Targeted inhibition of the methyltransferase SETD8 synergizes with the Wee1 inhibitor adavosertib in restraining glioblastoma growth.

Despite intense research efforts, glioblastoma remains an incurable brain tumor with a dismal median survival time of 15 months. Thus, identifying new therapeutic targets is an urgent need. Here, we show that the lysine methyltransferase SETD8 is overexpressed in 50% of high-grade gliomas. The small molecule SETD8 inhibitor UNC0379, as well as siRNA-mediated inhibition of SETD8, blocked glioblastoma cell proliferation, by inducing DNA damage and activating cell cycle checkpoints. Specifically, in p53-proficient glioblastoma cells, SETD8 inhibition and DNA damage induced p21 accumulation and G1/S arrest whereas, in p53-deficient glioblastoma cells, DNA damage induced by SETD8 inhibition resulted in G2/M arrest mediated by Chk1 activation. Checkpoint abrogation, by the Wee1 kinase inhibitor adavosertib, induced glioblastoma cell lines and primary cells, DNA-damaged by UNC0379, to progress to mitosis where they died by mitotic catastrophe. Finally, UNC0379 and adavosertib synergized in restraining glioblastoma growth in a murine xenograft model, providing a strong rationale to further explore this novel pharmacological approach for adjuvant glioblastoma treatment.

Epigenetic remodelling of Fxyd1 promoters in developing heart and brain tissues.

FXYD1 is a key protein controlling ion channel transport. FXYD1 exerts its function by regulating Na+/K+-ATPase activity, mainly in brain and cardiac tissues. Alterations of the expression level of the FXYD1 protein cause diastolic dysfunction and arrhythmias in heart and decreased neuronal dendritic tree and spine formation in brain. Moreover, FXYD1, a target of MeCP2, plays a crucial role in the pathogenesis of the Rett syndrome, a neurodevelopmental disorder. Thus, the amount of FXYD1 must be strictly controlled in a tissue specific manner and, likely, during development. Epigenetic modifications, particularly DNA methylation, represent the major candidate mechanism that may regulate Fxyd1 expression. In the present study, we performed a comprehensive DNA methylation analysis and mRNA expression level measurement of the two Fxyd1 transcripts, Fxyd1a and Fxyd1b, in brain and heart tissues during mouse development. We found that DNA methylation at Fxyd1a increased during brain development and decreased during heart development along with coherent changes in mRNA expression levels. We also applied ultra-deep methylation analysis to detect cell to cell methylation differences and to identify possible distinct methylation profile (epialleles) distribution between heart and brain and in different developmental stages. Our data indicate that the expression of Fxyd1 transcript isoforms inversely correlates with DNA methylation in developing brain and cardiac tissues suggesting the existence of a temporal-specific epigenetic program. Moreover, we identified a clear remodeling of epiallele profiles which were distinctive for single developmental stage both in brain and heart tissues.

HDAC class I inhibitor domatinostat sensitizes pancreatic cancer to chemotherapy by targeting cancer stem cell compartment via FOXM1 modulation.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) represents an unmet clinical need due to the very poor prognosis and the lack of effective therapy. Here we investigated the potential of domatinostat (4SC-202), a new class I histone deacetylase (HDAC) inhibitor, currently in clinical development, to sensitize PDAC to first line standard gemcitabine (G)/taxol (T) doublet chemotherapy treatment. METHODS: Synergistic anti-tumor effect of the combined treatment was assessed in PANC1, ASPC1 and PANC28 PDAC cell lines in vitro as well as on tumor spheroids and microtissues, by evaluating combination index (CI), apoptosis, clonogenic capability. The data were confirmed in vivo xenograft models of PANC28 and PANC1 cells in athymic mice. Cancer stem cells (CSC) targeting was studied by mRNA and protein expression of CSC markers, by limiting dilution assay, and by flow cytometric and immunofluorescent evaluation of CSC mitochondrial and cellular oxidative stress. Mechanistic role of forkhead box M1 (FOXM1) and downstream targets was evaluated in FOXM1-overexpressing PDAC cells. RESULTS: We showed that domatinostat sensitized in vitro and in vivo models of PDAC to chemotherapeutics commonly used in PDAC patients management and particularly to GT doublet, by targeting CSC compartment through the induction of mitochondrial and cellular oxidative stress. Mechanistically, we showed that domatinostat hampers the expression and function of FOXM1, a transcription factor playing a crucial role in stemness, oxidative stress modulation and DNA repair. Domatinostat reduced FOXM1 protein levels by downregulating mRNA expression and inducing proteasome-mediated protein degradation thus preventing nuclear translocation correlated with a reduction of FOXM1 target genes. Furthermore, by overexpressing FOXM1 in PDAC cells we significantly reduced domatinostat-inducing oxidative mitochondrial and cellular stress and abolished GT sensitization, both in adherent and spheroid cells, confirming FOXM1 crucial role in the mechanisms described. Finally, we found a correlation of FOXM1 expression with poor progression free survival in PDAC chemotherapy-treated patients. CONCLUSIONS: Overall, we suggest a novel therapeutic strategy based on domatinostat to improve efficacy and to overcome resistance of commonly used chemotherapeutics in PDAC that warrant further clinical evaluation.

