ERC FUNDED PROJECTS

As an onco-hematologist with a strong expertise in genomics, I significantly contributed to the understanding of multiple myeloma (MM) heterogeneity and its evolution over time, driven by genotypic and phenotypic features carried by different subpopulations of cells. MM is preceded by prevalent, asymptomatic stages that may evolve with variable frequency, not accurately captured by current clinical prognostic scores. Supported by preliminary data, my hypothesis is that the same heterogeneity is present early on the disease course, and identification of the biological determinants of evolution at this stage will allow better prediction of its evolutionary trajectory, if not its control. In this proposal I will therefore make a sharp change from conventional approaches and move to early stages of MM using unique retrospective sample cohorts and ambitious prospective sampling. To identify clonal MM cells in the elderly before a monoclonal gammopathy can be detected, I will collect bone marrow (BM) from hundreds of hip replacement specimens, and analyze archive peripheral blood samples of thousands of healthy individuals with years of annotated clinical follow-up. This will identify early genomic alterations that are permissive to disease initiation/evolution and may serve as biomarkers for clinical screening. Through innovative, integrated single-cell genotyping and phenotyping of hundreds of asymptomatic MMs, I will functionally dissect heterogeneity and characterize the BM microenvironment to look for determinants of disease progression. Correlation with clinical outcome and mini-invasive serial sampling of circulating cell-free DNA will identify candidate biological markers to better predict evolution. Last, aggressive modelling of candidate early lesions and modifier screens will offer a list of vulnerabilities that could be exploited for rationale therapies. These methodologies will deliver a paradigm for the use of molecularly-driven precision medicine in cancer.

1 998 781 €

Start date: 2019-03-01, End date: 2024-02-29

The fundamental limitation in our ability to dissect human diseases is the scarce availability of human tissues at relevant disease stages, which is particularly salient for neural disorders. Somatic cell reprogramming is overcoming this limitation through the derivation of patient-specific induced pluripotent stem cells (iPSC) that can be differentiated into disease-relevant cell-types. Despite these tantalizing possibilities, there are critical issues to be addressed in order to secure iPSC-modeling as a robust platform for the interrogation of disease aetiology and the development of new therapies. These concern the taming of human genetic variation, the identification of differentiation stages in which to uncover and validate phenotypes, and finally their translational into drug discovery assays. This project confronts these challenges focusing on the paradigmatic case of two rare but uniquely informative disorders caused by symmetric gene dosage imbalances at 7q11.23: Williams Beuren Syndrome and the subset of autism spectrum disorders associated to 7q11.23 microduplication. The hallmark of WBS is a unique behavioral-cognitive profile characterized by hypersociability and intellectual disability in the face of comparatively well-preserved language abilities. Hence, the striking symmetry in genotype and phenotype between WBS and 7dupASD points to the 7q11.23 cluster as a surprisingly small subset of dosage-sensitive genes affecting social behaviour and cognition. We build on a large panel of iPSC lines that we already reprogrammed from a unique cohort of WBS and 7dupASD patients and whose characterization points to specific derangements at the level of transcriptional/epigenetic control, protein synthesis and synaptic dysfunction. Through the integration of transcriptomic and epigenomic profiling with targeted mass spectrometry and gene network prediction we propose an innovative drug discovery pipeline for the identification of new therapeutic leads.

1 997 804 €

Start date: 2014-09-01, End date: 2019-08-31

Although tumor tissues can be infiltrated by T cells specific for tumor antigens, the effector functions of these lymphocytes are generally suppressed by CD4+ regulatory T cells (Tregs). Since tumor infiltrating Tregs can display function heterogeneity, depending on both the tumor type and the inflammatory milieu, only inhibition of the right Treg activity should result in the unleash of an effective anti-tumor T cell responses. Experimental plan: To identify the Tregs that truly inhibit anti-tumor T cells, we will profile by RNA-Seq the transcriptome of Tregs infiltrating both tumor and healthy tissues. In particular, we will focus on LncRNAs and the gene networks they modulate, since they have recently emerged as relevant epigenetic regulators of cell differentiation and identity. We will exploit this new knowledge to create a panel of regulatory transcripts, which will be assessed at single cell level on tumor infiltrating Tregs, so to determine the association of specific transcripts with different Treg populations. Since downregulation of specific lncRNAs might be an efficient way to inhibit the “unwanted” Tregs at tumor sites, we aim at targeting lncRNAs uniquely expressed in these Tregs and propose to develop AsiCs, chimeric molecules composed by an aptamer, single stranded oligonucleotides that bind to cell surface markers, and a siRNA, short RNAs downregulating specific lncRNAs. Deliverables and conclusions: this proposal will provide new knowledge on tumor infiltrating Tregs possibly allowing definition of molecular signatures of Tregs with either positive or negative effects on antitumor T cell responses. Moreover, we will develop new molecules that specifically target lncRNAs of interest and that will help identifying new antitumor therapeutic targets. In conclusion, the possibility to modulate Tregs effector functions may not only offer new anti-tumor therapy but more in general may be relevant to any immunomodulatory therapeutic strategies.

1 998 000 €

Start date: 2014-04-01, End date: 2019-03-31

G-quadruplexes (G-4) are polymorphic nucleic acid structures identified in gene promoters where they act as transcription regulators. G-4s have been found in eukaryotic and prokaryotic organisms, while very little information is available on viruses. The applicant research group has recently shown that HIV-1, which integrates into the human chromosomes and exploits cellular factors to activate transcription, takes advantage of G-4-mediated transcription regulation. G-4 disruption stimulates promoter activity while G-4 stabilization by small molecules inhibits it, showing a striking parallelism between HIV-1 LTR and eukaryotic promoter G-4s. Preliminary results indicate that similar G-4 structures form also in the viral RNA genome before retrotranscription. Available G-4 ligands, developed as anticancer drugs targeting DNA G-4, recognize both viral and cellular G-4s. Therefore, they cannot be straightforwardly used as anti-HIV compounds. The aim of this project is to develop highly specific anti-HIV-1 drugs targeting LTR DNA and/or RNA G-4s, using both reversible G-4 ligands and G-4-selective alkylating/cleaving agents, triggered by external stimuli. These approaches will be taken: a) to increase selectivity by 1) screening of ligands against LTR G-4s to select the best hits among libraries of G-4 ligands; 2) conjugation of the most promising leads to modified nucleic acids complementing LTR G-4 loop/flanking regions, to deliver the drug to its target; b) to stabilize binding by conjugation of the ligands to 3) an alkylating/cleaving subunit, and 4) an activable moiety (such as quinone methides) that alkylates the target only once the drug has reached it. Physico-chemical, biomolecular, cellular and viral assays will be used to tests the compounds. This approach should deliver reversible and irreversible ligands that selectively inhibit viral transcription and/or reverse transcription, thus preventing virus production and/or integration into the host genome.

1 989 471 €

Start date: 2014-05-01, End date: 2019-04-30