Abstracts

Acute myeloid leukaemia (AML) is a group of aggressive haematopoietic malignancies associated with adverse outcome. Fms-like tyrosine kinase 3 (FLT3) receptor mutations confer a particularly poor prognosis to AML patients. There is no satisfactory treatment against this disease, especially for the cases harbouring FLT3 mutations, and the quest for novel therapeutic options continues. Drug repurposing represents a powerful strategy to single out existing agents active in novel therapeutic contexts. We performed a high-throughput drug screening, designed to search for agents that inhibit the growth of AML cell lines with mutated FLT3 within libraries of FDA-approved compounds or molecules in advanced phases of clinical trials. Two compounds were identified and chosen from the list of 290 hits for in vitro and in vivo validation. We confirmed that in vitro treatment with the selected agents reduces AML cell growth through a cytotoxic or cytostatic effect. We identified the synergies/additivities of the two molecules with standard anti-AML drugs (e.g., cytarabine, doxorubicine) and a specific FLT3 inhibitor (quizartinib). Next, we determined that both compounds act through their reported mechanism of action. In addition, we identified a novel function for the two agents: the induction of the endoplasmic reticulum stress and the unfolded protein response that follows. Our results support the potential of the selected compounds for the treatment of AML patients, including those with FLT3 mutations, provided that the ongoing in vivo validation is successful.

Colorectal cancer arises from a multi-step process leading to the progressive accumulation of genetic and epigenetic mutations, causing deregulation in homeostasis and neoplastic transformation. Epigenetic and genetic alterations are able to induce a constitutive activation of the WNT signaling pathway, whose aberrant activity converges into deregulation of proliferation, differentiation and cell death pathways. Despite this knowledge of aberrant WNT activity, upstream interference with this signaling pathway induces adverse effects due to high cross-talk with other pathways, highlighting a need to find alternative ways to indirectly target the effectors of this pathway.

In recent years, several studies have been focused on epigenetic players, which act by depositing specific and reversible post-translational modifications. For this reason, they are being recognized as promising new targets for the development of cancer therapeutic strategies. In this context, my project takes advantage of 3D intestinal organoid cultures carrying oncogenic deregulations of the WNT pathway, as a platform for pooled and arrayed RNA interference screens to identify novel regulators controlling the transcriptional aspect of this oncogenic pathway. I also implemented the validation of selected targets in human metastatic colorectal cancer organoids to highlight their clinical relevance. Finally, this project generated important technical knowledge through this pioneering approach that will open up the possibility of performing functional screens in other tissues from which organoid cultures have already been established.

The Myc oncoprotein is a bHLH-LZ transcription factor that heterodimerizes with another bHLH-LZ protein, Max, in order to bind DNA and activate transcription. Max can also di- merize with a variety of alternative bHLH-LZ partners, including Mxd1-4, Mnt and Mga, that act as transcriptional repressors, and are thought to counteract Myc activity at common target genes. Max and Mga are also part of the variant Polycomb Repressive Complex PRC1.6, suggesting that their antagonistic effect on Myc activity may be mediated through PRC1.6. The role of Max has been extensively studied in neuroendocrine carcinomas, spe- cifically in Pheocromocytoma (PC) and Small Cell Lung Cancer (SCLC), where loss of Max can lead to tumor suppression. Beside loss of Max, Myc amplification or loss of Mga can also occur in SCLC: all of these events are mutually exclusive, pointing to a common functional consequence. In light of these observations, we hypothesized that Mga/Max may recruit the PRC1.6 complex which can then antagonize Myc/Max function at a common set of target genes. We further speculated that this may endow Mga and/or Pcgf6 with a general tumor suppressor activity, possibly extending to tumor types other than PC or SCLC.

With this project, we dissected a specific molecular mechanism of Eμ-myc lymphomagene- sis. Specifically, we unraveled a novel role of Pcgf6 as tumor suppressor in Myc-induced lymphoma development. B-cell specific Pcgf6 deletion leads to accelerated tumor for- mation, while, on the other hand, such deletion of Mga does not cause similar phenotype.

