Dampening DNA damage checkpoint signalling via coordinated BRCT domain interactions.

In response to DNA damage, checkpoint signalling protects genome integrity at the cost of repressing cell cycle progression and DNA replication. Mechanisms for checkpoint down-regulation are therefore necessary for proper cellular proliferation. We recently uncovered a phosphatase-independent mechanism for dampening checkpoint signalling, where the checkpoint adaptor Rad9 is counteracted by the repair scaffolds Slx4-Rtt107. Here, we establish the molecular requirements for this new mode of checkpoint regulation. We engineered a minimal multi-BRCT-domain (MBD) module that recapitulates the action of Slx4-Rtt107 in checkpoint down-regulation. MBD mimics the damage-induced Dpb11-Slx4-Rtt107 complex by synergistically interacting with lesion-specific phospho-sites in Ddc1 and H2A. We propose that efficient recruitment of Dpb11-Slx4-Rtt107 or MBD via a cooperative 'two-site-docking' mechanism displaces Rad9. MBD also interacts with the Mus81 nuclease following checkpoint dampening, suggesting a spatio-temporal coordination of checkpoint signalling and DNA repair via a combinatorial mode of BRCT-domains interactions.

Oncogenic Cells of Renal Embryonic Lineage Sensitive to the Small-Molecule Inhibitor QC6352 Display Depletion of KDM4 Levels and Disruption of Ribosome Biogenesis.

The histone lysine demethylases KDM4A-C are involved in physiologic processes including stem cell identity and self-renewal during development, DNA damage repair, and cell-cycle progression. KDM4A-C are overexpressed and associated with malignant cell behavior in multiple human cancers and are therefore potential therapeutic targets. Given the role of KDM4A-C in development and cancer, we aimed to test the potent, selective KDM4A-C inhibitor QC6352 on oncogenic cells of renal embryonic lineage. The anaplastic Wilms tumor cell line WiT49 and the tumor-forming human embryonic kidney cell line HEK293 demonstrated low nanomolar QC6352 sensitivity. The cytostatic response to QC6352 in WiT49 and HEK293 cells was marked by induction of DNA damage, a DNA repair-associated protein checkpoint response, S-phase cell-cycle arrest, profound reduction of ribosomal protein gene and rRNA transcription, and blockade of newly synthesized proteins. QC6352 caused reduction of KDM4A-C levels by a proteasome-associated mechanism. The cellular phenotype caused by QC6352 treatment of reduced migration, proliferation, tumor spheroid growth, DNA damage, and S-phase cell-cycle arrest was most closely mirrored by knockdown of KDM4A as determined by siRNA knockdown of KDM4A-C. QC6352 sensitivity correlated with high basal levels of ribosomal gene transcription in more than 900 human cancer cell lines. Targeting KDM4A may be of future therapeutic interest in oncogenic cells of embryonic renal lineage or cells with high basal expression of ribosomal protein genes.

Metabolic reprogramming of cancer cells by JMJD6-mediated pre-mRNA splicing associated with therapeutic response to splicing inhibitor.

Dysregulated pre-mRNA splicing and metabolism are two hallmarks of MYC-driven cancers. Pharmacological inhibition of both processes has been extensively investigated as potential therapeutic avenues in preclinical and clinical studies. However, how pre-mRNA splicing and metabolism are orchestrated in response to oncogenic stress and therapies is poorly understood. Here, we demonstrate that jumonji domain containing 6, arginine demethylase, and lysine hydroxylase, JMJD6, acts as a hub connecting splicing and metabolism in MYC-driven human neuroblastoma. JMJD6 cooperates with MYC in cellular transformation of murine neural crest cells by physically interacting with RNA binding proteins involved in pre-mRNA splicing and protein homeostasis. Notably, JMJD6 controls the alternative splicing of two isoforms of glutaminase (GLS), namely kidney-type glutaminase (KGA) and glutaminase C (GAC), which are rate-limiting enzymes of glutaminolysis in the central carbon metabolism in neuroblastoma. Further, we show that JMJD6 is correlated with the anti-cancer activity of indisulam, a 'molecular glue' that degrades splicing factor RBM39, which complexes with JMJD6. The indisulam-mediated cancer cell killing is at least partly dependent on the glutamine-related metabolic pathway mediated by JMJD6. Our findings reveal a cancer-promoting metabolic program is associated with alternative pre-mRNA splicing through JMJD6, providing a rationale to target JMJD6 as a therapeutic avenue for treating MYC-driven cancers.

