Publications

Abstract Purpose: Despite the remarkable activity of BTK inhibitors (BTKi) in relapsed B-cell non–Hodgkin lymphoma (B-NHL), no clinically-relevant biomarker has been associated to these agents so far. The relevance of phosphoproteomic profiling for the early identification of BTKi responders remains underexplored. Experimental Design: A set of six clinical samples from an ongoing phase I trial dosing patients with chronic lymphocytic leukemia (CLL) with TG-1701, a novel irreversible and highly specific BTKi, were characterized by phosphoproteomic and RNA sequencing (RNA-seq) analysis. The activity of TG-1701 was evaluated in a panel of 11 B-NHL cell lines and mouse xenografts, including two NF-κB- and BTKC481S-driven BTKi-resistant models. Biomarker validation and signal transduction analysis were conducted through real-time PCR, Western blot analysis, immunostaining, and gene knockout (KO) experiments. Results: A nonsupervised, phosphoproteomic-based clustering did match the early clinical outcomes of patients with CLL and separated a group of “early-responders” from a group of “late-responders.” This clustering was based on a selected list of 96 phosphosites with Ikaros-pSer442/445 as a potential biomarker for TG-1701 efficacy. TG-1701 treatment was further shown to blunt Ikaros gene signature, including YES1 and MYC, in early-responder patients as well as in BTKi-sensitive B-NHL cell lines and xenografts. In contrast, Ikaros nuclear activity and signaling remained unaffected by the drug in vitro and in vivo in late-responder patients and in BTKC481S, BTKKO, and noncanonical NF-κB models. Conclusions: These data validate phosphoproteomic as a valuable tool for the early detection of response to BTK inhibition in the clinic, and for the determination of drug mechanism of action.

Summary Recent findings are challenging the classical hematopoietic model in which long-term hematopoietic stem cells (LT-HSC) are the base of the hematopoietic system. Clonal dynamics analysis of the hematopoietic system indicate that LT-HSC are not the main contributors of normal hemapoiesis in physiological conditions and the hematopoietic system is mainly maintained by multipotent progenitors (MPPs, hereafter HPC) and LT-HSCs are mostly in a non-active state. The first HSCs emerge from the aorta-gonad and mesonephros (AGM) region along with hematopoietic progenitors (HPC) within hematopoietic clusters. Molecular pathways that determine the HSC fate instead of HPC are still unknown, although inflammatory signaling, including NF-κB has been implicated in the development of HSCs. Here, we identify a chromatin binding function for IκBα (also known as the inhibitor of NF-κB) that is Polycomb repression complex 2 (PRC2)-dependent and specifically determines dormant vs proliferating HSCs from the onset of their emergence in the AGM. We find a specific reduction of LT-HSCs in the IκBα knockout new-born pups. This defect is manifested at the FL stage already, and traceable to the first emerging HSCs in the E11.5 AGM, without affecting the general HPC population. IκBα deficient LT-HSCs express dormancy signature genes, are less proliferative and can robustly respond to activation stimuli such as in vitro culture and serial transplantation. At the molecular level, we find decreased PRC2-dependent H3K27me3 at the promoters of several retinoic acid signaling elements in the IκBα - deficient aortic endothelium and E14.5 FL LT-HSCs. Additionally, IκBα binding itself is found in the promoters of retinoic acid receptors rarα in the AGM, and rarγ in the LT-HSC of FL. Overall, we demonstrate that the retinoic acid pathway is over-activated in the hematopoietic clusters of IκBα-deficient AGMs leading to premature dormancy of LT-HSCs that persists in the FL LT-HSCs.

<div>Abstract<p>DNA hypomethylation is a common trait of colorectal cancer. Studies in tumor cell lines and animal models indicate that genome-wide demethylation may cause genetic instability and hence facilitate or accelerate tumor progression. Recent studies have shown that DNA hypomethylation precedes genomic damage in human gastrointestinal cancer, but the nature of this damage has not been clearly established. Here, we show a thorough analysis of DNA methylation and genetic alterations in two series of colorectal carcinomas. The extent of DNA demethylation but not of hypermethylation (both analyzed by amplification of intermethylated sites in near 200 independent sequences arbitrarily selected) correlated with the cumulated genomic damage assessed by two different techniques (arbitrarily primed PCR and comparative genomic hybridization). DNA hypomethylation–related instability was mainly of chromosomal nature and could be explained by a genome-wide effect rather than by the concurrence of the most prevalent genetic and epigenetic alterations. Moreover, the association of <i>p53</i> mutations with genomic instability was secondary to DNA hypomethylation and the correlation between DNA hypomethylation and genomic instability was observed in tumors with and without mutation in the <i>p53</i> gene. Our data support a direct link between genome-wide demethylation and chromosomal instability in human colorectal carcinogenesis and are consistent with the studies in model systems demonstrating a role of DNA demethylation in inducing chromosomal instability. (Cancer Res 2006; 66(17): 8462-8)</p></div>

