Tesi etd-01122025-160351
Link copiato negli appunti
Tipo di tesi
Dottorato
Autore
SALANI, FRANCESCA
Indirizzo email
f.salani1@gmail.com
URN
etd-01122025-160351
Titolo
Predictive and prognostic role of histone post-translational modifications in hepatocellular carcinoma
Settore scientifico disciplinare
MED/06
Corso di studi
Istituto di Scienze della Vita - PhD in Medicina Traslazionale - PON
Relatori
relatore Prof. PASSINO, CLAUDIO
Parole chiave
- Atezolizumab/Bevacizumab
- EED
- EZH2
- Hepatocellular carcinoma
- Histone Post-Translational Modifications
- liquid biopsy
Data inizio appello
19/02/2026;
Disponibilità
parziale
Riassunto analitico
Background: Advanced hepatocellular carcinoma (aHCC) is an aggressive primary liver cancer whose incidence and mortality are rapidly increasing in western Countries. First-line therapies landscape has been revolutionized since 2022 by the introduction of immune check-point inhibitors-based regimens, of which atezolizumab/bevacizumab (AtezoBev) combination is the most widely used. However, improvement of systemic therapies’ activity and efficacy are warranted. Epigenetic gene regulation is a main driver of cancer cell plasticity and acquired drug resistance. Among epigenetic alterations, histone post-translational modifications (HPTMs) control gene expression via chromatin accessibility. Of these, the circulating fraction of nucleosomal Histone 3 lysine-27 trimethylation (cf-H3K27me3) and Histone 3 lysine-36 trimethylation (cf-H3K36me3) showed a prognostic role in a retrospective cohort of aHCC patients treated with sorafenib. Moreover, several epigenetic enzymes (“writers”, “erasers”) are targetable by small molecule inhibitors, some of which are currently tested in clinical trials.
With this study we aimed at: a) exploring in-vitro the anti-cancer activity of two HPTMs-writers' inhibitors on HCC models; b) describing the prognostic and predictive ability of cf-H3K27me3 and cf-H3K36me3 in a cohort of aHCC patients treated with AtezoBev.
Methods: HPTMs-codifying genes were identified via HGNC database. The prognostic role of mRNA expression of the identified genes was enquired through cBioPortal and Kaplan Meier Plotter on a cohort of resected HCC from the TGCA database. The most statistically significant prognostic gene and its functional partner were selected for the in-vitro part of the study. Pharmacological inhibition of EZH2 and EED was performed in normoxic (02 21%) and hypoxic conditions (02 2%) on HCC models (HepG2, Huh7). Viability after treatment was assessed with trypan-blue cell count and caspase 3/7 assay. The drugs’ target engagement was verified with immunoblotting performed on extracted histones. Chromatin accessibility modifications and RNA expression were studied with ATAC and RNA sequencing, respectively. All the experiments were conducted as biological triplicates.
Regarding the translational study, 1.5 ml of plasma was obtained from aHCC patients receiving AtezoBev at 5 Italian oncological referral centers (AOU Pisana, AOU Careggi - Florence, AOU Federico II – Naples, IOV - Padua). The circulating fraction of histone 3 (cf-H3.1), cf-H3K27me3 and cf-H3K36me3 plasma concentrations were determined with Nu.Q® ELISA kits by Volition Rx and results were presented as normalized concentrations against cf-H3.1 (hereafter, “N-”). Patients’ baseline and on-treatment clinical and laboratory data were retrieved from medical records and correlated with activity and efficacy endpoints. Statistical analyses were performed with MedCalc v. 23.1.3 and Python.
Results: One hundred twenty-two genes encoding HPTMs-writers/erasers were identified. The expression of 14 genes showed a statistically significant correlation with overall survival (OS, p Benjamini-Hochberg <0.05): higher EZH2 mRNA was the most statistically significant predictor of shorter OS (p Benjamini-Hochberg, OS: 3.789*10-5). Similarly, higher expression of EED, EZH2 functional partner, was associated with shorter OS (p Benjamini-Hochberg: 0.01). Higher expressions of either EZH2 or EED were positively correlated with hypoxia scores (q-values < 0.05), suggesting hypoxia as a relevant characteristic to be further studied.
