an epigenetic modifier in cancer cells. This study contributes to the field by suggesting therapeutic strategies focusing on HDAC/HAT pharmacological targeting, thereby advancing our understanding of the role of HDAC6 in regulating cancer cell behavior and epigenetic landscape. Citation Format: Michela Gottardi Zamperla, Barbara Illi, Veronica Barbi, Chiara Cencioni, Stella Gagliardi, Maria Garofalo, Gabriele Antonio Zingale, Irene Pandino, Diego Sbardella, Lina Cipolla, Simone Sabbioneda, Antonella Farsetti, Chiara Ripamonti, Gianluca Fossati, Christian Steinkühler, Sandra Atlante, Carlo Gaetano. HDAC6 inhibition reshapes the epigenome of cancer via P300 stabilization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4582.

Supplementary Figure S4 shows the effect of obesogenic high-fat diet on glycolytic enzymes and HIF-1 alpha in vivo.

DNA polymerase eta (Polη) is the only translesion synthesis polymerase capable of error-free bypass of UV-induced cyclobutane pyrimidine dimers. A deficiency in Polη function is associated with the human disease Xeroderma pigmentosum variant (XPV). We hereby report the case of a 60-year-old woman known for XPV and carrying a Polη Thr191Pro variant in homozygosity. We further characterize the variant in vitro and in vivo, providing molecular evidence that the substitution abrogates polymerase activity and results in UV sensitivity through deficient damage bypass. This is the first functional molecular characterization of a missense variant of Polη, whose reported pathogenic variants have thus far been loss of function truncation or frameshift mutations. Our work allows the upgrading of Polη Thr191Pro from ‘variant of uncertain significance’ to ‘likely pathogenic mutant’, bearing direct impact on molecular diagnosis and genetic counseling. Furthermore, we have established a robust experimental approach that will allow a precise molecular analysis of further missense mutations possibly linked to XPV. Finally, it provides insight into critical Polη residues that may be targeted to develop small molecule inhibitors for cancer therapeutics.

Supplementary Figure S5 shows the output of ESTIMATE and PUREE methods to assess tumor purity.