High-risk neuroblastoma (NB) is an aggressive childhood tumor that originates from progenitor neural crest cells. Even if the therapeutic protocol for NB is articulate and aggressive, the outcome remains dismal, with the 5-year disease-free overall survival below rating 50%. A novel drug combination strategy can possibly provide a new solution to this unmet therapeutic need. 13-cis retinoic acid (13-cis-RA, isotretinoin) is an anti-proliferative and pro-differentiative agent currently used in the post-consolidation phase of NB therapy. To identify molecules able to potentiate the anti-proliferative activity of 13-cis-RA, NB cells were treated with a library of 169 naturally occurring polyphenols in combination with the retinoid. This in vitro screen led to the identification of isorhamnetin as a synergistic partner of 13-cis-RA, producing an 80% reduction in cell viability. At the molecular level, this synergistic effect is followed by a marked increase in the expression of a member of the catecholamine receptor superfamily: the adrenergic receptor alpha-1B (ADRA1B) suggesting that this receptor might represent a key mediator of the synergistic effect of 13-cis-RA and isorhamnetin observed in vitro. This finding redirected our attention to the class of adrenergic receptors (ARs) as novel targets in NB. To investigate the role of ADRA1B in the synergism, we generated CHP134 NB cell lines knocked-out (KO) for the receptor and observed that exposure of CHP134 KO cell to 13-cis-RA leads to a reduction of cell viability and neural differentiation. We, therefore, substituted the genetic KO strategy with the alpha-1B adrenergic antagonist, L765,314, obtaining the same results. Subsequently, we extended the analysis on the role of adrenergic receptors (AR) performing a biased screen using two libraries of AR-ligands. The screen results confirm that the molecules working as alpha-1-ARs antagonists are those that greatly increase cell sensitivity to 13-cis-RA with reduction of cell viability and increase in differentiation. We confirmed our observation in NB xenograft mice models in vivo, treating mice with a combination of 13-cis-RA and the FDA approved alpha-1 AR antagonist doxazosin. The proposed pharmacological treatment was effective in slowing tumor growth, leading to tumors of smaller size. From our results, we can conclude that the deletion or inhibition of alpha-1-AR sensitizes NB cells to 13-Cis-RA, both in terms of induction of apoptosis and neural differentiation. Since NB is a catecholamine-rich tumor, we propose that antagonization of alpha-1-AR disrupts the established autocrine pro-survival circuit generated by catecholamines in NB and restores the ability of the cells to follow the pro-differentiative and pro-apoptotic programs endorsed by 13-cis-RA. Considering the druggable nature of the alpha-1-AR receptors, we indicate this class of receptors as a novel pharmacological target for the treatment of neuroblastoma.

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Background: Neuroblastoma is the most common extracranial solid tumor in children. The survival rate in high-risk neuroblastoma is less than 50 % despite intensive multimodal therapy, and there is thus an immense need for new treatment options. In a previous preclinical study conducted at Uppsala University, treatment with sunitinib was found to inhibit tumor growth and angiogenesis in an orthotopic model of high-risk neuroblastoma.  Aim: The present study aimed to further explore the effect of sunitinib on tumor stroma, focusing on whether it was possible to detect and quantify tumor-associated neutrophils (TANs) in tumor sections from the above-mentioned study using immunohistochemistry (IHC). Methods: Tissue sections from formalin-fixated paraffin-embedded tumors were stained with anti-Ly-6G/Ly-6C, anti-ITGAM, or hematoxylin-eosin, and the number of granulocytes was quantified manually using a light microscope. An independent samples two-tailed t-test was used for statistical analysis. Results: The average number of granulocytes increased by 40 % in animals treated with sunitinib compared to control animals (p = 0.003) in hematoxylin-eosin stained tumor sections of orthotopic neuroblastomas. The results from the staining with anti-Ly-6G/Ly-6C or anti-ITGAM, on the other hand, were impossible to quantify due to the high background staining despite the concentration of antibody used. Conclusion: In conclusion, this report indicates that the density of TANs in an orthotopic murine neuroblastoma model is increased by treatment with sunitinib. However, to confirm this result, the study should be repeated once a reliable IHC method for the detection of TANs has been developed.