Mga and Pcgf6 are part of the same repressive complex (PRC1.6) and the presence of Mga is essential for Pcgf6 chromatin association. Considering this, our data strongly imply that in B-cell lymphomagenesis, Pcgf6 exerts its tumor suppressive function in PRC1.6- and Mga- independent manner.

Chromatin distribution of Myc, Max and Pcgf6 in Eμ-myc lymphomas suggest that Pcgf6 does not affect the association of Myc and Max with chromatin. Therefore, with these anal- yses we demonstrate that PRC1.6, as assessed by Pcgf6 distribution, does not limit Myc association with chromatin, but rather bind to common set of target genes.

Moreover, with transcriptomic profiling of Eμ-myc lymphomas, we showed that tumor sup- pressive role of Pcgf6 is conducted via non-transcriptional mechanism. Mild changes ob- served in tumors scored down regulation of genes involved in immune surveillance path- ways, which opened the door towards an alternative tumor suppressive role of Pcgf6, exe- cuted through immune surveillance mechanisms. Dissecting different populations of tumor- infiltrated cells in our Pcgf6 KO Eμ-myc tumors, we preliminary showed that the loss of Pcgf6 leads to a global downregulation of infiltrated T-cells, specifically effector CD8+ and CD4+, suggesting its non-cell autonomous function and its involvement in T-cell recruitment and/or activation.

In light of these data, we propose a novel mechanism of Pcgf6-mediated tumor suppression that is conducted in non-transcriptional PRC1.6-independent manner and most likely through immune surveillance pathways.

 

The histone demethylase LSD1 is deregulated in several tumors, including leukemias, providing the rationale for the clinical use of LSD1 inhibitors. Treatment of AML cells with LSD1 inhibitors shows a highly variable pattern of response and only a minority of AML cells are sensitive to LSD1 inhibition as single treatment. However, a strong cooperation of LSD1 inhbition and the differentiation agent retinoic acid (RA) can be observed in most of the AML subtypes, even in those resistant to either drug alone.

In acute promyelocytic leukemia (APL), pharmacological doses of RA induce differentiation of APL cells through degradation of the PML-RAR oncogene. APL cells are resistant to LSD1 inhibition or knock-out, but LSD1 inhibition sensitizes them to physiological doses of RA without altering the stability of PML-RAR, and extends survival of leukemic mice upon RA treatment. Non-enzymatic activities of LSD1 are essential to block differentiation of leukemic cells, while the combination of LSD1 inhibitors (or LSD1 knock-out) with low doses of RA releases a differentiation-associated gene expression program, not strictly dependent on changes in histone H3K4 methylation (known substrate of LSD1). An integrated proteomic/epigenomic/mutational analysis showed that LSD1 inhibitors alter the recruitment of LSD1-containing complexes to chromatin through inhibition of the interaction between LSD1 and GFI1, a relevant transcription factor in hematopoiesis.

Same experiments performed in non-APL AML cells confirmed the critical role of LSD1-GFI1 interaction in RA sensitization, beside the APL context.

Cell identity has to be maintained throughout life and its deregulation leads to several pathologies, primarily cancer. Polycomb Repressive Complex 1 (PRC1) are evolutionary-conserved multiprotein complexes that through the deposition of a Ubiquitin molecule on lysine 119 of histone H2A promotes transcriptional repression. We described the fundamental role of PRC1 in preserving intestinal stem cells identity through the inhibition of non-lineage specific transcription factors. However, tissue context plays a critical role in protein function, leading to the possibility that PRC1 could work differently among tissues. To investigate PRC1 role in adult stem cell maintenance, we examined its role in hair follicle stem cells during regeneration. We elucidated a general role of PRC1 in stem cell identity maintenance, accomplished through the regulation of the same targets. However, differently from intestine, PRC1 loss in the hair follicle leads to the activation of a specific epidermal program, showing that the pool of transcription factors present in different stem cell population alters the transcriptional outcome of PRC1 loss.