Current evidence supports the involvement of brain-derived neurotrophic factor (BDNF) Val66Met polymorphisms in psychiatric and neurodegenerative disorders, including Alzheimer disease (AD) [1]. Previous studies of the association of this genotype with whole hippocampal volume included patients of a variety of disorders [2-3]. However, the impact of BDNF Val66Met polymorphisms on the volumes of the hippocampal subfields in cognitively unimpaired individuals remains to be elucidated. Therefore, the aim of the present study is to evaluate the impact of Val66Met polymorphism on hippocampal subfield volumes in cognitively unimpaired participants. BDNF Val66Met polymorphisms were determined in a sample of 430 cognitively unimpaired late/middle-aged participants from the ALFA (ALzheimer & FAmilies study [4]). Participants underwent a brain 3D-T1-weighted MRI scan and volumes of the hippocampal subfields were determined using Freesurfer (v6.0). The dominant, recessive, codominant and additive effects of the Val66Met genotype on the hippocampal subfield volumes were assessed using general linear models corrected by age, gender, education, number of APOE-e4 alleles and total intracranial volume. We additionally evaluated whether the association between age and hippocampal subfields was modified by BDNF Val66Met genotypes. p-values below 0.05 (FDR-corrected) were considered as statistically significant. Characteristics of the study participants, and differences in hippocampal subfield volumes between Val66Met genotypes are shown in Table 1 and Figure 1, respectively. Association analysis revealed that BDNF Met carriers showed statistically significant larger bilateral volumes of the subiculum under dominant and additive models (bd =20.5, pd=0.003; ba = 19.4, pa = 0.013) [Tables 2-3]. No significant results after FDR-correction were found under recessive and codominant models [Tables 4-5]. Furthermore, subfield volume reductions with aging were not significantly moderated by the Val66Met status.

Abstract Epigenetic modifications are known to be critical in regulating normal developmental processes and cellular differentiation. DNA methylation of CpG dinucleotides is a well-characterized epigenetic marker frequently associated with transcriptional silencing. CpG dinucleotides are non-randomly distributed throughout the genome with regions enriched for CpGs being known as CpG islands. It is now well established that epigenetic inactivation of tumor suppressor genes can occur by CpG island promoter methylation. For example, BRCA1 promoter methylation occurs in approximately 10 - 15% of all breast cancers and is associated with gene silencing. Recent technological advances have enabled DNA methylation to be profiled genome-wide. Using the Infinium 450 thousand probe arrays, we have studied DNA methylation and copy number changes to discover new markers of potential clinical relevance in breast cancer. Expression analyses by immunohistochemisty on tissue arrays for subtype-specific markers (ER, PR, HER2, Ki-67, EGFR, CK5/6, CK8, CK18, MUC1 and Claudin-3) carried out previously were used to establish DNA methylation markers of breast cancer subtypes. This integrated approach is presented in clinical context with available data on histological grade, disease stage and breast cancer-specific survival outcomes. Citation Format: Olafur A. Stefansson, Sebastian Moran, Antonio Gomez, Sergi Sayols Puig, Jorunn Eyfjord, Manel Esteller. Genome-wide analysis of DNA methylation and copy number changes in breast cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2018. doi:10.1158/1538-7445.AM2013-2018

Cancer cells progress through the accumulation of genetic alterations. Familial adenomatous polyposis (FAP) tumors provide an excellent model to unravel the molecular steps underlying malignant transformation. Global genomic damage was assessed in 56 adenomas and 3 carcinomas from six FAP patients and compared with that of sporadic adenomas and carcinomas. Evolutive trees were traced after application of maximum likelihood clustering and split decomposition methods to the analysis of comprehensive genetic profiles generated by diverse molecular approaches: arbitrarily primed PCR, comparative genomic hybridization, and flow cytometry. Overall, genomic damage as assessed by arbitrarily primed PCR was lower in familial adenomas than in sporadic adenomas and carcinomas. Comparative genomic hybridization data also show a low number of alterations in the majority of FAP adenomas. Tumors of the same patient were likely to share specific genetic alterations and may be grouped into one or two clusters. Putative common pathways were also identified, which included tumors of up to three different patients. According to our data, FAP tumors accumulate specific genetic alterations and in a preferred order that is characteristic of each individual. Moreover, the particular genetic background and environmental conditions of a FAP patient restrain the molecular evolution portrait of synchronous tumors.

