Drug induced degradation of driver proteins: A novel approach to target MLL-fusions

Acute leukaemias in infants are associated with inferior outcomes. The majority of infant acute leukaemia is characterized by balanced chromosomal translocations involving the mixed lineage leukaemia (MLL) gene. Previous work in the department established that the novel formed MLL fusions are the proto-oncoproteins responsible for leukaemia initiation and maintenance so that inhibition of MLL-fusions, in conditional mouse models, resulted in complete disease remission. Therefore, a therapy that inactivates the MLL fusion protein, by protein degradation for example, would offer new hope to these patients. The aim of this study is to identify clinically approved drugs that are capable of inducing degradation of MLL leukaemic fusion proteins. An indicator cell line consisting of a THP1 cell clone expressing firefly luciferase fused to the MLL-AF9 protein, combined with renilla luciferase, was used to screen a collection of 1,280 approved drugs for their ability to induce proteolysis of MLL fusion proteins. 25 drugs lowered the luciferase to renilla ratio, of which 3 were confirmed by western blotting to decrease MLL-fusion protein levels. One drug was taken forward for further analysis. This drug was able to induce the proteolysis of various MLL fusion proteins in human MLL rearranged cell lines and primary patient material. Transcriptome profiling showed shut down of the MLL-fusion signature within 16hrs of addition of the drug. Proteolysis of MLL fusion proteins should also result in a block in self-renewal, as was previously shown in the conditional mouse model. While the drug had no significant impact on the colony formation of normal haematopoietic progenitor cells, it was able to block the colony formation ability of MLL rearranged cell lines. Finally, global alterations in the epigenetic landscape following drug treatment were analysed. This study highlights a new approach to identifying drugs that block driver oncogenes and has identified a potential novel treatment for a major subtype of acute leukaemias in infants

Frizzled receptor 6 marks rare, highly tumourigenic stem-like cells in mouse and human neuroblastomas

Copyright © 2011 Cantilena et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The article was made available through the Brunel Open Access Publishing Fund.Wnt signalling is an important component of vertebrate development, required for specification of the neural crest. Ten Wnt receptors [Frizzled receptor 1-10 (Fzd1-10)] have been identified so far, some of which are expressed in the developing nervous system and the neural crest. Here we show that expression of one such receptors, Fzd6, predicts poor survival in neuroblastoma patients and marks rare, HIF1/2 α-positive cells in tumour hypoxic areas. Fzd6 positive neuroblastoma cells form neurospheres with high efficiency, are resistant to doxorubicin killing and express high levels of mesenchymal markers such as Twist1 and Notch1. Expression of Fzd6 is required for the expression of genes of the noncanonical Wnt pathway and the spheres forming activity. When transplanted into immunodeficient mice, neuroblastoma cells expressing the Fzd6 marker grow more aggressively than their Fzd6 negative counterparts. We conclude that Fzd6 is a new surface marker of aggressive neuroblastoma cells with stem cell-like features.This work was sponsored by the Wellcome Trust, the RICC cancer fund, SPARKS, the Italian Association for Cancer Research, Regione Liguria and the Italian Ministry of Health

Physical interaction between MYCN oncogene and polycomb repressive complex 2 (PRC2) in neuroblastoma: Functional and therapeutic implications

This article is made available through the Brunel Open Access Publishing Fund. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.CLU (clusterin) is a tumor suppressor gene that we have previously shown to be negatively modulated by the MYCN proto-oncogene, but the mechanism of repression was unclear. Here, we show that MYCN inhibits the expression of CLU by direct interaction with the non-canonical E box sequence CACGCG in the 5′-flanking region. Binding of MYCN to the CLU gene induces bivalent epigenetic marks and recruitment of repressive proteins such as histone deacetylases and Polycomb members. MYCN physically binds in vitro and in vivo to EZH2, a component of the Polycomb repressive complex 2, required to repress CLU. Notably, EZH2 interacts with the Myc box domain 3, a segment of MYC known to be essential for its transforming effects. The expression of CLU can be restored in MYCN-amplified cells by epigenetic drugs with therapeutic results. Importantly, the anticancer effects of the drugs are ablated if CLU expression is blunted by RNA interference. Our study implies that MYC tumorigenesis can be effectively antagonized by epigenetic drugs that interfere with the recruitment of chromatin modifiers at repressive E boxes of tumor suppressor genes such as CLU.SPARKS, The Neuroblastoma Society, a Wellcome Trust grant (to A. S.), and the Italian Association for Cancer Research

Moneta Docet. Per l’avvio di un dialogo sull’insegnare a leggere la moneta antica

In difficult times for the humanities – and in particular for the antiquities – it is intended to present a reasoned note on the contribution that the study of coins brings today to historical and archaeological knowledge and on the situation, the critical points and the ways of teaching the Numismatics in Italian universities

Due studi sulla VII Olimpica di Pindaro: 2. I significati della pioggia d’oro

Analysis of the mythological and religious meaning of the golden rain in Pindar’s seventh Olympian ode, with reference to the birth of the island of Rhodes. For the first part, see “Prometheus” 13, 1987, 209-216

