Publications

Supplementary Information from A Tumor Suppressor Enhancer of <i>PTEN</i> in T-cell Development and Leukemia

<p>Supplementary Figures and Tables combined</p>

Epigenetic loss of the transfer RNA-modifying enzyme TYW2 induces ribosome frameshifts in colon cancer

Significance Defects in transfer RNA (tRNA) modifications occur in human pathologies such as cancer; however, how these alterations contribute to the disease is poorly understood. One example is the tumor-specific hypomodification of position 37 of tRNA Phe , which was first described 45 y ago, although its cause and consequences have remained unknown. Here we report that the tRNA Phe hypomodification is due to promoter CpG island hypermethylation-associated transcriptional silencing of TYW2, a key enzyme in the synthesis of wybutosine derivatives. Furthermore, epigenetic loss of TYW2 in transformed cells provokes hypomodified tRNA Phe -mediated ribosome frameshifting, dysregulating mRNA abundance via nonsense-mediated decay. Importantly, TYW2 silencing in cancer cells confers enhanced migration and epithelial-to-mesenchymal features that are associated in early-stage colorectal cancer patients with poor clinical outcome.

Epigenetic <i>SMAD3</i> Repression in Tumor-Associated Fibroblasts Impairs Fibrosis and Response to the Antifibrotic Drug Nintedanib in Lung Squamous Cell Carcinoma

Abstract The tumor-promoting fibrotic stroma rich in tumor-associated fibroblasts (TAF) is drawing increased therapeutic attention. Intriguingly, a trial with the antifibrotic drug nintedanib in non–small cell lung cancer reported clinical benefits in adenocarcinoma (ADC) but not squamous cell carcinoma (SCC), even though the stroma is fibrotic in both histotypes. Likewise, we reported that nintedanib inhibited the tumor-promoting fibrotic phenotype of TAFs selectively in ADC. Here we show that tumor fibrosis is actually higher in ADC-TAFs than SCC-TAFs in vitro and patient samples. Mechanistically, the reduced fibrosis and nintedanib response of SCC-TAFs was associated with increased promoter methylation of the profibrotic TGFβ transcription factor SMAD3 compared with ADC-TAFs, which elicited a compensatory increase in TGFβ1/SMAD2 activation. Consistently, forcing global DNA demethylation of SCC-TAFs with 5-AZA rescued TGFβ1/SMAD3 activation, whereas genetic downregulation of SMAD3 in ADC-TAFs and control fibroblasts increased TGFβ1/SMAD2 activation, and reduced their fibrotic phenotype and antitumor responses to nintedanib in vitro and in vivo. Our results also support that smoking and/or the anatomic location of SCC in the proximal airways, which are more exposed to cigarette smoke particles, may prime SCC-TAFs to stronger SMAD3 epigenetic repression, because cigarette smoke condensate selectively increased SMAD3 promoter methylation. Our results unveil that the histotype-specific regulation of tumor fibrosis in lung cancer is mediated through differential SMAD3 promoter methylation in TAFs and provide new mechanistic insights on the selective poor response of SCC-TAFs to nintedanib. Moreover, our findings support that patients with ADC may be more responsive to antifibrotic drugs targeting their stromal TGFβ1/SMAD3 activation. Significance: This study implicates the selective epigenetic repression of SMAD3 in SCC-TAFs in the clinical failure of nintedanib in SCC and supports that patients with ADC may benefit from antifibrotic drugs targeting stromal TGFβ1/SMAD3.

Supplementary Figure S1 from Targeted Exome Sequencing of Krebs Cycle Genes Reveals Candidate Cancer–Predisposing Mutations in Pheochromocytomas and Paragangliomas

&lt;p&gt;(A) Quantitative PCR analysis of RBP1 expression of the 49 tumors included in the study compared to controls comprising five tumors carrying mutation in Krebs cycle genes and five cases carrying mutations in RET or NF1. RU: relative units. (B) SDHB immunohistochemistry of tumor_4 compared to a positive control tumor carrying a non-SDH mutation. The scale bars represent 50μm. (C) Representation of DNA methylation (M_values) of the CpG island probes located within the SDHC locus of tumor_4 compared to a blood sample from the same patient and a control DNA. (D) Immunohistochemical staining of 5-hmC in IDH3B-mutated tumor. Nuclear 5-hmC was observed only in sustentacular and some stromal cells. The scale bar represents 50μm.&lt;/p&gt;

