Publications

<p>Differences in cell type abundance upon AZA treatment according to hematological improvement (HI) and marrow complete response (mCR)</p>

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Cytotoxicity is a key mechanism against tumor development. Cytotoxic cells (i.e.T cells and NK cells) are tightly regulated by immune checkpoint events, which dictate presenting cell destiny. In recent studies, the interaction between CD155 and TIGIT appeared to play an important role in cytotoxicity inhibition. Epigenetics is an important player in the regulation of gene expression, including immune checkpoint regulators. Novel immunotherapeutic strategies, such as Bispecific T-cell engagers (BiTE) and Chimeric antigen receptor (CAR) therapies, are being evaluated for MM treatment, with promising results. Aims: To validate the relevance of CD155 on cytotoxic cells (CD8+ T cells and NK cells) and its role in novel immunotherapies against malignant plasma cells, as well as to determine if patient stratification based on CD155 expression is associated with outcome. Methods: Methylation data was obtained from the Infinium Methylation450k BeadChip Kit (Illumina) for different hematological cell lines and the status of the promoter region of CD155 was studied in the context of MM. Bisulfite sequencing, quantitative PCR and azacytidine treatment were used to validate the in-silico data. We created CD155 depletion models in RPMI-8226 and JJN-3 cells using short hairpin RNA and developed an in vitro coculture system. Cytotoxic cells (either T-cells from healthy donors, anti-BCMA CAR-T, or anti-BCMA CAR-NK cells) faced MM cells. Surviving tumor cells were measured by flow cytometry. Moreover, T-cells were also incubated with anti-BCMA/CD3 BiTE. Subsequently, we used an anti-TIGIT neutralizing monoclonal antibody to determine whether the CD155-TIGIT interaction mediates the mechanism of action of CD155 in tumor escape. Finally, we analyzed the correlation between CD155 expression and survival in newly diagnosed MM samples from the CoMMpass project public data (N= 793). Results: After validating the methylation regulation of CD155, we studied CD155 participation in T-cell cytotoxicity and observed how CD155 reduced T-cell activation. RPMI-8226 and JJN-3 controls showed higher resistance to cytotoxicity (p=0.02 and p<0.001, respectively). The cytotoxic effect increased in the presence of 100pg/mL of anti-BCMA/CD3 BiTE, but the depleted models were more sensitive to it (p<0.001). When we incorporated anti-TIGIT into the system, we again observed expressing models with higher survival than depleted models on IgG controls and increased cytotoxicity in the presence of anti-TIGIT (RPMI-8226, p=0.005; JJN-3, p=0.004). When the models were treated with CAR-modified cells, the effects were always in the same direction; CD155-depleted models were more sensitive to cytotoxic effects either by 1:4 anti-BCMA CAR-T cells or by 1:8 anti-BCMA CAR-NK and 10ug/mL of anti-TIGIT. Supporting these results, the data obtained from the CoMMpass project showed that newly diagnosed MM patients with higher expression of CD155 had shorter overall survival (median overall survival of CD155 high: 56 months; CD155 low: not reached; p<0.001). Summary/Conclusion: In the context of MM, expression of CD155 effectively reduces the cytotoxic cell response, which is mediated by the interaction of CD155 on MM cells and TIGIT on cytotoxic cells. This interaction seems to impact patients treated with current therapies since patients’ outcomes correlate with CD155 expression. Furthermore, novel therapies focused on enhancing immune cytotoxicity benefit from CD155 depletion. These results warrant further investigation of CD155 as a biomarker and target for novel immunotherapies. Keywords: Tumor immunology, Epigenetic, Multiple myeloma, Immunotherapy

<title>Abstract</title> Tumor-intrinsic defects in immune recognition and interferon-gamma (IFNγ) signaling pathways facilitate immune evasion and limit the efficacy of immune checkpoint blockade (ICB). Here, we delineate the mutational landscape and functional consequences of amino acid substitutions in key immune-related genes, <italic>B2M</italic>, <italic>CALR</italic>, <italic>IFNGR1</italic>, <italic>IFNGR2</italic>, <italic>JAK1</italic>, and <italic>JAK2</italic>, across more than 12 000 primary tumors and cancer cell lines. Genomic alterations affecting the coding regions of at least one of these genes were identified in approximately 11% of cancers, with missense variants accounting for 55% of these events. <italic>B2M</italic> exhibited the highest mutation frequency per base pair, the mutations predominantly involving truncating variants. A curated set of 215 missense mutations in <italic>B2M</italic>, <italic>IFNGR1</italic>, <italic>IFNGR2</italic>, and <italic>JAK2</italic> was interrogated using SIFT, PolyPhen-2, and AlphaMissense, yielding predicted pathogenicity rates of 52%, 35%, and 27%, respectively. Functional assays revealed JAK2 and IFNGR1 variants that impaired IFNγ-mediated transcriptional activation and growth suppression, and B2M variants that disrupted HLA class I complex formation. Notably, AlphaMissense predictions showed the highest concordance with experimental data. These findings provide a detailed mutational map of antigen presentation and IFNγ-response components in cancer, offering a resource of specific mutations in immune pathways that compromise tumor immunogenicity and may influence the response to ICB.

