The novel SMYD3 inhibitor EM127 impairs DNA repair response to chemotherapy-induced DNA damage and reverses cancer chemoresistance.

BACKGROUND: SMYD3 has been found implicated in cancer progression. Its overexpression correlates with cancer growth and invasion, especially in gastrointestinal tumors. SMYD3 transactivates multiple oncogenic mechanisms, favoring cancer development. Moreover, it was recently shown that SMYD3 is required for DNA restoration by promoting homologous recombination (HR) repair. METHODS: In cellulo and in vivo models were employed to investigate the role of SMYD3 in cancer chemoresistance. Analyses of SMYD3-KO cells, drug-resistant cancer cell lines, patients' residual gastric or rectal tumors that were resected after neoadjuvant therapy and mice models were performed. In addition, the novel SMYD3 covalent inhibitor EM127 was used to evaluate the impact of manipulating SMYD3 activity on the sensitization of cancer cell lines, tumorspheres and cancer murine models to chemotherapeutics (CHTs). RESULTS: Here we report that SMYD3 mediates cancer cell sensitivity to CHTs. Indeed, cancer cells lacking SMYD3 functions showed increased responsiveness to CHTs, while restoring its expression promoted chemoresistance. Specifically, SMYD3 is essential for the repair of CHT-induced double-strand breaks as it methylates the upstream sensor ATM and allows HR cascade propagation through CHK2 and p53 phosphorylation, thereby promoting cancer cell survival. SMYD3 inhibition with the novel compound EM127 showed a synergistic effect with CHTs in colorectal, gastric, and breast cancer cells, tumorspheres, and preclinical colorectal cancer models. CONCLUSIONS: Overall, our results show that targeting SMYD3 may be an effective therapeutic strategy to overcome chemoresistance.

Uncoupling p38α nuclear and cytoplasmic functions and identification of two p38α phosphorylation sites on ß-catenin: implications for the Wnt signaling pathway in CRC models.

BACKGROUND: Activation of the Wnt pathway has been linked to colorectal cancer (CRC). Previous reports suggest that Wnt3a can activate p38. Besides, p38α feeds into the canonical Wnt/ß-catenin pathway by inhibiting GSK3ß through phosphorylation. Recently, we identified p38α as a new druggable member of ß-catenin chromatin-associated kinase complexes in CRC. METHODS: The functional relationship between p38α and ß-catenin was characterized in CRC cells, patient-derived CRC stem cells, patient-derived tumor intestinal organoids, and in vivo models (C57BL/6-APCMin/+ mice). The role of p38α in ß-catenin transcriptional activity was assessed by pharmacological inhibition with ralimetinib. RESULTS: We used the GSK3ß inhibitor TWS-119, which promotes the activation of Wnt signaling, to uncouple p38α nuclear/cytoplasmatic functions in the Wnt pathway. Upon GSK3ß inhibition, nuclear p38α phosphorylates ß-catenin at residues S111 and T112, allowing its binding to promoter regions of Wnt target genes and the activation of a transcriptional program implicated in cancer progression. If p38α is pharmacologically inhibited in addition to GSK3ß, ß-catenin is prevented from promoting target gene transcription, which is expected to impair carcinogenesis. CONCLUSIONS: p38α seems to play a dual role as a member of the ß-catenin destruction complex and as a ß-catenin chromatin-associated kinase in CRC. This finding may help elucidate mechanisms contributing to human colon tumor pathogenesis and devise new strategies for personalized CRC treatment.

SMYD3 Modulates AMPK-mTOR Signaling Balance in Cancer Cell Response to DNA Damage.

Cells respond to DNA damage by activating a complex array of signaling networks, which include the AMPK and mTOR pathways. After DNA double-strand breakage, ATM, a core component of the DNA repair system, activates the AMPK-TSC2 pathway, leading to the inhibition of the mTOR cascade. Recently, we showed that both AMPK and mTOR interact with SMYD3, a methyltransferase involved in DNA damage response. In this study, through extensive molecular characterization of gastrointestinal and breast cancer cells, we found that SMYD3 is part of a multiprotein complex that is involved in DNA damage response and also comprises AMPK and mTOR. In particular, upon exposure to the double-strand break-inducing agent neocarzinostatin, SMYD3 pharmacological inhibition suppressed AMPK cascade activation and thereby promoted the mTOR pathway, which reveals the central role played by SMYD3 in the modulation of AMPK-mTOR signaling balance during cancer cell response to DNA double-strand breaks. Moreover, we found that SMYD3 can methylate AMPK at the evolutionarily conserved residues Lys411 and Lys424. Overall, our data revealed that SMYD3 can act as a bridge between the AMPK and mTOR pathways upon neocarzinostatin-induced DNA damage in gastrointestinal and breast cancer cells.

