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Currently ~50% of patients with a diagnosis of high-risk neuroblastoma will not survive due to relapsing or refractory disease. Recent innovations in immunotherapy for solid tumors are highly promising, but the low MHC-I expression of neuroblastoma represents a major challenge for T cell-mediated immunotherapy. Here, we propose a novel T cell-based immunotherapy approach for neuroblastoma, based on the use of TEG002, αβ-T cells engineered to express a defined γδ-T cell receptor, which can recognize and kill target cells independent of MHC-I. In a co-culture killing assay, we showed that 3 out of 6 neuroblastoma organoids could activate TEG002 as measured by IFNγ production. Transcriptional profiling showed this effect correlates with an increased activity of processes involved in interferon signaling and extracellular matrix organization. Analysis of the dynamics of organoid killing by TEG002 over time confirmed that organoids which induced TEG002 activation were efficiently killed independent of their MHC-I expression. Of note, efficacy of TEG002 treatment was superior to donor-matched untransduced αβ-T cells or endogenous γδ-T cells. Our data suggest that TEG002 may be a promising novel treatment option for a subset of neuroblastoma patients.

Several in vivoand in vitro data demonstrated that the DNA damage response (DDR) plays a crucial role in tumorigenesisacting as an anti-cancer barrier. Indeed, cells respond to DNA breaks caused by both exogenous (e.g. chemicals, chemotherapeutics) and endogenous (e.g. deregulated function of oncogenes) sources of DNA damages by properly triggering the activation of the DDR. The latter involves the recruitment of DNA repair proteins to sites of damage and the activation of checkpoint mechanisms to slow down cell cycle progression, thus allowing DNA damage repair or, if the damage could not be repaired, leading to apoptosis or cellular senescence. As a consequence, alterations of DDR functionality have a deep impact on cell proliferation, survival, genomic instability and tumorprogression. microRNAs(miRNAs) are small non coding RNAs that finely regulate gene expression by binding the 3’UTR of their target mRNAs, thus altering theirtranslation, stability and localization. It has been shown that several miRNAsmodulate critical cellular processes deregulated in cancer, acting either as oncogenesor tumorsuppressors. In the present study, we focused on the identification of miRNAsas new modulators of DNA damage activated signalingin several normal and transformed breast cancer cells. In particular, we discovered that miR-181 family acts as a modulator of the levels of ATM (ataxia telangiectasiamutated) kinase, one of the most important proteins involved in sensing DNA double strand breaks and activating DDR. The role of miR-181 family in the DDR pathway, its involvement in the induction of cellular senescence triggered by oncogenicstress (OIS) and its relevance in breast cancer will be presented.