Prolonged cell cycle arrest by the CDK4/6 antagonist narazaciclib restores ibrutinib response in preclinical models of BTKi‐resistant mantle cell lymphoma

Introduction: Bruton tyrosine kinase inhibitors (BTKi) have transformed the therapeutic landscape of mantle cell lymphoma (MCL); however, primary and acquired resistance to these agents remains a challenge. Previous studies have suggested that narazaciclib (ON123300), a second-generation, orally bioavailable and clinical-stage CDK4/6 inhibitor (CDKi), may trigger cell cycle arrest and significant tumor growth inhibition (TGI) in BTKi-resistant MCL models. Methods: We compared the efficacy and safety of narazaciclib with the health authority approved CDKi in association with different BTKi, in a panel of 10 MCL cell lines with distinct sensitivity to the first-in-class BTKi, ibrutinib. We used the CellTiter-Glo proliferation assay, FACS-mediated quantification of cell cycle and apoptosis, and RNA sequencing followed by gene set enrichment analysis (GSEA), RT-PCR and western blot validations. In addition, we evaluated the safety and efficacy of narazaciclib/ibrutinib combo in vivo in an immune-competent chicken embryo chorioallantoic membrane (CAM) MCL xenograft. Results: Narazaciclib exhibited the highest antitumor activity among MCL cell lines (mean IC50: 3.61 ± 2.1 µM), regardless of their sensitivity to ibrutinib. Although there was no correlation between CDKi sensitivity and activation of the CDK4/CDK6-pRb pathway in MCL, transcriptomic and phenotypic analyses revealed a predominant downregulation of E2F target genes and G2/M checkpoint response (NES > 2.5) upon narazaciclib treatment. This feature was associated to intracellular accumulation of p21, p16, and phospho-p27, decreased mitotic index, G1 cell cycle blockade, and apoptosis onset. When combined with ibrutinib, but not with the second generation therapeutic acalabrutinib, narazaciclib achieved significant synergistic antitumor activity in both BTK-sensitive and BTK-resistant cells. The combination was not associated with improved apoptosis, but rather with a slight but constant (+10%–15%) augmentation in G1 phase blockade and the down-modulation of cell cycle-associated transcriptome. Both the downregulation of phospho-histone H3 and the upregulation of p-p27/p27 and p16, also underwent a 10%–15% improvement in combination-treated cells. In vivo, while narazaciclib single agent achieved a 28% TGI in the CAM model, the narazaciclib-ibrutinib combination reduced tumor spreading by 65% and allowed a 50% reduction in malignant B cell infiltration into the bone marrow, with no detectable toxicity. Conclusions: Narazaciclib, due to its completely distinct MoA from BTKi involving the direct modulation of the cell cycle, can achieve significant synergistic activity with ibrutinib in vitro and in vivo, especially in BTKi-resistant models of MCL. Ongoing phospho-proteomics and genetic edition assays will help deciphering the molecular bases of this unique drug cooperation at the cell cycle level. Encore Abstract - previously submitted to AACR 2023 The research was funded by: This study was financially supported by Onconova Therapeutics, Spanish Ministry of Science and Innovation (grant PID2021-123039OB-C21) (to GR). MFS holds a predoctoral fellowship (FI19/00338) from Instituto de Salud Carlos III. This work was carried out under the CERCA Program (Generalitat de Catalunya). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Combination Therapies, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. A. Makovski-Silverstein Employment or leadership position: Onconova Therapeutics S. Cosenza Employment or leadership position: Onconova Therapeutics G. Roué Research funding: Onconova Therapeutics

Figure S1 from Single-cell Multiomics Analysis of Myelodysplastic Syndromes and Clinical Response to Hypomethylating Therapy

&lt;p&gt;Mutational burden at diagnosis across patients and correlation between single-cell and bulk VAFs&lt;/p&gt;

