MYCN and the p53-MDM2/MDMX-p14ARF network in neuroblastoma amd response to MDM2-p53 antagonists

Background: MYCN-amplification is a major negative prognostic marker, occurring in 25-30% of neuroblastomas. MYCN plays contradictory roles in promoting cell growth and sensitizing cells to apoptosis, and we have recently shown that p53 is a direct transcriptional target of MYCN, and may be an important mechanism of MYCN-induced apoptosis. Although p53 mutations are rare in neuroblastoma at diagnosis, the p53/MDM2/p14ARF pathway is inactivated in 35% of cases through MDM2-amplification or p14ARF inactivation. Neuroblastoma is therefore an ideal target for p53 reactivation using MDM2-p53 antagonists. MDMX, a homologue of MDM2, is another negative regulator of p53 which is often overexpressed in cancers and has been shown to compromise the effects of MDM2-p53 antagonists in various cancer types. MDMX expression and the effect on MDM2-p53 antagonists has not been investigated in neuroblastoma. Hypotheses 1) Reactivation of p53 by inhibition of its negative regulator MDM2, using the MDM2-p53 antagonists Nutlin-3 and MI-63, will result in p53-mediated growth arrest and apoptosis preferentially in MYCN-amplified cells 2) MDMX knockdown increases and p14ARF knockdown decreases the sensitivity of neuroblastoma cell lines to MDM2-p53 antagonists. Methods: The effect of MYCN, MDM2, MDMX and p14ARF was investigated on the response to MDM2-p53 antagonists using siRNA in a panel of 21 neuroblastoma cell lines. Sensitivity was measured by growth inhibition, apoptosis assays including caspase activity and fluorescent activated cell sorting, and the effect on the p53 response measured by Western blotting. Results: Using the SHEP Tet21N MYCN regulatable system, MYCN(-) cells were more resistant to both Nutlin-3 and MI-63 mediated growth inhibition and apoptosis compared to MYCN(+) cells and siRNA mediated knockdown of MYCN in 4 MYCN-amplified cell lines resulted in decreased p53 expression and activation, as well as decreased levels of apoptosis following treatment with MDM2-p53 antagonists. In a panel of cell lines treated with Nutlin-3 and MI-63, the sub-set amplified for MYCN had a significantly lower mean GI50 value and increased caspase 3/7 activity compared to the non-MYCN-amplified group of cell lines, but p53 mutant cell lines were resistant to the antagonists regardless of MYCN status. Knockdown of MDM2 did not alter the apoptotic response to Nutlin-3 or MI-63 but surprisingly, knockdown of MDMX resulted in decreased levels of apoptosis. MDMX expression varied amongst the neuroblastoma cell lines and positively correlated with caspase 3/7 activity following MDM2-p53 antagonist treatment. p14ARF impaired cell lines underwent less apoptosis following MDM2-p53 antagonist treatment and following Nutlin-3 treatment, 3 of 4 p14ARF impaired cell lines underwent a pronounced G1 arrest. p14ARF knockdown alone resulted in decreased caspase 3/7 activity, and following MDM2-p53 antagonist treatment there was decreased caspase 3 cleavage and activity, and decreased PARP cleavage. Conclusions: Amplification or overexpression of MYCN sensitizes neuroblastoma cell lines with wildtype p53 to MDM2-p53 antagonists and these compounds may therefore be particularly effective in treating high risk MYCN-amplified disease. This data also suggests that neuroblastomas with high MDMX expression may be more susceptible to MDM2-p53 antagonist treatment, but that cells with inactivated p14ARF predominantly undergo a G1 arrest which may protect them from apoptosis. MDMX and p14ARF status may therefore be important in addition to MYCN in determining the outcome of neuroblastomas treated with MDM2-p53 antagonists.EThOS - Electronic Theses Online ServiceBACRCancer Research UKNewcastle UniversityGBUnited Kingdo

