Comparative molecular targets for anticancer chemotherapy: new insights and perspectives

The past thirty years have reported the introduction of multiple anticancer therapies targeting various aspects of the cancer hallmarks, which are essential for successful tumor propagation and dissemination. In this sense, the evolution of molecular-scale technology has been central to the identification of new cancer targets. The receptor tyrosine kinase (RTK) Hardy-Zuckerman 4 feline sarcoma virus homolog (c-KIT) is a critical regulator of growth, differentiation, migration and proliferation in the hematopoietic system, in germ cells and melanocytes. Since it activates a number of intracellular signaling pathways implicated in the tumor progression, it is one of the most studied proto-oncogenes as well as the target of drugs belonging to the family of tyrosine kinase inhibitors (TKIs). Actually, TKIs are employed for the treatment of human and canine c-KIT-dependent tumors as an alternative to standard chemotherapy. Nevertheless, multiple resistance phenomena frequently occur. Recently, the discovery of G-quadruplex (G4) structures highlighted a new role for DNA in cancer biology. DNA G4 are four-stranded globular nucleic acid secondary structures, formed in specific G-rich sequences with biological significance; among these ones, the human telomeres and the promotorial region of oncogenes such as c-KIT. In the first part of this dissertation, three compounds were proved to bind in silico c-KIT G4 and were tested in human and canine cell lines to check for their potential usefulness as therapeutic agents. Interesting results, e.g. c-KIT mRNA and protein inhibition, were obtained with an anthraquinone derivative (AQ1) that caused a block of cell proliferation. In another study, the occurrence of c-KIT mutations was investigated in matched primary and metastatic canine cutaneous mast cell tumor (MCT), to make a recommendation for the best therapeutic choice. In dogs, 10-30% of MCTs possess c-KIT mutations, and the relevance of the mutational status for the therapy with TKIs is nowadays accepted also in this species; however, little is known on c-KIT mutational status in metastatic MCTs. In all analyzed dogs, there was a perfect concordance between c-KIT mutational status in primary MCT and the relative lymph node metastasis. This has a relevant implication for clinical practices. Finally, during the Ph.D. program, a collaboration was established with the Centre de Recherche en Cancérologie de Marseille, and particularly with Dr. Patrice Dubreuil. In his most recent articles, he discovered a set of genes that are frequently mutated in human systemic mastocytosis (SM) and cooperate with c-KIT in the disease malignant evolution. In the last study illustrated in this Ph.D. thesis, the mutational profile of these hotspot genes in canine MCTs samples has been screened, in order to find molecular similarities between the two diseases, thereby justifying the use of domestic dog as an animal model in comparative oncology

Screening of candidate G-quadruplex ligands for the human c-KIT promotorial region and their effects in multiple in-vitro models

Stabilization of G-quadruplex (G4) structures in promoters is a novel promising strategy to regulate gene expression at transcriptional and translational levels. c-KIT proto-oncogene encodes for a tyrosine kinase receptor. It is involved in several physiological processes, but it is also dysregulated in many diseases, including cancer. Two G-rich sequences able to fold into G4, have been identified in c-KIT proximal promoter, thus representing suitable targets for anticancer intervention. Herein, we screened an \u201cin house\u201d library of compounds for the recognition of these G4 elements and we identified three promising ligands. Their G4-binding properties were analyzed and related to their antiproliferative, transcriptional and post-transcriptional effects in MCF7 and HGC27 cell lines. Besides c-KIT, the transcriptional analysis covered a panel of oncogenes known to possess G4 in their promoters. From these studies, an anthraquinone derivative (AQ1) was found to efficiently downregulate c-KIT mRNA and protein in both cell lines. The targeted activity of AQ1 was confirmed using c-KIT\u2013dependent cell lines that present either c-KIT mutations or promoter engineered (i.e., \u3b1155, HMC1.2 and ROSA cells). Present results indicate AQ1 as a promising compound for the target therapy of c-KIT-dependent tumors, worth of further and in depth molecular investigations

Comparative molecular targets for anticancer chemotherapy: new insights and perspectives

