Aberrant paracrine signalling for bone remodelling underlies the mutant histone-driven giant cell tumour of bone.

Oncohistones represent compelling evidence for a causative role of epigenetic perturbations in cancer. Giant cell tumours of bone (GCTs) are characterised by a mutated histone H3.3 as the sole genetic driver present in bone-forming osteoprogenitor cells but absent from abnormally large bone-resorbing osteoclasts which represent the hallmark of these neoplasms. While these striking features imply a pathogenic interaction between mesenchymal and myelomonocytic lineages during GCT development, the underlying mechanisms remain unknown. We show that the changes in the transcriptome and epigenome in the mesenchymal cells caused by the H3.3-G34W mutation contribute to increase osteoclast recruitment in part via reduced expression of the TGFß-like soluble factor, SCUBE3. Transcriptional changes in SCUBE3 are associated with altered histone marks and H3.3G34W enrichment at its enhancer regions. In turn, osteoclasts secrete unregulated amounts of SEMA4D which enhances proliferation of mutated osteoprogenitors arresting their maturation. These findings provide a mechanism by which GCTs undergo differentiation in response to denosumab, a drug that depletes the tumour of osteoclasts. In contrast, hTERT alterations, commonly found in malignant GCT, result in the histone-mutated neoplastic cells being independent of osteoclasts for their proliferation, predicting unresponsiveness to denosumab. We provide a mechanism for the initiation of GCT, the basis of which is dysfunctional cross-talk between bone-forming and bone-resorbing cells. The findings highlight the role of tumour/microenvironment bidirectional interactions in tumorigenesis and how this is exploited in the treatment of GCT.

Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants.

CRISPR/Cas9, base editors and prime editors comprise the contemporary genome editing toolbox. Many studies have optimized the use of CRISPR/Cas9, as the original CRISPR genome editing system, in substituting single nucleotides by homology directed repair (HDR), although this remains challenging. Studies describing modifications that improve editing efficiency fall short of isolating clonal cell lines or have not been validated for challenging loci or cell models. We present data from 95 transfections using a colony forming and an immortalized cell line comparing the effect on editing efficiency of donor template modifications, concentration of components, HDR enhancing agents and cold shock. We found that in silico predictions of guide RNA efficiency correlated poorly withactivity in cells. Using NGS and ddPCR we detected editing efficiencies of 5-12% in the transfected populations which fell to 1% on clonal cell line isolation. Our data demonstrate the variability of CRISPR efficiency by cell model, target locus and other factors. Successful genome editing requires a comparison of systems and modifications to develop the optimal protocol for the cell model and locus. We describe the steps in this process in a flowchart for those embarking on genome editing using any system and incorporate validated HDR-boosting modifications for those using CRISPR/Cas9.

Inhibition of Histone H3K27 Demethylases Inactivates Brachyury (TBXT) and Promotes Chordoma Cell Death.

Expression of the transcription factor brachyury (TBXT) is normally restricted to the embryo, and its silencing is epigenetically regulated. TBXT promotes mesenchymal transition in a subset of common carcinomas, and in chordoma, a rare cancer showing notochordal differentiation, TBXT acts as a putative oncogene. We hypothesized that TBXT expression is controlled through epigenetic inhibition to promote chordoma cell death. Screening of five human chordoma cell lines revealed that pharmacologic inhibition of the histone 3 lysine 27 demethylases KDM6A (UTX) and KDM6B (JMJD3) leads to cell death. This effect was phenocopied by dual genetic inactivation of KDM6A/B using CRISPR/Cas9. Inhibition of KDM6A/B with a novel compound KDOBA67 led to a genome-wide increase in repressive H3K27me3 marks with concomitant reduction in active H3K27ac, H3K9ac, and H3K4me3 marks. TBXT was a KDM6A/B target gene, and chromatin changes at TBXT following KDOBA67 treatment were associated with a reduction in TBXT protein levels in all models tested, including primary patient-derived cultures. In all models tested, KDOBA67 treatment downregulated expression of a network of transcription factors critical for chordoma survival and upregulated pathways dominated by ATF4-driven stress and proapoptotic responses. Blocking the AFT4 stress response did not prevent suppression of TBXT and induction of cell death, but ectopic overexpression of TBXT increased viability, therefore implicating TBXT as a potential therapeutic target of H3K27 demethylase inhibitors in chordoma. Our work highlights how knowledge of normal processes in fetal development can provide insight into tumorigenesis and identify novel therapeutic approaches. SIGNIFICANCE: Pharmacologic inhibition of H3K27-demethylases in human chordoma cells promotes epigenetic silencing of oncogenic TBXT, alters gene networks critical to survival, and represents a potential novel therapy.

