Oncogenic chimeric transcription factors drive tumor-specific transcription, processing, and translation of silent genomic regions.

Many cancers are characterized by gene fusions encoding oncogenic chimeric transcription factors (TFs) such as EWS::FLI1 in Ewing sarcoma (EwS). Here, we find that EWS::FLI1 induces the robust expression of a specific set of novel spliced and polyadenylated transcripts within otherwise transcriptionally silent regions of the genome. These neogenes (NGs) are virtually undetectable in large collections of normal tissues or non-EwS tumors and can be silenced by CRISPR interference at regulatory EWS::FLI1-bound microsatellites. Ribosome profiling and proteomics further show that some NGs are translated into highly EwS-specific peptides. More generally, we show that hundreds of NGs can be detected in diverse cancers characterized by chimeric TFs. Altogether, this study identifies the transcription, processing, and translation of novel, specific, highly expressed multi-exonic transcripts from otherwise silent regions of the genome as a new activity of aberrant TFs in cancer.

Functional loss of a noncanonical BCOR-PRC1.1 complex accelerates SHH-driven medulloblastoma formation.

Medulloblastoma is a malignant childhood brain tumor arising from the developing cerebellum. In Sonic Hedgehog (SHH) subgroup medulloblastoma, aberrant activation of SHH signaling causes increased proliferation of granule neuron progenitors (GNPs), and predisposes these cells to tumorigenesis. A second, cooperating genetic hit is often required to push these hyperplastic cells to malignancy and confer mutation-specific characteristics associated with oncogenic signaling. Somatic loss-of-function mutations of the transcriptional corepressor BCOR are recurrent and enriched in SHH medulloblastoma. To investigate BCOR as a putative tumor suppressor, we used a genetically engineered mouse model to delete exons 9/10 of Bcor (BcorΔE9-10 ) in GNPs during development. This mutation leads to reduced expression of C-terminally truncated BCOR (BCORΔE9-10). While BcorΔE9-10 alone did not promote tumorigenesis or affect GNP differentiation, BcorΔE9-10 combined with loss of the SHH receptor gene Ptch1 resulted in fully penetrant medulloblastomas. In Ptch1+/- ;BcorΔE9-10 tumors, the growth factor gene Igf2 was aberrantly up-regulated, and ectopic Igf2 overexpression was sufficient to drive tumorigenesis in Ptch1+/- GNPs. BCOR directly regulates Igf2, likely through the PRC1.1 complex; the repressive histone mark H2AK119Ub is decreased at the Igf2 promoter in Ptch1+/- ;BcorΔE9-10 tumors. Overall, our data suggests that BCOR-PRC1.1 disruption leads to Igf2 overexpression, which transforms preneoplastic cells to malignant tumors.

Molecular characterisation defines clinically-actionable heterogeneity within Group 4 medulloblastoma and improves disease risk-stratification.

Group 4 tumours (MBGrp4) represent the majority of non-WNT/non-SHH medulloblastomas. Their clinical course is poorly predicted by current risk-factors. MBGrp4 molecular substructures have been identified (e.g. subgroups/cytogenetics/mutations), however their inter-relationships and potential to improve clinical sub-classification and risk-stratification remain undefined. We comprehensively characterised the paediatric MBGrp4 molecular landscape and determined its utility to improve clinical management. A clinically-annotated discovery cohort (n = 362 MBGrp4) was assembled from UK-CCLG institutions and SIOP-UKCCSG-PNET3, HIT-SIOP-PNET4 and PNET HR + 5 clinical trials. Molecular profiling was undertaken, integrating driver mutations, second-generation non-WNT/non-SHH subgroups (1-8) and whole-chromosome aberrations (WCAs). Survival models were derived for patients ≥ 3 years of age who received contemporary multi-modal therapies (n = 323). We first independently derived and validated a favourable-risk WCA group (WCA-FR) characterised by ≥ 2 features from chromosome 7 gain, 8 loss, and 11 loss. Remaining patients were high-risk (WCA-HR). Subgroups 6 and 7 were enriched for WCA-FR (p < 0·0001) and aneuploidy. Subgroup 8 was defined by predominantly balanced genomes with isolated isochromosome 17q (p < 0·0001). While no mutations were associated with outcome and overall mutational burden was low, WCA-HR harboured recurrent chromatin remodelling mutations (p = 0·007). Integration of methylation and WCA groups improved risk-stratification models and outperformed established prognostication schemes. Our MBGrp4 risk-stratification scheme defines: favourable-risk (non-metastatic disease and (i) subgroup 7 or (ii) WCA-FR (21% of patients, 5-year PFS 97%)), very-high-risk (metastatic disease with WCA-HR (36%, 5-year PFS 49%)) and high-risk (remaining patients; 43%, 5-year PFS 67%). These findings validated in an independent MBGrp4 cohort (n = 668). Importantly, our findings demonstrate that previously established disease-wide risk-features (i.e. LCA histology and MYC(N) amplification) have little prognostic relevance in MBGrp4 disease. Novel validated survival models, integrating clinical features, methylation and WCA groups, improve outcome prediction and re-define risk-status for ~ 80% of MBGrp4. Our MBGrp4 favourable-risk group has MBWNT-like excellent outcomes, thereby doubling the proportion of medulloblastoma patients who could benefit from therapy de-escalation approaches, aimed at reducing treatment induced late-effects while sustaining survival outcomes. Novel approaches are urgently required for the very-high-risk patients.