Metabolic reprogramming of cancer cells by JMJD6-mediated pre-mRNA splicing is associated with therapeutic response to splicing inhibitor.

Dysregulated pre-mRNA splicing and metabolism are two hallmarks of MYC-driven cancers. Pharmacological inhibition of both processes has been extensively investigated as potential therapeutic avenues in preclinical and clinical studies. However, how pre-mRNA splicing and metabolism are orchestrated in response to oncogenic stress and therapies is poorly understood. Here, we demonstrate that Jumonji Domain Containing 6, Arginine Demethylase and Lysine Hydroxylase, JMJD6, acts as a hub connecting splicing and metabolism in MYC-driven neuroblastoma. JMJD6 cooperates with MYC in cellular transformation by physically interacting with RNA binding proteins involved in pre-mRNA splicing and protein homeostasis. Notably, JMJD6 controls the alternative splicing of two isoforms of glutaminase (GLS), namely kidney-type glutaminase (KGA) and glutaminase C (GAC), which are rate-limiting enzymes of glutaminolysis in the central carbon metabolism in neuroblastoma. Further, we show that JMJD6 is correlated with the anti-cancer activity of indisulam, a "molecular glue" that degrades splicing factor RBM39, which complexes with JMJD6. The indisulam-mediated cancer cell killing is at least partly dependent on the glutamine-related metabolic pathway mediated by JMJD6. Our findings reveal a cancer-promoting metabolic program is associated with alternative pre-mRNA splicing through JMJD6, providing a rationale to target JMJD6 as a therapeutic avenue for treating MYC-driven cancers.

The Genetic and Epigenetic Features of Bilateral Wilms Tumor Predisposition: A Report from the Children's Oncology Group AREN18B5-Q Study.

This study comprehensively evaluated the landscape of genetic and epigenetic events that predispose to synchronous bilateral Wilms tumor (BWT). We performed whole exome or whole genome sequencing, total-strand RNA-seq, and DNA methylation analysis using germline and/or tumor samples from 68 patients with BWT from St. Jude Children's Research Hospital and the Children's Oncology Group. We found that 25/61 (41%) of patients evaluated harbored pathogenic or likely pathogenic germline variants, with WT1 (14.8%), NYNRIN (6.6%), TRIM28 (5%) and the BRCA-related genes (5%) BRCA1, BRCA2, and PALB2 being most common. Germline WT1 variants were strongly associated with somatic paternal uniparental disomy encompassing the 11p15.5 and 11p13/WT1 loci and subsequent acquired pathogenic CTNNB1 variants. Somatic coding variants or genome-wide copy number alterations were almost never shared between paired synchronous BWT, suggesting that the acquisition of independent somatic variants leads to tumor formation in the context of germline or early embryonic, post-zygotic initiating events. In contrast, 11p15.5 status (loss of heterozygosity, loss or retention of imprinting) was shared among paired synchronous BWT in all but one case. The predominant molecular events for BWT predisposition include pathogenic germline variants or post-zygotic epigenetic hypermethylation at the 11p15.5 H19/ICR1 locus (loss of imprinting). This study demonstrates that post-zygotic somatic mosaicism for 11p15.5 hypermethylation/loss of imprinting is the single most common initiating molecular event predisposing to BWT. Evidence of somatic mosaicism for 11p15.5 loss of imprinting was detected in leukocytes of a cohort of BWT patients and long-term survivors, but not in unilateral Wilms tumor patients and long-term survivors or controls, further supporting the hypothesis that post-zygotic 11p15.5 alterations occurred in the mesoderm of patients who go on to develop BWT. Due to the preponderance of BWT patients with demonstrable germline or early embryonic tumor predisposition, BWT exhibits a unique biology when compared to unilateral Wilms tumor and therefore warrants continued refinement of its own treatment-relevant biomarkers which in turn may inform directed treatment strategies in the future.

Genetic and epigenetic features of bilateral Wilms tumor predisposition in patients from the Children's Oncology Group AREN18B5-Q.

