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

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

Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation.

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 modeling 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 modeling 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.

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

IκBs exert principal functions as cytoplasmic inhibitors of NF-kB transcription factors. Additional roles for IκB homologues have been described, including chromatin association and transcriptional regulation. Phosphorylated and SUMOylated IκBα (pS-IκBα) binds to histones H2A and H4 in the stem cell and progenitor cell compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unknown. Here, we show that serine 32-36 phosphorylation of IκBα favors its binding to nucleosomes and demonstrate that p-IκBα association with H4 depends on the acetylation of specific H4 lysine residues. The N-terminal tail of H4 is removed during intestinal cell differentiation by proteolytic cleavage by trypsin or chymotrypsin at residues 17-19, which reduces p-IκBα binding. Inhibition of trypsin and chymotrypsin activity in HT29 cells increases p-IκBα chromatin binding but, paradoxically, impaired goblet cell differentiation, comparable to IκBα deletion. Taken together, our results indicate that dynamic binding of IκBα to chromatin is a requirement for intestinal cell differentiation and provide a molecular basis for the understanding of the restricted nuclear distribution of p-IκBα in specific stem cell compartments.

IκBα deficiency imposes a fetal phenotype to intestinal stem cells.

The intestinal epithelium is a paradigm of adult tissue in constant regeneration that is supported by intestinal stem cells (ISCs). The mechanisms regulating ISC homeostasis after injury are poorly understood. We previously demonstrated that IκBα, the main regulator of NF-κB, exerts alternative nuclear functions as cytokine sensor in a subset of PRC2-regulated genes. Here, we show that nuclear IκBα is present in the ISC compartment. Mice deficient for IκBα show altered intestinal cell differentiation with persistence of a fetal-like ISC phenotype, associated with aberrant PRC2 activity at specific loci. Moreover, IκBα-deficient intestinal cells produce morphologically aberrant organoids carrying a PRC2-dependent fetal-like transcriptional signature. DSS treatment, which induces acute damage in the colonic epithelium of mice, results in a temporary loss of nuclear P-IκBα and its subsequent accumulation in early CD44-positive regenerating areas. Importantly, IκBα-deficient mice show higher resistance to damage, likely due to the persistent fetal-like ISC phenotype. These results highlight intestinal IκBα as a chromatin sensor of inflammation in the ISC compartment.

Revisiting ß-Catenin Signaling in T-Cell Development and T-Cell Acute Lymphoblastic Leukemia.

ß-Catenin/CTNNB1 is critical for leukemia initiation or the stem cell capacity of several hematological malignancies. This review focuses on a general evaluation of ß-catenin function in normal T-cell development and T-cell acute lymphoblastic leukemia (T-ALL). The integration of the existing literature offers a state-of-the-art dissection of the complexity of ß-catenin function in leukemia initiation and maintenance in both Notch-dependent and independent contexts. In addition, ß-catenin mutations are screened for in T-ALL primary samples, and it is found that they are rare and with little clinical relevance. Transcriptional analysis of Wnt family members (Ctnnb1, Axin2, Tcf7, and Lef1) and Myc in different publicly available T-ALL cohorts indicates that the expression of these genes may correlate with T-ALL subtypes and/or therapy outcomes.

Genome-Wide Patterns of Sequence Divergence of Protein-Coding Genes Between Drosophila buzzatii and D. mojavensis.

Evolutionary rates for protein-coding genes are determined not only by natural selection but also by multiple genomic factors including mutation rates, recombination, gene expression levels, and chromosomal location. To investigate the joint effects of different genomic determinants on protein evolution, we compared the coding sequences of 9017 single-copy orthologs between 2 cactophilic species from the Drosophila subgenus, Drosophila mojavensis and D. buzzatii, whose genomes have been previously sequenced. We assessed the impact of 7 genomic determinants, that is, chromosome type, recombination, chromosomal inversions, expression breadth, expression level, gene length, and the number of exons, on divergence rates of protein-coding genes to understand patterns of evolutionary variation. Integrative analysis of these factors revealed that 1) X-linked and autosomal genes evolve at significantly different rates in agreement with the faster-X hypothesis, 2) genes located on the dot chromosome and pericentromeric regions have higher divergence rates, 3) genes located at chromosomes with more fixed inversions have higher pairwise divergence than those located at nearly collinear chromosomes, and 4) gene expression patterns can be considered the strongest determinant of protein evolution. In addition, the number of exons and protein length had a significant effect on pairwise divergence at synonymous sites. All in all, our results show the relative importance of each genomic factor on the rates of protein evolution and functional constraint in these 2 cactophilic Drosophila species.