Class I HDAC inhibitor entinostat synergizes with PLK1 inhibitors in MYC-amplified medulloblastoma cells.

PURPOSE: We and others have demonstrated that MYC-amplified medulloblastoma (MB) cells are susceptible to class I histone deacetylase inhibitor (HDACi) treatment. However, single drug treatment with HDACi has shown limited clinical efficacy. We hypothesized that addition of a second compound acting synergistically with HDACi may enhance efficacy. METHODS: We used a gene expression dataset to identify PLK1 as a second target in MB cells and validated the relevance of PLK1 in MB. We measured cell metabolic activity, viability, and cycle progression in MB cells after treatment with PLK1-specific inhibitors (PLK1i). Chou-Talalay synergy calculations were used to determine the nature of class I HDACi entinostat and PLK1i interaction which was validated. Finally, the clinical potential of the combination was assessed in the in vivo experiment. RESULTS: MYC-amplified tumor cells are highly sensitive towards treatment with ATP-competitive PLK1i as a monotherapy. Entinostat and PLK1i in combination act synergistically in MYC-driven MB cells, exerting cytotoxic effects at clinically relevant concentrations. The downstream effect is exerted via MYC-related pathways, pointing out the potential of MYC amplification as a clinically feasible predictive biomarker for patient selection. While entinostat significantly extended survival of mice implanted with orthotopic MYC-amplified MB PDX, there was no evidence of the improvement of survival when treating the animals with the combination. CONCLUSION: The combination of entinostat and PLK1i showed synergistic interaction in vitro, but not in vivo. Therefore, further screening of blood-brain barrier penetrating PLK1i is warranted to determine the true potential of the combination as no on-target activity was observed after PLK1i volasertib treatment in vivo.

ClustMMRA v2: A Scalable Computational Pipeline for the Identification of MicroRNA Clusters Acting Cooperatively on Tumor Molecular Subgroups.

In recent cancer genomics programs, large-scale profiling of microRNAs has been routinely used in order to better understand the role of microRNAs in gene regulation and disease. To support the analysis of such amount of data, scalability of bioinformatics pipelines is increasingly important to handle larger datasets.Here, we describe a scalable implementation of the clustered miRNA Master Regulator Analysis (clustMMRA) pipeline, developed to search for genomic clusters of microRNAs potentially driving cancer molecular subtyping. Genomically clustered microRNAs can be simultaneously expressed to work in a combined manner and jointly regulate cell phenotypes. However, the majority of computational approaches for the identification of microRNA master regulators are typically designed to detect the regulatory effect of a single microRNA.We have applied the clustMMRA pipeline to multiple pediatric tumor datasets, up to a hundred samples in size, demonstrating very satisfying performances of the software on large datasets. Results have highlighted genomic clusters of microRNAs potentially involved in several subgroups of the different pediatric cancers or specifically involved in the phenotype of a subgroup. In particular, we confirmed the cluster of microRNAs at the 14q32 locus to be involved in multiple pediatric cancers, showing its specific downregulation in tumor subgroups with aggressive phenotype.

PML IV/ARF interaction enhances p53 SUMO-1 conjugation, activation, and senescence.