Details of the primers used for PCR amplifications in this study. (XLSX 27 kb)

&lt;p&gt;Supplementary Figure 2. Copy number and expression levels of six KATs in SCLC cell lines&lt;/p&gt;

Abstract Background: We previously reported that high-dose Vitamin C (VitC) can synergize with immune checkpoint blockade in immunocompetent mice bearing colorectal, pancreatic and breast tumors. Here, we aim to unveil the molecular mechanisms underlying the above preclinical observations. We hypothesized that VitC could trigger immunogenic cell death (ICD), thereby stimulating an immune-mediated anticancer response. Methods: Induction of ICD was assessed by the expression or emission of specific markers, including calreticulin, as well as by ex-vivo phagocytosis assays by co-culturing dendritic cells with breast or colon cancer cells treated with VitC. A calreticulin blocking antibody was administered in vivo in combination with VitC to functionally characterize the role of ICD induction in the anticancer effects of vitC. Ex-vivo spatial transcriptomic and immunofluorescence analyses were performed to characterize the impact of the treatment on tumor and immune cell populations. Results: Pharmacological doses of VitC were able to induce oxidative stress as indicated by spatial transcriptomic data and confirmed by ex vivo 8-hydroxyguanine staining in tumor sections. VitC treatment induced ICD markers and among these calreticulin and HMGB1. Although the induction of these danger markers was seen in tumors grown both in immunocompromised and immunocompetent VitC treated mice, anticancer activity was evident only in the presence of an intact immune system. In vivo, a calreticulin blocking antibody effectively prevented its translocation and inhibited HMGB1 release in murine tumors. Notably, preventing calreticulin exposure abrogated the immune mediated anticancer effect of VitC. VitC enhanced the infiltration of natural killers, activated CD8+ T lymphocytes, while it decreased the number of Tregs. Calreticulin blockade impaired the modulation of the immune tumor microenvironment induced by VitC. Conclusions: VitC treatment can mobilize calreticulin and HMGB1, which are among the established markers of immunogenic cell death. These results, alongside the observation that VitC exerts its maximal antitumor activity only in immunocompetent mice and its anticancer activity is abolished by anti-calreticulin blocking antibody demonstrated that VitC is an ICD inducer when given at pharmacological doses. These findings lend the preclinical rationale and inform the translational analyses planned within the ALFEO clinical trial (Clinical trial information: ECTR2022-502101-15-00), that is investigating pharmacological doses of vitC in combination with nivolumab and ipilimumab in mismatch repair proficient colon cancer patients. Funded by- Next Generation EU - PNRR M6C2 - PNRR-MAD-2022-12376593. Citation Format: Alessandro Cavaliere, Marco Macagno, Federica Maione, Vito Amodio, Marc Escobosa Olmo, Rosaria Chilà, Simona Lamba, Pietro Paolo Vitiello, Valeria Pessei, Daniela Grases, Elena Perez, Alice Bartolini, Noemi Congiusta, Giovanni Germano, Andrea Sartore-Bianchi, Teresa Troiani, Manel Esteller, Eduard Porta, Alberto Bardelli, Federica Di Nicolantonio. Immunogenic cell death mediates the anticancer efficacy of high dose vitamin C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6169.

Epigenomic changes may either cause disease or modulate its expressivity, adding a layer of complexity to mendelian diseases. X-linked adrenoleukodystrophy (X-ALD) is a rare neurometabolic condition exhibiting discordant phenotypes, ranging from a childhood cerebral inflammatory demyelination (cALD) to an adult-onset mild axonopathy in spinal cords (AMN). The AMN form may occur with superimposed inflammatory brain demyelination (cAMN). All patients harbor loss of function mutations in the ABCD1 peroxisomal transporter of very-long chain fatty acids. The factors that account for the lack of genotype-phenotype correlation, even within the same family, remain largely unknown. To gain insight into this matter, here we compared the genome-wide DNA methylation profiles of morphologically intact frontal white matter areas of children affected by cALD with adult cAMN patients, including male controls in the same age group. We identified a common methylomic signature between the two phenotypes, comprising (i) hypermethylation of genes harboring the H3K27me3 mark at promoter regions, (ii) hypermethylation of genes with major roles in oligodendrocyte differentiation such as MBP, CNP, MOG and PLP1 and (iii) hypomethylation of immune-associated genes such as IFITM1 and CD59. Moreover, we found increased hypermethylation in CpGs of genes involved in oligodendrocyte differentiation, and also in genes with H3K27me3 marks in their promoter regions in cALD compared with cAMN, correlating with transcriptional and translational changes. Further, using a penalized logistic regression model, we identified the combined methylation levels of SPG20, UNC45A and COL9A3 and also, the combined expression levels of ID4 and MYRF to be good markers capable of discriminating childhood from adult inflammatory phenotypes. We thus propose the hypothesis that an epigenetically controlled, altered transcriptional program may drive an impaired oligodendrocyte differentiation and aberrant immune activation in X-ALD patients. These results shed light into disease pathomechanisms and uncover putative biomarkers of interest for prognosis and phenotypic stratification.