Nur77 and FHL2: Novel players in vascular and immune disease

The nuclear receptor Nur77 is an early response gene that is induced by diverse extracellular signals in a wide range of tissues and cultured cells. It has been implicated in the regulation of genes involved in metabolic disease, adipogenesis, inflammation, and vascular disease. FHL2/DRAL/SLIM3 is a LIM-only protein that has been shown to interact with many proteins and acts as a co-activator or co- repressor depending on the cell-type and cellular context. It is a crucial adaptor protein and plays a pivotal role in a range of physiological and pathological processes, including proliferation, migration, differentiation and apoptosis. The aim of this thesis is to increase our understanding of fundamental pathways critical in vascular diseases including atherosclerosis, restenosis, coagulation, and immune diseases including asthma, airway inflammation and schistosomiasis. To achieve this goal, we performed numerous distinct studies on the role of nuclear receptor Nur77 and LIM-only protein FHL2 using mouse models and several cell types. In this thesis, functional properties of Nur77 and FHL2, as well as their impact on multiple signaling pathways in vascular and immune disease were studied. These research efforts are ultimately directed at designing novel therapeutic strategies that may aid in mitigating the development of vascular and immune disease

Advancing induced pluripotent stem cell (iPSC) technology by assessing genetic instability and immune response

[eng] Induced pluripotent stem cells (iPSC) can be made from adult somatic cells by reprogramming them with Oct4, Sox2, Klf4 and c-Myc. IPSC have given rise to a new technology to study and treat human disease (Takahashi et al., 2007). However, before iPSC clinical application, we need to step back and address two main challenges: (i) Genetic stability of iPSC. (ii) Immune response of iPSC-derived cells. To address these key issues, the overall mission of this PhD thesis is to advance iPSC technology by addressing two objectives. First, is to replace c-Myc with Cyclin D1 in the reprogramming cocktail (Oct4, Sox2, Klf4 and c-Myc or Cyclin D1) and second, to study the immune response of iPSC-derived cells. The quality of the starting iPSC determines the quality of the differentiated cells to be transplanted for clinical applications. In terms of genetic stability, aberrant cell reprogramming leads to genetic and epigenetic modifications that are the most significant barriers to clinical applications of patient iPSC derivatives (Gore et al., 2011). Such aberrations can result from the cellular stress that accompanies reprogramming or from the reprogramming factors themselves (Lee et al., 2012a). IPSC made with c-Myc are neoplastic in mouse models and have a higher tumorigenic potential than embryonic stem cells, prompting a search for new pluripotency factors that can replace the oncogenic factors Klf4 and c-Myc (Huangfu et al., 2008; Miura et al., 2009; Okita et al., 2007). We chose Cyclin D1 to replace c-Myc because of previous observation it can be used to reprogram cells to iPSC (Edel et al., 2010) and because of its DNA repair function (Chalermrujinanant et al., 2016). In this thesis we adopt a synthetic mRNA method to demonstrate that Cyclin D1 and c-Myc made iPSC have equal pluripotency using standard methods of characterisation. Moreover, no significant changes in copy number variation were found between starting skin cells and iPSC highlighting it is the method of choice for generating high quality iPSC. Further in- depth analysis revealed that Cyclin D1 made iPSC have reduced genetic instability assessed by: (i) reduced DNA double strand breaks (DSB), (ii) higher nuclear amount of the homologous recombination key protein Rad51, (iii) reduced multitelomeric signals (MTS) and (iv) reduced teratoma growth kinetics in vivo, compared to c-Myc made iPSC. Moreover, we demonstrate that Cyclin D1 iPSC derived neural stem cells engraft successfully, survive long term and differentiate into mature neuron cell types with high efficiency, with no evidence of pathology in a spinal cord injury rat model. As