Computational Sequence Analysis of Inversion Breakpoint Regions in the Cactophilic Drosophila mojavensis Lineage.

We investigated rates of chromosomal evolution in Drosophila mojavensis using whole-genome sequence information from D. mojavensis, Drosophila buzzatii, and Drosophila virilis. Drosophila mojavensis is a cactophilic species of the repleta group living under extreme ecological conditions in the deserts of the Southwestern United States and Northwestern México. The genome of D. buzzatii, another member of the repleta group, was recently sequenced and the largest scaffolds anchored to all chromosomes using diverse procedures. Chromosome organization between D. mojavensis and D. buzzatii was compared using MUMmer and GRIMM software. Our results corroborate previous cytological analyses that indicated chromosome 2 differed between these 2 species by 10 inversions, chromosomes X and 5 differed by one inversion each, and chromosome 4 was homosequential. In contrast, we found that chromosome 3 differed by 5 inversions instead of the expected 2 that were previously inferred by cytological analyses. Thirteen of these inversions occurred in the D. mojavensis lineage: 12 are fixed and one of them is a polymorphic inversion previously described in populations from Sonora and Baja California, México. We previously investigated the breakpoints of chromosome 2 inversions fixed in D. mojavensis. Here we characterized the breakpoint regions of the 5 inversions found in chromosome 3 in order to infer the molecular mechanism that generated each inversion and its putative functional consequences. Overall, our results reveal a number of gene alterations at the inversion breakpoints with putative adaptive consequences that point to natural selection as the cause for fast chromosomal evolution in D. mojavensis.

Exploration of the Drosophila buzzatii transposable element content suggests underestimation of repeats in Drosophila genomes.

BACKGROUND: Many new Drosophila genomes have been sequenced in recent years using new-generation sequencing platforms and assembly methods. Transposable elements (TEs), being repetitive sequences, are often misassembled, especially in the genomes sequenced with short reads. Consequently, the mobile fraction of many of the new genomes has not been analyzed in detail or compared with that of other genomes sequenced with different methods, which could shed light into the understanding of genome and TE evolution. Here we compare the TE content of three genomes: D. buzzatii st-1, j-19, and D. mojavensis. RESULTS: We have sequenced a new D. buzzatii genome (j-19) that complements the D. buzzatii reference genome (st-1) already published, and compared their TE contents with that of D. mojavensis. We found an underestimation of TE sequences in Drosophila genus NGS-genomes when compared to Sanger-genomes. To be able to compare genomes sequenced with different technologies, we developed a coverage-based method and applied it to the D. buzzatii st-1 and j-19 genome. Between 10.85 and 11.16 % of the D. buzzatii st-1 genome is made up of TEs, between 7 and 7,5 % of D. buzzatii j-19 genome, while TEs represent 15.35 % of the D. mojavensis genome. Helitrons are the most abundant order in the three genomes. CONCLUSIONS: TEs in D. buzzatii are less abundant than in D. mojavensis, as expected according to the genome size and TE content positive correlation. However, TEs alone do not explain the genome size difference. TEs accumulate in the dot chromosomes and proximal regions of D. buzzatii and D. mojavensis chromosomes. We also report a significantly higher TE density in D. buzzatii and D. mojavensis X chromosomes, which is not expected under the current models. Our easy-to-use correction method allowed us to identify recently active families in D. buzzatii st-1 belonging to the LTR-retrotransposon superfamily Gypsy.

IκBα controls dormancy in hematopoietic stem cells via retinoic acid during embryonic development.

Recent findings suggest that Hematopoietic Stem Cells (HSC) and progenitors arise simultaneously and independently of each other already in the embryonic aorta-gonad mesonephros region, but it is still unknown how their different features are established. Here, we uncover IκBα (Nfkbia, the inhibitor of NF-κB) as a critical regulator of HSC proliferation throughout development. IκBα balances retinoic acid signaling levels together with the epigenetic silencer, PRC2, specifically in HSCs. Loss of IκBα decreases proliferation of HSC and induces a dormancy related gene expression signature instead. Also, IκBα deficient HSCs respond with superior activation to in vitro culture and in serial transplantation. At the molecular level, chromatin regions harboring binding motifs for retinoic acid signaling are hypo-methylated for the PRC2 dependent H3K27me3 mark in IκBα deficient HSCs. Overall, we show that the proliferation index in the developing HSCs is regulated by a IκBα-PRC2 axis, which controls retinoic acid signaling.

