Structure and function of centromere

Kromosomi predstavljaju iznimno važan dio eukariotske stanice. Oni nose nasljednu uputu (DNA) i osiguravaju prijenos genetičke informacije s roditelja na potomstvo. Veoma važan dio kromosoma jesu centromeri koji zajedno s kinetohornim aparatom čine vezu između kromosoma i mikrotubula diobenog vretena. U ovom seminaru objašnjena je specifična građa centromera na razini nukleosoma. Također je opisana građa kinetohora te na koji se način osigurava kretanje kinetohora, a samim time i kromosoma uzduž mikrotubula diobenog vretena. Na kraju, dan je osvrt na proces neocentromerizacije (stvaranje novih centromera) te je objašnjena uloga centromera, kinetohora i neocentromera u mehanizmu nastanka poremećaja vezanih uz segregaciju kromosoma.Chromosomes are very important part of eukaryotic cells. They carry genetic information (DNA) and provide transfer from parents to the offspring. Centromere and kinetochore are very important part of eukaryotic chromosome because they make link between chromosome and mitotic spindle and thus provide chromosome segregation. In this seminar specific architecture of centromere is explained. It also describes kinetochore architecture that provides kinetochore movement and thus chromosomes along mitotic spindle microtubules. At the end are an overview of neocentromerisation process and the role of centromere, kinetochore and neocentromeres in the chromosomes segregation disorders

Structure and function of centromere

Kromosomi predstavljaju iznimno važan dio eukariotske stanice. Oni nose nasljednu uputu (DNA) i osiguravaju prijenos genetičke informacije s roditelja na potomstvo. Veoma važan dio kromosoma jesu centromeri koji zajedno s kinetohornim aparatom čine vezu između kromosoma i mikrotubula diobenog vretena. U ovom seminaru objašnjena je specifična građa centromera na razini nukleosoma. Također je opisana građa kinetohora te na koji se način osigurava kretanje kinetohora, a samim time i kromosoma uzduž mikrotubula diobenog vretena. Na kraju, dan je osvrt na proces neocentromerizacije (stvaranje novih centromera) te je objašnjena uloga centromera, kinetohora i neocentromera u mehanizmu nastanka poremećaja vezanih uz segregaciju kromosoma.Chromosomes are very important part of eukaryotic cells. They carry genetic information (DNA) and provide transfer from parents to the offspring. Centromere and kinetochore are very important part of eukaryotic chromosome because they make link between chromosome and mitotic spindle and thus provide chromosome segregation. In this seminar specific architecture of centromere is explained. It also describes kinetochore architecture that provides kinetochore movement and thus chromosomes along mitotic spindle microtubules. At the end are an overview of neocentromerisation process and the role of centromere, kinetochore and neocentromeres in the chromosomes segregation disorders

Structure and function of centromere

Kromosomi predstavljaju iznimno važan dio eukariotske stanice. Oni nose nasljednu uputu (DNA) i osiguravaju prijenos genetičke informacije s roditelja na potomstvo. Veoma važan dio kromosoma jesu centromeri koji zajedno s kinetohornim aparatom čine vezu između kromosoma i mikrotubula diobenog vretena. U ovom seminaru objašnjena je specifična građa centromera na razini nukleosoma. Također je opisana građa kinetohora te na koji se način osigurava kretanje kinetohora, a samim time i kromosoma uzduž mikrotubula diobenog vretena. Na kraju, dan je osvrt na proces neocentromerizacije (stvaranje novih centromera) te je objašnjena uloga centromera, kinetohora i neocentromera u mehanizmu nastanka poremećaja vezanih uz segregaciju kromosoma.Chromosomes are very important part of eukaryotic cells. They carry genetic information (DNA) and provide transfer from parents to the offspring. Centromere and kinetochore are very important part of eukaryotic chromosome because they make link between chromosome and mitotic spindle and thus provide chromosome segregation. In this seminar specific architecture of centromere is explained. It also describes kinetochore architecture that provides kinetochore movement and thus chromosomes along mitotic spindle microtubules. At the end are an overview of neocentromerisation process and the role of centromere, kinetochore and neocentromeres in the chromosomes segregation disorders

From Genes to Therapy : Modeling and Novel Therapeutic Strategies for Brain Tumors