PRC1 is composed by several subunits that define different biochemical sub-complexes specified by 6 different mutually exclusive PCGF proteins (PCGF1-6). Their role in embryonic development is widely studied, however their involvement in adult tissue maintenance is still obscure. Exploiting different PCGFs conditional knock out mouse models we aim to address the specific sub complexes roles in tissue homeostasis maintenance, in order to define their contribution in the phenotypic outcome observed in PRC1 loss of function intestinal and hair follicle LGR5 stem cells.

 

 

 

Mitotic spindle orientation is a prerequisite for the correct completion of mitosis, and is essential for tissue morphogenesis and maintenance. The core constituent of the spindle orientation machinery is represented by Gai:LGN:NuMA complexes, which orient the spindle by generating pulling forces on astral microtubules. Besides several studies identified the minimal binding domains of NuMA and LGN, how such interaction is organized and triggers microtubules-motor activation still remains largely unclear.

My PhD project focused on the characterization of the NuMA:LGN interaction and on the analysis of the role of the microtubule-binding domain of NuMA. Studies conducted during this thesis revealed that NuMA and LGN assemble in hetero-hexameric structures. Consistently, an LGN oligomerization-deficient mutant cannot rescue misorientation defects in HeLa cells and Caco-2 three-dimensional cysts. We provided evidence that LGN and NuMA assemble high-order oligomers in cells, and that the 3:3 stoichiometry of the NuMA:LGN complex combined with the dimeric state of NuMA promotes the formation of a large proteins network. Furthermore, we showed that the NuMA:LGN oligomers are compatible with the direct association of NuMA to microtubules, and that the microtubules-binding domain of NuMA is required to correctly localize NuMA at the poles and at the cortex, and to orient the spindle. Collectively, our findings suggest a model whereby cortical LGN:NuMA hetero-hexamers favor the accumulation of dynein motors at cortical sites. We speculate that direct binding of NuMA to astral microtubule plus-tips assists the movement of dynein along the depolymerizing astral microtubules to promote spindle placement.

Despite having been used for more than a century, the exact mechanisms of action, of resistance and the best treatment schedule of most chemotherapeutic agents remain elusive. Mitomycin C (MMC) is the gold standard adjuvant treatment for bladder cancer. However, it is effective only in a proportion of patients, suggesting that, aside from cytotoxicity, other mechanisms could be involved in mediating the success or failure of treatment. We hypothesized that MMC might induce immunogenic cell death (ICD), leading to an antitumor immune response. Here, we describe that MMC fosters ICD via the exposure of damage signals, increased phagocytosis by dendritic cells (DCs) and in vivo tumor protection. MMC-induced ICD relies on the cytoplasmic release of mitochondrial DNA that activates the inflammasome for efficient IL-1β secretion that promotes DC maturation. We found the ICD resistant cancer cells fail to generate an inflammatory microenvironment and display mitochondria dysfunction of the respiratory chain, which is associated with drug resistance in bladder cancer patients. The identification of ICD as a novel immune-related mechanism of action of MMC provides opportunities to optimize bladder cancer management and identify ICD-related biomarkers of treatment efficacy.

Lysine specific-demethylase 1 (LSD1) is aberrantly expressed in acute myeloid leukemia (AML) and is emerging as a promising target for the epigenetic therapy of different AML subtypes. The Experimental Therapeutic Unit at the IFOM-IEO Campus optimized potent and specific LSD1 inhibitors, already characterized in vitro for their selectivity and in vivo for their anti-proliferative effects on self-renewing AML cells. In my thesis project, I studied the effects of these compounds on the pattern of histone post-translational modifications (PTMs) and on the LSD1 interaction network in NB4-APL cells (an AML subtype), using a panel of quantitative mass-spectrometry strategies. We discovered that a 24-hour treatment with the inhibitors alters the levels of histone modifications (increases H3K4me2, H3K27me2 and H3K27me3, and decreases H3K27me1). LSD1 knock-out NB4 cells display similar changes in histone PTMs, strongly suggesting a specific association with the cellular response to LSD1 inhibition. We also identified the complete set of LSD1 interactors in our model using SILAC-based proteomics, most of which are involved in chromatin remodelling and transcription regulation activities. The analysis of the LSD1-interactome after drug treatment identified two LSD1 interactors (GFI1 and GSE1) with decreased binding. Inhibition of the LSD1- GFI1 interaction promoted reduction of cell proliferation and differentiation of NB4 cells. Regarding the LSD1-GSE1 interaction, we found that GSE1 down-regulation and LSD1 inhibition up-regulated a common set of genes involved in “cytokine-mediated signalling” and “regulation of apoptosis”, thus suggesting the existence of a regulatory LSD1-GSE1 axis controlling the transcription of these genes. Collectively, these data provide novel insights into the molecular activity of LSD1 and its inhibitors in APL cells.

