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Bigas, A.

Publications and source records attributed to Bigas, A..

16 recordsLinked to original sources

SMARCA4 MUTATIONS PROMOTE CHEMOTHERAPY RESISTANCE IN T-ACUTE LYMPHOBLASTIC LEUKEMIA THROUGH ALTERED CHROMATIN REMODELING AT AP1-ASSOCIATED REGULATORY REGIONS

Chromatin remodelers play a critical role in orchestrating chemotherapy responses in cancer cells. Components of the SWI/SNF complex are frequently mutated across cancer types, with the ATPase subunit SMARCA4 among the most commonly altered. In T-cell acute lymphoblastic leukemia (T-ALL), however, SMARCA4 mutations are rare at diagnosis but enriched in relapse samples, suggesting a potential role in therapy resistance. Using isogenic T-ALL cell lines with or without SMARCA4 mutations, we show that mutant SMARCA4 confers resistance to a broad range of chemotherapeutic agents. By combining ATAC-seq and RNA-seq, we demonstrate that SMARCA4-mutant cells display an attenuated transcriptional response to chemotherapy accompanied by impaired remodeling of AP-1-associated regulatory regions linked to stress response. These cells downregulate stress-related genes while maintaining expression programs associated with cellular fitness. Pharmacological inhibition of AP-1 phenocopies this response, further supporting a functional link between SWI/SNF-mediated remodeling at AP-1-associated regulatory regions, AP-1 activity, and chemotherapy sensitivity. Together, our findings indicate that SWI/SNF-mediated remodeling of these regions contributes to an effective chemotherapy-induced stress response, and that its failure provides T-ALL cells with a survival

cancer biology↗

Developmental programming of hematopoietic stem cell dormancy by evasion of Notch signaling

Dormant hematopoietic stem cells (HSCs) are a rare subset of deeply quiescent, metabolically inactive cells that preserve lifelong hematopoietic regeneration. In mice, these cells were thought to arise exclusively after birth in the BM, while fetal liver HSCs were considered uniformly proliferative and rapidly expanding. Here, using H2B-GFP label-retention strategies, we identify a previously unrecognized population of low-dividing HSCs that is specified during embryogenesis and persists into adulthood. We show that this dormant state is established in specific fetal HSCs that evade the activation of Notch signaling within the fetal liver niche, rather than through global loss of pathway activity. Genetic and transient perturbation of Notch signaling reveals a narrow developmental window in during which reduced Notch exposure promotes entry into a durable dormancy program, whereas irreversible pathway disruption compromises long-term function. Notably, transient Notch during fetal development, but not in adult BM, induces HSC quiescence while enhancing long-term repopulation capacity. Together, these findings demonstrate that HSC dormancy is developmentally programmed before birth and identify evasion of Notch signaling as a key mechanism establishing this state, fundamentally revising current models of how long-term regenerative potential is specified.

developmental biology↗

Diverse transcriptomic and mutational patterns but limited functional pathway alterations in patient-derived SS cells

Structured AbstractO_ST_ABSBackgroundC_ST_ABSEradication of SS is hampered by its genetic and molecular heterogeneity. A better understanding of the putative commonalities underlying SS oncogenicity may help to provide more efficient therapeutic strategies against this devastating disease. PurposeThe present work analyzes the whole transcriptome of different patient-derived SS cells to identify expression patterns, functional programs and expressed gene mutations that may provide clues on new therapeutic options for SS patients MethodsMononuclear cells were recovered by Ficoll gradient separation from fresh peripheral blood of SS patients (n=7). Selected pathway-based compounds and the MALT1 inhibitor MI2 were used for in vitro drug sensitivity testing. SS cells viability was evaluated using CellTiter-Glo_3D Cell Viability Assay and flow cytometry analysis. We validated the usefulness of MI2 using patient-derived SS cells xenotransplanted (PDX) into Nod Scid Gamma mice. ResultsIn vitro data indicated that cell lines and primary malignant SS cells all display different sensitivities against specific pathway inhibitors. However, MALT1 inhibition led to a robust effect in vitro that was partially reproduced in the in vivo NSG mice xenograft model. ConclusionOur investigations revealed the actual possibility of inhibiting the downstream TCR signaling complex form by CARD11, BCL10 and MALT1 in SS therapy. Key PointsPatient-derived SS cells are transcriptionally and mutationally heterogeneous but share some common pathway alterations. Inhibition of MALT1 reduces NF-{kappa}B signaling and cell growth in cell lines and patient-derived SS cells.