Micro-introducción a Homero

What do we talk about when we talk about Homer? It is difficult to conceive him as a man, a historical individuum, and the ancient Greeks knew about him as little as we do. Even if we question his poems, we cannot grasp any trait of an author's personality. But his language and his style tell us enough to understand that what we were searching in a single poet must be found in a tradition. The Iliad and the Odyssey must be understood as documents of oral-traditional poetry and not of literature: at least in the sense that they were not meant to be read. Although they have been considered for centuries the archetypes of all western literatures, their place is among the crowded company of the anonymous singers who gave us masterpieces like Beowulf, Mahabharata, Chanson de Roland.¿De qué hablamos cuando hablamos de Homero? Es difícil concebirlo como un hombre, como un individuo histórico, y los antiguos griegos conocían sobre él tan poco como nosotros. Incluso si examinamos sus poemas, no podemos captar ningún rasgo de la personalidad de un autor. Pero su lenguaje y su estilo nos dicen lo suficiente como para comprender que lo que hemos estado buscando en un solo poeta debe encontrarse en una tradición. La Ilíada y la Odisea deben entenderse como documentos de poesía oral tradicional y no de literatura: al menos en el sentido de que no estaban destinados a ser leídos. Aunque desde siglos se consideren los arquetipos de todas las literaturas occidentales, su lugar está entre la concurrida compañía de los cantores anónimos que nos legaron obras maestras como Beowulf, Mahabharata, Chanson de Roland

The Role of Endoglin in the Immunomodulatory Capacities of Mesenchymal Stem Cells and the Relationship to Hyperbaric Oxygen Therapy

Mesenchymal stem cells (MSCs), a type of “adult” stem cell found in most organs, represent an emerging tool in the field of regenerative medicine. In the setting of myocardial injury, for example, MSCs have been shown to promote repair and recovery. Studies have been hampered, however, by variation in MSC phenotypes, as defined by cell surface marker expression, and the absence of a clear consensus of what MSC phenotypes are best able to support regeneration. Providing insight into the regenerative capabilities of varying MSCs phenotypes is crucial to their continued success in therapies. One cell surface marker that has been shown to have functional relevance to regenerative medicine is endoglin (CD105). Endoglin is a type I transmembrane protein that functions as an auxiliary receptor for the TGFβ receptor complex. The absence of endoglin on MSCs reportedly defines a population with a greater propensity for cardiovascular differentiation and greater capacity to support myocardial repair. However, despite their ability to improve post-infarct cardiac function and reduce the size of resultant scars, the retention of these cells in myocardial tissue varies from only 3%-6%. Thus, transdifferentiation seems an unlikely explanation for the regenerative effects. In addition to their capacity for multipotential differentiation, MSCs also have a reported ability to suppress various immune responses, including suppression of stimulated T-cell proliferation. I hypothesized that the absence of CD105 may identify a population of MSCs with an altered capacity to modulate the immunologic milieu at the time of myocardial injury, and this difference in immunomodulatory function accounts for the improved outcomes. To investigate this idea, MSCs that either express endoglin (CD105+) or not (CD105-) were co-cultured with T-cells and effects on stimulated T-cell proliferation examined. Surprisingly, neither CD105+ nor CD105- MSCs were able to suppress proliferation of either CD4+ or CD8+ T-cells. In light of this observation, effects of MSCs on T-cell differentiation were assessed. Notably, co-culture of both CD105+ and CD105- MSCs with CD4+ T-cells showed a striking effect on T-cell differentiation. Syngeneic MSCs induced Th2 skewing, with increased expression of IL-4 and IL-10 and a marked decrease in IL-17 expression. The presence of CD105 in the MSCs influenced this outcome, with a more pronounced decrease in IL-17 expression and increased IL-4 secretion. Effects of allogeneic MSCs on T-cell differentiation were also examined. Due to the reported “immune privilege” of MSCs, it has been proposed that allogeneic MSCs may be utilized to expand the pool of MSCs available for clinical use. In an allogeneic co-culture system, both CD105+ and CD105- MSCs significantly affected T-cell differentiation. Compared to syngeneic MSCs, allogeneic MSCs stimulated higher expression of IL-4, IL-5, and IL-10, as well as increased secretion of IL-4 and IL-10. There were fewer differences between CD105+ and CD105- MSCs. However, CD105- MSCs induced much less IL-2 and IFNγ compared to CD105+ MSCs. These results indicate that MSCs influence T-cell differentiation, resulting in a Th2 skewing with increased production of the immunosuppressive cytokine IL-10 and, in the case of syngeneic cells, diminished Th17 differentiation. This effect could be essential for the previously described MSC-induced cardiac functional preservation, and could explain differences between CD105+ and CD105- phenotypes. In addition to these mechanistic studies, I also examined potential clinical relevance of CD105 expression in MSCs in umbilical cord blood transplantation. This question arose from a clinical study was underway at the University of Kansas Cancer Center that focused on using hyperbaric oxygen (HBO) for improving clinical outcomes post umbilical cord blood (UCB) transplant. In the observation of post-transplant transfusion requirements, HBO-treated patients required less supportive blood products than historic UCB-recipients. Furthermore, they experienced decreased time to transfusion independence. However, upon examining the correlation between levels of erythropoietin in these HBO-patients to their transfusion requirements, the data did not match with animal studies, which showed a reduction in erythropoietin was the causative method by which HBO improved engraftment. Therefore, we examined the supportive role of MSCs in response to HBO in the hematopoietic niche. This included observing changes in CD105 on MSCs after exposure to HBO, as endoglin is a known hypoxia response gene. While the observation of immunomodulatory factors produced by MSCs as a result of HBO is still underway, a downregulation of CD105 12 hours after exposure to HBO was observed. This correlates to the homing window of hematopoietic stem cells in the context of transplant and may be indicative of changes in MSCs providing a more supportive bone marrow microenvironment after exposure to HBO-therapy