EZH2 inhibition exerted a limited cytotoxic effect in terms of IC50 both in normoxic and hypoxic conditions, without significant time-dependency (IC50 comparison between 2- and 3-days treatment). RNA sequencing conducted on EZH2-inhibited HCC cells under hypoxia showed a significant increase in lipid metabolism reprogramming, compared to untreated cells.
EED inhibition under hypoxic conditions showed greater cytotoxic effect than in normoxic conditions (p for live cells count comparison after 10µM treatment in hypoxia vs normoxia: 0.01) and a significant time-dependency (7-days vs 4-days IC50 comparison: p 0.03), at least in one of the used models. Chromatin accessibility assay performed on EED-inhibited cells under hypoxia showed a significant increase in 148 gene-coding regions. Some of the up-regulated pathways comprised potentially targetable pro-oncogenes such as ALK, PIK3CA and AKT; the only onco-suppressor pathway identified was metallothionein one, which is functionally related to Wnt/Beta-catenin, a known determinant of HCC aggressiveness. These results seem to suggest the opportunity to explore EED inhibition in combination with potentially synergic partners.
On the clinical side of the study, from January 2022 to July 2024, 60 aHCC patients were enrolled, mainly characterized by HCV etiology. AtezoBev median progression-free survival (PFS) was 5.6 (95% IC: 8.1-13.2) months and median OS was 13.3 (95% IC: 11.6-16.6) months. Baseline concentration of cf-N-H3K27me3, the readout of PRC2 activity, significantly predicted PFS (ROC curve AUC 0.75, p 0.01). Baseline concentration of cf-N-H3K36me3, the readout of SETD2 which acts antagonistically to PRC2 in many cancer types, showed a positive prognostic role for both PFS (Log-Rank test, p: 0.01) and OS (Log-Rank test, p: 0.01).
Primary resistance to AtezoBev treatment was significantly anticipated by higher baseline cf-H3.1 concentration (ROC AUC: 0.7, p: 2.35e-03) and lower cf-N-H3K36me3 values (ROC AUC: 0.7, p: 4.38e-03), outperforming AFP.
Conclusion: In-silico, PRC2 subunits EZH2 and EED expression represents negative prognostic factors for OS in resected HCC. From our in-vitro results, EZH2 or EED inhibition does not seem to represent active monotherapy strategies for clinical trials, while sequencing data support EED inhibitor combination with other targets’ inhibitors in HCC. Some cf-HPTMs related to PRC2 activity showed a prognostic and predictive role for AtezoBev treatment in a cohort of aHCC patients, outperforming AFP.
With this study we aimed at: a) exploring in-vitro the anti-cancer activity of two HPTMs-writers' inhibitors on HCC models; b) describing the prognostic and predictive ability of cf-H3K27me3 and cf-H3K36me3 in a cohort of aHCC patients treated with AtezoBev.
Methods: HPTMs-codifying genes were identified via HGNC database. The prognostic role of mRNA expression of the identified genes was enquired through cBioPortal and Kaplan Meier Plotter on a cohort of resected HCC from the TGCA database. The most statistically significant prognostic gene and its functional partner were selected for the in-vitro part of the study. Pharmacological inhibition of EZH2 and EED was performed in normoxic (02 21%) and hypoxic conditions (02 2%) on HCC models (HepG2, Huh7). Viability after treatment was assessed with trypan-blue cell count and caspase 3/7 assay. The drugs’ target engagement was verified with immunoblotting performed on extracted histones. Chromatin accessibility modifications and RNA expression were studied with ATAC and RNA sequencing, respectively. All the experiments were conducted as biological triplicates.
