Final Report Summary - PLOIDYNET (The impact of chromosomal instability on health: Molecular causes and consequences of aneuploidy)
Chromosomal instability (CIN) has been an appreciated hallmark of cancer cells for over a century. Despite this long-standing knowledge, we still do not fully understand how CIN makes cells adopt a malignant fate, or what effect (beneficial/adverse) CIN has on non-transformed cells. Understanding this is important, as it will take us closer to therapies that exploit CIN as a feature that discriminates cancer cells from non-cancer cells, thus yielding better-targeted cancer therapies. To address these questions, a multidisciplinary approach is needed, which was the reason to start PloidyNet, a consortium of academic and industrial investigators, all with an interest in chromosomal instability, and all with a unique research angle.
PloidyNet’s main goal was to train 11 young scientists, 9 ESRs and 2 ERs, in the field of chromosomal instability. To achieve this, we brought together some of Europe’s leading scientists to train our fellows. Within PloidyNet, fellows were exposed to a wide variety of state of the art technologies and used several model systems to address how CIN emerges in cancer, to explore what the consequences of CIN are and to find ways of exploiting CIN to improve cancer therapy. PloidyNet consisted of 11 beneficiaries and 1 associated partner representing 4 member states and Switzerland including 3 commercial enterprises. 11 students of 9 nationalities were recruited (8 out 11 female).
To achieve the best possible training, we designed a high quality and highly complementary training program consisting of [1] training by doing research in at least two different labs, [2] industrial rotations and [3] a number of focused workshops, organized by the academic and industrial participants of the network. Our training program captured the multidisciplinary nature of PloidyNet’s participants while providing highly specialized training, as students all had an individual research project that exploits expertise of at least 2-3 participating labs. All students visited at least one other lab and one of our industrial partners as part of their project. Furthermore, through Annual Network Meetings, focused courses organized at the hosting institutes, through an interactive WebPortal and a strong social network, students had access to a wide variety of model systems, technologies and expertise, which will yield a next generation of scientists that are well prepared for a career in life sciences, either in industry or academia.
In addition to the Network’s training aims, a number of scientific aims were addressed.
WP4 Molecular Causes of Aneuploidy: In this workpackage, students worked together to better understand the molecular processes that lead to aneuploidy. To this aim, they developed human artificial chromosomes (HACs) that, when engineered into human cell lines, can be used to ‘fish’ for and quantify proteins that accumulate at (aneuploid) kinetochores. In addition, students have developed mass spectrometry protocols to quantify expression of proteins important for signaling aneuploidy in various aneuploid cell lines that are now being employed to further characterize how the chromosome segregation machinery copes with abnormal chromosome numbers.
WP5 Physiological consequences of Aneuploidy: In this WP we studied how cells respond to an aneuploid state. For this, students performed an RNA interference screen to identify several genes that signal abnormal karyotypes, for which candidate genes are currently being validated, and another screen to identify kinases that provoke apoptosis in response to CIN. In addition, we developed a biosensor for p53 activity (a key component in signaling aneuploidy) by endogenously tagging p53-protein. When validated, this tool will be translated into a mouse model (WP8-biosensors). Finally, we characterized primary non-small cell lung cancer (NSCLC) tumor samples and tumor-derived induced pluripotent stem cells to analyze aneuploid tumor genomes using next generation sequencing, which is yielding a number of potentially interesting candidate genes involved in aneuploid tumor survival. The first data from this were published August 2015 in Cancer Discovery (Cancer Discov. 2015 Aug; 5(8): 821-31).
WP6 The role of Aneuploidy in Cancer Development: The aim of WP6 was to better understand how aneuploidy leads to cancer. To this aim, students made use of mouse models in which chromosomal instability was provoked by various means in the mammary gland. So far, the mouse models have been crossed to get the correct genotypes to develop aneuploid mammary tumors. In addition, students employed recently developed protocols to culture tumors in a 3D setting in a dish (organoid culture) using colon carcinoma and mouse mammary tumor cells as a model system. Tumors and tumor cultures are and will be further characterized for karyotype heterogeneity using single cell sequencing and RNA sequencing.
WP7 Targeting Chromosome Segregation in Cancer Therapy: The aim of this work package was to train students how to drug mitotic pathways to best achieve efficient tumor cell death in vitro and in mice. As this work package depends on the success of new mitotic inhibitors being developed (e.g. based on the results on the RNA interference screen under WP5). The first step in this WP has been the further characterization of an Mps1 inhibitor revealing that cells can actually cope much better with the inhibitor-induced CIN than previously anticipated, raising several new interesting questions.
WP8 Assay Development: Students participating in this WP developed a number of new assays that will facilitate quantifying aneuploidy in vitro as well as in vivo. One tool through which PloidyNet research has progressed tremendously over the last years is single cell sequencing, by which we can now reliably assess full karyotypes of single non-dividing cells. Furthermore, we have engineered constructs and mouse ES cell lines to generate recombinant mice that express combinations of fluorescent mitotic markers (H2B-Centrin3-Tubulin or H2B-CenpB-Tubulin) in a conditional fashion, which will allow us to label (and lineage trace) cells in cell lineages of choice by intravital imaging. Finally, students developed a flow cytometry-based assay to detect aneuploidy using cell surface markers. For this, we are now validating ~70 candidates epitopes that appear to be differentially expressed in aneuploid cells, with a current focus on components of the integrin superfamily that appear to be deregulated as a consequence of aneuploidy.
