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Background The three-dimensional genome organization is critical for gene regulation and can malfunction in diseases like cancer. As a key regulator of genome organization, CCCTC-binding factor (CTCF) has been characterized as a DNA-binding protein with important functions in maintaining the topological structure of chromatin and inducing DNA looping. Among the prolific binding sites in the genome, several events with altered CTCF occupancy have been reported as associated with effects in physiology or disease. However, hitherto there is no comprehensive survey of genome-wide CTCF binding patterns across different human cancers. Results To dissect functions of CTCF binding, we systematically analyze over 700 CTCF ChIP-seq profiles across human tissues and cancers and identify cancer-specific CTCF binding patterns in six cancer types. We show that cancer-specific lost and gained CTCF binding events are associated with altered chromatin interactions, partially with DNA methylation changes, and rarely with sequence mutations. While lost bindings primarily occur near gene promoters, most gained CTCF binding events exhibit enhancer activities and are induced by oncogenic transcription factors. We validate these findings in T cell acute lymphoblastic leukemia cell lines and patient samples and show that oncogenic NOTCH1 induces specific CTCF binding and they cooperatively activate expression of target genes, indicating transcriptional condensation phenomena. Conclusions Specific CTCF binding events occur in human cancers. Cancer-specific CTCF binding can be induced by other transcription factors to regulate oncogenic gene expression. Our results substantiate CTCF binding alteration as a functional epigenomic signature of cancer.

Nowadays, personalized cancer therapy relies on small molecules, monoclonal antibodies, or antibody–drug conjugates (ADC). Many nanoparticle (NP)-based drug delivery systems are also actively investigated, but their advantage over ADCs has not been demonstrated yet. Here, using the Avidin-Nucleic-Acid-Nano-Assemblies (ANANAS), a class of polyavidins multifuctionalizable with stoichiometric control, we compare quantitatively anti-EGFR antibody(cetuximab)-targeted NPs to the corresponding ADC. We show that ANANAS tethering of cetuximab promotes a more efficient EGFR-dependent vesicle-mediated internalization. Cetuximab-guided ANANAS carrying doxorubicin are more cytotoxic in vitro and much more potent in vivo than the corresponding ADC, leading to 43% tumor reduction at low drug dosage (0.56 mg/kg). Advantage of cetuximab-guided ANANAS with respect to the ADC goes beyond the increase in drug-to-antibody ratio. Even if further studies are needed, we propose that NP tethering could expand application of the anti-EGFR antibody to a wider number of cancer patients including the KRAS-mutated ones, currently suffering from poor prognosis. The nature of the linker is known to affect the efficacy of antibody–drug conjugate (ADC). Here the authors show cetuximab-guided Avidin-Nucleic-Acid-Nanoassemblies to be superior to cetuximab-doxorubicin conjugate, and show its efficacy in KRAS mutant breast cancer, allowing for therapeutic expansion of anti-EGFR therapy.

Mutations in the cytosolic 5′-nucleotidase II gene accelerate the inactivation of chemotherapeutic nucleoside analogs in acute lymphoblastic leukemia (ALL)-promoting lymphoblasts. Increased nucleotide metabolism may therefore constitute an important resistance mechanism in chemotherapy-resilient ALL. Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric patients and over 50% of adult patients with ALL do not achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most substantial challenge in the treatment of this disease 1 , 2 . Using whole-exome sequencing, we identify mutations in the cytosolic 5′-nucleotidase II gene ( NT5C2 ), which encodes a 5′-nucleotidase enzyme that is responsible for the inactivation of nucleoside-analog chemotherapy drugs, in 20/103 (19%) relapse T cell ALLs and 1/35 (3%) relapse B-precursor ALLs. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside-analog metabolism in disease progression and chemotherapy resistance in ALL.