Neuroblastoma (NB) is a childhood cancer with a highly complex nature. High-risk NB patients undergo intensive treatment regimens that are often followed by long-term side effects. This, in addition to the emergence of resistant cancer cells, highlights a need for novel therapeutic targets and treatment strategies to improve outcome in NB. P21 is a cyclin-dependent kinase inhibitor considered to play a role in tumor resistance and aggressiveness due to its involvement in cell cycle and/or apoptosis. This project aimed to explore the expression of endogenous p21 in high-risk NB cell lines and whether p21 could be a therapeutic target for high-risk NB. Endogenous p21 levels were investigated using RT-qPCR and quantitative immunocytochemistry in eight high-risk NB cell lines. A small molecular inhibitor of p21, UC2288, was used in these cell lines to investigate tumour cell viability following p21 inhibition. In addition, combination treatment with UC2288 and the chemotherapy drug cisplatin was performed on resistant NB cell lines. Our results show variable expression of p21, where cell lines with high endogenous p21 expression showed sensitivity to single agent treatment with cisplatin or UC2288. Moreover, resistant NB cell lines showed lower endogenous p21 expression, however, combination treatment with UC2288 and cisplatin showed reduced viability, indicating sensitivity to combination treatment. This project highlights the potential of using p21 as a therapeutic target as well as a predictive biomarker in high-risk NB.

Gene expression regulation encompasses a wide range of mechanisms that govern cellular processes. Among these, post-transcriptional regulation, including translation control, plays a pivotal role in ensuring precise protein synthesis, timing, and quantity. Perturbations of mechanisms such as RNA modifications, and interactions between RNA-binding proteins (RBPs) and specific RNA motifs, can lead to dysregulation of essential cellular processes. These alterations contribute to the development of various disorders, including cancer, neurodegenerative diseases, and metabolic disorders. Many publicly available datasets and studies offer opportunities to investigate the link between alterations in these mechanisms and disease manifestations. However, the limited availability of datasets for certain conditions or notable inconsistencies among reported associations prevent complete understanding of the underlying processes. Therefore, extending the investigations to encompass a diverse range of genes and/or diseases will enhance our comprehension of these intricate regulatory and disease mechanisms, aiding in the identification of potential therapeutic targets and innovative interventions to mitigate pathological conditions. In particular, we focused on three separate aspects involved in gene expression regulation: RNA modifications, RBPs interactions with RNA secondary structures, and the Kozak consensus sequence as a translational modulator. Each part uncovers essential mechanisms that govern post-transcriptional control of gene expression, shedding light on their roles in cellular processes and disease contexts. At first, we performed a comprehensive exploration of 15 RBPs involved in the regulation of the N6-methyladenosine (m6A) methylation. Leveraging data from The Cancer Genome Atlas (TCGA), we conducted a pan-cancer analysis across 31 tumor types to uncover the distribution of alterations of these factors, and we developed a user-friendly web application to enable users to conduct similar analyses. Additionally, we performed a parallel analysis focused on neuroblastoma, using data from publicly available and in-house datasets. These investigations unveil the potential impact of a subset of m6A factors on cancer development and progression. While in the first case, VIRMA and YTHDF reader proteins, emerged as the most frequently altered genes with significant pan-cancer prognostic implications, in the context of neuroblastoma, the writer METTL14 and the reader ALKBH5, showed the most prominent roles. Subsequently, our focus shifted to a distinct subset of RBPs capable of interacting with RNA secondary structures, particularly with RNA G-quadruplexes (RG4s). We established a comprehensive database cataloging RBPs with potential RG4-binding capabilities. This resource represents a valuable tool for researchers aiming to explore the intricate interplays between RBPs and RG4s, and their putative implications in diverse biological processes and diseases. Finally, attention was directed to the Kozak sequence, a pivotal determinant of the regulation of translation initiation. Exploiting the power of base editors, we developed a method to optimize translation initiation by modifying the Kozak sequence. This strategy offers promise in addressing haploinsufficiency-related disorders, where enhancing the functional protein is essential. Overall, these findings present opportunities for the identification of potential therapeutic targets and precision medicine strategies to alleviate a spectrum of pathological conditions, thus fostering advancements in the field of molecular biology and disease management.