cancer biology↗

The Interferon gamma Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) requires activation of the pluripotency network and resetting of the epigenome by erasing the epigenetic memory of the somatic state. In female mouse cells, a critical epigenetic reprogramming step is the reactivation of the inactive X chromosome. Despite its importance, a systematic understanding of the regulatory networks linking pluripotency and X-reactivation is missing. Here we reveal the pathways important for iPSC reprogramming and X-reactivation using a genome-wide CRISPR screen. In particular, we discover that activation of the interferon {gamma} (IFN{gamma}) pathway early during reprogramming accelerates pluripotency acquisition and X-reactivation. IFN{gamma} stimulates STAT3 signaling and the pluripotency network and leads to enhanced TET-mediated DNA demethylation, which consequently boosts X-reactivation. We therefore gain a mechanistic understanding of the role of IFN{gamma} in reprogramming and X-reactivation and provide a comprehensive resource of the molecular networks involved in these processes.

genetics↗

Epigenetic modifications driving ground state pluripotency exit require an NF-κB-independent chromatin IκBα function

Maintenance of pluripotency is a multifactorial process in which NF-{kappa}B is a negative regulator. Our previous work identified a chromatin role for I{kappa}B, the master regulator of NF-{kappa}B signaling, that is critical for the proper regulation of various tissue stem cells. Here, we found that I{kappa}B accumulates specifically in the chromatin fraction of pluripotent embryonic stem cells. I{kappa}B depletion does not affect NF-kB-dependent transcription, but causes a profound epigenetic rewiring in pluripotent stem cells, including alterations in H3K27me3, a histone mark catalyzed by Polycomb repression complex 2. Chromatin changes induced by I{kappa}B depletion affect a subset of pluripotency genes and are associated with altered gene transcription. At the cellular level, I{kappa}B-deficient embryonic stem cells are arrested in a naive pluripotency state when cultured in serum/LIF conditions and fail to exit pluripotency under differentiation conditions. By constructing separation-of-function mutants, we show that the effects of I{kappa}B in regulating stem cell pluripotency are NF-{kappa}B-independent, but mainly rely on its chromatin-related function. Taken together, our results reveal a novel mechanism by which I{kappa}B participates in the regulation of the pluripotent state of embryonic stem cells and shed light on the interplay between inflammatory signals and the regulation of pluripotency.

cell biology↗

Separation-of-function mutants reveal the NF-κB-independent involvement of IκBα in the regulation of stem cell and oncogenic programs

We previously demonstrated that the NF-{kappa}B inhibitor I{kappa}B binds the chromatin together with PRC2 to regulate a subset of developmental- and stem cell-related genes. This alternative function has been elusive in both physiological and disease conditions because of the predominant role of I{kappa}B as a negative regulator of NF-{kappa}B. We here uniquely characterize specific residues of I{kappa}B that allow the generation of separation-of-function (SOF) mutants that are defective for either NF-{kappa}B-related (SOF{Delta}NF-{kappa}B) or chromatin-related (SOF{Delta}H2A,H4) activities. Expression of I{kappa}B SOF{Delta}NF-{kappa}B, but not SOF{Delta}H2A/H4, is sufficient to negatively regulate a specific stemness program in intestinal cells, thus rescuing the differentiation blockage imposed by I{kappa}B deficiency. In contrast, full I{kappa}B activity is required for regulating clonogenic/tumor-initiating activity of colorectal cancer cells. Our data indicate that SOF mutants represent an exclusive tool for studying I{kappa}B functions in physiology and disease, and identified I{kappa}B as a robust prognosis biomarker for human cancer.

cell biology↗

IKK1 kinase coordinates BRD4 and JAK/STAT signaling to subvert DNA damage-based anticancer therapy