Posttranscriptional control of gene expression is important for defining both normal and pathological cellular phenotypes. In vitro, RNA-binding proteins (RBPs) have recently been shown to play important roles in posttranscriptional regulation; however, the contribution of RBPs to cell specification is not well understood. Here, we determined that the RBP insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is specifically overexpressed in mixed lineage leukemia-rearranged (MLL-rearranged) B-acute lymphoblastic leukemia (B-ALL), which constitutes a subtype of this malignancy associated with poor prognosis and high risk of relapse. IGF2BP3 was required for the survival of B-ALL cell lines, as knockdown led to decreased proliferation and increased apoptosis. Enforced expression of IGF2BP3 provided murine BM cells with a strong survival advantage, led to proliferation of hematopoietic stem and progenitor cells, and skewed hematopoietic development to the B cell/myeloid lineage. Cross-link immunoprecipitation and high throughput sequencing uncovered the IGF2BP3-regulated transcriptome, which includes oncogenes MYC and CDK6 as direct targets. IGF2BP3 regulated transcripts via targeting elements within 3' untranslated regions (3'UTR), and enforced IGF2BP3 expression in mice resulted in enhanced expression of Myc and Cdk6 in BM. Together, our data suggest that IGF2BP3-mediated targeting of oncogenic transcripts may represent a critical pathogenetic mechanism in MLL-rearranged B-ALL and support IGF2BP3 and its cognate RNA-binding partners as potential therapeutic targets in this disease.

About a fifth of children with acute T-lymphoblastic leukaemia (T-ALL) succumb to the disease, suggesting an unrecognised biological heterogeneity that might contribute to drug resistance. We postulated that T-ALL originating from early T-cell precursors (ETPs), a recently defined subset of thymocytes that retain stem-cell-like features, would respond poorly to lymphoid-cell-directed therapy. We studied leukaemic cells, collected at diagnosis, to identify cases with ETP features and determine their clinical outcome. Leukaemic cells from 239 patients with T-ALL enrolled at St Jude Children's Research Hospital (n=139) and in the Italian national study Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) ALL-2000 (n=100) were assessed by gene-expression profiling, flow cytometry, and single nucleotide polymorphism array analysis. Probabilities of survival and treatment failure were calculated for subgroups considered to have ETP-ALL or typical T-ALL. 30 patients (12·6%) had leukaemic lymphoblasts with an ETP-related gene-expression signature or its associated distinctive immunophenotype (CD1a −, CD8 −, CD5 weak with stem-cell or myeloid markers). Cases of ETP-ALL showed increased genomic instability, in terms of number and size of gene lesions, compared with those with typical T-ALL. Patients with this form of leukaemia had high risk of remission failure or haematological relapse (72% [95% CI 40–100] at 10 years vs 10% [4–16] at 10 years for patients with typical T-ALL treated at St Jude Children's Research Hospital; and 57% [25–89] at 2 years vs 14% [6–22] at 2 years for patients treated in the AIEOP trial). ETP-ALL is a distinct, previously unrecognised, pathobiological entity that confers a poor prognosis with use of standard intensive chemotherapy. Its early recognition, by use of the gene expression and immunophenotypic criteria outlined here, is essential for the development of an effective clinical management strategy. US National Cancer Institute, Cariplo Foundation, Citta della Speranza Foundation, Italian Association for Cancer Research (AIRC), Italian Ministry for University and Research, and American Lebanese Syrian Associated Charities (ALSAC).

The RNA-binding protein LIN28B regulates developmental timing and determines stem cell identity by suppressing the let-7 family of microRNAs. Postembryonic reactivation of LIN28B impairs cell commitment to differentiation, prompting their transformation. In this study, we assessed the extent to which ectopic lin28b expression modulates the physiological behavior of neural crest cells (NCC) and governs their transformation in the trunk region of developing embryos. We provide evidence that the overexpression of lin28b inhibits sympathoadrenal cell differentiation and accelerates NCC migration in two vertebrate models, Xenopus leavis and Danio rerio. Our results highlight the relevance of ITGA5 and ITGA6 in the LIN28B-dependent regulation of the invasive motility of tumor cells. The results also establish that LIN28B overexpression supports neuroblastoma onset and the metastatic potential of malignant cells through let-7a-dependent and let-7a-independent mechanisms.