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) recruit multiple partners, including p53 and many of its regulators. NBs are believed to facilitate several posttranslational modifications and are key regulators of senescence. PML, the organizer of NBs, is expressed as a number of splice variants that all efficiently recruit p53 partners. However, overexpression of only one of them, PML IV, triggers p53-driven senescence. Here, we show that PML IV specifically binds ARF, a key p53 regulator. Similar to ARF, PML IV enhances global SUMO-1 conjugation, particularly that of p53, resulting in p53 stabilization and activation. ARF interacts with and stabilizes the NB-associated UBC9 SUMO-conjugating enzyme, possibly explaining PML IV-enhanced SUMOylation. These results unexpectedly link two key tumor suppressors, highlighting their convergence for global control of SUMO conjugation, p53 activation, and senescence induction.

E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification.

Ubiquitin and ubiquitin-like proteins (UBLs) are directed to targets by cascades of E1, E2, and E3 enzymes. The largest ubiquitin E3 subclass consists of cullin-RING ligases (CRLs), which contain one each of several cullins (CUL1, -2, -3, -4, or -5) and RING proteins (RBX1 or -2). CRLs are activated by ligation of the UBL NEDD8 to a conserved cullin lysine. How is cullin NEDD8ylation specificity established? Here we report that, like UBE2M (also known as UBC12), the previously uncharacterized E2 UBE2F is a NEDD8-conjugating enzyme in vitro and in vivo. Biochemical and structural analyses indicate how plasticity of hydrophobic E1-E2 interactions and E1 conformational flexibility allow one E1 to charge multiple E2s. The E2s have distinct functions, with UBE2M/RBX1 and UBE2F/RBX2 displaying different target cullin specificities. Together, these studies reveal the molecular basis for and functional importance of hierarchical expansion of the NEDD8 conjugation system in establishing selective CRL activation.

MRT-92 inhibits Hedgehog signaling by blocking overlapping binding sites in the transmembrane domain of the Smoothened receptor.

The Smoothened (Smo) receptor, a member of class F G protein-coupled receptors, is the main transducer of the Hedgehog (Hh) signaling pathway implicated in a wide range of developmental and adult processes. Smo is the target of anticancer drugs that bind to a long and narrow cavity in the 7-transmembrane (7TM) domain. X-ray structures of human Smo (hSmo) bound to several ligands have revealed 2 types of 7TM-directed antagonists: those binding mostly to extracellular loops (site 1, e.g., LY2940680) and those penetrating deeply in the 7TM cavity (site 2, e.g., SANT-1). Here we report the development of the acylguanidine MRT-92, which displays subnanomolar antagonist activity against Smo in various Hh cell-based assays. MRT-92 inhibits rodent cerebellar granule cell proliferation induced by Hh pathway activation through pharmacologic (half maximal inhibitory concentration [IC50] = 0.4 nM) or genetic manipulation. Using [(3)H]MRT-92 (Kd = 0.3 nM for hSmo), we created a comprehensive framework for the interaction of small molecule modulators with hSmo and for understanding chemoresistance linked to hSmo mutations. Guided by molecular docking and site-directed mutagenesis data, our work convincingly confirms that MRT-92 simultaneously recognized and occupied both sites 1 and 2. Our data demonstrate the existence of a third type of Smo antagonists, those entirely filling the Smo binding cavity from the upper extracellular part to the lower cytoplasmic-proximal subpocket. Our studies should help design novel potent Smo antagonists and more effective therapeutic strategies for treating Hh-linked cancers and associated chemoresistance.

SALL4 is a CRL3REN/KCTD11 substrate that drives Sonic Hedgehog-dependent medulloblastoma.

The Sonic Hedgehog (SHH) pathway is crucial regulator of embryonic development and stemness. Its alteration leads to medulloblastoma (MB), the most common malignant pediatric brain tumor. The SHH-MB subgroup is the best genetically characterized, however the molecular mechanisms responsible for its pathogenesis are not fully understood and therapeutic benefits are still limited. Here, we show that the pro-oncogenic stemness regulator Spalt-like transcriptional factor 4 (SALL4) is re-expressed in mouse SHH-MB models, and its high levels correlate with worse overall survival in SHH-MB patients. Proteomic analysis revealed that SALL4 interacts with REN/KCTD11 (here REN), a substrate receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and a tumor suppressor lost in ~30% of human SHH-MBs. We demonstrate that CRL3REN induces polyubiquitylation and degradation of wild type SALL4, but not of a SALL4 mutant lacking zinc finger cluster 1 domain (ΔZFC1). Interestingly, SALL4 binds GLI1 and cooperates with HDAC1 to potentiate GLI1 deacetylation and transcriptional activity. Notably, inhibition of SALL4 suppresses SHH-MB growth both in murine and patient-derived xenograft models. Our findings identify SALL4 as a CRL3REN substrate and a promising therapeutic target in SHH-dependent cancers.