Objetivos: Avaliar os resultados das coletas de células hematopoéticas CD34+, por aférese, em crianças portadoras de neuroblastoma tratadas no Serviço de Oncologia e Hematologia do Instituto da Criança do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo; estudar os fatores (idade, peso, estimulação com quimioterapia, dose do G-CSF, uso terapêutico de 131I-MIBG e tempo entre exposição à quimioterapia prévia) que influenciaram na mobilização e no rendimento da coleta de células CD34+ no sangue periférico e associar a quantidade de células CD34+ obtidas com a evolução clínica do paciente. Métodos: Trata-se de um estudo retrospectivo de pacientes com neuroblastoma submetidos à coleta de células-tronco hematopoéticas entre janeiro de1989 e junho 2012. Resultados: Avaliados 45 prontuários de crianças com idade mediana de 3,1 anos (0-12 anos), 26 (57%) apresentavam metástase em medula óssea ao diagnóstico. O tempo entre diagnóstico e o início da mobilização foi em média 19,7 ± 12 meses (mediana de 15,8 meses). Dos pacientes estudados, 11/45 (24,4%) receberam 131I-MIBG terapêutico antes da mobilização. Somente cinco pacientes (11,1%) receberam quimioterapia associada ao G-CSF para mobilização; as demais 40 crianças (88,9%) receberam exclusivamente G-CSF na dose média 26,5 ± 5,3 ug/kg/dia (mediana 28 ug/kg/dia). Não houve correlação entre o número máximo de células CD34+ no sangue periférico com a idade (p=0,9), com o peso (p=0,63), com a dose do G-CSF (p=0,46) ou com o intervalo entre o diagnóstico e o início da mobilização (p=0,09). A mediana da quantificação de células CD34+/uL no sangue periférico foi de 36,6 células, média de 45,2 ± 42,6 (mínimo 1,7 e máximo 236,3). Pacientes que haviam recebido 131I-MIBG previamente à mobilização apresentaram menor número de células CD34+/uL no sangue periférico (p=0,04). Em 26 pacientes (57,8%) foi possível coletar mais de 2,0x106 células CD34+/Kg na primeira coleta e em 19 pacientes (42,2%) foram necessárias mais de uma coleta, sendo que, oito pacientes (17,8%) apresentaram falha de mobilização. Os pacientes que apresentaram menor quantidade de células CD34+/uL no sangue periférico (<= 12) não conseguiram número maior ou igual a 2,0x106 células CD34+/Kg em 81,8% das coletas. O número mediano de células infundidas foi de 2,66 x106 células CD34+/Kg (média 3,38 ±1,6; mínimo 1,8; máximo 8,74 x106 CD34+/Kg). Os pacientes apresentaram contagem de leucócitos maior que 1000/mm3 e de plaquetas maior 50000/mm3 por dois dias consecutivos em média, no dia 13 ± 10 e no dia 46 ± 33, respectivamente, após infusão. Conclusões: A coleta de células-tronco hematopoéticas por aférese foi factível em todos os pacientes do estudo. Não houve influência significativa da idade, do peso, da dose do G-CSF e do tempo entre diagnóstico e inicio da mobilização, no número máximo de células. O uso prévio à coleta de 131I-MIBG terapêutico parece influenciar negativamente no pico de células CD34+ no sangue periférico (p=0,04). A contagem de células CD34+ no sangue periférico é importante fator preditivo do resultado das coletas de células progenitoras hematopoéticas CD34+ por aférese / Objectives: To evaluate the results of peripheral hematopoietic CD34+ stem cells harvesting in children with neuroblastoma treated at Serviço de Oncologia e Hematologia do Instituto da Criança do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo; regarding age, weight, stimulation with chemotherapy, G-CSF dose, time between diagnosis and the mobilization beginning and therapeutic 131I-MIBG use and the influence in mobilization and peripheral harvesting of autologous hematopoietic stem cells and to associate the amount of CD34+ cells obtained with the patient\'s clinical evolution. Methods: Between January 1989 and June 2012, children with neuroblastoma underwent to mobilization and peripheral hematopoietic stem cell harvesting and were retrospectively analyzed. Results: The charts of 45 children were reviewed. Median age was 3.1 years (0-12years), and 26 (57%) had metastases in bone marrow at diagnosis. Average time between diagnosis and mobilization was 19.7 ± 12 months (median, 15.8 months). 