ERC FUNDED PROJECTS

Monoclonal antibodies against the EGF receptor (EGFR) provide substantive benefit to colorectal cancer (CRC) patients. However, no genetic lesions that robustly predict ‘addiction’ to the EGFR pathway have been yet identified. Further, even in tumours that regress after EGFR blockade, subsets of drug-tolerant cells often linger and foster ‘minimal residual disease’ (MRD), which portends tumour relapse. Our preliminary evidence suggests that reliance on EGFR activity, as opposed to MRD persistence, could be assisted by genetically-based variations in transcription factor partnerships and activities, gene expression outputs, and biological fates controlled by the WNT/beta-catenin pathway. On such premises, BEAT (Beta-catenin and EGFR Abrogation Therapy) will elucidate the mechanisms of EGFR dependency, and escape from it, with the goal to identify biomarkers for more efficient clinical management of CRC and develop new therapies for MRD eradication. A multidisciplinary approach will be pursued spanning from integrative gene regulation analyses to functional genomics in vitro, pharmacological experiments in vivo, and clinical investigation, to address whether: (i) specific genetic alterations of the WNT pathway affect anti-EGFR sensitivity; (ii) combined neutralisation of EGFR and WNT signals fuels MRD deterioration; (iii) data from analysis of this synergy can lead to the discovery of clinically meaningful biomarkers with predictive and prognostic significance. This proposal capitalises on a unique proprietary platform for high-content studies based on a large biobank of viable CRC samples, which ensures strong analytical power and unprecedented biological flexibility. By providing fresh insight into the mechanisms whereby WNT/beta-catenin signalling differentially sustains EGFR dependency or drug tolerance, the project is expected to put forward an innovative reinterpretation of CRC molecular bases and advance the rational application of more effective therapies.

1 793 421 €

Start date: 2017-10-01, End date: 2022-09-30

The research I propose here will provide an enabling technology; spatially resolved transcriptomics, to address important problems in cell- and developmental-biology, in particular: How are stem cells in the skin and gut proliferating without turning into cancers? How are differentiated cells related, in their transcriptome and spatial positions, to their progenitors? To investigate these problems on a molecular level and open up paths to find completely new spatiotemporal interdependencies in complex biological systems, I propose to use our newly developed DNA-origami strategy (Benson et al, Nature, 523 p. 441 (2015) ), combined with a combinatorial cloning technique, to build a new method for deep mRNA sequencing of tissue with single-cell resolution. These new types of origami are stable in physiological salt conditions and opens up their use in in-vivo applications. In DNA-origami we can control the exact spatial position of all nucleotides. By folding the scaffold to display sequences for hybridization of fluorophores conjugated to DNA, we can create optical nano-barcodes. By using structures made out of DNA, the patterns of the optical barcodes will be readable both by imaging and by sequencing, thus enabling the creation of a mapping between cell locations in an organ and the mRNA expression of those cells. We will use the method to perform spatially resolved transcriptomics in small organs: the mouse hair follicle, and small intestine crypt, and also perform the procedure for multiple samples collected at different time points. This will enable a high-dimensional data analysis that most likely will expose previously unknown dependencies that would provide completely new knowledge about how these biological systems work. By studying these systems, we will uncover much more information on how stem cells contribute to regeneration, the issue of de-differentiation that is a common theme in these organs and the effect this might have on the origin of cancer.

1 923 263 €

Start date: 2017-08-01, End date: 2022-07-31

Acute myeloid leukemia (AML) is a group of hematologic malignancies which, due to their molecular and clinical heterogeneity, have been traditionally difficult to classify and treat. Recently, next-generation, whole-genome sequencing has uncovered several recurrent somatic mutations that better define the landscape of AML genomics. Despite these advances in deciphering AML molecular subsets, there have been no concurrent improvements in AML therapy which still relies on the ‘antracycline+cytarabine’ scheme. Hereto, only about 40-50% of adult young patients are cured whilst most of the elderly succumb to their disease. Therefore, new therapeutic approaches which would take advantage of the new discoveries are clearly needed. In the past years, we discovered and characterized nucleophosmin (NPM1) mutations as the most frequent genetic alteration (about 30%) in AML, and today NPM1-mutated AML is a new entity in the WHO classification of myeloid neoplasms. However, mechanisms of leukemogenesis and a specific therapy for this leukemia are missing. Here, I aim to unravel the complex network of molecular interactions that take place in this distinct genetic subtype, and find their vulnerabilities to identify new targets for therapy. To address this issue, I will avail of relevant pre-clinical models developed in our laboratories and propose two complementary strategies: 1) a screening-based approach, focused either on the target, by analyzing synthetic lethal interactions through CRISPR-based genome-wide interference, or on the drug, by high-throughput chemical libraries screenings; 2) a hypothesis-driven approach, based on our recent gained novel insights on the role of specific intracellular pathways/genes in NPM1-mutated AML and on pharmacological studies with ‘old’ drugs, which we have revisited in the specific AML genetic context. I expect our discoveries will lead to find novel therapeutic approaches and make clinical trials available to patients as soon as possible.

1 883 750 €

Start date: 2017-04-01, End date: 2022-03-31

The activities of the Polycomb group (PcG) of repressive chromatin modifiers are required to maintain correct transcriptional identity during development and differentiation. These activities are altered in a variety of tumours by gain- or loss-of-function mutations, whose mechanistic aspects still remain unclear. PcGs can be classified in two major repressive complexes (PRC1 and PRC2) with common pathways but distinct biochemical activities. PRC1 catalyses histone H2A ubiquitination of lysine 119, and PRC2 tri-methylation of histone H3 lysine 27. However, PRC1 has a more heterogeneous composition than PRC2, with six mutually exclusive PCGF subunits (PCGF1–6) essential for assembling distinct PRC1 complexes that differ in subunit composition but share the same catalytic core. While up to six different PRC1 forms can co-exist in a given cell, the molecular mechanisms regulating their activities and their relative contributions to general PRC1 function in any tissue/cell type remain largely unknown. In line with this biochemical heterogeneity, PRC1 retains broader biological functions than PRC2. Critically, however, no molecular analysis has yet been published that dissects the contribution of each PRC1 complex in regulating transcriptional identity. We will take advantage of newly developed reagents and unpublished genetic models to target each of the six Pcgf genes in either embryonic stem cells or mouse adult tissues. This will systematically dissect the contributions of the different PRC1 complexes to chromatin profiles, gene expression programs, and cellular phenotypes during stem cell self-renewal, differentiation and adult tissue homeostasis. Overall, this will elucidate some of the fundamental mechanisms underlying the establishment and maintenance of cellular identity and will allow us to further determine the molecular links between PcG deregulation and cancer development in a tissue- and/or cell type–specific manner.

2 000 000 €

Start date: 2017-11-01, End date: 2022-10-31