Regulation of primary cilia disassembly through HUWE1-mediated TTBK2 degradation plays a crucial role in cerebellar development and medulloblastoma growth.

Development of the cerebellum requires precise regulation of granule neuron progenitor (GNP) proliferation. Although it is known that primary cilia are necessary to support GNP proliferation, the exact molecular mechanism governing primary cilia dynamics within GNPs remains elusive. Here, we establish the pivotal roles for the centrosomal kinase TTBK2 (Tau tubulin kinase-2) and the E3 ubiquitin ligase HUWE1 in GNP proliferation. We show that TTBK2 is highly expressed in proliferating GNPs under Sonic Hedgehog (SHH) signaling, coinciding with active GNP proliferation and the presence of primary cilia. TTBK2 stabilizes primary cilia by inhibiting their disassembly, thereby promoting GNP proliferation in response to SHH. Mechanistically, we identify HUWE1 as a novel centrosomal E3 ligase that facilitates primary cilia disassembly by targeting TTBK2 degradation. Disassembly of primary cilia serves as a trigger for GNP differentiation, allowing their migration from the external granule layer (EGL) of the cerebellum to the internal granule layer (IGL) for subsequent maturation. Moreover, we have established a link between TTBK2 and SHH-type medulloblastoma (SHH-MB), a tumor characterized by uncontrolled GNP proliferation. TTBK2 depletion inhibits SHH-MB proliferation, indicating that TTBK2 may be a potential therapeutic target for this cancer type. In summary, our findings reveal the mechanism governing cerebellar development and highlight a potential anti-cancer strategy for SHH-MB.

Cancer-specific epigenome identifies oncogenic hijacking by nuclear factor I family proteins for medulloblastoma progression.

Normal cells coordinate proliferation and differentiation by precise tuning of gene expression based on the dynamic shifts of the epigenome throughout the developmental timeline. Although non-mutational epigenetic reprogramming is an emerging hallmark of cancer, the epigenomic shifts that occur during the transition from normal to malignant cells remain elusive. Here, we capture the epigenomic changes that occur during tumorigenesis in a prototypic embryonal brain tumor, medulloblastoma. By comparing the epigenomes of the different stages of transforming cells in mice, we identify nuclear factor I family of transcription factors, known to be cell fate determinants in development, as oncogenic regulators in the epigenomes of precancerous and cancerous cells. Furthermore, genetic and pharmacological inhibition of NFIB validated a crucial role of this transcription factor by disrupting the cancer epigenome in medulloblastoma. Thus, this study exemplifies how epigenomic changes contribute to tumorigenesis via non-mutational mechanisms involving developmental transcription factors.

Medulloblastomas with ELP1 pathogenic variants: A weakly penetrant syndrome with a restricted spectrum in a limited age window.

Background: ELP1 pathogenic variants (PV) have been recently identified as the most frequent variants predisposing to Sonic Hedgehog (SHH) medulloblastomas (MB); however, guidelines are still lacking for genetic counseling in this new syndrome. Methods: We retrospectively reviewed clinical and genetic data of a French series of 29 ELP1-mutated MB. Results: All patients developed SHH-MB, with a biallelic inactivation of PTCH1 found in 24 tumors. Other recurrent alterations encompassed the TP53 pathway and activation of MYCN/MYCL signaling. The median age at diagnosis was 7.3 years (range: 3-14). ELP1-mutated MB behave as sporadic cases, with similar distribution within clinical and molecular risk groups and similar outcomes (5 y - OS = 86%); no unusual side effect of treatments was noticed. Remarkably, a germline ELP1 PV was identified in all patients with available constitutional DNA (n = 26); moreover, all tested familial trio (n = 11) revealed that the PVs were inherited. Two of the 26 index cases from the French series had a family history of MB; pedigrees from these patients and from 1 additional Dutch family suggested a weak penetrance. Apart from MB, no cancer was associated with ELP1 PVs; second tumors reported in 4 patients occurred within the irradiation fields, in the usual time-lapse for expected radiotherapy-induced neoplasms. Conclusions: The low penetrance, the "at risk' age window limited to childhood and the narrow tumor spectrum, question the actual benefit of genetic screening in these patients and their family. Our results suggest restricting ELP1 germline sequencing to patients with SHH-MB, depending on the parents" request.