The chromatin remodeling factors EP300 and TRRAP are novel SMYD3 interactors involved in the emerging 'nonmutational epigenetic reprogramming' cancer hallmark.

SMDY3 is a histone-lysine N-methyltransferase involved in several oncogenic processes and is believed to play a major role in various cancer hallmarks. Recently, we identified ATM, BRCA2, CHK2, MTOR, BLM, MET, AMPK, and p130 as direct SMYD3 interactors by taking advantage of a library of rare tripeptides, which we first tested for their in vitro binding affinity to SMYD3 and then used as in silico probes to systematically search the human proteome. Here, we used this innovative approach to identify further SMYD3-interacting proteins involved in crucial cancer pathways and found that the chromatin remodeling factors EP300 and TRRAP interact directly with SMYD3, thus linking SMYD3 to the emerging 'nonmutational epigenetic reprogramming' cancer hallmark. Of note, we validated these interactions in gastrointestinal cancer cell lines, including HCT-116 cells, which harbor a C-terminal truncating mutation in EP300, suggesting that EP300 binds to SMYD3 via its N-terminal region. While additional studies are required to ascertain the functional mechanisms underlying these interactions and their significance, the identification of two novel SMYD3 interactors involved in epigenetic cancer hallmark pathways adds important pieces to the puzzle of how SMYD3 exerts its oncogenic role.

SMYD3 Modulates the HGF/MET Signaling Pathway in Gastric Cancer.

Gastric cancer (GC) is the third most deadly cancer worldwide. Considerable efforts have been made to find targetable drivers in order to improve patient outcomes. MET is one of the most important factors involved in GC initiation and progression as it plays a major role in GC invasiveness and is related to cancer stemness. Unfortunately, treatment strategies targeting MET are still limited, with a proportion of patients responding to therapy but later developing resistance. Here, we showed that MET is a molecular partner of the SMYD3 methyltransferase in GC cells. Moreover, we found that SMYD3 pharmacological inhibition affects the HGF/MET downstream signaling pathway. Extensive cellular analyses in GC models indicated that EM127, a novel active site-selective covalent SMYD3 inhibitor, can be used as part of a synergistic approach with MET inhibitors in order to enhance the targeting of the HGF/MET pathway. Importantly, our data were confirmed in a 3D GC cell culture system, which was used as a surrogate to evaluate stemness characteristics. Our findings identify SMYD3 as a promising therapeutic target to impair the HGF/MET pathway for the treatment of GC.

Tumor Testing and Genetic Analysis to Identify Lynch Syndrome Patients in an Italian Colorectal Cancer Cohort.

Lynch syndrome (LS) is an inherited cancer susceptibility syndrome caused by germline mutations in a DNA mismatch repair (MMR) gene or in the EPCAM gene. LS is associated with an increased lifetime risk of colorectal cancer (CRC) and other malignancies. The screening algorithm for LS patient selection is based on the identification of CRC specimens that have MMR loss/high microsatellite instability (MSI-H) and are wild-type for BRAFV600. Here, we sought to clinically and molecularly characterize patients with these features. From 2017 to 2023, 841 CRC patients were evaluated for MSI and BRAFV600E mutation status, 100 of which showed MSI-H. Of these, 70 were wild-type for BRAFV600. Among these 70 patients, 30 were genetically tested for germline variants in hereditary cancer predisposition syndrome genes. This analysis showed that 19 of these 30 patients (63.3%) harbored a germline pathogenic or likely pathogenic variant in MMR genes, 2 (6.7%) harbored a variant of unknown significance (VUS) in MMR genes, 3 (10%) harbored a VUS in other cancer-related genes, and 6 (20%) were negative to genetic testing. These findings highlight the importance of personalized medicine for tailored genetic counseling, management, and surveillance of families with LS and other hereditary cancer syndromes.

The somatic p.T81dup variant in AKT3 gene underlies a mild cerebral phenotype and expands the spectrum including capillary malformation and lateralized overgrowth.