2008/2009 A vast body of evidence from clinical and basic research studies has demonstrated that the p53 pathway acts as an essential barrier in preventing cancer onset and development. ------------------------ A vast body of evidence from clinical and basic research studies has demonstrated that the p53 pathway acts as an essential barrier in preventing cancer onset and development. p53 receives and integrates a wide variety of cytotoxic and genotoxic stress signals from upstream sensors translating them into different cellular outcomes, ranging from apoptosis, cell cycle arrest, se-nescence, DNA repair or other tumor-suppressive responses. p53 exerts its role mainly at the transcriptional level and its timely activation/inactivation in response to stress depends on a complex repertoire of post-translational modifications and interactions with proteins. The crucial role of the p53 pathway in tumor suppression is highlighted by the fact that almost all tumors select for its functional inactivation, either by directly mutating the p53 gene or by altering the expression and functions of key p53 regulators and effectors. Therefore, identification of cellular factors that modulate this pathway and that could be altered in cancer cells, thus allowing to eva-de p53 control, is crucial for understanding cancer development and for designing novel effecti-ve therapeutic approaches. In this context, the aberrant expression or function of microRNAs (miRNAs) might be highly relevant. microRNAs are small non coding RNAs that finely regulate gene expression by binding the 3’UTR of their target mRNAs, thus altering their translation, stability and localization. It has been shown that several miRNAs modulate critical cellular processes deregulated in cancer, ac-ting either as oncogenes or tumor suppressors. On this basis, the aim of this thesis has been the identification and characterization of novel miRNAs able to modulate p53 functions by altering either upstream regulators (i.e. stress-activated kinases) or cofactors of p53. With this purpose we initially selected a panel of twenty-one candidate oncogenic miRNAs from the literature. First, we tested these miRNAs in a functional screening for their ability to modula-te p53-dependent functions in response to cisplatin (apoptosis) and nutlin-3 (cell cycle arrest). Second, miRNAs were also screened for their ability to impair p53 transcriptional activity through a reporter-based assay. We identified five candidate miRNAs from the first approach, and three from the second. miR-26a was identified through both approaches, suggesting that it might repersent a critical modulator of p53 functions. We demonstrated that miR-26a overe-xpression is able to dampen p53 transcriptional activity towards several p53 target promoters (Bax, Pig3 and p21). Moreover, we have shown that miR-26a strongly reduces p53-dependent apoptosis upon DNA damage in different cell lines, to a similar extent as obtained by RNAi-mediated p53 knock-down. At the molecular level, we observed that miR-26a impairs p53 activation by targeting multiple stress-activated kinases that phosphorylate p53, such as ATM, HIPK2 and PKCδ. Accordingly, miR-26a reduces p53 phosphorylation on Ser15 and Ser46 upon DNA damage. As a consequen-ce, miR-26a overexpression allows cells to proliferate in the presence of oncogenic stress, bypassing the induction of senescence (OIS) driven by RASV12 oncogene similar to what obtai-ned by knockdown of either p53 or its activationg kinase ATM. Considering our data and the reports describing miR-26a overexpression in several tumors (e.g. glioblastomas), we speculate that aberrant expression of miR-26a might represent an oncogenic process by preventing activation of the p53 pathway and thus relieving a primary barrier against transformation. For all these reasons, the perspective to block miR-26a expression/functions could represent a new important way to tackle tumors. XXI Ciclo 1981

Breast cancer is a heterogeneous tumor type characterized by a complex spectrum of molecular aberrations, resulting in a diverse array of malignant features and clinical outcomes. Deciphering the molecular mechanisms that fuel breast cancer development and act as determinants of aggressiveness is a primary need to improve patient management. Among other alterations, aberrant expression of microRNAs has been found in breast cancer and other human tumors, where they act as either oncogenes or tumor suppressors by virtue of their ability to finely modulate gene expression at the post-transcriptional level. In this study, we describe a new role for miR-181a/b as negative regulators of the DNA damage response in breast cancer, impacting on the expression and activity of the stress-sensor kinase ataxia telangiectasia mutated (ATM). We report that miR-181a and miR-181b were overexpressed in more aggressive breast cancers, and their expression correlates inversely with ATM levels. Moreover we demonstrate that deregulated expression of miR-181a/b determines the sensitivity of triple-negative breast cancer cells to the poly-ADP-ribose-polymerase1 (PARP1) inhibition. These evidences suggest that monitoring the expression of miR-181a/b could be helpful in tailoring more effective treatments based on inhibition of PARP1 in breast and other tumor types.

About half of all human tumors contain an inactivating mutation of p53, while in the remaining tumors, the p53 pathway is frequently abrogated by alterations of other components of its signaling pathway. In humans, the p53 tumor suppressor is part of a small gene family that includes two other members, p73 and p63, structurally and functionally related to p53. Accumulating evidences indicate that all p53-family proteins function as molecular hubs of a highly interconnected signaling network that coordinates cell proliferation, differentiation and death in response to physiological inputs and oncogenic stress. Therefore, not only the p53-pathway but the entire "p53-family pathway" is a primary target for cancer drug development. In particular, the p53-related protein p73 has a crucial role in determining cellular responses to chemotherapy, and can vicariate p53 functions in triggering cell death after DNA damage in multiple experimental models. The biology and regulation of p73 is complex, since the TP73 gene incorporates both tumor-suppressive and proto-oncogenic functions. However, the p73 gene is rarely mutated in tumors, so appropriate pharmacological manipulation of the p73 pathway is a very promising approach for cancer therapy. Here we provide an overview of the principal mechanism of p73 regulation, and describe several examples of pharmacological tools that can induce p73 accumulation and function by acting on upstream p73 modulators or displacing inhibitory p73 interactors. A better understanding of how the p73 pathway works is mandatory to discover additional players intervening in this pathway and has important implications for the improvement of cancer treatment with the development of new molecules or with the reposition of currently available drugs.