Developmental basis of SHH medulloblastoma heterogeneity.

Many genes that drive normal cellular development also contribute to oncogenesis. Medulloblastoma (MB) tumors likely arise from neuronal progenitors in the cerebellum, and we hypothesized that the heterogeneity observed in MBs with sonic hedgehog (SHH) activation could be due to differences in developmental pathways. To investigate this question, here we perform single-nucleus RNA sequencing on highly differentiated SHH MBs with extensively nodular histology and observed malignant cells resembling each stage of canonical granule neuron development. Through innovative computational approaches, we connect these results to published datasets and find that some established molecular subtypes of SHH MB appear arrested at different developmental stages. Additionally, using multiplexed proteomic imaging and MALDI imaging mass spectrometry, we identify distinct histological and metabolic profiles for highly differentiated tumors. Our approaches are applicable to understanding the interplay between heterogeneity and differentiation in other cancers and can provide important insights for the design of targeted therapies.

Age-dependent in vivo conversion of mouse cochlear pillar and Deiters' cells to immature hair cells by Atoh1 ectopic expression.

Unlike nonmammalian vertebrates, mammals cannot convert inner ear cochlear supporting cells (SCs) into sensory hair cells (HCs) after damage, thus causing permanent deafness. Here, we achieved in vivo conversion of two SC subtypes, pillar cells (PCs) and Deiters' cells (DCs), into HCs by inducing targeted expression of Atoh1 at neonatal and juvenile ages using novel mouse models. The conversion only occurred in ∼10% of PCs and DCs with ectopic Atoh1 expression and started with reactivation of endogenous Atoh1 followed by expression of 11 HC and synaptic markers, a process that took approximately 3 weeks in vivo. These new HCs resided in the outer HC region, formed stereocilia, contained mechanoelectrical transduction channels, and survived for >2 months in vivo; however, they surprisingly lacked prestin and oncomodulin expression and mature HC morphology. In contrast, adult PCs and DCs no longer responded to ectopic Atoh1 expression, even after outer HC damage. Finally, permanent Atoh1 expression in endogenous HCs did not affect prestin expression but caused cell loss of mature HCs. Together, our results demonstrate that in vivo conversion of PCs and DCs into immature HCs by Atoh1 is age dependent and resembles normal HC development. Therefore, combined expression of Atoh1 with additional factors holds therapeutic promise to convert PCs and DCs into functional HCs in vivo for regenerative purposes.

Hypermethylated in cancer 1 (HIC1) recruits polycomb repressive complex 2 (PRC2) to a subset of its target genes through interaction with human polycomb-like (hPCL) proteins.

HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene epigenetically silenced or deleted in many human cancers. HIC1 is involved in regulatory loops modulating p53- and E2F1-dependent cell survival, growth control, and stress responses. HIC1 is also essential for normal development because Hic1-deficient mice die perinatally and exhibit gross developmental defects throughout the second half of development. HIC1 encodes a transcriptional repressor with five C(2)H(2) zinc fingers mediating sequence-specific DNA binding and two repression domains: an N-terminal BTB/POZ domain and a central region recruiting CtBP and NuRD complexes. By yeast two-hybrid screening, we identified the Polycomb-like protein hPCL3 as a novel co-repressor for HIC1. Using multiple biochemical strategies, we demonstrated that HIC1 interacts with hPCL3 and its paralog PHF1 to form a stable complex with the PRC2 members EZH2, EED, and Suz12. Confirming the implication of HIC1 in Polycomb recruitment, we showed that HIC1 shares some of its target genes with PRC2, including ATOH1. Depletion of HIC1 by siRNA interference leads to a partial displacement of EZH2 from the ATOH1 promoter. Furthermore, in vivo, ATOH1 repression by HIC1 is associated with Polycomb activity during mouse cerebellar development. Thus, our results identify HIC1 as the first transcription factor in mammals able to recruit PRC2 to some target promoters through its interaction with Polycomb-like proteins.