The past thirty years have reported the introduction of multiple anticancer therapies targeting various aspects of the cancer hallmarks, which are essential for successful tumor propagation and dissemination. In this sense, the evolution of molecular-scale technology has been central to the identification of new cancer targets. The receptor tyrosine kinase (RTK) Hardy-Zuckerman 4 feline sarcoma virus homolog (c-KIT) is a critical regulator of growth, differentiation, migration and proliferation in the hematopoietic system, in germ cells and melanocytes. Since it activates a number of intracellular signaling pathways implicated in the tumor progression, it is one of the most studied proto-oncogenes as well as the target of drugs belonging to the family of tyrosine kinase inhibitors (TKIs). Actually, TKIs are employed for the treatment of human and canine c-KIT-dependent tumors as an alternative to standard chemotherapy. Nevertheless, multiple resistance phenomena frequently occur. Recently, the discovery of G-quadruplex (G4) structures highlighted a new role for DNA in cancer biology. DNA G4 are four-stranded globular nucleic acid secondary structures, formed in specific G-rich sequences with biological significance; among these ones, the human telomeres and the promotorial region of oncogenes such as c-KIT. In the first part of this dissertation, three compounds were proved to bind in silico c-KIT G4 and were tested in human and canine cell lines to check for their potential usefulness as therapeutic agents. Interesting results, e.g. c-KIT mRNA and protein inhibition, were obtained with an anthraquinone derivative (AQ1) that caused a block of cell proliferation. In another study, the occurrence of c-KIT mutations was investigated in matched primary and metastatic canine cutaneous mast cell tumor (MCT), to make a recommendation for the best therapeutic choice. In dogs, 10-30% of MCTs possess c-KIT mutations, and the relevance of the mutational status for the therapy with TKIs is nowadays accepted also in this species; however, little is known on c-KIT mutational status in metastatic MCTs. In all analyzed dogs, there was a perfect concordance between c-KIT mutational status in primary MCT and the relative lymph node metastasis. This has a relevant implication for clinical practices. Finally, during the Ph.D. program, a collaboration was established with the Centre de Recherche en Cancérologie de Marseille, and particularly with Dr. Patrice Dubreuil. In his most recent articles, he discovered a set of genes that are frequently mutated in human systemic mastocytosis (SM) and cooperate with c-KIT in the disease malignant evolution. In the last study illustrated in this Ph.D. thesis, the mutational profile of these hotspot genes in canine MCTs samples has been screened, in order to find molecular similarities between the two diseases, thereby justifying the use of domestic dog as an animal model in comparative oncology.Negli ultimi trent’anni, l’evoluzione delle tecnologie in campo medico-scientifico ha permesso la più profonda conoscenza dei meccanismi molecolari alla base dello sviluppo, della crescita e della diffusione del tumore. Tutto ciò ha permesso di sviluppare le cosiddette terapie mirate, identificando nuovi bersagli terapeutici. Il recettore tirosin-chinasico c-KIT è un fattore critico per la regolazione della crescita, differenziazione, migrazione e proliferazione delle cellule germinali, di quelle del sistema ematopoietico e dei melanociti. c-KIT è anche coinvolto nell’attivazione di numerosi meccanismi intracellulari implicati nella progressione tumorale e, allo stesso tempo, è uno dei proto-oncogeni più studiati ed il bersaglio di farmaci appartenenti alla famiglia degli inibitori tirosin-chinasici (TKIs). Attualmente, i TKIs sono approvati come trattamento alternativo alla chemioterapia tradizionale in tumori c-KIT dipendenti in uomo e cane tuttavia, fenomeni di resistenza a questi farmaci si verificano frequentemente. Negli ultimi anni, la scoperta di strutture secondarie del DNA chiamate G-quadruplex (G4) ha evidenziato un nuovo ruolo degli acidi nucleici nella biologia tumorale. Tali conformazioni si formano in specifiche sequenze del DNA ricche in residui di guanina, localizzate principalmente nei telomeri e nelle regioni promotoriali di alcuni oncogeni come c-KIT. Nella prima parte di questa tesi di dottorato, tre composti scelti sulla base della loro capacità di legare e stabilizzare le conformazioni G4 sono stati testati in linee cellulari stabilizzate di uomo e cane al fine di determinare la loro efficacia come potenziali agenti terapeutici. In questo senso, alcuni risultati interessanti in termini di blocco della proliferazione nonché della trascrizione e traduzione di c-KIT si sono ottenuti con un derivato della famiglia degli antrachinoni chiamato AQ1. In un altro studio proposto, il profilo mutazionale di c-KIT è stato analizzato in una coorte di campioni di mastocitoma del cane composti da tumore primitivo e relativa metastasi linfonodale. Nel cane infatti, la percentuale tra il 10 ed il 30% dei mastocitomi presenta almeno una mutazione di c-KIT nel tumore primitivo, tuttavia, poche conoscenze si hanno relativamente al profilo mutazionale nelle metastasi. Dai risultati ottenuti, tutti i cani analizzati hanno dimostrato avere una perfetta concordanza tra tumore primitivo e metastasi in termini di status mutazionale di c-KIT con rilevanti implicazioni cliniche per la scelta della miglior terapia da attuare da parte degli oncologi veterinari. Infine, nel corso del secondo anno di dottorato, è nata una collaborazione con il Dr. Patrice Dubreuil del Centre de Recherche en Cancérologie de Marseille, a Marsiglia. Nei suoi recenti articoli sulla mastocitosi dell’uomo, egli ha scoperto un set di geni che presentano mutazioni in talune percentuali di casi e cooperano con c-KIT nello sviluppo delle forme più gravi ed aggressive della malattia. Nell’ultima pubblicazione illustrata in questa dissertazione, è stato eseguito lo screening del profilo mutazionale di questi nuovi geni in campioni di mastocitoma di cane al fine di trovare analogie molecolari che possano giustificare l’uso del cane come animale modello nell’oncologia comparata