Abstract Introduction. The family of ten-eleven translocation dioxygenases (TETs) regulates dioxygenase activity-dependent DNA demethylation and dioxygenase activity-independent histone modification, controlling the dynamic differentiation and lineage commitment of hematopoietic stem/progenitor cells, regulating the access of key transcription factors to the enhancers of target genes. TET2 somatic mutations, recurrent in marginal zone lymphoma (MZL) as well as in other hematological cancers, are associated with abnormal epigenetic landscape changes. To uncover the role of mutated (mut) TET2, we performed CRISPR editing, pharmacological screening, omics analyses, and in vivo experiments in MZL models. Methods. We used two bona fide MZL cell lines, VL51 and HC1. Genetic editing was done with CRISPR/Cas9. Monoclonal cell lines were obtained from isogenic populations by limiting dilution. In vitro and in vivo proliferation of edited cells was analyzed using Incucyte live cell imaging and xenograft models in NSG mice. Single-cell RNA sequencing (scRNA-Seq), bulk RNA-Seq, and DNA methylation profiling were also performed on edited cells. Additionally, the anti-proliferation effects of TETi76 (Guan et al., 2020) and a custom library of 474 anti-cancer compounds were assessed in edited cells. Results. Using CRISPR/Cas9, we reverted the HC1 cell line from mut-TET2 to wild-type (wt) and inserted the same mutation (P594X) in TET2 wt VL51. In isogenic models, mut-TET2 cells had increased proliferation in vitro and in vivo. In vivo, mice with mut-TET2 VL51 xenografts also showed a trend for poorer survival compared to those with wt xenografts. The TET inhibitor TETi76 was more active in mut-TET2 cells (HC1 and VL51 with CRISPR-generated TET2 mutation). Bulk RNA-Seq and scRNA-Seq showed differences at the transcriptome level between TET2 wt and TET2 edited cells, including immune signaling pathways, PI3K signaling, and MYC targets. Integration of RNA-Seq with DNA methylation data revealed that differential gene expression was associated with differential methylation of the corresponding specific gene promoter regions. Pharmacological screening in wt and mut-TET2 monoclonal cells showed that several drugs, such as pictilisib, rapamycin, romidepsin, tucidinostat, and umbralisib, were more active in mut-TET2 cells, and their targets largely agreed with what was observed at the transcriptome level. Conclusions. Mut-TET2 MZL cell lines generated by CRISPR gene editing exhibited different growth profiles and altered transcriptomes and DNA methylation patterns compared to their wt counterparts. Further, mut-TET2 conferred increased sensitivity to the TET inhibitor TETi76 and to multiple targeted agents, particularly those targeting PI3K, MTOR, and histone deacetylases. Our findings provide novel insights into the role of mut-TET2 in lymphomagenesis and reveal potential drug-targetable pathways for lymphomas with mut-TET2. Citation Format: Fangwen Zhang, Luciano Cascione, Sara Napoli, Chiara Tarantelli, Elisa Civanelli, Giulio Sartori, Francesca Guidetti, Alberto J. Arribas, Filippo Spriano, Anand Tiwari, Emanuele Zucca, Andrea Alimonti, Davide Rossi, Babal K. Jha, Manel Esteller, Afua Adjeiwaa. Mensah, Francesco Bertoni. Development of isogenic models of marginal zone lymphoma for therapeutic target discovery in TET2-deficient disease [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C023.

<p>Epigenetic signature in lung cancer and non-tumoral paraffins classified based on histological subtypes.</p>

Abstract Background Gene‐environment interactions are important in understanding Alzheimer’s disease (AD) etiology. Current research is limited, possibly due to weak effects of individual genetic variants. We analysed interaction between genetics of hippocampal volume, environmental exposures and levels of AD biomarkers in cognitively unimpaired individuals at increased risk of AD. Method A total of 319 cognitively unimpaired, middle‐age participants from the Alzheimer’s and Families (ALFA) study with data for genotyping, environmental exposures, and cerebrospinal fluid (CSF) biomarkers were included. Genome‐wide genotyping was performed using Infinium Neuro Consortium (NeuroChip) Array. Genetic variants that passed a quality control/imputation procedure were used to calculate polygenic risk scores (PRS) using PRSice version 2. Land use regression estimated residential exposure to particulate matter (PM 2.5 , PM 10 ), and nitrogen dioxide (NO 2 ). NeuroToolKit robust prototype assays and Elecsys® immunoassays (both Roche Diagnostics International Ltd, Rotkreuz, Switzerland) were used to measure CSF biomarkers, and amyloid positivity was defined as amyloid‐beta(Aβ)42/40 ratio <0.071. Associations between PRSs and environmental factors were investigated and estimated using multiple logistic/linear regression models adjusted for age and sex; CSF biomarkers were included as outcomes. Result PRS‐hippocampal volume (HV)×PM 10 and PRS‐HV×NO 2 were significantly associated with p‐tau (p = 0.045 and p = 0.025, respectively) (Figure 1). Individuals at high genetic predisposition for larger hippocampal volumes had lower levels of p‐tau at low levels of air pollution compared to individuals at low/null genetic predisposition; as the level of exposure to PM 10 and NO 2 increased, higher values of p‐tau were observed compared to low‐risk individuals. Conclusion Our results showed that air pollution may have a deleterious effect on biological mechanisms of AD, in cognitively unimpaired individuals with higher genetic liability for larger hippocampal volumes. Further research is needed to elucidate the biological mechanism involved in such associations.