Medulloblastoma is the most common malignant pediatric brain tumor and is molecularly divided into four subgroups – WNT, SHH, Group 3 and Group 4. Two thirds of medulloblastoma patients survive, but survivors often suffer from severe, lifelong side-effects. The MYCN oncogene is deregulated in many medulloblastoma patients. Glioblastoma is the most common malignant brain tumor in adults, with a median survival of about one year. Glioblastoma is a highly heterogeneous tumor where targeted therapy has, so far, not been successful and most glioblastoma patients unfortunately die. In our first study we developed three novel humanized models of MYCN-driven SHH medulloblastoma. Histologically and molecularly these models closely resembled the infant class of SHH tumors. Further, we identified a set of clinically relevant genes that had prognostic significance among patients. mTOR signaling pathway was identified as a major contributor to invasion and dissemination, and we showed that mTOR specific inhibition suppressed migration and viability in vitro. In the second study we performed a forward genetic screen of retrovirally-induced murine PDGFB-driven gliomas and identified more than fifty candidate cancer-causing genes, of which many were mutated or deregulated in glioblastoma. One of the genes identified in this screen was PPFIBP1, found to be differentially expressed from obstructive retroviral integrations in PDGFB-driven glioma clones. Lower PPFIBP1 expression significantly decreased survival of mice and was found to be suppressed in glioblastoma patients. We propose PPFIBP1 to be a novel tumor suppressor gene that contributes to glioblastoma development. In the last study we used a panel of 19 patient-derived glioblastoma cell lines and identified a characteristic expression signature that predicts sensitivity to BET inhibition. BET inhibition resulted in apoptosis and senescence, cell cycle arrest and modulation of DNA damage response. The inhibitory effects of BET inhibition were further enhanced in combination with temozolomide, suggesting a promising future therapy for distinct subgroups of glioblastoma patients. This thesis addresses novel molecular findings in medulloblastoma and glioblastoma development, presents clinically relevant brain tumor models, and promising therapeutic approaches that can be used in future clinical trials in malignant pediatric and adult brain tumors

From Genes to Therapy : Modeling and Novel Therapeutic Strategies for Brain Tumors

Medulloblastoma is the most common malignant pediatric brain tumor and is molecularly divided into four subgroups – WNT, SHH, Group 3 and Group 4. Two thirds of medulloblastoma patients survive, but survivors often suffer from severe, lifelong side-effects. The MYCN oncogene is deregulated in many medulloblastoma patients. Glioblastoma is the most common malignant brain tumor in adults, with a median survival of about one year. Glioblastoma is a highly heterogeneous tumor where targeted therapy has, so far, not been successful and most glioblastoma patients unfortunately die. In our first study we developed three novel humanized models of MYCN-driven SHH medulloblastoma. Histologically and molecularly these models closely resembled the infant class of SHH tumors. Further, we identified a set of clinically relevant genes that had prognostic significance among patients. mTOR signaling pathway was identified as a major contributor to invasion and dissemination, and we showed that mTOR specific inhibition suppressed migration and viability in vitro. In the second study we performed a forward genetic screen of retrovirally-induced murine PDGFB-driven gliomas and identified more than fifty candidate cancer-causing genes, of which many were mutated or deregulated in glioblastoma. One of the genes identified in this screen was PPFIBP1, found to be differentially expressed from obstructive retroviral integrations in PDGFB-driven glioma clones. Lower PPFIBP1 expression significantly decreased survival of mice and was found to be suppressed in glioblastoma patients. We propose PPFIBP1 to be a novel tumor suppressor gene that contributes to glioblastoma development. In the last study we used a panel of 19 patient-derived glioblastoma cell lines and identified a characteristic expression signature that predicts sensitivity to BET inhibition. BET inhibition resulted in apoptosis and senescence, cell cycle arrest and modulation of DNA damage response. The inhibitory effects of BET inhibition were further enhanced in combination with temozolomide, suggesting a promising future therapy for distinct subgroups of glioblastoma patients. This thesis addresses novel molecular findings in medulloblastoma and glioblastoma development, presents clinically relevant brain tumor models, and promising therapeutic approaches that can be used in future clinical trials in malignant pediatric and adult brain tumors

From Genes to Therapy : Modeling and Novel Therapeutic Strategies for Brain Tumors

Medulloblastoma is the most common malignant pediatric brain tumor and is molecularly divided into four subgroups – WNT, SHH, Group 3 and Group 4. Two thirds of medulloblastoma patients survive, but survivors often suffer from severe, lifelong side-effects. The MYCN oncogene is deregulated in many medulloblastoma patients. Glioblastoma is the most common malignant brain tumor in adults, with a median survival of about one year. Glioblastoma is a highly heterogeneous tumor where targeted therapy has, so far, not been successful and most glioblastoma patients unfortunately die. In our first study we developed three novel humanized models of MYCN-driven SHH medulloblastoma. Histologically and molecularly these models closely resembled the infant class of SHH tumors. Further, we identified a set of clinically relevant genes that had prognostic significance among patients. mTOR signaling pathway was identified as a major contributor to invasion and dissemination, and we showed that mTOR specific inhibition suppressed migration and viability in vitro. In the second study we performed a forward genetic screen of retrovirally-induced murine PDGFB-driven gliomas and identified more than fifty candidate cancer-causing genes, of which many were mutated or deregulated in glioblastoma. One of the genes identified in this screen was PPFIBP1, found to be differentially expressed from obstructive retroviral integrations in PDGFB-driven glioma clones. Lower PPFIBP1 expression significantly decreased survival of mice and was found to be suppressed in glioblastoma patients. We propose PPFIBP1 to be a novel tumor suppressor gene that contributes to glioblastoma development. In the last study we used a panel of 19 patient-derived glioblastoma cell lines and identified a characteristic expression signature that predicts sensitivity to BET inhibition. BET inhibition resulted in apoptosis and senescence, cell cycle arrest and modulation of DNA damage response. The inhibitory effects of BET inhibition were further enhanced in combination with temozolomide, suggesting a promising future therapy for distinct subgroups of glioblastoma patients. This thesis addresses novel molecular findings in medulloblastoma and glioblastoma development, presents clinically relevant brain tumor models, and promising therapeutic approaches that can be used in future clinical trials in malignant pediatric and adult brain tumors

