New molecular approaches to control rhabdomyosarcoma onset and metastasis in preclinical systems

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. The aim of this study was to identify molecular events involved in rhabdomyosarcoma onset for the development of new therapeutic approaches against specific molecular targets. BALB-p53neu mice develop pelvic rhabdomyosarcoma and combines the activation of HER-2/neu oncogene with the inactivation of an allele of p53 oncosuppressor gene. Gene expression profiling led to the identification of genes potentially involved in rhabdomyosarcoma genesis and therefore of candidate targets. The pattern of expression of p53, HER-2/neu, CDKN2A/p19ARF and IGF-2 suggested that these alterations might be involved in gender-, site- and strain-specific development of rhabdomyosarcoma. Other genes such as CDKN1A/p21 might be involved. The role of IGF-2, CDKN2A/p19ARF and CDKN1A/p21 in tumor growth was investigated with siRNA in murine rhabdomyosarcoma cells. Silencing of p19ARF and p21 induced inhibition of growth and of migration ability, indicating a possible pro-tumor and pro-metastatic role in rhabdomyosarcoma in absence of p53. In addition the autocrine IGF-2/IGF-1R loop found in early phases of cancer progression strengthens its key role in sustaining rhabdomyosarcoma growth. As rhabdomyosarcoma displays defective myogenic differentiation, a therapeutic approach aimed at enhancing myogenic differentiation of rhabdomyosarcoma cells. Forced expression of myogenin was able to restore myogenic differentiation, significantly reduced cell motility and impaired tumor growth and metastatic spread. IL-4 treatment increased rhabdomyosarcoma cell growth, decreased myogenin expression and promoted migration of cells lacking myogenin. Another approach was based on small kinase inhibitors. Agents specifically targeting members of the HER family (Lapatinib), of the IGF system (NVP-AEW541) or downstream signal transducers (NVP-BEZ235) were investigated in vitro in human rhabdomyosarcoma cell lines as therapeutic anti-tumor and anti-metastatic tools. The major effects were obtained with NVP-BEZ235 treatment that was able to strongly inhibit cell growth in vitro and showed anti-metastatic effects in vivo.

New Synthetic Polyamines as Multi-Target-Directed Ligands for the Treatment of Alzheimer's Disease and Cancer: Design, Synthesis and Biological Evaluation

Alzheimer's disease (AD) and cancer represent two of the main causes of death worldwide. They are complex multifactorial diseases and several biochemical targets have been recognized to play a fundamental role in their development. Basing on their complex nature, a promising therapeutical approach could be represented by the so-called "Multi-Target-Directed Ligand" approach. This new strategy is based on the assumption that a single molecule could hit several targets responsible for the onset and/or progression of the pathology. In particular in AD, most currently prescribed drugs aim to increase the level of acetylcholine in the brain by inhibiting the enzyme acetylcholinesterase (AChE). However, clinical experience shows that AChE inhibition is a palliative treatment, and the simple modulation of a single target does not address AD aetiology. Research into newer and more potent anti-AD agents is thus focused on compounds whose properties go beyond AChE inhibition (such as inhibition of the enzyme β-secretase and inhibition of the aggregation of beta-amyloid). Therefore, the MTDL strategy seems a more appropriate approach for addressing the complexity of AD and may provide new drugs for tackling its multifactorial nature. In this thesis, it is described the design of new MTDLs able to tackle the multifactorial nature of AD. Such new MTDLs designed are less flexible analogues of Caproctamine, one of the first MTDL owing biological properties useful for the AD treatment. These new compounds are able to inhibit the enzymes AChE, beta-secretase and to inhibit both AChE-induced and self-induced beta-amyloid aggregation. In particular, the most potent compound of the series is able to inhibit AChE in subnanomolar range, to inhibit β-secretase in micromolar concentration and to inhibit both AChE-induced and self-induced beta-amyloid aggregation in micromolar concentration. Cancer, as AD, is a very complex pathology and many different therapeutical approaches are currently use for the treatment of such pathology. However, due to its multifactorial nature the MTDL approach could be, in principle, apply also to this pathology. Aim of this thesis has been the development of new molecules owing different structural motifs able to simultaneously interact with some of the multitude of targets responsible for the pathology. The designed compounds displayed cytotoxic activity in different cancer cell lines. In particular, the most potent compounds of the series have been further evaluated and they were able to bind DNA resulting 100-fold more potent than the reference compound Mitonafide. Furthermore, these compounds were able to trigger apoptosis through caspases activation and to inhibit PIN1 (preliminary result). This last protein is a very promising target because it is overexpressed in many human cancers, it functions as critical catalyst for multiple oncogenic pathways and in several cancer cell lines depletion of PIN1 determines arrest of mitosis followed by apoptosis induction. In conclusion, this study may represent a promising starting pint for the development of new MTDLs hopefully useful for cancer and AD treatment.