Activation of the IKK kinase complex has recurrently been linked to colorectal cancer (CRC) initiation and progression. However, identification of downstream effectors other than NF-{kappa}B has remained elusive. Analysis of IKK-dependent substrates after UV-treatment revealed that BRD4 phosphorylation by IKK is required for chromatin-binding dynamics upon damage. Moreover, IKK induces the NF-{kappa}B-dependent transcription of LIF leading to STAT3 activation, association of BRD4 to STAT3 and recruitment to specific target genes. IKK abrogation results in defective BRD4 and STAT3 function leading to irreparable DNA damage and apoptotic cell death upon different stimuli. Simultaneous inhibition of BRAF-dependent IKK activity or BRD4 and the JAK/STAT pathway enhanced the therapeutic potential of 5-FU plus irinotecan in CRC cells, and is curative in a chemotherapy-resistant CRC xenograft model. Coordinated expression of LIF and IKK is a poor prognosis marker for CRC patients. Our data uncover a functional link between IKK, BRD4 and JAK/STAT signaling with clinical relevance.

cancer biology↗

Radical fringe facilitates NOTCH1 and JAG1 cis interactions to sustain Hematopoietic stem cell fate

Hematopoietic stem cells (HSCs) develop within a short time window from the hemogenic endothelium in the aorta- gonads-and mesonephros (AGM) region during embryonic development. The first HSCs reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). The signalling mechanisms that divert HSCs from HPCs are unknown. Notch signaling is essential for arterial specification, IAHC formation and HSC activity, but current studies on how Notch drives these different fates are inconsistent. To determine the role of Notch in the specification of hemogenic endothelium, HSC and/or HPCs, we extensively analyzed Notch dynamics in the period of HSC generation. We defined the expression pattern of Notch signalling molecules at the gene and protein level and established a molecular mechanism that reconcile previous studies demonstrating the loss of HSC activity in NOTCH1, JAG1 and RBPJ null mutants, the enhanced HSC generation by blocking specific Notch activities or the abrogation of emerging HSCs by high Notch activation. We now demonstrate that Notch activity is highest in a subset of Gfi1+ hemogenic endothelial cells and is gradually lost with HSC maturation. We uncover that the HSC phenotype is maintained through loss of Notch activity due to increasing levels of NOTCH1 and JAG1 interactions on the surface of the same cell (cis) that renders the NOTCH1 receptor from being activated. Forcing activation of the NOTCH1 receptor in IAHC cells activates a hematopoietic differentiation program and supports a cis-inhibitory function for JAG1 and NOTCH1. Furthermore, we demonstrate that this cis-inhibitory interaction is enabled by RADICAL FRINGE (RFNG), a glycosyltransferase that enhances the affinity of NOTCH1 to JAG1 in cis. Finally, our results indicate that NOTCH1-JAG1 cis-inhibition is necessary for preserving the HSC phenotype in the hematopoietic clusters of the aorta.

developmental biology↗

IκBα controls dormancy induction in Hematopoietic stem cell development via retinoic acid

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-{kappa}B has been implicated in the development of HSCs. Here, we identify a chromatin binding function for I{kappa}B (also known as the inhibitor of NF-{kappa}B) 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 I{kappa}B 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. I{kappa}B 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 I{kappa}B - deficient aortic endothelium and E14.5 FL LT-HSCs. Additionally, I{kappa}B binding itself is found in the promoters of retinoic acid receptors rar in the AGM, and rar{gamma} in the LT-HSC of FL. Overall, we demonstrate that the retinoic acid pathway is over-activated in the hematopoietic clusters of I{kappa}B-deficient AGMs leading to premature dormancy of LT-HSCs that persists in the FL LT-HSCs.

cell biology↗

Dissecting infant leukemia developmental origins with a hemogenic gastruloid model

