UOSD SAFU

Metastatic melanomas harboring BRAF-V600 mutations are currently treated with combinations of BRAF and MEK inhibitors (MAPKi) increasing the objective responses, disease free survival and overall survival over monotherapy with BRAF inhibitors. Unfortunately, several patients suffer from ab initio or acquired resistance to these agents. Several efforts have been directed in recent years to understand mechanisms of resistance to MAPK inhibitors. These studies have shown a prominent involvement of non-mutational adaptive events, among which also deregulation of a class of small non-coding RNAs, namely microRNAs.

Dr. Fattore’s research activities are focused on the study of the role of miRNAs in this phenomenon. His work stems from a comprehensive analysis of the entire miRNome using an in vitro model of acquired drug resistance to BRAF inhibitors represented by several melanoma cell lines mutated in the BRAF oncogene. In this way, it was highlighted a general dysregulation of a large network of miRNAs, both oncosuppressors and oncomiRs involved in several signaling pathways responsible for the modification of cell intrinsic and cell extrinsic features characteristic of drug resistant tumors. In particular, the focus has been directed to four miRNAs, two known oncosuppressor miRNAs, miR-199b-5p and miR-204-5p, and two novel oncomiRs, namely miR-4443 and miR-4488. Their characterization as therapeutic and diagnostic tools has been the focus of the work of Dr. Fattore in the last year. The first activity has taken advantage of Lipid Nanoparticles (LNPs) carrying oncosuppressive miRNAs (i.e. miR-199b-5p and miR-204-5p), which have been tested in vitro in different melanoma cell lines alone or in combination with target therapy. Results have demonstrated the capability of LNPs to reduce melanoma cell growth; these effects are strongly improved when they have been combined with BRAFi+MEKi. These findings have paved the way to perform in vivo validation using melanoma cell lines xenografted in nude mice. The goal will be to determine the capability of LNPs+BRAFi+MEKi to reduce tumor growth and delay tumor recurrence in vivo. As to the diagnostic applications, they have been measured the circulating levels of miR-204-5p, miR-199b-5p, miR-4443 and miR-4488 in the serum of 51 BRAF-mutated melanoma patients before the beginning of therapy with MAPKi. Results demonstrated that miR-204-5p circulating levels before starting therapy have a strong predictive value for OS and PFS. Concerning OS, patients with a ΔCt value under the ROC cut-off show a shorter median time to death in comparison to patients with a ΔCt value over the ROC cut-off (10 months 95% confidence interval (95%CI): (3.9-16.1) vs 34 months 95%CI: (25.7-42.3); p-value=0.013). Concerning PFS analysis, patients with a ΔCt value under the ROC cut-off have a shorter median time to progression in comparison to patients with a ΔCt value over the ROC cut-off (5 months 95%CI: (4.1-5.9) vs 18 months 95%CI: (7.9-28.1); p-value=0.006). Furthermore, this miRNA is also able to predict both OS and PFS in combination with miR-199b-5p and miR-4488. These promising findings justify further efforts to: 1) increase the predictive value of the circulating miRNAs and 2) confirm their relevance on a larger cohort of patients. The goal is to develop this signature as a companion diagnostic in the clinic.

By using advanced preclinical models, Leonetti’s group investigated new therapeutic approaches for the treatment of human solid cancers. Preliminary in vitro experiments performed in breast cancer cells, demonstrated that G-quadruplex (G4) compounds, which are able to interact with G4 DNA structures present in cancer cells, synergizes with antineoplastic drugs in killing cancer cells. The in vivo results, obtained in an orthotopic model of breast cancer, which recapitulate clinical situation, confirmed that G4 ligands were able to increase the efficacy of Paclitaxel, thus suggesting a possible clinical application of this combination. The recent advances in organoids, in vitro 3D culture of human cancer, have opened new avenues for a more efficient translation of basic cancer research into novel treatment regimens. To this purpose, the same group have established orthotopic models of colon cancer by implanting organoids possessing KRAS mutations, which are generally associated with clinical aggressiveness of this cancer and reduced survival of the patients. This model, which closely mimic the clinical setting, will be used for investigating more efficacy therapies in the context of KRAS mutated colon cancer for which no effective therapies exist. Finally, analysis with the Open Array Real-Time PCR platform which include 624 cancer genes, permitted to identify genes regulated by G4 ligands. In particular, the analysis evidentiated 5 genes up-regulated and 19 genes down-regulated after the treatment with the G4 ligand. Interestingly, these genes have a relevant role in colon cancer progression and 3 out of 5 genes up-regulated and in 11 out of 19 down-regulated, possess G4 sequences, thus confirming the transcriptional control of the G4 ligand. Future studies are planned to establish the functional relevance of these genes with the aim to identify new therapeutic targets.