Rare and complicated forms of infantile hemangiomas: new therapeutic outlooks

Infantile hemangiomas (IHs) are the most common benign neoplastic pathology of childhood; their natural history generally involves three phases: after the onset, which usually occurs in the first weeks of life, there is the proliferation phase where the IH reaches its maximum development and it is followed by the spontaneous involution which leads to the IH regression. The duration and the extent of these phases may vary widely even though in most of the cases the involution process begins around twelve months of life and the regression, complete or partial, is completed around seventh-ninth year of life. The majority of the IHs does not require any treatment. However, 10%-20% is likely to develop serious complications, functional impairments or aesthetic alterations and entail a timely treatment. Although there is no treatment protocol currently shared, therapies usually used in cases with a complication risk consist in: systemic or intralesional steroids as a first choice; interferon α, vincristine and/or bleomicin as second or third choice and/or surgical treatment. Propranolol, a non-selective beta-blocker, has been used for cardiovascular diseases even in childhood for decades. Since 2008 it has been widely used in the IHs treatment, although it is still "off-label". In literature there are hundreds of cases and some clinical studies that show the effectiveness and safety of this drug for this indication. Thanks to a multidisciplinary team (Dermatologists, Cardiologists, Paediatricians, and Radiologists) of S. Orsola-Malpighi Hospital, a clinical study, which has been previously approved by the ethics committee, is carried out in order to evaluate the efficacy and safety of systemic propranolol in the treatment of IHs in paediatric age. At the end of 2012, 78 patients underwent this treatment: the results we have obtained so far show a good efficacy and safety profile in agreement with the data provided by the literature.

Generation and characterization of mouse models of Small cell lung cancer and Basal cell carcinoma for the preclinical evaluation of new therapies

Background. Human small cell lung cancer (SCLC) accounting for approximately 15-20% of all lung cancers, is an aggressive tumor with high propensity for early regional and distant metastases. Although the initial tumor rate response to chemotherapy is very high, SCLC relapses after approximately 4 months in ED and 12 months in LD. Basal cell carcinoma (BCC) is the most prevalent cancer in the western world, and its incidence is increasing worldwide. This type of cancer rarely metastasizes and the death rate is extraordinary low. Surgery is curative for most of the patients, but for those that develop locally advanced or metastatic BCC there is currently no effective treatment. Both types of cancer have been deeply investigated and genetic alterations, MYCN amplification (MA) among the most interesting, have been found. These could become targets of new pharmacological therapies. Procedures. We created and characterized novel BLI xenograft orthotopic mouse models of SCLC to evaluate the tumor onset and progression and the efficacy of new pharmacological strategies. We compared an in vitro model with a transgenic mouse model of BCC, to investigate and delineate the canonical HH signalling pathway and its connections with other molecular pathways. Results and conclusions. The orthotopic models showed latency and progression patterns similar to human disease. Chemotherapy treatments improved survival rates and validated the in vivo model. The presence of MA and overexpression were confirmed in each model and we tested the efficacy of a new MYCN inhibitor in vitro. Preliminar data of BCC models highlighted Hedgehog pathway role and underlined the importance of both in vitro and in vivo strategies to achieve a better understanding of the pathology and to evaluate the applicability of new therapeutic compounds

Effect of loss of CDKL5 on brain development in a new Cdkl5 knockout mouse model

Rett's Syndrome (RTT) is a severe neurodevelopmental disorder, characterized by cognitive disability that appears in the first months/years of life. Recently, mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been detected in RTT patients characterized by early-onset seizures. CDKL5 is highly expressed in the brain starting from early postnatal stages to adulthood, suggesting the importance of this kinase for proper brain maturation and function. However, the role/s of CDKL5 in brain development and the molecular mechanisms whereby CDKL5 exerts its effects are still largely unknown. In order to characterize the role of CDKL5 on brain development, we created a mice carrying a targeted conditional knockout allele of Cdkl5. A first behavioral characterization shows that Cdkl5 knockout mice recapitulate several features that mimic the clinical features described in CDKL5 patients and are a useful tool to investigate phenotypic and functional aspects of Cdkl5 loss. We used the Cdkl5 knockout mouse model to dissect the role of CDKL5 on hippocampal development and to establish the mechanism/s underlying its actions. We found that Cdkl5 knockout mice showed increased precursor cell proliferation in the hippocampal dentate gyrus. Interestingly, this region was also characterized by an increased rate of apoptotic cell death that caused a reduction in the final neuron number in spite of the proliferation increase. Moreover, loss of Cdkl5 led to decreased dendritic development of new generated granule cells. Finally, we identified the Akt/GSK3-beta signaling as a target of Cdkl5 in the regulation of neuronal precursor proliferation, survival and maturation. Overall our findings highlight a critical role of CDKL5/AKT/GSK3-beta signaling in the control of neuron proliferation, survival and differentiation and suggest that CDKL5-related alterations of these processes during brain development underlie the neurological symptoms of the CDKL5 variant of RTT.