Genetic predisposition to Alzheimer’s disease is differentially associated with joint volumetric variations in the disease continuum

Abstract Background Imaging genetics studies commonly assess the individual association between genetic information and brain features without considering joint brain modulations. The aim of this study was to explore the structural variation of the brain at different stages of Alzheimer’s disease (AD), depending on the genetic predisposition to AD. Method A total of 351 cognitively unimpaired (CU) middle‐age participants from the ALFA (Alzheimer’s and FAmilies) study and 330 cognitively impaired participants from ADNI (Alzheimer’s Disease Neuroimaging Initiative) with magnetic resonance imaging scans, genetic information and CSF‐amyloid status were included. They were classified as CU Aβ‐, CU Aβ+, (mild cognitive impairment) MCI Aβ+ and AD Aβ+[ Table 1 ]. Freesurfer was used to obtain cortical and subcortical parcellations using the Desikan‐Killiany atlas. A polygenic risk score estimating each participant’s genetic predisposition to AD (PRS‐AD) was calculated including genetic variants at the genome‐wide suggestive level (5×10‐6). Individuals were classified into high/low genetic predisposition groups. Statistical analysis was based on compositional data analysis. First, we computed the optimal multivariate brain structural variation signature (elastic net selection). Then, a logistic regression was performed to determine the association between the optimal signature and the PRS‐AD. Models were stratified by disease stage and adjusted for age and sex. Result Individuals at different stages on the AD continuum displayed different brain volumetric modulations associated with higher genetic predisposition to AD [ Figure 1 ]. For instance, the optimal signature associated with an increased risk of AD was mainly characterized by increased hippocampal volumes in Aβ‐ individuals, but decreased volumes in MCI individuals along with the modulation of other temporal regions. Moreover, in AD patients at higher genetic predisposition of AD, the optimal signature was characterized by increased and decreased volumes in frontal regions (e.g. pars triangularis, lateral orbitofrontal vs. medial orbitofrontal, respectively) [ Figure 2 ]. Conclusion Results showed AD stage‐specific multivariate volumetric variation associated with an increased genetic risk of AD. Main results suggested that regions that modulated together, although differing according to AD stage, were implicated in the structural brain topology of specific memory networks. The analysis of the joint volumetric variation of brain subregions brings an innovative modeling perspective for neurogenetic studies of AD.

Additional file 2: Table S2. of Cancer network activity associated with therapeutic response and synergism

Normalized CNA values for all cancer cell lines. (XLSX 55 kb)

Supplementary data from Orthoxenografts of Testicular Germ Cell Tumors Demonstrate Genomic Changes Associated with Cisplatin Resistance and Identify PDMP as a Resensitizing Agent

&lt;p&gt;Supplementary materials and methods, supplementary tables and supplementary figure legends&lt;/p&gt;

Abnormal PcG protein expression in Hodgkin's lymphoma. Relation with E2F6 and NFκB transcription factors

The Polycomb group (PcG) of proteins comprises a family of repressors of homeobox genes that play key roles in body formation, haematopoiesis and cell cycle control. In this study, a large-scale analysis of PcG protein expression (BMI1, MEL18, PH1, RNF2, RING1, and RYBP) was performed in 321 Hodgkin's lymphoma (HL) biopsies and in reactive lymphoid tissues using tissue microarrays. The relevance of PcG proteins in HL was also investigated by the simultaneous analysis of PcG and other proteins involved in the control of cell cycle, transcription machinery and lymphoid differentiation. The analysis revealed increased expression of a set of PcG proteins (particularly RYBP and BMI1) in tumour cells in comparison with reactive lymphoid tissue. One of the most striking findings was anomalous RYBP expression in 55% of classical HL cases associated with an unfavourable response to treatment and shorter survival. The data obtained in this study also show an association of PcG proteins with E2F6 and NFkappaB transcription factors. The statistical relationship between PcG and NFkappaB activation was further explored in HL-derived cell lines treated with curcumin, an NFkappaB inhibitor, and TNFalpha. Up- or downregulation of MEL18 was paralleled by loss or gain of activated NFkappaB, which suggests that NFkappaB may regulate expression of this protein. Investigation of the relationship between E2F6 and RING1 by immunofluorescence and confocal analysis, in HL cell lines and paraffin sections, revealed co-expression of both proteins in the same tumour cells. These results allow us to propose that the formation of transcription complexes with E2F6 may modify the functional status of PcG proteins in HSR cells.

