Phd in INNOVATIVE BIOMEDICAL TECHNOLOGIES IN CLINICAL MEDICINE

Thesis title: Targeting the DNA-repair chromatin-modifier protein SMYD3 as a potential epigenetics-based therapy for gastric cancer

Gastric cancer (GC) is the third most common cause of cancer-related death worldwide. Since it is often diagnosed only at advanced stages, the prognosis is generally poor, with a median overall survival of less than one year. In the current clinical setting, chemotherapy remains the mainstay of treatment; however, its clinical effectiveness is frequently undermined by disease recurrence and the emergence of resistance, highlighting the urgent need for novel therapeutic strategies. The histone methyltransferase SMYD3 is overexpressed in approximately 50% of GCs, where it exerts oncogenic functions that contribute to tumor progression. We investigated the role of SMYD3 in the regulation of DNA damage repair mechanisms in GC. Through co-immunoprecipitation (Co-IP) assays, we identified specific interactions between SMYD3 and homologous recombination (HR) proteins, suggesting its involvement in multiple stages of the HR repair cascade. This finding also pointed to a potential role of SMYD3 in the earliest steps of the DNA damage response, likely through its function as a chromatin remodeler. To explore this hypothesis, we used the ER-AsiSI system in GC cells and performed chromatin immunoprecipitation (ChIP) analyses to assess histone modifications associated with DNA damage signaling, as well as the recruitment of HR proteins to the break site. Both in silico and in vitro analyses demonstrated that ATM, a critical kinase of the DNA damage response, phosphorylates SMYD3 at threonine 22. The functional relevance of this phosphorylation was established in GC cell lines and further confirmed in vivo, using protein extracts from GC patients treated with neoadjuvant chemotherapy, and then subjected to surgical resection. Moreover, Co-IP experiments revealed that ATM-dependent phosphorylation of SMYD3 is required for the assembly of the HR repair complex. Functionally, inhibition of SMYD3 led to a complete suppression of HR repair and a partial impairment of non-homologous end joining (NHEJ). In this light, we evaluated a therapeutic strategy based on the combined inhibition of SMYD3 and PARP, aiming to achieve complete suppression of DNA repair through synthetic lethality. This combinatorial approach was validated in GC cells, spheroids, and patient-derived organoids. Finally, to overcome the challenge of acquired resistance to PARP inhibitors, we generated GC cells resistant to Olaparib and demonstrated that inhibition of SMYD3 effectively restored drug sensitivity. Altogether, these findings uncover a previously unrecognized role of SMYD3 as a key regulator of the DNA damage response in GC. By integrating mechanistic insights with translational approaches, this work highlights SMYD3 inhibition, alone or in combination with PARP inactivation, as a promising therapeutic strategy to target DNA repair vulnerabilities in this tumor model. Remarkably, from a therapeutic perspective, our findings provided compelling evidence that SMYD3 represents a promising druggable target in GC.