Regarding the translational study, 1.5 ml of plasma was obtained from aHCC patients receiving AtezoBev at 5 Italian oncological referral centers (AOU Pisana, AOU Careggi - Florence, AOU Federico II – Naples, IOV - Padua). The circulating fraction of histone 3 (cf-H3.1), cf-H3K27me3 and cf-H3K36me3 plasma concentrations were determined with Nu.Q® ELISA kits by Volition Rx and results were presented as normalized concentrations against cf-H3.1 (hereafter, “N-”). Patients’ baseline and on-treatment clinical and laboratory data were retrieved from medical records and correlated with activity and efficacy endpoints. Statistical analyses were performed with MedCalc v. 23.1.3 and Python.
Results: One hundred twenty-two genes encoding HPTMs-writers/erasers were identified. The expression of 14 genes showed a statistically significant correlation with overall survival (OS, p Benjamini-Hochberg <0.05): higher EZH2 mRNA was the most statistically significant predictor of shorter OS (p Benjamini-Hochberg, OS: 3.789*10-5). Similarly, higher expression of EED, EZH2 functional partner, was associated with shorter OS (p Benjamini-Hochberg: 0.01). Higher expressions of either EZH2 or EED were positively correlated with hypoxia scores (q-values < 0.05), suggesting hypoxia as a relevant characteristic to be further studied.
EZH2 inhibition exerted a limited cytotoxic effect in terms of IC50 both in normoxic and hypoxic conditions, without significant time-dependency (IC50 comparison between 2- and 3-days treatment). RNA sequencing conducted on EZH2-inhibited HCC cells under hypoxia showed a significant increase in lipid metabolism reprogramming, compared to untreated cells.
EED inhibition under hypoxic conditions showed greater cytotoxic effect than in normoxic conditions (p for live cells count comparison after 10µM treatment in hypoxia vs normoxia: 0.01) and a significant time-dependency (7-days vs 4-days IC50 comparison: p 0.03), at least in one of the used models. Chromatin accessibility assay performed on EED-inhibited cells under hypoxia showed a significant increase in 148 gene-coding regions. Some of the up-regulated pathways comprised potentially targetable pro-oncogenes such as ALK, PIK3CA and AKT; the only onco-suppressor pathway identified was metallothionein one, which is functionally related to Wnt/Beta-catenin, a known determinant of HCC aggressiveness. These results seem to suggest the opportunity to explore EED inhibition in combination with potentially synergic partners.
On the clinical side of the study, from January 2022 to July 2024, 60 aHCC patients were enrolled, mainly characterized by HCV etiology. AtezoBev median progression-free survival (PFS) was 5.6 (95% IC: 8.1-13.2) months and median OS was 13.3 (95% IC: 11.6-16.6) months. Baseline concentration of cf-N-H3K27me3, the readout of PRC2 activity, significantly predicted PFS (ROC curve AUC 0.75, p 0.01). Baseline concentration of cf-N-H3K36me3, the readout of SETD2 which acts antagonistically to PRC2 in many cancer types, showed a positive prognostic role for both PFS (Log-Rank test, p: 0.01) and OS (Log-Rank test, p: 0.01).
Primary resistance to AtezoBev treatment was significantly anticipated by higher baseline cf-H3.1 concentration (ROC AUC: 0.7, p: 2.35e-03) and lower cf-N-H3K36me3 values (ROC AUC: 0.7, p: 4.38e-03), outperforming AFP.
Conclusion: In-silico, PRC2 subunits EZH2 and EED expression represents negative prognostic factors for OS in resected HCC. From our in-vitro results, EZH2 or EED inhibition does not seem to represent active monotherapy strategies for clinical trials, while sequencing data support EED inhibitor combination with other targets’ inhibitors in HCC. Some cf-HPTMs related to PRC2 activity showed a prognostic and predictive role for AtezoBev treatment in a cohort of aHCC patients, outperforming AFP.
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