Differential effects of tofacitinib on macrophage activation contribute to lack of response in ulcerative colitis patients

Abstract Background and Aims Tofacitinib, a Janus kinase inhibitor, is approved for the treatment of moderate-to-severe ulcerative colitis. Nonetheless, 40-60% of patients will not respond adequately. The mechanisms underlying responses to tofacitinib remain unknown. Methods We applied single-cell and/or bulk RNA analysis to biopsies (n=23 and 63, respectively) from ulcerative colitis patients (n= 31) before and after tofacitinib treatment. Response was assessed using endoscopic and clinical criteria. In vitro-derived macrophages and primary intestinal fibroblasts were used to validate our findings. Results Forty percent of patients responded to tofacitinib. Responders exhibited higher baseline JAK-STAT activity, while non-responders had increased baseline NF-kB pathway activation. Response was associated with significant changes in the abundance and/or activation of immune, epithelial, and stromal cells and the downregulation of S100A9, FCGR3A, MMP12 in resident macrophages. In contrast, non-responders showed a significant increase in the number and activation of macrophages and fibroblasts following tofacitinib treatment, including upregulation of MMP9, IL1B, IL6, CXCL1, CXCL8 and S100A9 compared to baseline. In monocyte-derived macrophages tofacitinib drove the hyperactivation of macrophages in response to lipopolysaccharide, but not TNF or IFNγ. This effect is dependent on the inhibition of IL-10 signaling, which is abundantly induced in response to LPS, but not to TNF or IFNγ. In contrast, cultured fibroblasts, which produced no IL-10 regardless of the stimuli, showed no hyperactivation when pre-treated with tofacitinib. Conclusions We conclude that resistance to tofacitinib is mediated by the hyperactivation of myeloid cells and we identify IL-10-dependent macrophages as one cellular subset contributing to this resistance.

Supplementary Table 1 from CD137 (4-1BB) Costimulation Modifies DNA Methylation in CD8&lt;sup&gt;+&lt;/sup&gt; T Cell–Relevant Genes

&lt;p&gt;DNA methylation array results with urelumab&lt;/p&gt;

P695: SINGLE-CELL MULTIOMICS ANALYSIS OF MYELODYSPLASTIC SYNDROME PREDICTS CLINICAL RESPONSE TO TREATMENT WITH DNA DEMETHYLATION AGENT

Background: Epigenetic defects, such as DNA methylation alterations, are a common feature of human malignancies. This finding has supported the use of hypomethylating agents (HMAs) as potential agents to treat cancer patients, being accepted the clinical approval of these drugs in the treatment of myeloid malignancies as one of the great accomplishments of this research area. The best case is myelodysplastic syndrome (MDS), a pre-leukemia disease that can progress to acute myeloid leukemia (AML), where limited treatment repertoire was available until the emergence of HMAs. However, it has not been elucidated how we can predict if an MDS patient will be a responder or non-responder to the epigenetic compound and the duration of the effect. Using the awesome analytical resolution of the recently developed single-cell methodologies, we have herein addressed this point. Aims: To employ novel single-cell technologies to characterize mutational and immunophenotypic profiles that predict the response to HMAs and the duration of their effect in MDS patients. Methods: To evaluate the evolution of the clonal cellular and molecular composition of MDS upon the therapy with HMAs, we carried out single-cell DNA sequencing (scDNA-seq) of an amplicon panel for 53 genes frequently mutated in myeloid malignancies and single-cell protein sequencing (scProt-seq) of 45 cell-surface proteins to yield a simultaneous profile of the genetic context and immunophenotype. We sequenced hundreds of thousands of cells from these MDS samples where paired bone marrow samples were analyzed at the time of diagnosis and at the end of at least six cycles of azacitidine use. Results: The aforementioned single-cell multiomics approach led us to identify not only genetic mutations present in bone marrow samples and their clonal evolution throughout the course of azacitidine treatment, but also the cell types that harbor such mutations, which were defined by a unique combination of surface markers. More precisely, we proved the co-occurrence of particular missense, nonsense and frameshift mutations in the same myeloid progenitor cell and the linear or multibranched development of the mutant clones depending on the patient. Furthermore, we observed the persistence of cell lineages with specific mutations (such as non-classical monocytes and CD8+ effector T-cells) after the treatment with this DNA demethylation agent. Summary/Conclusion: Overall, our work shows that the co-occurrence of particular truncating/stop codon mutations in the same myeloid progenitor cell; the linear or multibranched evolution of the mutant clones; and the persistence of unique cell lineages with specific mutational profiles can predict if HMA treatment will provoke a clinical response in MDS patients. Keywords: Immunophenotype, Hypomethylating agents, Myelodysplastic syndrome, Somatic mutation