Might canine mast cell tumors share some molecular pathways with human systemic mastocytosis besides c-KIT?

Introduction Human systemic mastocytosis (SM) and canine cutaneous mast cell tumor (MCT) are both characterized by proliferation and the accumulation of abnormal mast cells in tissues and the presence of activating mutations in the receptor tyrosine kinase c-KIT. Recently, it has been shown that additional cooperating events are likely to contribute to SM pathogenesis and/or phenotype. In present study, hypothesizing analogies in molecular mechanisms and gene dysfunctions between SM and canine MCT, the mutational profile of some genes, commonly mutated in human myeloid malignancies and involved in splicing machinery, DNA methylation and signal transduction processes, was investigated in canine MCTs. Materials and Methods Genomic DNA was extracted from 75 MCT biopsies. Canine TET2, IDH2, IDH1, SRSF2, SF3B1, KRAS and NRAS DNA sequences were identified and the presence of \u201chuman-like\u201d hot spot mutations was checked by using a ABI 3730 sequencer. Results No mutations were ever identified in target genes except for TET2, for which one point-mutation and one amino-acid deletion, detected in 2 samples (3.7%), and a single nucleotide polymorphism (SNP) found out in TET2 exon 11 (a tandem repetition of a glutamine amino-acid). Conclusion In SM, TET2 mutations occur at a frequency ten times higher than the one observed in the dog. Therefore, it looks like that the two pathologies might differ in molecular pathways involved in the disease pathogenesis; despite this, the TET2 tandem SNP is actually under investigation in a population of healthy dogs to verify any predisposition in disease development

Mutational Hotspot of TET2, IDH1, IDH2, SRSF2, SF3B1, KRAS, and NRAS from Human Systemic Mastocytosis Are Not Conserved in Canine Mast Cell Tumors.

Both canine cutaneous mast cell tumor (MCT) and human systemic mastocytosis (SM) are characterized by abnormal proliferation and accumulation of mast cells in tissues and, frequently, by the presence of activating mutations in the receptor tyrosine kinase V-Kit Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog (c-KIT), albeit at different incidence (>80% in SM and 10-30% in MCT). In the last few years, it has been discovered that additional mutations in other genes belonging to the methylation system, the splicing machinery and cell signaling, contribute, with c-KIT, to SM pathogenesis and/or phenotype. In the present study, the mutational profile of the Tet methylcytosine dioxygenase 2 (TET2), the isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2), the serine/arginine-rich splicing factor 2 (SRSF2), the splicing factor 3b subunit 1 (SF3B1), the Kirsten rat sarcoma viral oncogene homolog (KRAS) and the neuroblastoma RAS viral oncogene homolog (NRAS), commonly mutated in human myeloid malignancies and mastocytosis, was investigated in canine MCTs.Using the Sanger sequencing method, a cohort of 75 DNA samples extracted from MCT biopsies already investigated for c-KIT mutations were screened for the "human-like" hot spot mutations of listed genes.No mutations were ever identified except for TET2 even if with low frequency (2.7%). In contrast to what is observed in human TET2 no frame-shift mutations were found in MCT samples.Results obtained in this preliminary study are suggestive of a substantial difference between human SM and canine MCT if we consider some target genes known to be involved in the pathogenesis of human SM