Developing synchronous bilateral Wilms tumor suggests an underlying (epi)genetic predisposition. Here, we evaluate this predisposition in 68 patients using whole exome or genome sequencing (n = 85 tumors from 61 patients with matched germline blood DNA), RNA-seq (n = 99 tumors), and DNA methylation analysis (n = 61 peripheral blood, n = 29 non-diseased kidney, n = 99 tumors). We determine the predominant events for bilateral Wilms tumor predisposition: 1)pre-zygotic germline genetic variants readily detectable in blood DNA [WT1 (14.8%), NYNRIN (6.6%), TRIM28 (5%), and BRCA-related genes (5%)] or 2)post-zygotic epigenetic hypermethylation at 11p15.5 H19/ICR1 that may require analysis of multiple tissue types for diagnosis. Of 99 total tumor specimens, 16 (16.1%) have 11p15.5 normal retention of imprinting, 25 (25.2%) have 11p15.5 copy neutral loss of heterozygosity, and 58 (58.6%) have 11p15.5 H19/ICR1 epigenetic hypermethylation (loss of imprinting). Here, we ascertain the epigenetic and genetic modes of bilateral Wilms tumor predisposition.

TERT Expression in Wilms Tumor Is Regulated by Promoter Mutation or Hypermethylation, WT1, and N-MYC.

Increased TERT mRNA is associated with disease relapse in favorable histology Wilms tumor (WT). This study sought to understand the mechanism of increased TERT expression by determining the association between TERT and WT1 and N-MYC, two proteins important in Wilms tumor pathogenesis that have been shown to regulate TERT expression. Three out of 45 (6.7%) WTs and the corresponding patient-derived xenografts harbored canonical gain-of-function mutations in the TERT promoter. This study identified near ubiquitous hypermethylation of the TERT promoter region in WT compared to normal kidney. WTs with biallelic inactivating mutations in WT1 (7/45, 15.6%) were found to have lower TERT expression by RNA-seq and qRT-PCR and lower telomerase activity determined by the telomerase repeat amplification protocol. Anaplastic histology and increased percentage of blastema were positively correlated with higher TERT expression and telomerase activity. In vitro shRNA knockdown of WT1 resulted in decreased expression of TERT, reduced colony formation, and decreased proliferation of WiT49, an anaplastic WT cell line with wild-type WT1. CRISPR-Cas9-mediated knockout of WT1 resulted in decreased expression of telomere-related gene pathways. However, an inducible Wt1-knockout mouse model showed no relationship between Wt1 knockout and Tert expression in normal murine nephrogenesis, suggesting that WT1 and TERT are coupled in transformed cells but not in normal kidney tissues. N-MYC overexpression resulted in increased TERT promoter activity and TERT transcription. Thus, multiple mechanisms of TERT activation are involved in WT and are associated with anaplastic histology and increased blastema. This study is novel because it identifies potential mechanisms of TERT activation in Wilms tumor that could be of therapeutic interests.

Surprising phenotypic diversity of cancer-associated mutations of Gly 34 in the histone H3 tail.

Sequencing of cancer genomes has identified recurrent somatic mutations in histones, termed oncohistones, which are frequently poorly understood. Previously we showed that fission yeast expressing only the H3.3G34R mutant identified in aggressive pediatric glioma had reduced H3K36 trimethylation and acetylation, increased genomic instability and replicative stress, and defective homology-dependent DNA damage repair. Here we show that surprisingly distinct phenotypes result from G34V (also in glioma) and G34W (giant cell tumors of bone) mutations, differentially affecting H3K36 modifications, subtelomeric silencing, genomic stability; sensitivity to irradiation, alkylating agents, and hydroxyurea; and influencing DNA repair. In cancer, only 1 of 30 alleles encoding H3 is mutated. Whilst co-expression of wild-type H3 rescues most G34 mutant phenotypes, G34R causes dominant hydroxyurea sensitivity, homologous recombination defects, and dominant subtelomeric silencing. Together, these studies demonstrate the complexity associated with different substitutions at even a single residue in H3 and highlight the utility of genetically tractable systems for their analysis.

Stand-alone rolling circle amplification combined with capillary electrophoresis for specific detection of small RNA.

Noncoding small RNAs play diverse, important biological roles through gene expression regulation. However, their low expression levels make it difficult to identify new small RNA species and study their functions, calling for the development of detection schemes with higher simplicity, sensitivity, and specificity. Herein, we reported a straightforward assay that combined the stand-alone rolling circle amplification (RCA) with capillary electrophoresis (CE) for specific and sensitive detection of small RNAs in biological samples. In order to enhance the overall reaction efficiency and simplify the procedure, RCA was not preceded with ligation, and a preformed circular probe was employed as the template for the target small RNA-primed isothermal amplification. The long RCA product was digested and analyzed by CE. Two DNA polymerases, the Phi29 and Bst, were compared for their detection performance. Bst is superior in the aspects of specificity, procedure simplicity, and reproducibility, while Phi29 leads to a 5-fold lower detection limit and is able to detect as low as 35 amol of the target small RNA. Coamplification of an internal standard with the target and employment of the RNase A digestion step allow accurate and reproducible quantification of low amounts of small RNA targets spiked into hundreds of nanograms of the plant total RNA extract with a recovery below 110% using either enzyme. Our assay can be adapted to a capillary array system for high-throughput screening of small RNA expression in biological samples. Also, the one-step isothermal process has the potential to conveniently amplify a very limited amount of the RNA samples, e.g., RNA extracted from only a few cells, inside the capillary column or on a microchip.