Cannabinoid system combined to classic targets for a new MTDL strategy: design and synthesis of natural inspired molecules for Alzheimer's disease

In this thesis is described the design and synthesis of potential agents for the treatment of the multifactorial Alzheimer’s disease (AD). Our multi-target approach was to consider cannabinoid system involved in AD, together with classic targets. In the first project, designed modifications were performed on lead molecule in order to increase potency and obtain balanced activities on fatty acid amide hydrolase and cholinesterases. A small library of compounds was synthesized and biological results showed increased inhibitory activity (nanomolar range) related to selected target. The second project was focused on the benzofuran framework, a privileged structure being a common moiety found in many biologically active natural products and therapeutics. Hybrid molecules were designed and synthesized, focusing on the inhibition of cholinesterases, Aβ aggregation, FAAH and on the interaction with CB receptors. Preliminary results showed that several compounds are potent CB ligands, in particular the high affinity for CB2 receptors, could open new opportunities to modulate neuroinflammation. The third and the fourth project were carried out at the IMS, Aberdeen, under the supervision of Prof. Matteo Zanda. The role of the cannabinoid system in the brain is still largely unexplored and the relationship between the CB1 receptors functional modification, density and distribution and the onset of a pathological state is not well understood. For this reasons, Rimonabant analogues suitable as radioligands were synthesized. The latter, through PET, could provide reliable measurements of density and distribution of CB1 receptors in the brain. In the fifth project, in collaboration with CHyM of York, the goal was to develop arginine analogues that are target specific due to their exclusively location into NOS enzymes and could work as MRI contrasting agents. Synthesized analogues could be suitable substrate for the transfer of polarization by p-H2 molecules through SABRE technique transforming MRI a more sensitive and faster technique.

Development of new chemical entities to target neuroinflammatory pathways.

Neuroinflammatory pathways are main culprits of neurodegenerative diseases' onset and progression, including Alzheimer’s disease (AD). On this basis, several anti-inflammatory drugs were repurposed in clinical trials. However, they have failed, probably because neuroinflammation is a complex network, still not fully understood. From these evidences, this thesis focused on the design and synthesis of new chemical entities as potential neuroinflammatory drugs or chemical probes. Projects 1 and 2 aimed to multi-target-directed ligand (MTDL) development to target neuroinflammation in AD. Polypharmacology by MTDLs is considered one of the most promising strategies to face the multifactorial nature of neurodegenerative diseases. Particularly, Project 1 took inspiration from a cromolyn-ibuprofen drug combination polypharmacological approach, which was recently investigated in AD clinical trials. Based on that, two cromolyn-(S)-ibuprofen codrug series were designed and synthesized. Parent drugs were combined via linking or fusing strategies in 1:2 or 1:1 ratio, by means of hydrolyzable bonds. Project 2 started from a still ongoing AD clinical trial on investigational drug neflamapimod. It is a selective inhibitor of p38α-MAPK, a kinase strictly involved in neuroinflammatory pathways. On the other side, rasagiline, an anti-Parkinson drug, was also repurposed as AD treatment. Indeed, rasagiline’s propargylamine fragment demonstrated to be responsible not only for the MAO-B selective inhibition, but also for the neuroprotective activity. Thus, to synergistically combine these two effects into single-molecules, a small set of neflamapimod-rasagiline hybrids was developed. In the end BMX, a poorly investigated kinase, which seems to be involved in pro-inflammatory mediator production, was explored for the development of new chemical probes. High-quality chemical probes are a powerful tool in target validation and starting points for the development of new drug candidates. Thus, Project 3 focused on the design and synthesis of two series of optimized BMX covalent inhibitors as selective chemical probes.

Study of genes involved in her2-positive breast cancer progression for identification of new therapeutic targets

HER2 overexpression is observed in 20-30% of invasive breast carcinomas and it is correlated with poor prognosis. Although targeted therapies have revolutionized the treatment of HER2-positive breast cancer, a high number of patients presented primary or acquired resistance to monoclonal antibodies and tyrosine kinase inhibitors. Tumor heterogenicity, epithelial to mesenchymal transition (EMT) and cancer stem cells are key factors in target therapy resistance and tumor progression. The aim of this project was to discover alternative therapeutic strategies to over-come tumor resistance by harnessing immune system and looking for new targetable molecules. The results reported introduce a virus-like particles-based vaccine against HER2 as promising therapeutic approach to treat HER2-positive tumors. The high and persistent anti-HER2 antibody titers elicited by the vaccine significantly inhibited tumor growth and metastases onset. Furthermore, the polyclonal response induced by the vaccine also inhibited human HER2-positive breast cancer cells resistant to trastuzumab in vitro, suggesting its efficacy also on trastuzumab resistant tumors. To identify new therapeutic targets to treat progressed breast cancer, we took advantage from a dynamic model of HER2 expression obtained in our laboratory, in which HER2 loss and cancer progression were associated with the acquisition of EMT and stemness features. Targeting EMT-involved molecules, such as PDGFR-β, or the induction of epithelial markers, like E-cadherin, proved to be successful strategy to impair HER2-negative tumor growth. Density alterations, which might be induced by anti-HER2 target therapies, in cell culture condition of a cell line with a labile HER2 expression, caused HER2 loss probably as consequence of more aggressive subpopulations which prevail over the others. These subpopulations showed an increased EMT and stemness profile, confirming that targeting EMT-involved molecules or antigen expressed by cancer stem cells together with anti-HER2 target therapies is a valid strategy to inhibit HER2-positive cells and simultaneously prevent selection of more aggressive clone.