Abstract Background Early detection has proven to be the most effective strategy to reduce the incidence and mortality of colorectal cancer (CRC). Nevertheless, most current screening programs suffer from low participation rates. A blood test may improve both the adherence to screening and the selection to colonoscopy. In this study, we conducted a serum-based discovery and validation of cfDNA methylation biomarkers for CRC screening in a multicenter cohort of 433 serum samples including healthy controls, benign pathologies, advanced adenomas (AA), and CRC. Results First, we performed an epigenome-wide methylation analysis with the MethylationEPIC array using a sample pooling approach, followed by a robust prioritization of candidate biomarkers for the detection of advanced neoplasia (AN: AA and CRC). Then, candidate biomarkers were validated by pyrosequencing in independent individual cfDNA samples. We report GALNT9 , UPF3A , WARS , and LDB2 as new noninvasive biomarkers for the early detection of AN. The combination of GALNT9 / UPF3A by logistic regression discriminated AN with 78.8% sensitivity and 100% specificity, outperforming the commonly used fecal immunochemical test and the methylated SEPT9 blood test. Conclusions Overall, this study highlights the utility of cfDNA methylation for CRC screening. Our results suggest that the combination methylated GALNT9 / UPF3A has the potential to serve as a highly specific and sensitive blood-based test for screening and early detection of CRC.

B136 Background: Global hypomethylation and CpG island promoter hypermethylation have been identified as hallmarks of human cancer. The genome of the transformed cell undergoes a global genomic hypomethylation and a dense hypermethylation of CpG islands associated with gene regulatory regions. Although the temporal association between these two events has not been elucidated, it is widely recognized that global hypomethylation is one of the earliest epigenetic changes in the transformation to a diseased cell, followed by a progressive increase in gene specific hypermethylation. There are no studies that examine the relationship between these two events in liver cancer as a potential indicator of early carcinogenesis. Previously, we reported hypermethylation of PGP9.5 and NMDAR2B in human esophageal cancer.
 >Objective: In this study we aim to validate the use of PGP9.5 and NMDAR2B together with global methylation levels as a potential biomarker panel for early detection of liver cancer.
 >Methods: Ten frozen tumor tissues from liver cancer patients and 10 frozen liver tissue control samples from non-cancerous patients were obtained from the Cooperative Human Tissue Network. DNA was extracted using standard methods. Genomic DNA samples were boiled and treated with nuclease P1 and alkaline phosphatase. Global DNA hypomethylation was quantified using HPLC for fraction separation and Mass Spectrometry for quantification. We compared the promoter methylation status of PGP9.5 and NMDAR2B in pre-malignant liver with that in liver tumor tissues by using quantitative methylation-specific PCR (Q-MSP). The methylation status of p16, p15, and RASSF1A was also examined in parallel.
 >Results: A global methylation index measuring methylated cytidine relative to global cytidine in the genome was significantly lower (p-value = 0.0001) for all tumor tissue, mean = 2.39 (95% CI, 1.99, 2.80), when compared to tissue from non-cancerous liver, mean = 3.54 (95% CI, 3.16, 3.93). In liver tumor, PGP9.5 methylation was observed with highest frequency (100 %). The methylation frequency in NMDAR2B and RASSF1A was also very high (83%), but p16 or p15 methylation was relatively low (33% in p16 and 0% in p15), Interestingly, RASSF1A methylation was detected in all non-cancerous liver tissue tested (100%). NMDAR2B and p15 methylation was observed in (57%), and (14%) non-cancerous liver. PGP9.5 and p16 methylation was not detected in non-cancerous liver tissue.
 >Conclusion: The results of this proof-of-principle study suggest that an epigenetic panel of global and gene specific methylation changes may be a useful biomarker of early detection in cancer. RASSF1A and NMDAR2B are possibly more sensitive markers than p15 or p16 in detection of liver cancer.