In sum, PloidyNet has accomplished its mission. Management has successfully been implemented and training started and was completed according to the plan. Research has yield promising data, some of which has been published in international peer reviewed papers with significant impact. All E(S)Rs presented their work at (inter)national meetings, contributing to various outreach events and benefiting from the multidisciplinary nature of PloidyNet.
Information about the PloidyNet project can be found on our website (http://www.aneuploidy.nl/).
PloidyNet’s main goal was to train 11 young scientists, 9 ESRs and 2 ERs, in the field of chromosomal instability. To achieve this, we brought together some of Europe’s leading scientists to train our fellows. Within PloidyNet, fellows were exposed to a wide variety of state of the art technologies and used several model systems to address how CIN emerges in cancer, to explore what the consequences of CIN are and to find ways of exploiting CIN to improve cancer therapy. PloidyNet consisted of 11 beneficiaries and 1 associated partner representing 4 member states and Switzerland including 3 commercial enterprises. 11 students of 9 nationalities were recruited (8 out 11 female).
To achieve the best possible training, we designed a high quality and highly complementary training program consisting of [1] training by doing research in at least two different labs, [2] industrial rotations and [3] a number of focused workshops, organized by the academic and industrial participants of the network. Our training program captured the multidisciplinary nature of PloidyNet’s participants while providing highly specialized training, as students all had an individual research project that exploits expertise of at least 2-3 participating labs. All students visited at least one other lab and one of our industrial partners as part of their project. Furthermore, through Annual Network Meetings, focused courses organized at the hosting institutes, through an interactive WebPortal and a strong social network, students had access to a wide variety of model systems, technologies and expertise, which will yield a next generation of scientists that are well prepared for a career in life sciences, either in industry or academia.
In addition to the Network’s training aims, a number of scientific aims were addressed.
WP4 Molecular Causes of Aneuploidy: In this workpackage, students worked together to better understand the molecular processes that lead to aneuploidy. To this aim, they developed human artificial chromosomes (HACs) that, when engineered into human cell lines, can be used to ‘fish’ for and quantify proteins that accumulate at (aneuploid) kinetochores. In addition, students have developed mass spectrometry protocols to quantify expression of proteins important for signaling aneuploidy in various aneuploid cell lines that are now being employed to further characterize how the chromosome segregation machinery copes with abnormal chromosome numbers.
WP5 Physiological consequences of Aneuploidy: In this WP we studied how cells respond to an aneuploid state. For this, students performed an RNA interference screen to identify several genes that signal abnormal karyotypes, for which candidate genes are currently being validated, and another screen to identify kinases that provoke apoptosis in response to CIN. In addition, we developed a biosensor for p53 activity (a key component in signaling aneuploidy) by endogenously tagging p53-protein. When validated, this tool will be translated into a mouse model (WP8-biosensors). Finally, we characterized primary non-small cell lung cancer (NSCLC) tumor samples and tumor-derived induced pluripotent stem cells to analyze aneuploid tumor genomes using next generation sequencing, which is yielding a number of potentially interesting candidate genes involved in aneuploid tumor survival. The first data from this were published August 2015 in Cancer Discovery (Cancer Discov. 2015 Aug; 5(8): 821-31).
WP6 The role of Aneuploidy in Cancer Development: The aim of WP6 was to better understand how aneuploidy leads to cancer. To this aim, students made use of mouse models in which chromosomal instability was provoked by various means in the mammary gland. So far, the mouse models have been crossed to get the correct genotypes to develop aneuploid mammary tumors. In addition, students employed recently developed protocols to culture tumors in a 3D setting in a dish (organoid culture) using colon carcinoma and mouse mammary tumor cells as a model system. Tumors and tumor cultures are and will be further characterized for karyotype heterogeneity using single cell sequencing and RNA sequencing.
WP7 Targeting Chromosome Segregation in Cancer Therapy: The aim of this work package was to train students how to drug mitotic pathways to best achieve efficient tumor cell death in vitro and in mice. As this work package depends on the success of new mitotic inhibitors being developed (e.g. based on the results on the RNA interference screen under WP5). The first step in this WP has been the further characterization of an Mps1 inhibitor revealing that cells can actually cope much better with the inhibitor-induced CIN than previously anticipated, raising several new interesting questions.
WP8 Assay Development: Students participating in this WP developed a number of new assays that will facilitate quantifying aneuploidy in vitro as well as in vivo. One tool through which PloidyNet research has progressed tremendously over the last years is single cell sequencing, by which we can now reliably assess full karyotypes of single non-dividing cells. Furthermore, we have engineered constructs and mouse ES cell lines to generate recombinant mice that express combinations of fluorescent mitotic markers (H2B-Centrin3-Tubulin or H2B-CenpB-Tubulin) in a conditional fashion, which will allow us to label (and lineage trace) cells in cell lineages of choice by intravital imaging. Finally, students developed a flow cytometry-based assay to detect aneuploidy using cell surface markers. For this, we are now validating ~70 candidates epitopes that appear to be differentially expressed in aneuploid cells, with a current focus on components of the integrin superfamily that appear to be deregulated as a consequence of aneuploidy.
In sum, PloidyNet has accomplished its mission. Management has successfully been implemented and training started and was completed according to the plan. Research has yield promising data, some of which has been published in international peer reviewed papers with significant impact. All E(S)Rs presented their work at (inter)national meetings, contributing to various outreach events and benefiting from the multidisciplinary nature of PloidyNet.
Information about the PloidyNet project can be found on our website (http://www.aneuploidy.nl/).