11/45 (24.4%) received therapeutic 131I-MIBG prior to mobilization. The average G-CSF dose was 26.5 ± 5.3mg/kg/day (mean 28mg/kg/day). There was no correlation between the absolute number of peripheral CD34+ cells and age (p=0.9), weight (p=0.63), G-CSF dose (p=0.46) or the range between diagnosis and early mobilization (p=0.09). The median quantification of CD34+ cells/uL in peripheral blood was 36.6, average 45.2 ±42.6 (minimum 1.7 and maximum 236.3 CD34+ cells/uL). Patients who had received therapeutic 131I-MIBG prior to mobilization, showed fewer absolute amount of CD34+/uL cells in peripheral blood (p=0.04). In 26 patients (57.8%) it was possible to harvest more than 2.0 x106 CD34+ cells/kg at first apheresis and in 19 patients (42.2%) more than one collection were necessary, and eight patients (17.8 %) failure to mobilize. Patients presenting less than 12 CD34+ cells/uL in peripheral blood on the harvesting day failed to reach more then 2.0x106 cells CD34+/kg in 81.8% of the apheresis procedures. It was infused a median number of 2.66 x106 CD34+ cells/kg (mean 1.6 ± 3.38; min 1.8; max 8.74 x106 CD34+ cells/kg). After the stem cells infusion, patients had white blood cells count greater than 1000/mm3 and platelet greater than 50,000/mm3 for two consecutive days on average after 13 ±10 and 46 ± 33 days, respectively. Conclusions: The hematopoietic stem cells harvesting was feasible in all patients included in this report. The G-CSF dose, age, weight and the period between harvesting and diagnosis showed no influence in mobilization and harvesting of autologous hematopoietic stem cells, however the absolute number of peripheral blood CD34+ cells/uL is an important predictive factor for the harvesting outcome. Additionally our findings support for the first time the notion that the use of therapeutic 131I-MIBG could have a negative impact in mobilization of peripheral blood stem cells in children with neuroblastoma

Objetivos: Avaliar os resultados das coletas de células hematopoéticas CD34+, por aférese, em crianças portadoras de neuroblastoma tratadas no Serviço de Oncologia e Hematologia do Instituto da Criança do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo; estudar os fatores (idade, peso, estimulação com quimioterapia, dose do G-CSF, uso terapêutico de 131I-MIBG e tempo entre exposição à quimioterapia prévia) que influenciaram na mobilização e no rendimento da coleta de células CD34+ no sangue periférico e associar a quantidade de células CD34+ obtidas com a evolução clínica do paciente. Métodos: Trata-se de um estudo retrospectivo de pacientes com neuroblastoma submetidos à coleta de células-tronco hematopoéticas entre janeiro de1989 e junho 2012. Resultados: Avaliados 45 prontuários de crianças com idade mediana de 3,1 anos (0-12 anos), 26 (57%) apresentavam metástase em medula óssea ao diagnóstico. O tempo entre diagnóstico e o início da mobilização foi em média 19,7 ± 12 meses (mediana de 15,8 meses). Dos pacientes estudados, 11/45 (24,4%) receberam 131I-MIBG terapêutico antes da mobilização. Somente cinco pacientes (11,1%) receberam quimioterapia associada ao G-CSF para mobilização; as demais 40 crianças (88,9%) receberam exclusivamente G-CSF na dose média 26,5 ± 5,3 ug/kg/dia (mediana 28 ug/kg/dia). Não houve correlação entre o número máximo de células CD34+ no sangue periférico com a idade (p=0,9), com o peso (p=0,63), com a dose do G-CSF (p=0,46) ou com o intervalo entre o diagnóstico e o início da mobilização (p=0,09). A mediana da quantificação de células CD34+/uL no sangue periférico foi de 36,6 células, média de 45,2 ± 42,6 (mínimo 1,7 e máximo 236,3). Pacientes que haviam recebido 131I-MIBG previamente à mobilização apresentaram menor número de células CD34+/uL no sangue periférico (p=0,04). Em 26 pacientes (57,8%) foi possível coletar mais de 2,0x106 células CD34+/Kg na primeira coleta e em 19 pacientes (42,2%) foram necessárias mais de uma coleta, sendo que, oito pacientes (17,8%) apresentaram falha de mobilização. Os pacientes que apresentaram menor quantidade de células CD34+/uL no sangue periférico (<= 12) não conseguiram número maior ou igual a 2,0x106 células CD34+/Kg em 81,8% das coletas. O número mediano de células infundidas foi de 2,66 x106 células CD34+/Kg (média 3,38 ±1,6; mínimo 1,8; máximo 8,74 x106 CD34+/Kg). Os pacientes apresentaram contagem de leucócitos maior que 1000/mm3 e de plaquetas maior 50000/mm3 por dois dias consecutivos em média, no dia 13 ± 10 e no dia 46 ± 33, respectivamente, após infusão. Conclusões: A coleta de células-tronco hematopoéticas por aférese foi factível em todos os pacientes do estudo. Não houve influência significativa da idade, do peso, da dose do G-CSF e do tempo entre diagnóstico e inicio da mobilização, no número máximo de células. O uso prévio à coleta de 131I-MIBG terapêutico parece influenciar negativamente no pico de células CD34+ no sangue periférico (p=0,04). A contagem de células CD34+ no sangue periférico é importante fator preditivo do resultado das coletas de células progenitoras hematopoéticas CD34+ por aférese / Objectives: To evaluate the results of peripheral hematopoietic CD34+ stem cells harvesting in children with neuroblastoma treated at Serviço de Oncologia e Hematologia do Instituto da Criança do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo; regarding age, weight, stimulation with chemotherapy, G-CSF dose, time between diagnosis and the mobilization beginning and therapeutic 131I-MIBG use and the influence in mobilization and peripheral harvesting of autologous hematopoietic stem cells and to associate the amount of CD34+ cells obtained with the patient\'s clinical evolution. Methods: Between January 1989 and June 2012, children with neuroblastoma underwent to mobilization and peripheral hematopoietic stem cell harvesting and were retrospectively analyzed. Results: The charts of 45 children were reviewed. Median age was 3.1 years (0-12years), and 26 (57%) had metastases in bone marrow at diagnosis. Average time between diagnosis and mobilization was 19.7 ± 12 months (median, 15.8 months). 11/45 (24.4%) received therapeutic 131I-MIBG prior to mobilization. The average G-CSF dose was 26.5 ± 5.3mg/kg/day (mean 28mg/kg/day). There was no correlation between the absolute number of peripheral CD34+ cells and age (p=0.9), weight (p=0.63), G-CSF dose (p=0.46) or the range between diagnosis and early mobilization (p=0.09). The median quantification of CD34+ cells/uL in peripheral blood was 36.6, average 45.2 ±42.6 (minimum 1.7 and maximum 236.3 CD34+ cells/uL). Patients who had received therapeutic 131I-MIBG prior to mobilization, showed fewer absolute amount of CD34+/uL cells in peripheral blood (p=0.04). In 26 patients (57.8%) it was possible to harvest more than 2.0 x106 CD34+ cells/kg at first apheresis and in 19 patients (42.2%) more than one collection were necessary, and eight patients (17.8 %) failure to mobilize. Patients presenting less than 12 CD34+ cells/uL in peripheral blood on the harvesting day failed to reach more then 2.0x106 cells CD34+/kg in 81.8% of the apheresis procedures. It was infused a median number of 2.66 x106 CD34+ cells/kg (mean 1.6 ± 3.38; min 1.8; max 8.74 x106 CD34+ cells/kg). After the stem cells infusion, patients had white blood cells count greater than 1000/mm3 and platelet greater than 50,000/mm3 for two consecutive days on average after 13 ±10 and 46 ± 33 days, respectively. Conclusions: The hematopoietic stem cells harvesting was feasible in all patients included in this report. The G-CSF dose, age, weight and the period between harvesting and diagnosis showed no influence in mobilization and harvesting of autologous hematopoietic stem cells, however the absolute number of peripheral blood CD34+ cells/uL is an important predictive factor for the harvesting outcome. Additionally our findings support for the first time the notion that the use of therapeutic 131I-MIBG could have a negative impact in mobilization of peripheral blood stem cells in children with neuroblastoma