Current in vitro models of developmental blood formation lack spatio-temporal accuracy and weakly replicate successive waves of hematopoiesis. Herein, we describe a mouse embryonic stem cell (SC)-derived 3D hemogenic gastruloid (haemGx) that captures multi-wave blood formation, progenitor specification from hemogenic endothelium (HE), and generates hematopoietic progenitors capable of short-term engraftment of immunodeficient mice upon maturation in an in vivo niche. We took advantage of the haemGx model to interrogate the origins of infant acute myeloid leukemia (infAML). We focused on MNX1-driven leukemia, representing the commonest genetic abnormality unique to the infant group. Enforced MNX1 expression in haemGx promotes the expansion and in vitro transformation of yolk sac-like erythroid-myeloid progenitors at the HE-to-hematopoietic transition to faithfully recapitulate patient transcriptional signatures. By combining phenotypic, functional and transcriptional profiling, including at the single-cell level, we establish the haemGx as a useful new model for the study of normal and leukemic embryonic hematopoiesis.

developmental biology↗

β-catenin activity induces an RNA biosynthesis program promoting therapy resistance in T Acute Lymphoblastic Leukemia

Understanding the molecular mechanisms that contribute to the appearance of chemotherapy resistant cell populations is necessary to improve cancer treatment. We have now investigated the role of {beta}-catenin/CTNNB1 in the evolution of T-Acute Lymphoblastic Leukemia (T-ALL) patients and its involvement in therapy resistance. We have identified a specific gene signature that is directly regulated by {beta}-catenin, TCF/LEF factors and ZBTB33/Kaiso in T-ALL cell lines, which is highly and significantly represented in 5 out of 6 refractory patients from a cohort of 40 children with T-ALL. By subsequent refinement of this gene signature, we found that a subset of {beta}-catenin target genes involved with RNA-processing function are sufficient to segregate T-ALL refractory patients in three independent cohorts. We demonstrate the implication of {beta}-catenin in RNA and protein synthesis in T-ALL and provide experimental evidence that {beta}-catenin is crucial for the cellular response to chemotherapy, mainly in the cellular recovery phase after treatment. We propose that combination treatments involving chemotherapy plus {beta}-catenin inhibitors will enhance chemotherapy response and prevent disease relapse in T-ALL patients.

cancer biology↗

Chemotherapy induces a YAP1-dependent fetal conversion to human Colorectal Cancer cells that is predictive of poor patient outcome

Current therapy against colorectal cancer is based on DNA-damaging agents that eradicate highly proliferative malignant cells. Whether sublethal chemotherapy affects tumor cell behavior and impacts on patient outcome is primarily unstudied. We now show that sublethal chemotherapy imposes a quiescent-like state to p53 wildtype human colorectal cancer (CRC) cells that is linked to the acquisition of a fetal phenotype downstream of YAP1, similar to that observed after intestinal damage. CRC cells displaying this fetal phenotype exhibit tumor- initiating activity comparable to untreated cells but superior metastatic capacity. Notably, nuclear YAP1 accumulation, or detection of the fetal signature in tumors predict poor prognosis in CRC patients carrying p53 wildtype tumors. Collectively, our results uncover a potential adverse response of tumor cells to suboptimal chemotherapy, and identify nuclear YAP1 and fetal conversion of colorectal tumors as biomarkers for prognosis and therapy prescription. Statement of significanceChemotherapy induces a quiescent-like phenotype to colorectal cancer cells that is linked to the acquisition of a YAP1-dependent fetal signature. Notably, this signature is predictive of patient outcome in different cohorts of human colorectal cancer.

cancer biology↗

Dynamic association of IκBα to chromatin is regulated by acetylation and cleavage of histone H4

I{kappa}Bs exert a principal function as cytoplasmic inhibitors of the NF-kB transcription factors. Additional functions for I{kappa}B homologues have been described including association to chromatin and transcriptional regulatioin. Phosphorylated and SUMOylated I{kappa}B (pS-I{kappa}B) binds histones H2A and H4 in the stem and progenitor compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unstudied. We here show that serine 32-36 phosphorylation of I{kappa}B favors its binding with nucleosomes and demonstrated that p-I{kappa}B association to H4 is favored by acetylation at specific H4 lysine residues. N-terminal tail of H4 is lost during intestinal cell differentiation by proteolytic cleavage at residues 17-19 imposed ny trypsin or chymotrypsin, which interferes p-I{kappa}B binding. Paradoxically, inhibition of trypsin and chymotrypsin activity in HT29 cells increased p-I{kappa}B chromatin binding and impaired goblet cell differentiation, comparable to I{kappa}B deletion. Together our results indicate that dynamic binding of I{kappa}B to chromatin is a requirement for intestinal cell differentiation and provide a molecular base for the restricted nuclear distribution of p-I{kappa}B at specific stem cell compartments.