Background Long non-coding RNAs (lncRNAs) play a variety of cellular roles, including regulation of transcription and translation, leading to alterations in gene expression. Some lncRNAs modulate the expression of chromosomally adjacent genes. Here, we assess the roles of the lncRNA CASC15 in regulation of a chromosomally nearby gene, SOX4, and its function in RUNX1/AML translocated leukemia. Results CASC15 is a conserved lncRNA that was upregulated in pediatric B-acute lymphoblastic leukemia (B-ALL) with t (12; 21) as well as pediatric acute myeloid leukemia (AML) with t (8; 21), both of which are associated with relatively better prognosis. Enforced expression of CASC15 led to a myeloid bias in development, and overall, decreased engraftment and colony formation. At the cellular level, CASC15 regulated cellular survival, proliferation, and the expression of its chromosomally adjacent gene, SOX4 . Differentially regulated genes following CASC15 knockdown were enriched for predicted transcriptional targets of the Yin and Yang-1 (YY1) transcription factor. Interestingly, we found that CASC15 enhances YY1-mediated regulation of the SOX4 promoter. Conclusions Our findings represent the first characterization of this CASC15 in RUNX1-translocated leukemia, and point towards a mechanistic basis for its action.

Through negative regulation of gene expression, microRNAs (miRNAs) can function as oncosuppressors in cancers, and can themselves show altered expression in various tumor types. Here, we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many of the cell-fate-determining stages. Notch regulates a subset of MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulate these phenomena, and can be used in anti-cancer therapies. In a screening of potential targets within Notch signaling, miR-34a was seen to be a regulator of the Notch pathway through its targeting of Notch ligand Delta-like 1 (Dll1). Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. Using an inducible tetracycline on-off model of miR-34a expression, we show that in Daoy MB cells, Dll1 is the first target that is regulated in MB, as compared to the other targets analyzed here: Cyclin D1, cMyc and CDK4. MiR-34a expression negatively affects CD133(+)/CD15(+) tumor-propagating cells, then we assay through reverse-phase proteomic arrays, Akt and Stat3 signaling hypo-phosphorylation. Adenoviruses carrying the precursor miR-34a induce neurogenesis of tumor spheres derived from a genetic animal model of MB (Patch1(+/-) p53(-/-)), thus providing further evidence that the miR-34a/Dll1 axis controls both autonomous and non autonomous signaling of Notch. In vivo, miR-34a overexpression carried by adenoviruses reduces tumor burden in cerebellum xenografts of athymic mice, thus demonstrating an anti-tumorigenic role of miR-34a in vivo. Despite advances in our understanding of the pathogenesis of MB, one-third of patients with MB remain incurable. Here, we show that stable nucleic-acid-lipid particles carrying mature miR-34a can target Dll1 in vitro and show equal effects to those of adenovirus miR-34a cell infection. Thus, this technology forms the basis for their therapeutic use for the delivery of miR-34a in brain-tumor treatment, with no signs of toxicity described to date in non-human primate trials.

Glioblastoma multiforme (GBM) is a highly vascularized and aggressive brain tumor, with a strong ability to disseminate and invade the surrounding parenchyma. In addition, a subpopulation of GBM stem cells has been reported to possess the ability to transdifferentiate into tumor-derived endothelial cells (TDECs), supporting the resistance to anti-angiogenic treatments of newly formed blood vessels. Bone Morphogenetic Protein 9 (BMP9) is critically involved in the processes of cancer cell differentiation, invasion and metastasis, representing a potential tool in order to impair the intrinsic GBM aggressiveness. Here we demonstrate that BMP9 is able to trigger the activation of SMADs in patient-derived GBM cells, and to strongly inhibit proliferation and invasion by reducing the activation of PI3K/AKT/MAPK and RhoA/Cofilin pathways, respectively. Intriguingly, BMP9 treatment is sufficient to induce a strong differentiation of GBM stem-like cells and to significantly counteract the already reported process of GBM cell transdifferentiation into TDECs not only in in vitro mimicked TDEC models, but also in vivo in orthotopic xenografts in mice. Additionally, we describe a strong BMP9-mediated inhibition of the whole angiogenic process engaged during GBM tumor formation. Based on these results, we believe that BMP9, by acting at multiple levels against GBM cell aggressiveness, can be considered a promising candidate, to be further developed, for the future therapeutic management of GBM.