Frequent alterations in p16/CDKN2A identified by immunohistochemistry and FISH in chordoma.

The expression of p16/CDKN2A, the second most commonly inactivated tumour suppressor gene in cancer, is lost in the majority of chordomas. However, the mechanism(s) leading to its inactivation and contribution to disease progression have only been partially addressed using small patient cohorts. We studied 384 chordoma samples from 320 patients by immunohistochemistry and found that p16 protein was lost in 53% of chordomas and was heterogeneously expressed in these tumours. To determine if CDKN2A copy number loss could explain the absence of p16 protein expression we performed fluorescence in situ hybridisation (FISH) for CDKN2A on consecutive tissue sections. CDKN2A copy number status was altered in 168 of 274 (61%) of samples and copy number loss was the most frequent alteration acquired during clinical disease progression. CDKN2A homozygous deletion was always associated with p16 protein loss but only accounted for 33% of the p16-negative cases. The remaining immunonegative cases were associated with disomy (27%), monosomy (12%), heterozygous loss (20%) and copy number gain (7%) of CDKN2A, supporting the hypothesis that loss of protein expression might be achieved via epigenetic or post-transcriptional regulatory mechanisms. We identified that mRNA levels were comparable in tumours with and without p16 protein expression, but other events including DNA promoter hypermethylation, copy number neutral loss of heterozygosity and expression of candidate microRNAs previously implicated in the regulation of CDKN2A expression were not identified to explain the protein loss. The data argue that p16 loss in chordoma is commonly caused by a post-transcriptional regulatory mechanism that is yet to be defined.

CDK1 and PLK1 coordinate the disassembly and reassembly of the nuclear envelope in vertebrate mitosis.

Micronuclei (MN) arise from chromosomes or fragments that fail to be incorporated into the primary nucleus after cell division. These structures are a major source of genetic instability caused by DNA repair and replication defects coupled to aberrant Nuclear Envelope (NE). These problems ultimately lead to a spectrum of chromosome rearrangements called chromothripsis, a phenomenon that is a hallmark of several cancers. Despite its importance, the molecular mechanism at the origin of this instability is still not understood. Here we show that lagging chromatin, although it can efficiently assemble Lamin A/C, always fails to recruit Nuclear Pore Complexes (NPCs) proteins and that Polo-Like Kinase (PLK1) negatively regulates NPC assembly. We also provide evidence for the requirement of PLK1 activity for the disassembly of NPCs, but not Lamina A/C, at mitotic entry. Altogether this study reveals the existence of independent regulatory pathways for Lamin A/C and NPC reorganization during mitosis where Lamin A/C targeting to the chromatin is controlled by CDK1 activity (a clock-based model) while the NPC loading is also spatially monitored by PLK1.

Repo-Man/PP1 regulates heterochromatin formation in interphase.

Repo-Man is a protein phosphatase 1 (PP1) targeting subunit that regulates mitotic progression and chromatin remodelling. After mitosis, Repo-Man/PP1 remains associated with chromatin but its function in interphase is not known. Here we show that Repo-Man, via Nup153, is enriched on condensed chromatin at the nuclear periphery and at the edge of the nucleopore basket. Repo-Man/PP1 regulates the formation of heterochromatin, dephosphorylates H3S28 and it is necessary and sufficient for heterochromatin protein 1 binding and H3K27me3 recruitment. Using a novel proteogenomic approach, we show that Repo-Man is enriched at subtelomeric regions together with H2AZ and H3.3 and that depletion of Repo-Man alters the peripheral localization of a subset of these regions and alleviates repression of some polycomb telomeric genes. This study shows a role for a mitotic phosphatase in the regulation of the epigenetic landscape and gene expression in interphase.