we move towards the clinic with iPSC-derived cells for cell transplantation, the immunogenic response is thought to be one of the main advantages of iPSC technology for clinical application, because of its perceived lack of immune rejection of autologous cell therapy. We hypothesize that iPSC derived cells are unlikely to provoke an immune response. Here we have performed an analysis of the innate and adaptive immune response of human skin cells (termed F1) reprogramed to iPSC and then compared to iPSC-derived cells (termed F2) using proteomic and methylome arrays. We found little differences between MHCI expression and function; however, we discovered a short isoform of the Toll-like receptor 3 (TLR3), essential for viral dsRNA innate immune recognition, which is predominantly upregulated in all iPSC derived cells analysed and not seen in normal endogenous cells. High levels of the TLR3 isoform is associated with unresponsiveness to viral stimulation measured by lack of IL6 secretion in iPSC derived neural stem cells. We propose a new model that TLR3 short isoform competes with the full length wild type isoform destabilizing the essentially required TLR3 dimerization process. These differences could result in supressed inflammatory effects for transplanted human iPSC-derived cells in response to viral or bacterial insult. Further work to determine the in vivo effects is warranted and calls for screening of iPSC lines for TLR3 isoform expression levels before clinical use. In conclusion, this thesis has advanced iPSC technology by defining a new method that is a significant advance with novel insights that has immediate impact on current methods to generate iPSC for clinical application and more accurate disease modelling.[cat] Les cèl·lules mare pluripotents induïdes (iPSC) es poden derivar de cèl·lules somàtiques adultes mitjançant la reprogramació amb Oct4, Sox2, Klf4 i c-Myc. Les iPSC han donat lloc a una nova tecnologia per estudiar i tractar malalties humanes (Takahashi et al., 2007). No obstant, abans de la aplicació clínica de les iPSC, dos problemes principals han de ser adreçats: (i) Estabilitat genètica de les iPSC. (ii) Resposta immune de les cèl·lules derivades de iPSC. Per adreçar aquests dos qüestions cabdals, la missió principal d’aquest doctorat és avançar la tecnologia de les iPSC adreçant dos objectius. El primer, és la substitució de c-Myc per Ciclina D1 al còctel de reprogramació (Oct4, Sox2, Klf4 and c-Myc o Ciclina D1) i segon, estudiar la resposta immune de les cèl·lules derivades de iPSC. Hem escollit Ciclina D1 per substituir c-Myc atès a observacions prèvies que pot ser emprat per reprogramar (Edel et al., 2010) i donada la seva funció en reparació de l’ADN (Chalermrujinanant et al., 2016). Les iPSC reprogramades amb Ciclina D1 presenten una pluripotència similar a les reprogramades amb c-Myc, l’anàlisi en profunditat mostra però, que les iPSC reprogramades amb Cyclin D1 tenen una reduïda inestabilitat genètica adreçada per: (i) reducció en ruptures de doble cadena de DNA, (ii) major quantitat nuclear de la proteïna clau en la recombinació homòloga Rad51, (iii) reducció en senyals multitelomèriques (MTS) i (iv) reducció en la cinètica de creixement de teratomas in vivo, en comparació amb iPSC reprogramades amb c-Myc. A més a més, demostrem que les cèl·lules mare neuronals derivades d’aquestes iPSC son capaces de implantar-se exitosament, sobreviure a llarg termini i diferenciar a neurones madures sense evidències de patologia en un model de dany medul·lar. També hem realitzat un anàlisi del sistema immune innat i adaptatiu de cèl·lules humanes de la pell (nomenades F1) reprogramades a iPSC i comparades amb cèl·lules derivades de iPSC (nomenades F2). Hem descobert una isoforma curta del Toll-Like Receptor 3 (TLR3), essencial en el reconeixement de RNA de doble cadena d’origen víric, que està predominantment sobreexpresada en totes les cèl·lules derivades de iPSC analitzades i no trobat en cèl·lules endògenes. Nosaltres proposem un nou model per el qual la isoforma curta del TLR3 competeix amb la isoforma llarga wild type desestabilitzant el procés essencial de dimerització del TLR3