KDM5A Regulates a Translational Program that Controls p53 Protein Expression.

The p53 tumor suppressor pathway is frequently inactivated in human cancers. However, there are some cancer types without commonly recognized alterations in p53 signaling. Here we report that histone demethylase KDM5A is involved in the regulation of p53 activity. KDM5A is significantly amplified in multiple types of cancers, an event that tends to be mutually exclusive to p53 mutation. We show that KDM5A acts as a negative regulator of p53 signaling through inhibition of p53 translation via suppression of a subgroup of eukaryotic translation initiation genes. Genetic deletion of KDM5A results in upregulation of p53 in multiple lineages of cancer cells and inhibits tumor growth in a p53-dependent manner. In addition, we have identified a regulatory loop between p53, miR-34, and KDM5A, whereby the induction of miR-34 leads to suppression of KDM5A. Thus, our findings reveal a mechanism by which KDM5A inhibits p53 translation to modulate cancer progression.

Histone H3G34R mutation causes replication stress, homologous recombination defects and genomic instability in S. pombe.

Recurrent somatic mutations of H3F3A in aggressive pediatric high-grade gliomas generate K27M or G34R/V mutant histone H3.3. H3.3-G34R/V mutants are common in tumors with mutations in p53 and ATRX, an H3.3-specific chromatin remodeler. To gain insight into the role of H3-G34R, we generated fission yeast that express only the mutant histone H3. H3-G34R specifically reduces H3K36 tri-methylation and H3K36 acetylation, and mutants show partial transcriptional overlap with set2 deletions. H3-G34R mutants exhibit genomic instability and increased replication stress, including slowed replication fork restart, although DNA replication checkpoints are functional. H3-G34R mutants are defective for DNA damage repair by homologous recombination (HR), and have altered HR protein dynamics in both damaged and untreated cells. These data suggest H3-G34R slows resolution of HR-mediated repair and that unresolved replication intermediates impair chromosome segregation. This analysis of H3-G34R mutant fission yeast provides mechanistic insight into how G34R mutation may promote genomic instability in glioma.

Termination of Replication Stress Signaling via Concerted Action of the Slx4 Scaffold and the PP4 Phosphatase.

In response to replication stress, signaling mediated by DNA damage checkpoint kinases protects genome integrity. However, following repair or bypass of DNA lesions, checkpoint signaling needs to be terminated for continued cell cycle progression and proliferation. In budding yeast, the PP4 phosphatase has been shown to play a key role in preventing hyperactivation of the checkpoint kinase Rad53. In addition, we recently uncovered a phosphatase-independent mechanism for downregulating Rad53 in which the DNA repair scaffold Slx4 decreases engagement of the checkpoint adaptor Rad9 at DNA lesions. Here we reveal that proper termination of checkpoint signaling following the bypass of replication blocks imposed by alkylated DNA adducts requires the concerted action of these two fundamentally distinct mechanisms of checkpoint downregulation. Cells lacking both SLX4 and the PP4-subunit PPH3 display a synergistic increase in Rad53 signaling and are exquisitely sensitive to the DNA alkylating agent methyl methanesulfonate, which induces replication blocks and extensive formation of chromosomal linkages due to template switching mechanisms required for fork bypass. Rad53 hypersignaling in these cells seems to converge to a strong repression of Mus81-Mms4, the endonuclease complex responsible for resolving chromosomal linkages, thus explaining the selective sensitivity of slx4Δ pph3Δ cells to alkylation damage. Our results support a model in which Slx4 acts locally to downregulate Rad53 activation following fork bypass, while PP4 acts on pools of active Rad53 that have diffused from the site of lesions. We propose that the proper spatial coordination of the Slx4 scaffold and PP4 action is crucial to allow timely activation of Mus81-Mms4 and, therefore, proper chromosome segregation.