Multiple myeloma (MM) is a neoplasm characterized by the accumulation of proliferating antibodies producing plasma cells in the bone marrow. Despite several therapeutical improvements, MM remains incurable with patients subject to relapses. This disease is characterized by a high frequency of structural variants (SVs) and copy-number abnormalities (CNAs). In addition, an increasing number of studies provides evidence of numerous regulatory shifts in the genomic organization during the development of MM. Importantly, several undifferentiated MMs show reorganization of the chromatin including euchromatic histone marks up-regulation. Consistent with these findings, in MM cells an increase in the accessibility of chromatin compared to normal plasma cells was observed, with a significant conversion of heterochromatic regions into accessible “active chromatin”. Che-1/AATF (Che-1) is a protein identified by its ability to bind RNA polymerase II (Pol II)12. Several studies have demonstrated its involvement in the regulation of gene transcription and tumor cell proliferation. Che-1 is present in histone acetyltransferase complexes SAGA and ATAC through its interaction with the transcriptional co-activators ADA2, ADA3 and GCN5 and it acts as an endogenous HDAC1 inhibitor through its ability to disrupt the binding of pRb and Sp1 proteins to this enzyme. In addition, Che-1 plays an important role in the cellular response to the DNA damage (DDR) or to other cellular stressors, and sustains cell survival in MM cells by inhibiting mTORC1 activity and inducing autophagy. During this year, we demonstrated that Che-1 plays a crucial role in the transformation and proliferation of MM cells by increasing chromatin accessibility at both proximal and distal regulatory elements in in vitro and in vivo MM models. Using a comprehensive plethora of low- and high- throughput approaches coupled with ad hoc bioinformatic analysis, we observed a linear relationship between Che-1 and general histone acetylation in MM patients. Strikingly, Che-1 depletion induces a global transcription shut-off by systematically reducing histone acetylation. These results contribute to further elucidate the role of Che-1 as an essential component of the transcription machinery in MM, and in confirming Che-1 as a possible target for enhancing the efficacy of anti-tumor agents.

Cancer stem cells (CSCs) are tumor subpopulations driving disease development, progression, relapse and therapy resistance, and their targeting ensures tumor eradication. CSCs display heterogeneous replication stress (RS), but the functionality/relevance of the RS response (RSR) centered on the ATR-CHK1 axis is debated. In collaboration with Prof. Ilio Vitale, during this year we show that the RSR is efficient in primary CSCs from colorectal cancer (CRC-SCs), and describe unique roles for PARP1 and MRE11/RAD51. We demonstrated that PARP1 is upregulated in CRC-SCs resistant to several replication poisons and RSR inhibitors (RSRi). In these cells, PARP1 modulates replication fork speed resulting in low constitutive RS. Moreover, we showed that MRE11 and RAD51 cooperate in the genoprotection and mitosis execution of PARP1-upregulated CRC-SCs. These roles represent therapeutic vulnerabilities for CSCs. Indeed, PARP1i sensitized CRC-SCs to ATRi/CHK1i, inducing replication catastrophe, and prevented the development of resistance to CHK1i. Furthermore, MRE11i + RAD51i selectively killed PARP1-upregulated CRC-SCs via mitotic catastrophe. These results provide the rationale for biomarker-driven clinical trials in CRC using distinct RSRi combinations.

Strano’s group is actively investigating the involvement of the HIPPO transducers, YAP and TAZ, in the chemoresistance of diverse types of human cancers. The targeting of the transcriptional axis YAP/TAZ/TEAD holds promising therapeutic value as novel approach for cancer treatment. Moreover, Drs. Strano is focusing her efforts on the identification of non-coding RNA circulating biomarkers predicting mucositis as major side effect of chemo-radiotherapeutic treatment of human cancers.

The solar ultraviolet (UV) radiation is a most powerful environmental carcinogen. Skin tumours are steadily increasing at any latitude and among any racial and social group and such a trend is expected worsen in the next decades. Through protein oxidation studies our group has demonstrated that UV radiation preferentially oxidizes a subset of cellular proteins involved in stress response; protein folding/refolding and quality control; proteasomal function; DNA damage repair; cell architecture; adhesion/migration; proliferation and oncosuppression; as a consequence intercurrent genetic alteration are inefficiently removed and transformant clones are increasingly generated. Thus, environmental UV radiation is both a cancer initiating and a strong cancer promoting agent. Finally, UV act a selective agent to for those clones with increased fitness to Oxidative Stress thus increasing the cell genomic instability and promoting an accelerated rate of neoplastic evolution.

Papillomaviruses are a first-class single carcinogen whose relevance for human oncology is going to stay despite the vaccine availability. The recent identification of a mouse Papillomavirus paves the way to the setup of a convenient animal model for human HPV related neoplastic diseases. Our preliminary results indicate that in mouse newborn keratinocytes, following “in vitro” infection with MoPV The IFN type 1 response is poorly activated and takes place with a rather slow RNA kinetics. Conversely the IFN type 3 response occurs, as expected, within a few hours since the challenge. In both IFN type1 and type 3 response a rather poor TLR3 and TLR9 induction is seen. Such a lack of activation may be a crucial reason for establishment of persistent infection. These data deserve to be confirmed in more large and comprehensive experiments

The pandemic in 2020 has strongly influenced the Piaggio’s group research activities. Indeed, from the beginning of the pandemic in March 2020, the group has been involved in the development of protocols to study the impact of the SARS-CoV-2 virus on the immunological response of cancer patients. In the last month of the year, the group was also involved in projects on vaccine efficacy, which are being continued in 2021. Alongside the activities related to COVID-19, the group also continued the projects already started in previous years, also in collaboration with other groups, aimed at the identification of novel features of the neoplasia micro and macroenvironments.

Dr. Aymone Gurtner studies aim at identifying mutp53-dependent miRNAs and their target genes as useful prognostic and/or predictive biomarkers of response to therapy in patients with CRC. Since for many genes putatively regulated by mutp53 identified in these studies there are drugs approved by the FDA or undergoing clinical trials, these results open the way to identify “vulnerabilities” of mutp53 with studies on organoids derived from patients. Furthermore, her research interests are based on understanding the molecular mechanisms of miRNA deregulation in cancer. In this regard she is obtaining data that demonstrate a new GOF of mutp53 that resides in its ability to inhibit the expression of miRNA at the level of biogenesis interfering with Dicer activity. Moreover, in collaboration with Giulia Piaggio’s group, she is characterizing the functional role of NF-Y on transcriptional deregulation of miRNAs during epithelial mesenchymal transition of colon tumor cells.