Concurrent expression of HP-NAP enhances antitumor efficacy of oncolytic vaccinia virus but not for Semliki Forest virus

Oncolytic viruses (OVs) represent promising therapeutic agents for cancer therapy by selective oncolysis and induction of anti-tumor immunity. OVs can be engineered to express tumor-associated antigens and immune-modulating agents to provoke stronger antitumor immunity. Here, we engineered vaccinia virus (VV) and Semliki Forest virus (SFV) to express neuroblastoma-associated antigen disialoganglioside (GD2) and the immune modulator Helicobacter pylori neutrophil-activating protein (NAP) and compared their therapeutic potency. Oncolytic VV did not exhibit any antitumor benefits, whereas SFV was able to delay subcutaneous neuroblastoma (NXS2) tumor growth. Additional expression of the GD2 mimotope (GD2m) by VV-GD2m or SFV-GD2m did not improve their anti-tumor capacity compared to the parent viruses. Further arming these OVs with NAP resulted in contrasting anti-tumor efficacy. VV (VV-GD2m-NAP) significantly improved therapeutic efficacy compared to VV-GD2m, which was also associated with a significantly elevated anti-GD2 antibody, whereas there was no additive antitumor efficacy for SFV-GD2m-NAP compared to SFV-GD2m, nor was the anti-GD2 antibody response improved. Instead, NAP induced higher neutralizing antibodies against SFV. These observations suggest that distinct immune stimulation profiles are elicited when the same immunostimulatory factor is expressed by different OVs. Therefore, careful consideration and detailed characterization are needed when engineering OVs with immune-modulators

Tat‐PTD‐modified Oncolytic Adenovirus Driven by the SCG3 Promoter and ASH1 Enhancer for Neuroblastoma Therapy

Secretogranin III (SGC3) belongs to the granin family and is highly expressed in endocrine and neural tissues. The human SCG3 promoterhas not yet been characterized. We identified that a 0.5 kb DNA fragment upstream of the SCG3 gene can selectively drivetransgene expression in neuroblastoma cell lines. The strength of transgene expression was further increased and specificity maintained,by addition of the human achaete‐scute complex homolog 1 (ASH1) enhancer. We developed an oncolytic serotype 5‐basedadenovirus, where the SCG3 promoter and ASH1 enhancer drive E1A gene expression. The virus was further modified with a cellpenetratingpeptide (Tat‐PTD) in the virus capsid, which we have previously shown results in increased adenovirus transductionefficiency of many neuroblastoma cell lines. The virus, Ad5PTD(ASH1‐SCG3‐E1A), shows selective and efficient killing of neuroblastomacell lines in vitro, including cisplatin‐, etoposide‐ and doxorubicin‐insensitive neuroblastoma cells. Furthermore, it delays tumorgrowth and thereby prolonged survival for nude mice harboring subcutaneous human neuroblastoma xenograft. In conclusion, wereport a novel oncolytic adenovirus with potential use for neuroblastoma therapy.De två (2) första författarna delar förstaförfattarskapet.Other funds:TheSwedish Cancer Society (10‐0105 and 10‐0552), the Swedish ChildrenCancer Foundation (PROJ10/027, NBCNSPDHEL10/013,JIN C. ET AL. 20138PROJ11/062), Gunnar Nilsson’s Cancer Foundation, the SwedishResearch Council (K2013‐55X‐22191‐01‐3) and the Marcus andMarianne Wallenberg’s Foundation.</p

Novel cancer gene discovery using a forward genetic screen in RCAS-PDGFB-driven gliomas

Background Malignant gliomas, the most common malignant brain tumors in adults, represent a heterogeneous group of diseases with poor prognosis. Retroviruses can cause permanent genetic alterations that modify genes close to the viral integration site. Methods Here we describe the use of a high-throughput pipeline coupled to the commonly used tissue-specific retroviral RCAS-TVA mouse tumor model system. Utilizing next-generation sequencing, we show that retroviral integration sites can be reproducibly detected in malignant stem cell lines generated from RCAS-PDGFB-driven glioma biopsies. Results A large fraction of common integration sites contained genes that have been dysregulated or misexpressed in glioma. Others overlapped with loci identified in previous glioma-related forward genetic screens, but several novel putative cancer-causing genes were also found. Integrating retroviral tagging and clinical data, Ppfibp1 was highlighted as a frequently tagged novel glioma-causing gene. Retroviral integrations into the locus resulted in Ppfibp1 upregulation, and Ppfibp1-tagged cells generated tumors with shorter latency on orthotopic transplantation. In human gliomas, increased PPFIBP1 expression was significantly linked to poor prognosis and PDGF treatment resistance. Conclusions Altogether, the current study has demonstrated a novel approach to tagging glioma genes via forward genetics, validating previous results, and identifying PPFIBP1 as a putative oncogene in gliomagenesis