Evaluation of MGMT Gene Methylation in Neuroendocrine Neoplasms.

Unresectable neuroendocrine neoplasms (NENs) often poorly respond to standard therapeutic approaches. Alkylating agents, in particular temozolomide, commonly used to treat high-grade brain tumors including glioblastomas, have recently been tested in advanced or metastatic NENs, where they showed promising response rates. In glioblastomas, prediction of response to temozolomide is based on the assessment of the methylation status of the MGMT gene, as its product, O 6-methylguanine-DNA methyltransferase, may counteract the damaging effects of the alkylating agent. However, in NENs, such a biomarker has not been validated yet. Thus, we have investigated MGMT methylation in 42 NENs of different grades and from various sites of origin by two different approaches: in contrast to methylation-specific PCR (MSP), which is commonly used in glioblastoma management, amplicon bisulfite sequencing (ABS) is based on high-resolution, next-generation sequencing and interrogates several additional CpG sites compared to those covered by MSP. Overall, we found MGMT methylation in 74% (31/42) of the NENs investigated. A higher methylation degree was observed in well-differentiated tumors and in tumors originating in the gastrointestinal tract. Comparing MSP and ABS results, we demonstrate that the region analyzed by the MSP test is sufficiently informative of the MGMT methylation status in NENs, suggesting that this predictive parameter could routinely be interrogated also in NENs.

Prevalence of GLA gene mutations and polymorphisms in patients with multiple sclerosis: A cross-sectional study.

PURPOSE: Fabry Disease (FD) has been frequently proposed as possible underestimated differential diagnosis of Multiple Sclerosis (MS), but no study has been performed to test prevalence of GLA gene mutations in a population fulfilling diagnostic criteria of MS. Aim of this study is to determine the prevalence of GLA gene mutations in a large and representative population diagnosed with MS, simultaneously providing a critical revision of current literature reports of coexistence or misdiagnosis between these two conditions. METHODS: In this mono-centric cross-sectional study, 927 patients fulfilling McDonald diagnostic criteria and encompassing all MS phenotypes were enrolled. Patients underwent evaluation of α-GalA activity and genotyping. Both genetic variants annotated as pathogenic and GVUS were considered. Estimated alleles frequencies were then compared to the ones reported in the gnomAD database. RESULTS: GLA gene variants were found in seven individuals. Five patients carried variants previously described having controversial impact on FD phenotype, and the analysis of exome database revealed that they are not rare among healthy individuals. One patient showed a new variant never described before, and another one carried a late-onset FD cardiac variant. CONCLUSIONS: The overall prevalence of GLA gene variants in MS patients is comparable to the one estimated in healthy population. This result is further supported by critical revision of current literature evidences of misdiagnosis between MS and FD, arguing in favour of independence between these disorders.

Age and dPCR can predict relapse in CML patients who discontinued imatinib: the ISAV study.

Imatinib is effective for the treatment of chronic myeloid leukemia (CML). However even undetectable BCR-ABL1 by Q-RT-PCR does not equate to eradication of the disease. Digital-PCR (dPCR), able to detect 1 BCR-ABL1 positive cell out of 10(7) , has been recently developed. The ISAV study is a multicentre trial aimed at validating dPCR to predict relapses after imatinib discontinuation in CML patients with undetectable Q-RT-PCR. CML patients under imatinib therapy since more than 2 years and with undetectable PCR for at least 18 months were eligible. Patients were monitored by standard Q-RT-PCR for 36 months. Patients losing molecular remission (two consecutive positive Q-RT-PCR with at least 1 BCR-ABL1/ABL1 value above 0.1%) resumed imatinib. The study enrolled 112 patients, with a median follow-up of 21.6 months. Fifty-two of the 108 evaluable patients (48.1%), relapsed; 73.1% relapsed in the first 9 months but 14 late relapses were observed between 10 and 22 months. Among the 56 not-relapsed patients, 40 (37.0% of total) regained Q-RT-PCR positivity but never lost MMR. dPCR results showed a significant negative predictive value ratio of 1.115 [95% CI: 1.013-1.227]. An inverse relationship between patients age and risk of relapse was evident: 95% of patients <45 years relapsed versus 42% in the class ≥45 to <65 years and 33% of patients ≥65 years [P(χ(2) ) < 0.0001]. Relapse rates ranged between 100% (<45 years, dPCR+) and 36% (>45 years, dPCR-). Imatinib can be safely discontinued in the setting of continued PCR negativity; age and dPCR results can predict relapse.