Cis inhibition of NOTCH1 through JAGGED1 sustains embryonic hematopoietic stem cell fate.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium (HE) in the aorta- gonads-and mesonephros (AGM) region and reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). The signalling mechanisms that distinguish HSCs from HPCs are unknown. Notch signaling is essential for arterial specification, IAHC formation and HSC activity, but current studies on how Notch segregates these different fates are inconsistent. We now demonstrate that Notch activity is highest in a subset of, GFI1 + , HSC-primed HE cells, and is gradually lost with HSC maturation. We uncover that the HSC phenotype is maintained 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. Forced activation of the NOTCH1 receptor in IAHC activates a hematopoietic differentiation program. Our results indicate that NOTCH1-JAG1 cis-inhibition preserves the HSC phenotype in the hematopoietic clusters of the embryonic aorta.

ß-Catenin activity induces an RNA biosynthesis program promoting therapy resistance in T-cell 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 ß-catenin/CTNNB1 in the evolution of T-cell Acute Lymphoblastic Leukemia (T-ALL) patients and its involvement in therapy resistance. We have identified a specific gene signature that is directly regulated by ß-catenin, TCF/LEF factors and ZBTB33/Kaiso in T-ALL cell lines, which is highly and significantly represented in five out of six refractory patients from a cohort of 40 children with T-ALL. By subsequent refinement of this gene signature, we found that a subset of ß-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 ß-catenin in RNA and protein synthesis in T-ALL and provide in vitro and in vivo experimental evidence that ß-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 ß-catenin inhibitors will enhance chemotherapy response and prevent disease relapse in T-ALL patients.

Gene alterations at Drosophila inversion breakpoints provide prima facie evidence for natural selection as an explanation for rapid chromosomal evolution.

BACKGROUND: Chromosomal inversions have been pervasive during the evolution of the genus Drosophila, but there is significant variation between lineages in the rate of rearrangement fixation. D. mojavensis, an ecological specialist adapted to a cactophilic niche under extreme desert conditions, is a chromosomally derived species with ten fixed inversions, five of them not present in any other species. RESULTS: In order to explore the causes of the rapid chromosomal evolution in D. mojavensis, we identified and characterized all breakpoints of seven inversions fixed in chromosome 2, the most dynamic one. One of the inversions presents unequivocal evidence for its generation by ectopic recombination between transposon copies and another two harbor inverted duplications of non-repetitive DNA at the two breakpoints and were likely generated by staggered single-strand breaks and repair by non-homologous end joining. Four out of 14 breakpoints lay in the intergenic region between preexisting duplicated genes, suggesting an adaptive advantage of separating previously tightly linked duplicates. Four out of 14 breakpoints are associated with transposed genes, suggesting these breakpoints are fragile regions. Finally two inversions contain novel genes at their breakpoints and another three show alterations of genes at breakpoints with potential adaptive significance. CONCLUSIONS: D. mojavensis chromosomal inversions were generated by multiple mechanisms, an observation that does not provide support for increased mutation rate as explanation for rapid chromosomal evolution. On the other hand, we have found a number of gene alterations at the breakpoints with putative adaptive consequences that directly point to natural selection as the cause of D. mojavensis rapid chromosomal evolution.

Sézary syndrome patient-derived models allow drug selection for personalized therapy.

Current therapeutic approaches for Sézary syndrome (SS) do not achieve a significant improvement in long-term survival of patients, and they are mainly focused on reducing blood tumor burden to improve quality of life. Eradication of SS is hindered by its genetic and molecular heterogeneity. Determining effective and personalized treatments for SS is urgently needed. The present work compiles the current methods for SS patient-derived xenograft (PDX) generation and management to provide new perspectives on treatment for patients with SS. Mononuclear cells were recovered by Ficoll gradient separation from fresh peripheral blood of patients with SS (N = 11). A selected panel of 26 compounds that are inhibitors of the main signaling pathways driving SS pathogenesis, including NF-kB, MAPK, histone deacetylase, mammalian target of rapamycin, or JAK/STAT, was used for in vitro drug sensitivity testing. SS cell viability was evaluated by using the CellTiter-Glo_3D Cell Viability Assay and flow cytometry analysis. We validated one positive hit using SS patient-derived Sézary cells xenotransplanted (PDX) into NOD-SCID-γ mice. In vitro data indicated that primary malignant SS cells all display different sensitivities against specific pathway inhibitors. In vivo validation using SS PDX mostly reproduced the responses to the histone deacetylase inhibitor panobinostat that were observed in vitro. Our investigations revealed the possibility of using high-throughput in vitro testing followed by PDX in vivo validation for selective targeting of SS tumor cells in a patient-specific manner.

p53 wild-type colorectal cancer cells that express a fetal gene signature are associated with metastasis and poor prognosis.