A chemical screen identifies the chemotherapeutic drug topotecan as a specific inhibitor of the B-MYB/MYCN axis in neuroblastoma

The transcription factor MycN is the prototypical neuroblastoma oncogene and a potential therapeutic target. However, its strong expression caused by gene amplification in about 30% of neuroblastoma patients is a considerable obstacle to the development of therapeutic approaches aiming at eliminating its tumourigenic activity. We have previously reported that B-Myb is essentially required for transcription of the MYCN amplicon and have also shown that B-MYB and MYCN are engaged in a feed forward loop promoting the survival/proliferation of neuroblastoma cells. We postulated that pharmacological strategies breaking the B-MYB/MYCN axis should result in clinically desirable effects. Thus, we implemented a high throughput chemical screen, using a curated library of ~1500 compounds from the National Cancer Institute, whose endpoint was the identification of small molecules that inhibited B-Myb. At the end of the screening, we found that the compounds pinafide, ellipticine and camptothecin inhibited B-Myb transcriptional activity in luciferase assays. One of the compounds, the topoisomerase-1 inhibitor camptothecin, is of considerable clinical interest since its derivatives topotecan and irinotecan are currently used as first and second line treatment agents for various types of cancer, including neuroblastoma. We found that neuroblastoma cells with amplification of MYCN are more sensitive than MYCN negative cells to camptothecin and topotecan killing. Campothecin and topotecan caused selective down-regulation of B-Myb and MycN expression in neuroblastoma cells. Notably, forced overexpression of B-Myb could antagonize the killing effect of topotecan and camptothecin, demonstrating that the transcription factor is a key target of the drugs. These results suggest that camptothecin and its analogues should be more effective in patients whose tumours feature amplification of MYCN and/or overexpression of B-MYB

Polyphenon E enhances the antitumor immune response in neuroblastoma by inactivating myeloid suppressor cells

This is the author's accepted manuscript. The final published article is available from the link below. Note: In this manuscript as well as in the original published version of this article the word "Polyphenon" was incorrectly spelled in the title as "Polyphenol."Purpose: Neuroblastoma is a rare childhood cancer whose high risk, metastatic form has a dismal outcome in spite of aggressive therapeutic interventions. The toxicity of drug treatments is a major problem in this pediatric setting. In this study, we investigated whether Polyphenon E, a clinical grade mixture of green tea catechins under evaluation in multiple clinical cancer trials run by the National Cancer Institute (Bethesda, MD), has anticancer activity in mouse models of neuroblastoma. Experimental Design: We used three neuroblastoma models: (i) transgenic TH-MYCN mouse developing spontaneous neuroblastomas; (ii) nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenotransplanted with human SHSY5Y cells; and (iii) A/J mice transplanted with syngeneic Neuro 2A cells. Mice were randomized in control and Polyphenon E–drinking groups. Blood from patients with neuroblastoma and normal controls was used to assess the phenotype and function of myeloid cells. Results: Polyphenon E reduced the number of tumor-infiltrating myeloid cells, and inhibited the development of spontaneous neuroblastomas in TH-MYCN transgenic mice. In therapeutic models of neuroblastoma in A/J, but not in immunodeficient NOD/SCID mice, Polyphenon E inhibited tumor growth by acting on myeloid-derived suppressor cells (MDSC) and CD8 T cells. In vitro, Polyphenon E impaired the development and motility of MDSCs and promoted differentiation to more neutrophilic forms through the 67 kDa laminin receptor signaling and induction of granulocyte colony-stimulating factor. The proliferation of T cells infiltrating a patient metastasis was reactivated by Polyphenon E. Conclusions: These findings suggest that the neuroblastoma-promoting activity of MDSCs can be manipulated pharmacologically in vivo and that green tea catechins operate, at least in part, through this mechanism.SPARKS, Research in Childhood Cancer, the CGD Research Trust, and the Wellcome Trust