Heterozygous germline or somatic variants in AKT3 gene can cause isolated malformations of cortical development (MCDs) such as focal cortical dysplasia, megalencephaly (MEG), Hemimegalencephaly (HME), dysplastic megalencephaly, and syndromic forms like megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome, and megalencephaly-capillary malformation syndrome. This report describes a new case of HME and capillary malformation caused by a somatic AKT3 variant that differs from the common p.E17K variant described in literature. The patient's skin biopsy from the angiomatous region revealed an heterozygous likely pathogenic variant AKT3:c.241_243dup, p.(T81dup) that may affect the binding domain and downstream pathways. Compared to previously reported cases with a common E17K mosaic variant, the phenotype is milder and patients showed segmental overgrowth, an uncommon characteristic in AKT3 variant cases. These findings suggest that the severity of the disease may be influenced not only by the level of mosaicism but also by the type of variant. This report expands the phenotypic spectrum associated with AKT3 variants and highlights the importance of genomic analysis in patients with capillary malformation and MCDs.

Genotypes and phenotypes heterogeneity in PIK3CA-related overgrowth spectrum and overlapping conditions: 150 novel patients and systematic review of 1007 patients with PIK3CA pathogenetic variants.

BACKGROUND: Postzygotic activating PIK3CA variants cause several phenotypes within the PIK3CA-related overgrowth spectrum (PROS). Variant strength, mosaicism level, specific tissue involvement and overlapping disorders are responsible for disease heterogeneity. We explored these factors in 150 novel patients and in an expanded cohort of 1007 PIK3CA-mutated patients, analysing our new data with previous literature to give a comprehensive picture. METHODS: We performed ultradeep targeted next-generation sequencing (NGS) on DNA from skin biopsy, buccal swab or blood using a panel including phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin pathway genes and GNAQ, GNA11, RASA1 and TEK. Additionally, 914 patients previously reported were systematically reviewed. RESULTS: 93 of our 150 patients had PIK3CA pathogenetic variants. The merged PROS cohort showed that PIK3CA variants span thorough all gene domains, some were exclusively associated with specific PROS phenotypes: weakly activating variants were associated with central nervous system (CNS) involvement, and strongly activating variants with extra-CNS phenotypes. Among the 57 with a wild-type PIK3CA allele, 11 patients with overgrowth and vascular malformations overlapping PROS had variants in GNAQ, GNA11, RASA1 or TEK. CONCLUSION: We confirm that (1) molecular diagnostic yield increases when multiple tissues are tested and by enriching NGS panels with genes of overlapping 'vascular' phenotypes; (2) strongly activating PIK3CA variants are found in affected tissue, rarely in blood: conversely, weakly activating mutations more common in blood; (3) weakly activating variants correlate with CNS involvement, strong variants are more common in cases without; (4) patients with vascular malformations overlapping those of PROS can harbour variants in genes other than PIK3CA.

Understanding the Genetic Landscape of Pancreatic Ductal Adenocarcinoma to Support Personalized Medicine: A Systematic Review.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal malignancies worldwide. While population-wide screening recommendations for PDAC in asymptomatic individuals are not achievable due to its relatively low incidence, pancreatic cancer surveillance programs are recommended for patients with germline causative variants in PDAC susceptibility genes or a strong family history. In this study, we sought to determine the prevalence and significance of germline alterations in major genes (ATM, BRCA1, BRCA2, CDKN2A, EPCAM, MLH1, MSH2, MSH6, PALB2, PMS2, STK11, TP53) involved in PDAC susceptibility. We performed a systematic review of PubMed publications reporting germline variants identified in these genes in PDAC patients. Overall, the retrieved articles included 1493 PDAC patients. A high proportion of these patients (n = 1225/1493, 82%) were found to harbor alterations in genes (ATM, BRCA1, BRCA2, PALB2) involved in the homologous recombination repair (HRR) pathway. Specifically, the remaining PDAC patients were reported to carry alterations in genes playing a role in other cancer pathways (CDKN2A, STK11, TP53; n = 181/1493, 12.1%) or in the mismatch repair (MMR) pathway (MLH1, MSH2, MSH6, PMS2; n = 87/1493, 5.8%). Our findings highlight the importance of germline genetic characterization in PDAC patients for better personalized targeted therapies, clinical management, and surveillance.