molecular biology↗

A novel role of MNT as a negative regulator of REL and the NF-κB pathway

MNT, a transcription factor of the MXD family, is an important modulator of the oncoprotein MYC. Both MNT and MYC are basic-helix-loop-helix proteins that heterodimerize with MAX in a mutually exclusive manner, and bind to E-boxes within regulatory regions of their target genes. While MYC generally activates transcription, MNT represses it. However, the molecular interactions involving MNT as a transcriptional regulator beyond the binding to MAX remain unexplored. Here we demonstrate a novel MAX-independent protein interaction between MNT and c-REL (REL), the oncogenic member of the REL/NF-{kappa}B family. REL is involved in important biological processes and it is found altered in a variety of tumors. REL is a transcription factor that remains inactive in the cytoplasm in an inhibitory complex with I{kappa}B and translocates to the nucleus when the NF-{kappa}B pathway is activated. In the present manuscript, we show that MNT knockdown triggers REL translocation into the nucleus and thus the activation of the NF-{kappa}B pathway. Meanwhile, MNT overexpression results in the repression of I{kappa}B, a bona-fide REL target. Indeed, both MNT and REL bind to the I{kappa}B gene at a region mapping in the first exon, suggesting its regulation as a MNT-REL complex. Altogether our data indicate that MNT acts as a repressor of the NF-{kappa}B pathway by two different mechanisms: 1) retention of REL in the cytoplasm by MNT protein interaction and 2) MNT-driven repression of REL-target genes through a MNT-REL complex. These results widen our knowledge about MNT biological roles and reveal a novel connection between the MYC/MXD and the NF-{kappa}B pathways, two of the most prominent pathways involved in cancer.

cancer biology↗

Ancestral function of Inhibitors-of-kappaB regulates Caenorhabditis elegans development

Mammalian I{kappa}B proteins (I{kappa}Bs) exert their main function as negative regulators of NF-{kappa}B, a central signaling pathway controlling immunity and inflammation. An alternative chromatin role for I{kappa}Bs has been shown to affect stemness and cell differentiation. However, the involvement of NF-{kappa}B in this function has not been excluded. NFKI-1 and IKB-1 are I{kappa}B homologs in Caenorhabditis elegans, which lacks NF-{kappa}B nuclear effectors. We found that nfki-1 and ikb-1 mutants display developmental defects that phenocopy mutations in Polycomb and UTX-1 histone demethylase, suggesting a role for C. elegans I{kappa}Bs in chromatin regulation. Further supporting this possibility (i) we detected NFKI-1 in the nucleus of cells; (ii) NFKI-1 and IKB-1 bind to histones and Polycomb proteins, (iii) and associate with chromatin in vivo, and (iv) mutations in nfki-1 and ikb-1 alter chromatin marks. Based on these results, we propose that ancestral I{kappa}B inhibitors modulate Polycomb activity at specific gene subsets with an impact on development.

developmental biology↗

Notch ligand Dll4 impairs cell recruitment into aortic clusters and limits hematopoietic stem cells

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in cluster structures that protrude into the embryonic aortic lumen. Although much is known about the molecular characteristics of the developing hematopoietic cells, we lack a complete understanding of their origin and the three-dimensional organization of the niche. Here we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modelling to show the two-step process of intra-aortic hematopoietic cluster (IACH) formation. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the IAHC. Next, surrounding hemogenic cells are recruited into the IAHC, increasing their size and heterogeneity. We identified the Notch ligand Dll4 as a negative regulator of the recruitment phase of IAHC. Blocking of Dll4 promotes the entrance of new hemogenic Gfi1+ cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modelling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions.

developmental biology↗