DNA methylation-associated dysregulation of transfer RNA expression in human cancer

Bromodomain inhibitors and cancer therapy: From structures to applications

Aberrations in the epigenetic landscape are a hallmark of cancer. Alterations in enzymes that are “writers,” “erasers,” or “readers” of histone modification marks are common. Bromodomains are “readers” that bind acetylated lysines in histone tails. Their most important function is the regulation of gene transcription by the recruitment of different molecular partners. Moreover, proteins containing bromodomains are also epigenetic regulators, although little is known about the specific function of these domains. In recent years, there has been increasing interest in developing small molecules that can target specific bromodomains. First, this has helped clarify biological functions of bromodomain-containing proteins. Secondly, it opens a new front for combatting cancer. In this review we will describe the structures and mechanisms associated with Bromodomain and Extra-Terminal motif (BET) inhibitors and non-BET inhibitors, their current status of development, and their promising role as anti-cancer agents.

Figure S8 from Single-cell Multiomics Analysis of Myelodysplastic Syndromes and Clinical Response to Hypomethylating Therapy

&lt;p&gt;Distribution of WT and mutant cells per gene&lt;/p&gt;

Supplementary Figure 2 from A Novel Epigenetic Signature for Early Diagnosis in Lung Cancer

&lt;p&gt;Epigenetic and expression analysis based on histological subtypes from primary tissues of the TCGA database.&lt;/p&gt;

Additional file 1: of Epigenomic analysis detects aberrant super-enhancer DNA methylation in human cancer

Comparison of spatial transcriptomics technologies across six cancer types

Abstract Spatial biology experiments integrate the molecular and histological landscape of tissues to provide a previously inaccessible view of tissue biology, unlocking the architecture of complex multicellular tissues. Within spatial biology, spatial transcriptomics platforms are among the most advanced, allowing researchers to characterize the expression of thousands of genes across space. These new technologies are transforming our understanding of how cells are organized in space and communicate with each other to determine emergent phenotypes with unprecedented granularity. This is particularly important in cancer research, as it is becoming evident that tumor evolution is shaped not only by the genetic properties of cancer cells but also by how they interact with the tumor microenvironment and their spatial organization. While many platforms can generate spatial transcriptomics profiles, it is still unclear in which context each platform better suits the needs of its users. Here we compare the results obtained using 4 different spatial transcriptomics (VISIUM, VISIUM CytAssist, Xenium and CosMx) and one spatial proteomics (VISIUM CytAssist) platforms across serial sections of 6 FFPE samples from primary human tumors covering some of the most common forms of the disease (lung, breast, colorectal, bladder, lymphoma and ovary). We observed that the VISIUM platform with CytAssist chemistry yielded superior data quality. Xenium consistently produced more reliable results for in situ platforms, with better gene clustering and fewer false positives than CosMx. Interestingly, these platform-based variations didn’t significantly affect cell type identification. Finally, by comparing VISIUM protein profiles with the spatial transcriptomics data from all four platforms on each sample, we identified several genes with mismatched RNA and protein expression patterns, highlighting the importance of multi-omics profiling to reveal the true biology of human tumors.