The spectrin cytoskeleton is a major component of the mammalian cell cortex. While long known and ubiquitously expressed, its dynamic behaviour and cooperation with other major components of the cell cortex is poorly understood. Here we investigated spectrin reactions upon different mechanical cues, such as cell-driven perturbations, like cell adhesion, spreading and contraction, or environmentally driven ones, like compression, stretch and osmotic changes. Upon all of these challenges we observed that spectrin meshwork spatially adapts and reorganizes under the plasma membrane together with the acto-myosin cytoskeleton. Working together to maintain cell integrity, both cytoskeletons define specific membrane territories. Actin-rich regions control protrusions, adhesions and stress fibers, whereas spectrin-rich regions concentrate in retractile zones, covering low actin density territories of the cortex. Given this interplay, we wondered if spectrin could be potentially involved in the spatial and temporal regulation of membrane trafficking. We followed spectrin, actin and clathrin dynamics through live TIRF microscopy and observed an inverted correlation between spectrin and actin densities and endocytic capacities, suggesting a spectrin contribution to clathrin- mediated endocytosis. Our results pinpoint a role for spectrin in the support of the lipid bilayer in regions where actin cytoskeleton is not established, creating a fencing mechanism for actin remodelling and cargoes internalization. All these mechanisms potentially unveil why the spectrin family of protein is evolutionary highly conserved and ubiquitously expressed in eukaryotic cells, and might explain its involvement in a broad range of pathological condition.

The development of human brain is a fascinating and complex process that still needs to be uncovered at the molecular resolution. Even though animal studies have revealed a lot of its unfolding, the fine regulation of cellular differentiation trajectories that characterizes humans has become only recently open to experimental tractability, thanks to the development of organoids, human cellular models that are able to recapitulate the spatiotemporal architecture of the brain in a 3D fashion. Here we first benchmarked human brain organoids at the level of transcriptomic and structural architecture of cell composition along several stages of differentiation. Then we harnessed their properties to probe the longitudinal impact of GSK3 on human corticogenesis, a pivotal regulator of both proliferation and polarity, that we revealed having a direct impact on early neurogenesis with a selective role in the regulation of glutamatergic lineages and outer radial glia output. Moreover, we spearheaded the use of organoids for regulatory toxicology through the study of Endocrine disrupting chemicals (EDC), pervasive compounds that can interfere with human hormonal systems. Early life exposure to EDC is associated with human disorders, but the molecular events triggered remain unknown. We developed a novel approach, integrating epidemiological with experimental biology to study the mixtures of EDC that were associated with neurodevelopmental and metabolic adverse effects in the biggest pregnancy cohort profiled so far. Our experiments were carried out on two complementary models i) human fetal primary neural stem cells, and ii) 3-dimensional cortical brain organoids and we identified the genes specifically dysregulated by EDC mixture exposure, unravelling a significant enrichment for autism spectrum disorders causative genes, thereby proposing a convergent paradigm of neurodevelopmental disorders pathophysiology between genetic and environmental factors. Finally, while EDCs are everywhere, their impact on adverse health outcomes can vary substantially among individuals, suggesting that other genetic factors may play a pivotal role for the onset of the disorders. We took advantage of organoids multiplexing to recapitulate, at the same time, neurodevelopmental trajectories on multiple genetic backgrounds, and showed that chimeric organoids preserved the overall morphological organization and transcriptomic signatures of the ones generated from single lines. In conclusion our work shows the possibility to perform population level studies in vitro and use the deep resolution of molecular biology to dissect key aspects of human neurodevelopment.





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