Lateralized overgrowth with vascular malformation caused by a somatic PTPN11 pathogenic variant: Another piece added to the puzzle of mosaic RASopathies.

Lateralized/segmental overgrowth disorders (LOs) encompass a heterogeneous group of congenital conditions with excessive body tissue growth. Documented molecular alterations in LOs mostly consist of somatic variants in genes of the PI3KCA/AKT/mTOR pathway or of chromosome band 11p15.5 imprinted region anomalies. In some cases, somatic pathogenic variants in genes of the RAS/MAPK pathway have been reported. We present the first case of a somatic pathogenic variant (T507K) in PTPN11 causing a LO phenotype characterized by severe lateralized overgrowth, vascular proliferation, and cerebral astrocytoma. The T507K variant was detected in DNA from overgrown tissue in a leg with capillary malformation. The astrocytoma tissue showed a higher PTPN11 variant allele frequency. A pathogenic variant in FGFR1 was also found in tumor tissue, representing a second hit on the RAS/MAPK pathway. These findings indicate that RAS/MAPK cascade overactivation can cause mosaic overgrowth phenotypes resembling PIK3CA-related overgrowth disorders (PROS) with cancer predisposition and are consistent with the hypothesis that RAS/MAPK hyperactivation can be involved in the pathogenesis of astrocytoma. This observation raises the issue of cancer predisposition in patients with RAS/MAPK pathway gene variants and expands genotype spectrum of LOs and the treatment options for similar cases through inhibition of the RAS/MAPK oversignaling.

Beyond BRCA1/2: Homologous Recombination Repair Genetic Profile in a Large Cohort of Apulian Ovarian Cancers.

BACKGROUND: Pathogenic variants in homologous recombination repair (HRR) genes other than BRCA1/2 have been associated with a high risk of ovarian cancer (OC). In current clinical practice, genetic testing is generally limited to BRCA1/2. Herein, we investigated the mutational status of both BRCA1/2 and 5 HRR genes in 69 unselected OC, evaluating the advantage of multigene panel testing in everyday clinical practice. METHODS: We analyzed 69 epithelial OC samples using an NGS custom multigene panel of the 5 HRR pathways genes, beyond the genetic screening routine of BRCA1/2 testing. RESULTS: Overall, 19 pathogenic variants (27.5%) were detected. The majority (21.7%) of patients displayed a deleterious mutation in BRCA1/2, whereas 5.8% harbored a pathogenic variant in one of the HRR genes. Additionally, there were 14 (20.3%) uncertain significant variants (VUS). The assessment of germline mutational status showed that a small number of variants (five) were not detected in the corresponding blood sample. Notably, we detected one BRIP1 and four BRCA1/2 deleterious variants in the low-grade serous and endometrioid histology OC, respectively. CONCLUSION: We demonstrate that using a multigene panel beyond BRCA1/2 improves the diagnostic yield in OC testing, and it could produce clinically relevant results.

Spectrum of Germline Pathogenic Variants in BRCA1/2 Genes in the Apulian Southern Italy Population: Geographic Distribution and Evidence for Targeted Genetic Testing.

BRCA1/2-associated hereditary breast and ovarian cancer is the most common form of hereditary breast and ovarian cancer and occurs in all ethnicities and racial populations. Different BRCA1/BRCA2 pathogenic variants (PVs) have been reported with a wide variety among populations. In this study, we retrospectively analyzed prevalence and geographic distribution of pathogenic germline BRCA1/2 variants in families from Apulia in southern Italy and evaluated the genotype-phenotype correlations. Data were collected from Oncogenetic Services present in Apulian hospitals and a shared database was built containing Apulian native probands (n = 2026) that had undergone genetic testing from 2004 to 2019. PVs were detected in 499 of 2026 (24.6%) probands and 68.5% of them (342 of 499) were in the BRCA1 gene. We found 65 different PVs in BRCA1 and 46 in BRCA2. There were 10 most recurrent PVs and their geographical distribution appears to be significantly specific for each province. We have assumed that these PVs are related to the historical and geopolitical changes that occurred in Apulia over time and/or to a "founder effect". Broader knowledge of BRCA1/2 prevalence and recurring PVs in specific geographic areas could help establish more flexible genetic testing strategies that may enhance our ability to detect high-risk subjects.