Current therapy against colorectal cancer (CRC) is based on DNA-damaging agents that remain ineffective in a proportion of patients. Whether and how non-curative DNA damage-based treatment affects tumor cell behavior and patient outcome is primarily unstudied. Using CRC patient-derived organoids (PDO)s, we show that sublethal doses of chemotherapy (CT) does not select previously resistant tumor populations but induces a quiescent state specifically to TP53 wildtype (WT) cancer cells, which is linked to the acquisition of a YAP1-dependent fetal phenotype. Cells displaying this phenotype exhibit high tumor-initiating and metastatic activity. Nuclear YAP1 and fetal traits are present in a proportion of tumors at diagnosis and predict poor prognosis in patients carrying TP53 WT CRC tumors. We provide data indicating the higher efficacy of CT together with YAP1 inhibitors for eradication of therapy resistant TP53 WT cancer cells. Together these results identify fetal conversion as a useful biomarker for patient prognosis and therapy prescription.

Gut microbiome signatures linked to HIV-1 reservoir size and viremia control.

BACKGROUND: The potential role of the gut microbiome as a predictor of immune-mediated HIV-1 control in the absence of antiretroviral therapy (ART) is still unknown. In the BCN02 clinical trial, which combined the MVA.HIVconsv immunogen with the latency-reversing agent romidepsin in early-ART treated HIV-1 infected individuals, 23% (3/13) of participants showed sustained low-levels of plasma viremia during 32 weeks of a monitored ART pause (MAP). Here, we present a multi-omics analysis to identify compositional and functional gut microbiome patterns associated with HIV-1 control in the BCN02 trial. RESULTS: Viremic controllers during the MAP (controllers) exhibited higher Bacteroidales/Clostridiales ratio and lower microbial gene richness before vaccination and throughout the study intervention when compared to non-controllers. Longitudinal assessment indicated that the gut microbiome of controllers was enriched in pro-inflammatory bacteria and depleted in butyrate-producing bacteria and methanogenic archaea. Functional profiling also showed that metabolic pathways related to fatty acid and lipid biosynthesis were significantly increased in controllers. Fecal metaproteome analyses confirmed that baseline functional differences were mainly driven by Clostridiales. Participants with high baseline Bacteroidales/Clostridiales ratio had increased pre-existing immune activation-related transcripts. The Bacteroidales/Clostridiales ratio as well as host immune-activation signatures inversely correlated with HIV-1 reservoir size. CONCLUSIONS: The present proof-of-concept study suggests the Bacteroidales/Clostridiales ratio as a novel gut microbiome signature associated with HIV-1 reservoir size and immune-mediated viral control after ART interruption. Video abstract.

Host Transcriptome and Microbiota Signatures Prior to Immunization Profile Vaccine Humoral Responsiveness.

The identification of new biomarkers is essential to predict responsiveness to vaccines. We investigated the whole-blood transcriptome and microbiome prior to immunization, in order to assess their involvement in induction of humoral responses two months later. We based our analyses on stool and skin microbiota, and blood transcriptome prior to immunization, in a randomized clinical study in which participants were vaccinated with the MVA-HIV clade B vaccine (MVA-B). We found that the levels of neutralizing antibody responses were correlated with abundance of Eubacterium in stool and Prevotella in skin. In addition, genus diversity and bacterial species abundance were also correlated with the expression of genes involved in B cell development prior to immunization and forecast strong responders to MVA-B. To our knowledge, this is the first study integrating host blood gene expression and microbiota that might open an avenue of research in this field and to optimize vaccination strategies and predict responsiveness to vaccines.

Evolution of intestinal microbiome in a process of faecal microbiota transfer (FMT) in a patient with Clostridioides difficile infection: NGS analysis with different software programs. / Evolución del microbioma intestinal en un proceso de transferencia de microbiota fecal (TMF) en un paciente con infección por Clostridioides difficile: análisis por NGS con diferentes programas bioinformáticos.