Whole-Transcriptome Profiling of Canine and Human in Vitro Models Exposed to a G-Quadruplex Binding Small Molecule

G-quadruplexes (G4) are secondary nucleic acid structures that have been associated with genomic instability and cancer progression. When present in the promoter of some oncogenes, G4 structures can affect gene regulation and, hence, represent a possible therapeutic target. In this study, RNA-Seq was used to explore the effect of a G4-binding anthraquinone derivative, named AQ1, on the whole-transcriptome profiles of two common cell models for the study of KIT pathways; the human mast cell leukemia (HMC1.2) and the canine mast cell tumor (C2). The highest non-cytotoxic dose of AQ1 (2 mu M) resulted in 5441 and 1201 differentially expressed genes in the HMC1.2 and C2 cells, respectively. In both cell lines, major pathways such as cell cycle progression, KIT- and MYC-related pathways were negatively enriched in the AQ1-treated group, while other pathways such as p53, apoptosis and hypoxia-related were positively enriched. These findings suggest that AQ1 treatment induces a similar functional response in the human and canine cell models, and provide news insights into using dogs as a reliable translational model for studying G4-binding compounds

Concordance of c-kit Mutational Status in Matched Primary and Metastatic Cutaneous Canine Mast Cell Tumors at Baseline

BACKGROUND: Mutation analysis of proto-oncogene c-kit (c-kit) is advisable before starting treatment with tyrosine kinase inhibitors in dogs with mast cell tumor (MCT), including those with metastatic disease. Testing is usually performed on primary tumors, assuming that c-kit mutation status does not change in metastasis. HYPOTHESIS/OBJECTIVES: To give an insight into the mutational processes and to make a recommendation on the use of c-kit mutational analysis in the clinical setting. ANIMALS: Twenty-one client-owned dogs with metastatic MCT. METHODS: Dogs undergoing resection or biopsy for both primary and matched metastatic MCT were prospectively enrolled. Total RNA or DNA was extracted from primary MCT and corresponding metastases. Exons 8, 9, and 11 were amplified by PCR and sequenced. Genetic features between primary MCT and metastases were compared. Their correlation with clinicopathologic features was investigated. RESULTS: Concordance (mutated or wild-type) of mutational status, evaluable in 21 primary and matched metastatic (20 nodal and 1 splenic) MCTs, was 100%. Three new c-kit mutations were identified. No significant correlation was detected between c-kit mutation and clinicopathologic features. CONCLUSIONS AND CLINICAL IMPORTANCE: Proto-oncogene c-kit mutational status is conserved between any primary and its matched secondary tumor, suggesting that both can be used for c-kit mutational testing. Targeted therapies might be also used to treat metastatic disease

G-rich sequences within proto-oncogene KIT promoter region as targets for anticancer therapy.

In last decades the role of non-canonical DNA structures as switching on/off mechanism of gene transcription has been extensively investigated. In particular, downregulation of gene expression by induction of G-quadruplex DNA structures at promotorial level is an attractive anticancer strategy. In humans, two guanine-rich sequences (h_kit1 and h_kit2) were identified in the promotorial region of the proto-oncogene KIT. Their stabilization into G-quadruplex structures can find applications in the treatment of leukemias, mastocytosis, gastrointestinal stromal tumor, lung carcinomas, which are often associated to c-kit mis-regulation. Here we screeneed libraries of G-quadruplex binders to identify novel hits highly selective for the G-quadruplexes of KIT promoter. To provide the biological rationale for the molecular and cellular mechanisms of action of this novel therapeutic approach, a full characterization of target selectivity, DNA complex formation and cellular effects has been performed. To ultimately provide the required knowledge to convert the identified hits into new species-specific targeted drugs, we searched for a robust translational model too. Interestingly, the most common skin cancer in domestic dog, mast cell tumor, is also linked to a mutation and/or to an over-expression of c-kit. Moreover, a general conserved promotorial sequence between the two species has been confirmed, thus supporting dog as an excellent animal model. In particular, the conformational features of the canine kit1 sequence are substantially shared with the human one. Conversely, two isoforms of the kit2 sequences were identified and, in comparison with the human counterpart, both of them showed an altered distribution among several folded conformations. Here we compared our results on human and canine cells, in order to provide a more comprehensive description of the physiological pathways supported by c-kit mis-regulation in carcinogenesis and to highlight the similarities/differences between the two species