Cancer cells possess aberrant proteomes that can arise by the disruption of genes involved in physiological protein degradation. Here we demonstrate the presence of promoter CpG island hypermethylation-linked inactivation of DERL3 (Derlin-3), a key gene in the endoplasmic reticulum-associated protein degradation pathway, in human tumours. The restoration of in vitro and in vivo DERL3 activity highlights the tumour suppressor features of the gene. Using the stable isotopic labelling of amino acids in cell culture workflow for differential proteome analysis, we identify SLC2A1 (glucose transporter 1, GLUT1) as a downstream target of DERL3. Most importantly, SLC2A1 overexpression mediated by DERL3 epigenetic loss contributes to the Warburg effect in the studied cells and pinpoints a subset of human tumours with greater vulnerability to drugs targeting glycolysis.

Abstract Tumors are dynamic ecosystems where interactions between cancer cells and the tumor microenvironment (TME) shape malignancy. Using spatial transcriptomics, we analyzed 63 untreated tumors from 10 cancer types to investigate the spatial distribution of 13 cancer hallmarks. Our findings reveal that hallmark activity is spatially organized, with distinct compartmentalization of hallmark phenotypes across neoplastic and TME regions. Hallmarks such as "Resisting Cell Death" and "Enabling Replicative Immortality" were enriched in cancer-dominant areas, while "Inducing Angiogenesis" and "Activating Invasion and Metastasis" were primarily localized to the TME. Genomic analysis showed that subclonal diversity influences hallmark specialization, with genetically distinct clones often occupying specific ecological niches. These insights suggest that hallmark activities are not uniformly distributed but instead reflect the interplay between genetic heterogeneity and tumor spatial organization. We further modeled the spatial interdependence of hallmarks between the neoplastic and TME compartments, demonstrating that hallmark activities are shaped by ecological dynamics across the tumor. For example, hallmarks such as "Sustaining Proliferative Signaling" in the TME interface strongly influenced cancer cell hallmark expression. Application of this framework to 33 bladder cancer patients from the DUTRENEO trial revealed correlations between hallmark spatial patterns and treatment response. Notably, differences in the spatial localization of "Inducing Angiogenesis" and "Resisting Cell Death" were linked to neoadjuvant chemotherapy and immunotherapy sensitivity. These findings underscore the importance of spatial hallmark distribution in determining drug efficacy and suggest that spatial hallmark signatures could serve as predictive biomarkers for personalized cancer therapy. Citation Format: Eduard Porta-Pardo, Mustafa Sibai, Sergi Cervilla, Daniela Grases, Manel Esteller. The spatial landscape of cancer hallmarks reveals patterns of tumor ecological dynamics and drug sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB265.

Some but not all neonates are affected by prenatal exposure to serotonin reuptake inhibitor antidepressants (SRI) and maternal mood disturbances. Distinguishing the impact of these 2 exposures is challenging and raises critical questions about whether pharmacological, genetic, or epigenetic factors can explain the spectrum of reported outcomes. Using unbiased DNA methylation array measurements followed by a detailed candidate gene approach, we examined whether prenatal SRI exposure was associated with neonatal DNA methylation changes and whether such changes were associated with differences in birth outcomes. Prenatal SRI exposure was first associated with increased DNA methylation status primarily at <i>CYP2E1</i>(β_Non-exposed = 0.06, β_SRI-exposed = 0.30, FDR = 0); however, this finding could not be distinguished from the potential impact of prenatal maternal depressed mood. Then, using pyrosequencing of <i>CYP2E1</i> regulatory regions in an expanded cohort, higher DNA methylation status—both the mean across 16 CpG sites (<i>P</i> &lt; 0.01) and at each specific CpG site (<i>P</i> &lt; 0.05)—was associated with exposure to lower 3rd trimester maternal depressed mood symptoms only in the SRI-exposed neonates, indicating a maternal mood x SRI exposure interaction. In addition, higher DNA methylation levels at CpG2 (<i>P</i> = 0.04), CpG9 (<i>P</i> = 0.04) and CpG10 (<i>P</i> = 0.02), in the interrogated <i>CYP2E1</i> region, were associated with increased birth weight independently of prenatal maternal mood, SRI drug exposure, or gestational age at birth. Prenatal SRI antidepressant exposure and maternal depressed mood were associated with altered neonatal <i>CYP2E1</i> DNA methylation status, which, in turn, appeared to be associated with birth weight.