INTRODUCTION: Clostridioides difficile infection (CDI) has become a global healthcare challenge due to increases in its incidence and mortality rates. Faecal microbiota transfer (FMT) is postulated as a protocol to prevent CDI recurrence. MATERIAL AND METHODS: A donor faecal sample and patient faecal samples (pre-FMT and post-FMT) were analysed. The r16S gene was amplified and sequenced by NGS, and its diversity and taxonomy composition were examined. RESULTS: Microbial richness increased in post-FMT samples, and the ß diversity studies grouped the samples into two clusters. One included the non-pathological samples (donor and pre-FMT samples), and the other included the pathological sample. The results obtained by Qiime2 and Bioconductor were similar. CONCLUSION: The analysis showed an increase in taxonomic diversity after the FMT, which suggests its usefulness. Moreover, these results showed that standardisation of bioinformatics analysis is key.

Accumulation of Paneth Cells in Early Colorectal Adenomas Is Associated with Beta-Catenin Signaling and Poor Patient Prognosis.

BACKGROUND: Previous studies in mice indicated that Paneth cells and c-Kit-positive goblet cells represent the stem cell niche of the small intestine and colon, respectively, partly by supporting Wnt and Notch activation. Whether these cell populations play a similar role in human intestinal cancer remains unexplored. METHODS: We performed histopathological evaluation and immunohistochemical analysis of early colorectal adenomas and carcinoma adenoma from patients at the Hospital del Mar in Barcelona. We then determined the possible correlation between the different parameters analyzed and with patient outcomes. RESULTS: Paneth cells accumulate in a subset of human colorectal adenomas directly associated with Notch and Wnt/ß-catenin activation. Adenoma areas containing Paneth cells display increased vessel density in the lamina propria and higher levels of the stem cell marker EphB2. In an in-house cohort of 200 colorectal adenoma samples, we also observed a significant correlation between the presence of Paneth cells and Wnt activation. Kaplan-Meier analysis indicated that early adenoma patients carrying Paneth cell-positive tumors display reduced disease-free survival compared with patients with Paneth cell-free lesions. CONCLUSIONS: Our results indicate that Paneth cells contribute to the initial steps of cancer progression by providing the stem cell niche to adenoma cells, which could be therapeutically exploited.

Epigenetic Silencing of Tumor Suppressor miR-124 Directly Supports STAT3 Activation in Cutaneous T-Cell Lymphoma.

Increasing evidence supports a potential role for STAT3 as a tumor driver in cutaneous T-cell lymphomas (CTCL). The mechanisms leading to STAT3 activation are not fully understood; however, we recently found that miR-124, a known STAT3 regulator, is robustly silenced in MF tumor-stage and CTCL cells. OBJECTIVE: We studied here whether deregulation of miR-124 contributes to STAT3 pathway activation in CTCL. METHODS: We measured the effect of ectopic mir-124 expression in active phosphorylated STAT3 (p-STAT3) levels and evaluated the transcriptional impact of miR-124-dependent STAT3 pathway regulation by expression microarray analysis. RESULTS: We found that ectopic expression of miR-124 results in massive downregulation of activated STAT3 in different CTCL lines, which resulted in a significant alteration of genetic signatures related with gene transcription and proliferation such as MYC and E2F. CONCLUSIONS: Our study highlights the importance of the miR-124/STAT3 axis in CTCL and demonstrates that the STAT3 pathway is regulated through epigenetic mechanisms in these cells. Since deregulated STAT3 signaling has a major impact on CTCL initiation and progression, a better understanding of the molecular basis of the miR-124/STAT3 axis may provide useful information for future personalized therapies.

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

Mammalian IκB proteins (IκBs) exert their main function as negative regulators of NF-κB, a central signaling pathway controlling immunity and inflammation. An alternative chromatin role for IκBs has been shown to affect stemness and cell differentiation. However, the involvement of NF-κB in this function has not been excluded. NFKI-1 and IKB-1 are IκB homologs in Caenorhabditis elegans, which lacks NF-κB 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 IκBs in chromatin regulation. Further supporting this possibility (1) we detected NFKI-1 in the nucleus of cells; (2) NFKI-1 and IKB-1 bind to histones and Polycomb proteins, (3) and associate with chromatin in vivo, and (4) mutations in nfki-1 and ikb-1 alter chromatin marks. Based on these results, we propose that ancestral IκB inhibitors modulate Polycomb activity at specific gene subsets with an impact on development.